Designed and printed by AIMPRINT 01799 510101 2006 European Fission Yeast Meeting

Wellcome Trust Conference Centre, Hinxton, UK

16-18 March 2006 Organizers:

Jürg Bähler, Sanger Institute, Hinxton, UK Valerie Wood, Sanger Institute, Hinxton, UK Mitsuhiro Yanagida, Kyoto University, Japan Paul Nurse, Rockefeller University, USA

Cover picture: Photo mosaic of double helix made from various S. pombe pictures (J. Bähler)

i Sponsors Schedule Overview Main sponsor: Wellcome Trust (http://www.wellcome.ac.uk/) Thu 16th March We thank the following organizations and companies for their help with from 1400 Registration sponsoring this meeting: Cancer Research UK ( h t t p : / / s c i e n c e . c a n c e rre s e a rc h u k . o rg / ) 1500-1630 Reception 1630-1805 Session 1 Biobase ( h t t p : / / w w w. b i o b a s e . d e / ) 1805-2000 Dinner 2000-2130 Session 2 The Genetics Society ( h t t p : / / w w w. g e n e t i c s . o rg . u k / ) Discussion Session: Resources for the The Journal Yeast Fission Yeast ( h t t p : / / w w w 3 . i n t e r s c i e n c e . w i l e y. c o m / Community c g i - b i n / j h o m e / 3 8 9 5 ) Sat 18th March Blackwell Publishing ( h t t p : / / w w w. b l a c k w e l l p u b l i s h i n g . c o m / ) from 0800 Breakfast

Singer Instruments Fri 17th March 0930-1100 Session 6 ( h t t p : / / w w w. s i n g e r i n s t . c o . u k / ) 1100-1130 Break Springer from 0730 Breakfast 1130-1300 Session 7 (http://www.springer.com/) 0900-1030 Session 3 1300-1400 Lunch 1030-1100 Break 1100-1230 Session 4 1230-1400 Lunch 1400-1530 Session 5 1530-1600 Break 1600-1900 Poster Session 1900-1930 Pre-dinner drinks 1930 Banquet Dinner from 2130 Entertainment with IMMposters

ii iii Programme Thu 16th March, 2000-2130 Fri 17th March, 0900-1030 Fri 17th March, 1100-1230 Session 2: DNA Metabolism II Session 3: Environmental Responses Session 4: Gene Expression Control: Chairs: Tony Carr, Julie Cooper, & Signalling From Chromatin to Proteins Thu 16th March, 1630-1805 Paul Russell Chairs: Paul Young, Olaf Nielsen Chairs: Nic Jones, Karl Ekwall Introduction: Jürg Bähler, Val Wood Anna Hebden (Cooper lab, Søren Kjærrulff Introduction by Nic Jones Session 1: DNA Metabolism I Cancer Research UK) T7 (Nielsen lab, University of (Paterson Institute, UK) Chairs: Tony Carr, Julie Cooper, Surviving DNA damage with dys Copenhagen, Denmark) T10 Paul Russell functional telomeres Cdk-phosphorylation of the HMG- Daniel Lackner domain protein Ste11 controls the (Bähler lab, Zoi Lygerou Edgar Hartsuiker switch from mitosis to meiosis in Sanger Institute, UK) T16 (University of Patras, Greece) T1 (University of Sussex, UK) T8 fission yeast Genome-wide translational control in A stochastic hybrid model for The role of the S. pombe MRN fission yeast DNA replication complex in the removal of covalently Neil Bone linked protein from the DNA (Armstrong lab, Karl Ekwall Chris Norbury University of Sussex, UK) T11 (Karolinska Institute, Sweden) T17 (University of Oxford, UK) T2 Ramsay McFarlane Vtc4p: a regulator of vacuolar size Genome wide roles for histone Involvement of Cid1 poly(A) (University of Wales Bangor, UK) T9 and trafficking in S. pombe modifications and RNAi directed polymerase and the sub-telomeric S. pombe meiotic linear elements: chromatin silencing rqh2+ DNA helicase gene in a the relationship with pre-meiotic Paul Young common pathway following DNA replication and meiotic (Queen’s University, Canada) T12 Anthony Wright inhibition of DNA replication genetic recombination NatB N·-acetylation activity is (Karolinska Institute, Sweden) T18 required for Bsu1p pyridoxine Comparative studies of molecular Benoit Arcangioli transporter function in S. pombe mechanisms that program (Pasteur Institute, France) T3 Discussion Session: transcription The essential role of the homologous Resources for the Fission Yeast Clare Lawrence recombination for replication fork Community (Jones lab, Assen Roguev restart Chairs: Paul Nurse, Mitsuhiro Yanagida Paterson Institute, UK) T13 (Stewart lab, Regulation of S. pombe Atf1 protein The University of Technology Ken’ichi Mizuno Jacky Hayles levels by Sty1-mediated Dresden, Germany) T19 (Carr lab, (Cancer Research UK) phosphorylation and Comparative proteomic analysis of University of Sussex, UK) T4 Status of the S. pombe deletion heterodimerisation with Pcr1 chromatin related complexes in Aberrant chromosome collection S. cerevisiae and S. pombe rearrangement induced by Alison Day replication fork stalling at Mitsuhiro Yanagida (Veal lab, University of José Ayté a palindrome (Kyoto University, Japan) Newcastle, UK) T14 (Universitat Pompeu Fabra, Strain resources in Japan A conserved cysteine of Sty1 has an Spain) T20 Osami Niwa important role in oxidative stress Rem1 expression is regulated at the (Kazusa DNA Research Institute, Val Wood, Jürg Bähler resistance level of transcription and splicing Japan) T5 (Sanger Institute, UK) On the instability of aneuploidy in The future of the S. pombe Janni Petersen Xuefeng Xhu fission yeast GeneDB database (University of Manchester, UK) T15 (Gustafsson lab, Karolinska Institute, Control of cell size at division in Sweden) T21 Paul Russell Paul Nurse response to nutrient availability Genome wide occupancy profile of (The Scripps Research Institute, (Rockefeller University, USA) Mediator and the Srb8-11 module USA) T6 General discussion reveals interactions with Pathways that control the Possible future initiatives? coding regions recruitment of Crb2 to sites of DNA damage

iv v Amanda Greenall Iva Tolic-Nørrelykke (Max Planck Inst. Agnes Grallert Hiro Yamano (Whitehall lab, University of of Molecular Cell Biology and (Hagan lab, (Marie Curie Research Newcastle, UK) T22 Genetics, Germany) T28 Paterson Institute, UK) T33 Institute, UK) T37 Hip3 interacts with the HIRA proteins Optical tweezers in fission yeast: Characterization of the fission yeast Mechanisms of the APC/C- Hip1 and Slm9 and is required for mechanism of nuclear and septum CLASP, Peg1 dependent ubiquitylation transcriptional silencing and positioning and proteolysis accurate chromosome segregation Itaru Samejima (Sawin lab, Koji Nagao Fri 17th March, 1600-1900 University of Edinburgh, UK) T34 (Yanagida lab, Fri 17th March, 1400-1530 Identification of an Kyoto University, Japan) T38 Poster Session MTOC-localisation signal domain The domain of securin required for Session 5: Mitotic Spindle in Mto1 stabilization and inhibition of Odd numbers: 1600-1730 separase can have a separase Chairs: Takashi Toda, Iain Hagan Even numbers: 1730-1900 cleavage site by substitutions Sat 18th March, 1130-1300 Anne Kerres Yuko Tonami (Fleig lab, Heinrich-Heine- Session 7: Mitotic and (Murakami lab, Nagoya City Universität, Germany) T23 Sat 18th March, 0930-1100 Meiotic Cell-Division Cycle University, Japan) T39 The conserved kinetochore The mechanism of cell cycle component Spc7 regulates Session 6: Cytoskeleton and Cell Chairs: Shelley Sazer, Chris Norbury progression through meiosis I by microtubule-kinetochore association Morphogenesis the meiosis-specific forkhead and is linked to the Sim4-complex Jérome Wuarin transcription factor Mei4p in Chairs: Jacky Hayles, Ken Sawin (University of Dundee, UK) T35 fission yeast Masamitsu Sato Cell growth and cell cycle: (Toda lab, Cancer Research UK) T24 Introduction by Ken Sawin Cdc2 meets ribosome biogenesis Akira Yamashita Microtubule organisation via (University of Edinburgh, UK) (Yamamoto lab, University of regulation of microtubule-associated Tonje Tvegård Tokyo, Japan) T40 proteins TACC/Alp7 and TOG/Alp14 Stefania Castagnetti (Boye lab, Inst. for Cancer Suppressor mutants of the throughout the cell cycle (Nurse lab, Research, Norway) T36 meiosis I arrest caused by loss of Cancer Research UK) T29 A novel checkpoint reveals a the Mei2p dot Matylda Sczaniecka Generating cell form in fission yeast coupling between general cell (Hardwick lab, growth and the cell cycle Closing remarks University of Edinburgh, UK) T25 Dai Hirata Interactions between the spindle (Hiroshima University, Japan) T30 checkpoint proteins and the Control of growth polarity upon Anaphase Promoting Complex perturbed DNA replication in S. pombe Yolanda Sanchez John Meadows (Universidad de Salamanca, (Millar lab, National Institute for Spain) T31 Medical Research, UK) T26 Rgf1p is a specific Rho1-GEF that Dissociation of mitotic spindle coordinates cell polarization with cell position from the timing of anaphase wall biogenesis in fission yeast onset in fission yeast Anne Paoletti Sylvie Tournier (Curie Intitute, France) T32 (Université Paul Sabatier, Spatial regulation of mid1p cortical France) T27 distribution by pom1p kinase Mechanism controlling perpendicular alignment of the spindle to the axis of cell division in fission yeast

vi vii Talk Abstracts We develop here a stochastic hybrid Involvement of Cid1 poly(A) disorder Bloom’s syndrome. The rqh2 model of DNA replication in the cell polymerase and the sub- gene appears to be present in four cycle of the fission yeast. The model + sub-telomeric copies; at least one of A stochastic hybrid model for captures the interplay between discrete telomeric rqh2 DNA helicase these is expressed, following telomere 3 DNA replication. dynamics associated with the firing of gene in a common pathway erosion or replication stress, to origins of replication (pre-replicative, following inhibition of generate an unspliced 7 kb mRNA with K. Koutroumpas1, S. Dimopoulos1, replicating and post-replicative states), a long ORF that includes centromere- 2 1 DNA replication I. Legouras , P. Kouretas , continuous dynamics that arise out of related dh repeat sequences upstream 3 3 C. Heichinger , P. Nurse , the replication process itself 1 from sequences encoding the RecQ 2 1 (continuous progression of the Olivia S Rissland , helicase domain. Transcription of the Z. Lygerou , I. Lygeros Abigail Stevenson1, Li Phing Liew1, replication forks along the genome), 2 3 dh sequences within rqh2 is important and stochastic events due to the Shao-Win Wang , Daniel Lackner , in establishing sub-telomeric 1. Department of Electrical Juan Mata3, Jürg Bähler3 and Computer Engineering, probabilistic firing process (not all 1 heterochromatin via the RNAi-RITS and Chris J Norbury 4, 5 University of Patras, Greece potential origins will fire in each cell pathway . A highly distinctive pattern 2. Department of Medicine, and not all will fire at the same time). 1 of 3’ RNA processing indicates that The model is coded using data recently Sir William Dunn School of Pathology rqh2 transcripts may be direct Cid1 University of Patras, Greece and 2Department of Zoology, University 3. The Rockefeller University, USA obtained in fission yeast by some of the 3 targets. We propose a model in which authors, and tested by Monte Carlo of Oxford and Wellcome Trust Sanger Cid1-mediated polyadenylation of rqh2 Institute, Hinxton, UK. Modelling of biological processes has simulation. The statistics collected mRNA following replication stress been a very active research area in through the simulations provide + allows Rqh2 translation. Rqh2 may be valuable insight into DNA replication We previously identified S. pombe cid1 important for stabilisation and/or recent years, in the hope that through a screen for S-phase cell cycle mathematical models will provide in fission yeast and suggest new resolution of stalled DNA replication biological experiments that could checkpoint genes, and showed that the forks, particularly telomere-proximal insight into the underlining biological Cid1 protein is a cytoplasmic principles and guide the development be carried out to improve our 1, 2 forks, and maintenance of the S-M understanding of the process. non-canonical poly(A) polymerase . checkpoint signal. of new experiments. Many types of Deletion of cid1 results in a specific models have been proposed for subset of checkpoint defects when 1. Wang et al. Mol Cell Biol 20, biological processes in the literature, replicative DNA polymerases are including purely discrete, graph 3234-44. (2000). inhibited. To test the hypothesis that 2. Read et al. Proc Natl Acad Sci U S A theoretical models, continuous ODE Cid1 regulates the polyadenylation of 99, 12079-84 (2002). and PDE models, hybrid and stochastic checkpoint gene mRNAs, we have 3. Mandell et al. J Biol Chem 280, models. Among these, stochastic used microarray hybridisation to 5249-57 (2005). hybrid models, which combine identify RNA changes that result from 4. Cam et al. Nat Genet 37, continuous dynamics to discrete cid1 deletion. Most strikingly, we found 809-19 (2005). switches of states and to events 5. Kanoh et al. Curr Biol 15, cid1-dependent expression of 1808-19 (2005). determined probabilistically, appear rqh2+/SPAC212.11 specifically in ideally suited to describe biological S phase arrested cds1A or DNA pol c phenomena. (cdc27) mutants. Rqh2 is the second member of the RecQ DNA helicase family to be described in S. pombe; its closest relative in human cells is the BLM helicase, mutation of which causes the cancer predisposition

T1 T2 The essential role of the replication fork. We show that Swi10 Aberrant chromosome reduced when replication fork stalling is homologous recombination and Mus81 (XPF nuclease family) rearrangement induced by induced. Southern blot analysis and participate in two alternative resolution PFGE show that RFS at the palindrome for replication fork restart. pathways. Swi10 is essential for MT- replication fork stalling at leads to rapid chromosomal switching but is dispensable in the a palindrome rearrangement and the formation of Laura Roseaulin, Allyson Holmes, absence of donors, conversely Mus81 dicentric and acentric sister Yufuko Akamatsu, Iroshi Iwasaki and is dispensable for MT switching, but Ken’ichi Mizuno, Johanne Murray chromatids. The cells progress through Benoit Arcangioli essential when the sister chromatid is and Antony Carr G2 and enter mitosis with normal used for repair. The mechanism of kinetics suggesting that the Unite de la Dynamique du Génome, choice/exclusion of one or the other Genome Damage and Stability Centre, rearrangements occur during S phase Structures and Dynamics of the nucleases is unknown; however, the University of Sussex, Brighton, and the products do not activate the Genomes, Institut Pasteur, 25 rue du bacterial Holliday junction resolvase BN1 9RQ, UK. G2/M DNA damage checkpoint. Dr. Roux, 75724 Paris, Cedex 15, RusA partially suppress the absence of The dicentrics align correctly at France. Mus81. The Rqh1, Srs2 and Fbh1 Genomic rearrangements linked to metaphase with bipolar attachment helicases, intra-S and G2 checkpoints aberrant recombination are associated to the spindle but are then torn apart The role of recombination in the recov- are dispensable. Molecular with cancer and human genetic at anaphase leading to a ery of stalled/collapsed replication forks approaches, in several mutant back- diseases. Such recombination has catastrophic mitosis. is an essential process in maintaining grounds, have been used to detect indirectly been linked to replication fork genomic stability and defects in this and follow the fate of the recombination stalling. We have previously established The generation of the non-equivalent pathway cause predisposition to many intermediates at mat1. Altogether, a system, designated RuraR, to induce sister chromatids is dependent on the forms of diseases. In fission yeast the these work demonstrate the essential replication fork stalling (RFS) at a RTS1-Rtf1 system and a subset of situation becomes critical when the function of the HR machinery in the specific site in a controllable manner, recombination and checkpoint proteins. replication fork collides with the imprint repair of a single broken replication fork using a defined replication fork barrier These results lead us to conclude (unrepaired single-strand break or arising during vegetative growth. sequence RTS1 and trans-acting 1) RFS at the palindrome triggers the ribonucleotide) at the mat1 locus, and Since the HR proteins have been replication fork block protein Rtf1 chromosomal rearrangements through converts it into a one-ended double- shown to be also essential during (Lambert et al, 2005). In the RuraR homologous recombination; strand break. The repair process must meiosis. Our results imply a circular system, RFS is overcome by homolo- 2) the generation of non-equivalent restore a fork structure that is suitable relationship between MT switching and gous recombination between RTS1 sister chromatids is dependent upon to restart replication. We show that a meiosis, in which the HR proteins play sites leading to inversion of the inter- the palindromic sequences, which single collapsed replication fork a central role, extending the question of vening ura4 marker and rarely to are able to self-pair, forming intrastrand required the homologous recombination how genetic recombination works, site-specific gross chromosome hairpin structures; and (HR) enzymes for viability in fission to why it exists. rearrangements. 3) the palindrome system provides yeast. Whereas the Rad22, Rhp51 and an inducible system with which to Rhp54 are essential, Rad50 is not albeit We have constructed a derivative analyse the behaviour of dicentric grow very slowly and Swi5 and Rhp57 palindrome system, Rura-aruR, by chromosomes. are interchangeable. Following strand inserting another copy of the ura4 gene invasion with either the donor loci or in reverse orientation. In contrast to the the sister chromatid the joint molecule RuraR system, the viability of the must be resolved to restart the palindrome system is dramatically

T3 T4 On the instability of i.e., 75% of the meiotic segregants Pathways that control the yeast checkpoint protein Crb2 at aneuploidy in fission yeast were aneuploid. Fluorescence recruitment of Crb2 to sites ionizing radiation-induced DSBs microscopy of living cells revealed that requires histone H2A C-terminal of DNA damage Osami Niwa, Yoshie Tange, abnormal or uneven nuclear divisions phosphorylation and H4-K20 occurred in microcolonies from a methylation. However, the relevance of and Atsushi Kurabayashi Li-Lin Du1, Toru Nakamura2 portion of apparently aneuploid spores. 1 Crb2 relocalization remains uncertain In some cases, haploid or diploid cells and Paul Russell because mutants lacking either of these Kazusa DNA Research Institute, Japan emerged from microcolonies that also 1 histone modifications lack strong contained many dying cells. Department of Molecular Biology, checkpoint defects. I will describe our In many organisms, like plants and Genetically defined diploid spores did The Scripps Research Institute, latest studies on the semi-redundant yeasts where polyploidy is common, not have such abnormalities. La Jolla, California, 90237, USA. mechanisms that control the the generation of aneuploids is 2 We suggest that anomalous nuclear Department of Biochemistry and localization of Crb2 at DSBs and inevitable; that is, it occurs without divisions occurring in aneuploid cells Molecular Genetics, University of Illinois explore their functional significance. erroneous chromosome transmission. at Chicago, Chicago, IL 60607, USA. Hence, there must be unknown might, on one hand, lead to the amplification of aneuploidy to enhance mechanisms that eliminate aneuploids cell death, and, on the other hand, Cellular responses to DNA double- from the population. Growth strand breaks (DSBs) involve the disadvantage, which is thought to be lead to the resolution of aneuploids into stable euploids. relocalization of checkpoint proteins to generally intrinsic to aneuploids, might DSBs. The focal assembly of fission not sufficiently account for the elimination. Findings from our previous To pursue the aneuploid issue more systematically, we constructed a genetic study suggested that at least selection system for the disomy of some types of aneuploids in fission yeast can eventually survive as diploids chromosome 1 or 2 using intragenic complementation of ade6-210 and or haploids after a period of anomalous –216 alleles. Each of these alleles was growth during which unbalanced chromosome compositions must be integrated into a site near the respective centromere. This genetic corrected. In the present study, we selection system revealed that fission reexamined this issue to elucidate the mechanisms underlying aneuploid yeast aneuploids can apparently be stabilized through structural changes instability. of chromosomes, including partial We first demonstrated that aneuploids duplication, translocation, and circular minichromosomes. between n and 2n were produced from triploid meioses at the expected ratio;

T5 T6 Surviving DNA damage with missing a telomere binding protein and The role of the S. pombe MRN element formation and meiosis specific dysfunctional telomeres the other lacking telomeric DNA. complex in the removal of chromatin remodelling at ade6-M26 are not affected in rad50S but strongly covalently linked protein from A Anna K. Hebden, Kyle M. Miller, Reintroduction of trt1 to circular strains defective in rad50 . We have found Toru Nakamura and Julia P. Cooper causes linearization of chromosome III the DNA that the rad32-D25A nuclease-dead with the addition of telomere repeats. mutant is also unable to remove Rec12 Interestingly, this partially suppresses from the DNA, and that this defect is Cancer Research UK, 44 Lincoln’s Inn Edgar Hartsuiker, Kenichi Mizuno, A Fields, London WC2A 3PX, UK the drug sensitivity. To further distin- Tony Carr. epistatic with rad50S and rad50 guish between the topological issue of mutations, suggesting that the Rad32 chromosome circularity and the (endo)nuclease activity is reponsible Telomerase maintains telomeres by GDSC, University of Sussex, adding DNA repeats to chromosome presence or absence of telomere Brighton, UK for Rec12 removal. sequence, we created strains ends. Disruption of this enzyme leads containing telomere repeats lacking Because rad50S is specifically to loss of telomeric DNA with Meiotic recombination is initiated by a successive rounds of DNA replication, ends, either plasmid based (to allow DSB which is repaired using the defective in Rec12 removal, but high copy number) or integrated within proficient for downstream repair events, and in most cases, senescence. homologous chromosome as a the genome. In most cases the we have used it as a tool to study the Following loss of Trt1, the catalytic template. This meiosis-specific DSB is subunit of telomerase in fission yeast, presence of telomere repeats did not created by Rec12 (Spo11 homologue) potential role of the MRN complex in affect the drug sensitivity. Intriguingly, Top1 and Top2 removal in vegetative a population of cells survive having lost through a non-reversible topoiso- however, we observed rare survivors cells. In line with rad50S not being all telomeric DNA. These cells survive merase-like mechanism. Covalently by maintaining each of the three with greatly suppressed drug sensitivity bound Rec12 has to be removed from defective in general DNA repair, we upon disruption of telomerase. found that the mutant is not sensitive chromosomes as individual circles. the DNA to allow subsequent DSB end Analysis of these strains shows that resection and meiotic recombination. to MMS or x-radiation (at either While circular strains are viable, they they have survived by a novel Here we present evidence that the permissive or restrictive temperature), mechanism. While they appear to lack whereas rad50A is extremely sensitive grow slowly and exhibit DAPI staining S. pombe MRN complex the majority of telomeric DNA, they to these agents. However, the patterns suggestive of chromosome (Rad32/Rad50/Nbs1) is directly segregation problems. Reminiscent of show behaviours distinct from those of involved in this removal. sensitivity of rad50S against camp- typical circular chromosome-containing tothecin (locks Top1 on the DNA) and strains lacking the telomere binding survivors. Our data suggest that one of TOP-53 (locks Top2 on the DNA) protein Taz1, circular strains are hyper- We have created an S. pombe rad50S sensitive to agents that induce DNA these strains survives by amplifying mutant and characterised its meiotic mimics the temperature sensitive subtelomeric repeat sequences, and meiotic phenotype: at permissive double strand breaks. Interestingly, phenotypes. Using a physical assay, the other by amplifying rDNA temperature rad50S is only slightly upon chronic treatment with damaging we have shown that the removal of agents, the circular strains are more sequences. covalently bound Rec12 from the DNA sensitive to these reagents, but it becomes extremely sensitive at sensitive than strains lacking the is defective in a rad50S mutation as We present our progress in restrictive temperature. The sensitivity checkpoint protein, Rad3. However, well as in rad50A. Unlike S. cerevisiae, - A upon acute treatment, rad3 strains dis- understanding the nature of this strain S. pombe rad50S is temperature of rad50 and rad50S to these and the roles of telomeres and topology reagents is epistatic with a rad32-D25A play greater sensitivity. The similarities sensitive for meiotic DSB repair (PFGE) - in the survival of DNA double strand nuclease-dead mutation. These data between circular and taz1 strains and meiotic spore viability. prompted us to investigate the basis for breaks. Furthermore, I found that S. pombe strongly suggest that the MRN complex is also directly involved in Top1 the defects in these two strains, one rad50S separates the Rec12 removal defect from downstream meiotic and Top2 removal. recombination and repair related functions: meiotic recombination, linear

T7 T8 Schizosaccharomyces pombe demonstrated that Rec10 is a critical Cdk-phosphorylation of the S-phase. Ste11 contains a single meiotic linear elements: the component of LinEs and LinEs do not HMG-domain protein Ste11 consensus site for Cdc2 form in mutants with an inactivated phosphorylation at threonine-82 and relationship with pre-meiotic rec10 gene. Moreover, Rec10 appears controls the switch from mutation of this residue to alanine DNA replication and meiotic to be the functional counterpart of the mitosis to meiosis in causes constitutive expression of mfm2 genetic recombination. Saccharomyces cerevisiae AE protein fission yeast and, hence, loss of its cell-cycle Red1. The function(s) of Rec10 / LinEs regulated transcription. When 1Wells, J.L., 1Dafydd, H. F., (and AEs) remains unclear, although it Søren Kjærulff, Nicoline Resen threonine-82 is changed to an 1Pryce, D.W., 2Estreicher, A., has been suggested that they are Andersen, Mia Trolle Borup aspartate, mimicking constant 2Loidl, J. 1McFarlane, R.J.* required to mediate meiotic genetic and Olaf Nielsen. phosphorylation, the level of Ste11 recombination possible by generating a protein and mfm2 transcript is down- 1North West Cancer Research Fund mechanical context in which crossing Institute of Molecular Biology and regulated in mitotically growing cells as Institute, University of Wales Bangor, over is controlled. Physiology, University of Copenhagen, well as nitrogen starved cells and Memorial Building, Deiniol Road, DK-1353 Copenhagen K, Denmark sexual differentiation is severely Bangor, Gwynedd, LL57 2UW, In this study we have explored the impaired. In vitro, Ste11 can be relationship of LinEs with DNA replica- phosphorylated by either Cig2-Cdc2 United Kingdom. At a point in G1 called start in tion and meiotic recombination further. Schizosaccharomyces pombe the cell or Cdc13-Cdc2 complexes. 2Department of Chromosome Biology, We find that LinE structures form in the decides whether to stay in the mitotic Cdk phosphorylation of Ste11 does not University of Vienna, A-1030, Vienna, absence of pre-meiotic DNA replication cell cycle or to exit the mitotic cell lead to ubiquitination and degradation, Austria and that significant levels of meiotic cycle in order to differentiate. When but instead to inhibition of its DNA- recombination occur in the absence of deprived of nutrients S. pombe under- binding activity. In G1, when Cdc2- *[email protected] LinEs, an observation supported by the goes sexual differentiation. cyclin kinase activity is low, fact that Rad51 foci are observed in the A prerequisite for this process is unphosphorylated Ste11 can bind its The chromosomes of most organisms absence of LinEs. These data suggest induction of the HMG-box transcription target sequences and, therefore, undergo a process of inter homologue that Rec10 has more than one function factor Ste11 and down regulation of activate transcription of the genes synapsis during meiosis I. In the during meiosis and that at best LinEs cyclin dependent kinase (Cdk) activity. involved in differentiation. In the rest of majority of cases this culminates in the function as recombination enhancers. Ste11 controls many genes required for the cell cycle, phosphorylation of Ste11 formation of a tripartite proteinaceous We also expose a more complex this developmental pathway including by Cdc2-cyclin complexes impairs the structure known as the synaptonemal relationship between Rec10 and the itself through a positive feed-back loop. DNA binding activity of Ste11 and, complex (SC). Linear structures, known control of genetic recombination. We have found that Ste11 function is hence, transcription of Ste11 as axial elements (AE), are the In a specific rec10 mutant, which is directly antagonized by Cdk-dependent responsive genes. Thus, specific and precursory structures to the fully defective in LinE formation, there is a phosphorylation. Ste11 and mfm2, a timely cell cycle-dependent interactions formed SC. S. pombe does not form regional difference in control of Ste11-responsive gene, are periodically of Ste11 with Cdc2 may contribute to detectable SC structures analogous to crossing over and gene conversion expressed through the cell cycle. Ste11 the periodicity of expression of Ste11 those of other organisms, but does events, the implication of these protein and mfm2 transcript begin to and its target genes, thereby restricting observations are discussed. sexual development to G1. form proteinacious thread-like accumulate at anaphase, persist in G1 structures, known as linear elements and are down-regulated during (LinEs), which have many features in common with AEs. Recently it has been

T9 T10 Vtc4p: a regulator of vacuolar normal. The affected gene was ` proteins beginning with M-N- and M- NatB N -acetylation activity is size and trafficking in identified as vtc4, which encodes an 83 M-. Putative substrates of NatB in kD protein with a hydrophobic required for Bsu1p pyridoxine fission yeast comprise approximately Schizosaccharomyces pombe C-terminus. The homologous protein in transporter function in 500 proteins. S.cerevisiae is a member of a complex, N. Bone, P. Nurse* and J. Armstrong for which several functions have been S. pombe. Loss of Arm1p results in a failure of proposed, on the vacuolar membrane. pyridoxine transport as assessed by School of Life Sciences, JMS Building, Expression of a GFP-Vtc4p fusion in Nam, A., Chua, G., Owens, 3H-pyridoxine uptake experiments. University of Sussex, Falmer, S.pombe confirms localisation to the B., Freitag, S. and P. G. Young This occurs both at 25 C where the Brighton, BN1 9QG vacuolar membrane, while the mutant cells grow well and form colonies and phenotype can be rescued by expres- Department of Biology, at 36 C. Using Bsu1-GFP expressed *The Rockefeller University, 1250 York sion of the un-fused gene. Disruption of Queen’s University, Kingston, chromosomally from its native promoter Avenue, New York, NY10021, USA the vtc4 gene results in an enlarged ON K7L 3N6, Canada. as a measure of effective transcription, phenotype indistinguishable from the translation and cellular localization, The fission yeast S.pombe normally has original mutant. We propose that Vtc4p Sensitivity to amiloride, a diuretic drug the Bsu1p pyridoxine transporter is a complement of 40-80 small vacuoles, has a function in regulating vacuolar toxic to the fission yeast, expressed in arm1 disruptions and the equivalent of lysosomes in higher size. Although the usual trafficking Schizosaccharomyces pombe, requires localizes appropriately to the plasma cells. As well as functioning in storage pathways are normal, we found a the activity of the plasma membrane membrane. By western blotting the and protein turnover, we have shown requirement for Vtc4p in the Cvt vitamin B6 transporter, Bsu1p (formerly Bsu1-GFP protein is expressed at that vacuoles are dynamic structures, pathway, a route of delivery for two Car1p). Amiloride is a competitive similar levels in wild type and mutant involved in response to osmotic stress. vacuolar proteins which overlaps with inhibitor of pyridoxine uptake and thus backgrounds. Based on the Bsu1p N- In many cases, disruption or mutation the mechanism of autohpagy. affects thiamine metabolism and genes terminal sequence, Bsu1p is not a of genes involved in membrane traffic affected by it. bsu1 loss-of-function direct substrate of the NatB complex. pathways leads to altered vacuolar Sequencing the vtc4 gene from the mutants are amiloride resistant due to a A presently unknown NatB substrate morphology: for example, disruption of mutant revealed no change from the presumed failure to transport amiloride presumably requires acetylation for ypt7, involved in the last stage of database. A stop codon was, however, into the cell. The internal target(s) of Bsu1p transport activities to occur. endocytosis, results in vacuolar found to have been introduced in the 3’ amiloride toxicity in fission yeast have fragmentation. region of the adjacent, convergent ade6 not yet been well-defined (Jia et al., Disruption of arm1+ results in a broad locus: the strain is also ade-. RT PCR 1993; Niederberger et al., 1996; Stolz et range of other phenotypes many of From a collection of temperature shows that the levels of both vtc4 and al., 2005). which, based on data from the sensitive strains, we have examined a ade6 transcripts are significantly S. cerevisiae system, are likely the strain in which the normal vacuolar reduced in the mutant line. We have identified a new locus confer- result of failure of tropomyosin-actin morphology is replaced by a single, We found that this single point mutation ring amiloride resistance, termed arm1 interaction. Both actin and tropomyosin or a small number, of extremely large confers two distinct phenotypes via (for amiloride resistant mutant). Apart are NatB substrates in S. cerevisiae vacuoles. Cells containing these two adjacent genes. from growth on amiloride containing and mammalian cells and predicted to aberrant giant vacuoles can have plates, the mutant (and its gene disrup- be by N-terminal sequence in fission difficulty in dividing, but the trafficking tion) has a distinct temperature-sensi- yeast. The arm1 deletion strain pathways of endocytosis and delivery tive cell separation phenotype. At 25 C displays a disruption of actin cables, of most vacuolar enzymes appear cells grow well but include a population the formation of thick disoriented septa with multiple septa and cell compart- and failure to undergo fluid-phase ments. At 36 C all cells have mis- endocytosis. Disruption of the actin aligned thick septa, multiple cell com- cytoskeleton using the temperature partments, and fail to form colonies. sensitive cdc8 tropomyosin mutant By sequence similarity, Arm1p is the ` causes a failure of pyridoxine transport. auxiliary subunit of the NatB ·-acetyl- It is possible that the correct function of transferase complex. The NatA, B and the transporter although not its C complexes in budding yeast are membrane localization is dependent reported to acetylate more than half of directly or indirectly upon a fully all proteins. NatB is thought to acety- functional actin cytoskeleton. late all proteins with M-D- or M-E- amino termini as well as a subset of

T11 T12 Regulation of S.pombe Atf1 protein accumulates following stress A conserved cysteine of Sty1 response to different stresses are not protein levels by and this accumulation is lost in a strain has an important role in well understood. defective in the Sty1 signalling 2-cysteine peroxiredoxins are Sty1-mediated pathway. In addition, accumulation of a oxidative stress resistance thioredoxin peroxidases that in addition phosphorylation and mutant Atf1 protein that can no longer to detoxification of peroxides also have heterodimerisation with Pcr1 be phosphorylated is lost. Alison M. Day, Stephanie M. Bozonet, roles in peroxide signalling. Indeed, Measurement of the half-life of Atf1 Brian A Morgan & Elizabeth A. Veal we have shown that the 2-cys peroxire- Clare L. Lawrence, demonstrates that this accumulation is doxin in S. pombe, Tpx1, regulates the due to changes in Atf1 stability. Institute of Cell and Molecular Sty1 SAPK pathway via a mechanism Jessica L. Worthington, Biosciences, The Medical School, Hiromi Maekawa, Wolfgang Reiter, Atf1 stability is also regulated by Pcr1, involving a peroxide induced disulphide a heterodimeric partner of Atf1. University of Newcastle upon Tyne, complex between Tpx1 and Sty1. Caroline R.M. Wilkinson Newcastle upon Tyne, NE2 4HH, UK. and Nic Jones Similarly, Pcr1 levels are regulated by We have identified conserved cysteines Atf1. Thus there exist multiple in Tpx1 and Sty1 that are essential for In mammalian cells the JNK and p38 Cancer Research UK, Paterson Institute pathways that ensure that Atf1 levels formation of a disulphide bond between are appropriately regulated. families of stress-activated protein Tpx1 and Sty1. Using a serine for Cancer Research, Wilmslow Road, kinases (SAPK) are activated in Manchester M20 4BX Surprisingly, loss of Atf1 substitution mutant we have examined phosphorylation does not result in a response to a diverse range of further the role of this cysteine in Sty1. stresses. A SAPK homologue in the The Atf1 transcription factor plays a significant loss of stress-activated Here we will present data revealing the expression of Atf1 target genes, fission yeast Schizosaccharomyces mechanism underlying the role of this vital role in the ability of S. pombe cells pombe, Sty1 (also known as Spc1 and to respond appropriately to a variety of except in the case of oxidative stress. cysteine in the oxidative stress Accordingly, the phosphorylation Phh1) is also activated by response. The conservation of this different stress conditions including phosphorylation in response to a similar nutritional, osmotic and oxidative mutant strain does not display the cysteine in proteins homologous to same stress sensitivities as the atf1A range of stresses including oxidative Sty1 suggests that a similar mechanism stress. It regulates the expression of and osmotic stress. The sensing many genes in a stress-dependent mutant. The significance of these may regulate other MAPK. findings will be discussed. mechanisms involved in the regulation manner and its function is dependent of these SAPK signalling pathways in upon the stress-activated MAP kinase, Sty1. Previous studies have demonstrated that Atf1 is directly phosphorylated by Sty1. In this study we have investigated the role of such phosphorylation. The level of Atf1

T13 T14 Control of cell size at division the stress MAP kinase pathway (SRP) Genome-wide Translational be associated with many more ribo- in response to nutrient has a level of constitutive signaling that Control in Fission Yeast somes than less abundant transcripts, is required to control the timing of although ribosome density seems to availability. mitotic commitment in response to Daniel H. Lackner1, only correlate weakly with transcript nutritional availability (Shiozaki and 2 levels. We also found a strong inverse Janni Petersen1,2 and Paul Nurse1 Traude H. Beilharz , Russell 1995). The Plo1 kinase links the Samuel Marguerat1, correlation between ribosome density SRP to this control (Petersen and 2 1 and gene length: shorter genes tend to 1 Thomas Preiss and Jürg Bähler The Rockefeller University Box 5 1230 Hagan 2005), because SRP dependent be much tighter packed with ribosomes York Avenue, New York NY 10021 USA Phosphorylation on Plo1 Serine 402 1 than longer genes. 2 Wellcome Trust Sanger Institute, University of Manchester, Michael (S402) targets it to the SPB, where it Smith Building, Oxford Rd, M13 9PT, Fission Yeast Functional Genomics modulates mitotic entry. We find that Group, Hinxton, Cambridge, We are now testing whether these Manchester U.K. mutation of this site to block trends depend on other mRNA features CB10 1SA, U.K. [email protected] phosphorylation, or deletion of the 2 such as stability or polyA length. To Molecular Genetics Program, stress MAP kinase Sty1/Spc1, stops Victor Chang Cardiac Research obtain global data on polyA length, Cell growth and cell cycle progression cells from responding to changes in transcripts are fractionated using polyU Institute, Darlinghurst (Sydney) are generally tightly coupled, allowing available nutrients. Furthermore, Sty1 is columns and fractions of different tail NSW 2010, Australia cells to proliferate continuously while activated and Plo1 is phosphorylated lengths are quantified with DNA maintaining their size. Nutritional shift on S402 in response to nutritional microarrays. Transcript stability is We are interested in global roles of down experiments has shown that cell shifts. Thus nutrient signaling impact on determined by measuring mRNA levels size at cell division is controlled by size translational regulation and its Sty1/Spc1 activity that then impacts on coordination with other levels of gene at different times after blocking rather than time (Fantes and Nurse Plo1 phosphorylation in order to transcription. expression control. 1977). The critical cell size is modulated promote mitosis. by the growth conditions and is reset To obtain translational profiles for all Integration of these varied data sets will within a short time after changes in Fantes and Nurse, Exp Cell Research. provide insight into global mechanisms mRNAs, polysome preparations are these conditions. In all eukaryotes MAP 107:377-386 of post-transcriptional regulation. kinase cascades play an important role Shiozaki and Russell, 1995 Nature. separated according to their size using a sucrose gradient and the mRNAs in Moreover, combining translational and in modulating signaling to adapt a 378:739-43. expression profiling of cells in different Petersen and Hagan, 2005 Nature. each fraction (or pools of fractions) are range of processes, including transcrip- cell-cycle stages or subjected to tional control with changes in the cell 435:507-512. identified and quantified with DNA microarrays. Starting with exponentially various genetic and environmental environment. In fission yeast, perturbations will provide a genome- growing cells, we analyzed 12 in addition to responding to stresses, wide view of translational regulation in polysome fractions using DNA microarrays containing elements for all fission yeast, complementing our expression profiling data. known and predicted genes of fission yeast. This approach provided data on average numbers of associated ribosomes for most transcripts. Integration with data on mRNA steady- state levels revealed an interesting bias: the most abundant transcripts seem to

T15 T16 Genome wide roles for his- also affects subtelomeric regions which Comparative Studies of histones, histone deacetylases (HDACs) tone modifications and RNAi contain clustered stress and meiosis Molecular Mechanisms that and the RNA Polymerase II mediator induced genes. Thus, this combined complex. Gcn5 is a histone directed chromatin silencing genomic approach has uncovered Program Transcription. acetyltransferase found within the different roles for fission yeast HDACs SAGA complex and other related Karl Ekwall at the silent regions in repression and Fredrik Fagerström-Billai, Anna co-activator complexes. activation of gene expression. Johnsson, Yongtao Xue-Franzén, Karolinska Institutet, Dept of Maria Lundin and Anthony Wright S. pombe is the only fungus that Biosciences/School of Life Sciences, Reference: M. Wirén, R. A. Silverstein, contains two Tup1-like proteins (Tup11 Univ. I. Sinha, J. Walfridsson, H. Lee, P. School of Life Sciences, and Tup12) as a result of gene duplica- College Sodertorn, Alfred Nobel’s Allé Laurenson, L. Pillus, D. Robyr, M. Grunstein, Södertörns Högskola and Department tion. While Tup11 and Tup12 have 7, S-141 89, Huddinge, Sweden. and K. Ekwall. EMBO J 24:2906-18. of Biosciences and Nutrition, redundant functions at some genes we E-mail: [email protected], Karolinska Institutet, SE-141 89 have shown that Tup12 plays a specific Telephone: +46 8 6084713, In RNA interference (RNAi) Dicer Huddinge, Sweden. role in regulating genes involved in the Fax: +46 8 6084510. processes double stranded RNA to KCl-stress response (4). Unlike the produce short siRNAs that act post- Differential gene expression is an Tup11 and Tup12, Ssn6 is an essential Histone decatylases (HDACs) are transcriptionally to silence gene important mechanism by which the protein in S. pombe, suggesting the evolutionary conserved enzymes which expression. In several organisms functional diversity of different organ- existence of Ssn6-specific target are important for transcription and including fission yeast, RNAi also medi- isms is generated from their relatively genes. Using genome-wide expression other chromatin related processes. ates transcriptional silencing via forma- conserved genomes. The flexible profiling we have identified classes of We have conducted a genome wide tion of heterochromatin. Fission yeast mechanism by which activator proteins target genes that are specific for Ssn6 investigation into the enzymatic centromeric repeats are transcribed interact with target proteins makes or Tup11/12 as well as a class that specificity, gene expression profiles, into siRNA precursors (pre-siRNAs), recruitment of transcriptional requires both components of the co- and binding locations of four histone which are processed by Dicer to direct co-regulator proteins a potentially repressor. Genome-wide localisation deacetylases (HDACs), representing the heterochromatin formation. Recently, evolvable process whereby the con- studies show that the location of the three different phylogenetic classes in Rpb1 and Rpb2 subunits of RNA Pol II served gene expression machinery three proteins at different parts of the fission yeast (Schizosaccharomyces were shown to mediate RNAi-directed could be adapted to perform new tasks genome is highly correlated. Thus the pombe). By directly comparing chromatin modification but did during evolutionary diversification (1-3). differential requirements for the different nucleosome density, histone acetylation not affect pre-siRNA levels. Here we Co-regulator proteins would thus repre- co-repressor subunits for repression of patterns and HDAC binding in both show that another Pol II subunit, Rpb7 sent a class of proteins with conserved different target genes must represent a intergenic and coding regions with has a specific role in pre-siRNA tran- structure and molecular mechanism but post-recruitment facet of the co-repres- gene expression profiles, we found that scription. We define a centromeric pre- with diverged physiological function. sor. Interestingly, recent experiments Sir2 (class III) and Hos2 (class I) have a siRNA promoter from which using tiling arrays show that the co- role in preventing histone loss, Clr6 initiation is exquisitely sensitive to the We are performing comparative studies repressor is independenly located in (class I) is the principal enzyme in rpb7-G150D mutation. In contrast to of co-regulator proteins using both the promoters (as expected) and promoter-localized repression. Hos2 other Pol II subunits which affect down- S. pombe as a model system for coding regions of target genes. has an unexpected role in promoting stream events in the pathway, Rpb7 comparison with S. cerevisiae. We have high expression of growth-related promotes pre-siRNA transcription focused on the Gcn5 co-activator and Gcn5 is specifically required for required for RNAi-directed genes by deacetylating H4K16Ac in the Tup1-Ssn6 corepressor, both of adaptation to KCl- and CaCl2- mediated their open reading frames. Clr3 (class II) chromatin silencing. which have been extensively studied in stress in both S. pombe and act cooperatively with Sir2 throughout S. cerevisiae and are required for adap- S. cerevisiae. Adaptation to KCl-medi- the genome including the silent regions: Reference: I. Djupedal, M. Portoso, tation of S. pombe to KCl-mediated H. Spahr, C. Bonilla, C. M. Gustafsson, ated stress involved regulation of over- rDNA, centromeres, mat2/3 and telom- stress. In S. cerevisiae DNA-bound lapping but distinct sets of response eres. The most significant acetylation R. C. Allshire and K. Ekwall repressor proteins have been reported Genes&Development 19:2301-2306 genes in the two organisms. sites are H3K14Ac for Clr3 and H3K9Ac to recruit Ssn6 and thereby Tup1, which Interestingly, genes that require Gcn5 for Sir2 at their genomic targets. Clr3 subsequently represses transcription during adaptation to KCl are highly via interactions with hypoacetylated over-represented in the class of

T17 T18 species-specific response genes. Thus 3. Arabi A, Wu S, Ridderstrale K, Bierhoff H, Comparative proteomic approach a number of proteins were the role of Gcn5 in S. pombe and S. Shiue C, Fatyol K, Fahlén S, Hydbring P, analysis of chromatin related found to be shared between two or cerevisiae is correlated with gene Söderberg O, Grummt I, Larsson L-G, more different protein complexes and expression programs that have been Wright APH (2005) Nature Cell Biol. 7, complexes in S. Cerevisiae were termed ‘proteomic hyperlinks’. established since their divergence in 303-10. Eight of these serve as links in a 4. Fagerström-Billai F and Wright APH and S. Pombe evolution. (2005) Mol. Cell. Biol. 25, 716-27. network of protein complexes including Assen Roguev1, Daniel Schaft1,2, the major yeast histone deacetylase 1. Hermann S, Bernt KD, Wright APH (2001) Anna Shevchenko3, Rein Aasland4, complex (Rpd3C), the major H4 J. Biol. Chem. 276, 40127-32. Andrej Shevchenko3, A. Francis acetyltransferase complex 2. Ferreira ME, Hermann S, Prochasson P, Stewart1 (Esa1/NuA4C) and two chromatin Workman JL, Berndt KD, Wright APH remodeling complexes, one of which (2005) J. Biol. Chem., 280, 21779-84. 1 BIOTEC TU-Dresden, Tatzberg 47-51, (Swr1C) is responsible for incorporation 01307 Dresden, Germany of H2A.Z into chromatin. 2present address: The Victor Chang Cardiac Research Institute, Level 6, 384 A similar approach was then applied Victoria Street, Darlinghurst NSW 2010, then in S. pombe. S. pombe was Australia chosen mainly because of its evolution- 3Max Planck Institute for Molecular Cell ary distance from budding yeast and Biology and Genetics, vertebrates, its simplicity and ease of Pfotenhauerstrasse 108, 01307 handling. The S. pombe homologs of Dresden, Germany the proteomic hyperlinks from 4Department of Molecular Biology, S. cerevisiae were TAP-tagged, purified University of Bergen, HiB, P.O. box and protein complex members 7800, N-5020 Bergen, Norway identified. While this analysis revealed remarkably conserved proteomic cores We have applied sequential epitope of the complexes in the two yeasts, tagging and mass spectrometry (SEAM) important aspects of the network’s approach in Saccharomyces cerevisiae topology differed. The network in and Schizosaccharomyces pombe to S. pombe appears to be more complex determine and compare the proteomic and more closely related to partial environments of chromatin related pro- networks derived from higher organ- tein complexes. isms. To our knowledge this is the first systematic comparative proteomic Initially 29 proteins in S. cerevisiae were attempt between different species. selected bioinformaticaly on the basis The nature of the proteomic hyperlinks of presence of chromatin related connecting complexes of related but domains (SET, SANT, chromo, HDACs seemingly antagonizing functions raises and Sir2-homology deacetylase pro- many questions in regards to the teins). They were then TAP-tagged and targeting and regulation of such purified. Of the initial 29, 14 proteins important gene expression machinery were found to exist in multi-protein especially in the light of the recent complexes. Co-immunoprecipitating findings on the role of Eaf3 in H3- proteins were identified by K36Me recognition. mass-spectrometry, TAP-tagged and purified themselves. Through this

T18 T19 Rem1 expression is regulated meiosis. When rem1 is expressed Genome wide occupancy activation of transcription. The Srb8-11 at the level of transcription during mitotic cycle, even at very low profile of Mediator and the containing Mediator represses basal level, induces mitotic catastrophes. transcription in vitro and genetic and splicing rem1 expression is regulated at the Srb8-11 module reveals analysis also indicates that the Srb8–11 level of both transcription and splicing. interactions with coding module is involved in the negative 1 1 Alberto Moldón , Jordi Malapeira , rem1 transcription during meiosis is regions regulation of genes in vivo. Gerald R. Smith2, Paul Nurse3, regulated by forkhead transcription 1 1 Elena Hidalgo and José Ayté factors, being induced during Xuefeng Zhu1, Marianna Wirén2, Here we use chromatin pre-meiotic S phase and with a peak in 2 3 immunoprecipitation and DNA 1 Indranil Sinha , Nina N. Rasmussen , Cell Signalling Unit. Universitat transcription before the onset of Tomas Linder1, Steen Holmberg3, microarrays to investigate the genome Pompeu Fabra. Barcelona, Spain. meiosis I. On the other hand, Karl Ekwall2, Claes M. Gustafsson1 wide localization of Mediator and the 2Fred Hutchinson Cancer Research splicing is regulated independently of Srb8-11 module in fission yeast. 3 Center. Seattle, USA. The Rockefeller transcription, with Mei4 as a positive 1Deparptment of Laboratory Medicine, Mediator and the Srb8-11 module University. New York, USA. and Cig2 a negative factor of this Karolinska Institute, Novum, S-141 86 display very similar binding patterns, process. Furthermore, there are Huddinge, Sweden, 2Department of and interactions with promoters and Gene expression controls at the level of elements in the promoter of rem1 that Biosciences/Dept of Natural Sciences, upstream activating sequences RNA are crucial for meiosis. In the last control the meiotic-specific splicing Univ. College Sodertorn, Alfred Nobel’s correlates with increased transcription few years, splicing regulation is of this gene. Allé 7, S-141 89, Huddinge, activity. Unexpectedly, Mediator also emerging as an important key step for 3Department of Genetics, Institute of interacts with the downstream coding regulating different cellular processes, Molecular Biology, Oester region of many genes. including differentiation program. Farimagsgade 2A, DK-1353 These interactions display a negative Here we show that a fission yeast Copenhagen K, Denmark bias for positions closer to the 5’ ends cyclin, Rem1, is present only during of the open reading frame and appear The Mediator complex supposedly functionally important, since down functions as a bridge between gene regulation of transcription in a specific transcription factors and the temperature sensitive med17 mutant general transcription machinery at the strain, correlates with changes in promoter. Together, CDK8, CycC, Mediator binding in the coding region. Med11, and Med12, are parts of a We propose that Mediator coordinates repressive module (the Srb8-11 transcription initiation with module), which prevents RNA transcriptional events in the coding polymerase II interactions with core region of eukaryotic genes. Mediator and that is displaced upon

T20 T21 Hip3 interacts with the HIRA kDa protein, with similarity to The conserved kinetochore but plays no role in the transcriptional S. cerevisiae Hir3. Consistent with this, silencing of genes placed within this proteins Hip1 and Slm9 and is + component Spc7 regulates cells disrupted for hip3 exhibit a range region. The Spc7 protein was found to required for transcriptional of growth defects that are similar to microtubule-kinetochore interact with two conserved kineto- silencing and accurate those associated with loss of Hip1 and association and is linked to chore complexes, namely the Ndc80 chromosome segregation Slm9. These include temperature the Sim4-complex and the Mis12 complex; an interaction sensitivity, a cell cycle delay and also conserved in higher eukaryotes Amanda Greenall synthetic lethality with cdc25-22. Anne Kerres and Ursula Fleig (Cheeseman et al., 2004; Obuse et al., Furthermore, genetic analysis also 2004; Kerres et al., 2004; Liu et al., and Simon Whitehall + indicates that disruption of hip3 is Lehrstuhl für funktionelle 2005, EMBO J, 24:2919-30). epistatic with mutation of hip1+ and We have recently shown that Spc7 also Institute of Cell and Molecular + + Genomforschung der Mikroorganismen, Biosciences University of Newcastle, slm9 . Mutation of hip3 alleviates Heinrich-Heine-Universität, 40225 plays a role in the function of another Newcastle upon Tyne, NE2 4HH transcriptional silencing at several Düsseldorf, Germany kinetochore complex, the 12 compo- heterochromatic loci including in the email: [email protected] nent Sim4-Mal2-Mis6 complex (Liu et HIRA proteins are members of an evo- outer (otr) centromeric repeats, al., 2005). One of the components of lutionarily conserved family of histone indicating that Hip3 is required for the A critical aspect of mitosis is the the Sim4-Mal2-Mis6 complex was iso- chaperones that mediate nucleosome integrity of pericentric heterochromatin. capture and correct attachment of the lated as a suppressor of the non- assembly. We have previously demon- As a result loss of Hip3 function leads kinetochores by the dynamic micro- growth-phenotype of a spc7 tempera- strated that the S. pombe HIRA pro- to high levels of minichromosome loss tubules (MTs) of the spindle. ture sensitive mutant. Furthermore teins Hip1 and Slm9 are required for and an increased frequency of lagging Attachment is carried out between the these two proteins can be co-immuno- the propagation of pericentric hete- chromosomes during mitosis. plus-ends of MTs and the kinetochore, precipitated. In addition the correct rochromatin and thus accurate chromo- Importantly, the function of Hip1, a proteinaceous structure assembled localisation of at least one member of some segregation. HIRA proteins are Slm9 and Hip3 is not restricted to on the centromeric DNA. A large num- the Sim4-Mal2-Mis6 complex is known to function as components of constitutive heterochromatic loci as ber of proteins are known to be dependent on the presence of function- large protein complexes but the com- these proteins also repress the required for the complex process of al Spc7. We therefore propose that position of these complexes has not expression of a number of association between the kinetochore Spc7 has a central role in serving as a been defined. Therefore we have used euchromatic genes. and the plus-ends of spindle MTs. connecting element between various single-step affinity purification and However the precise interaction part- kinetochore complexes. MALDI-TOF mass spectrometry to ners at the MT-kinetochore interface are identify factors that interact with both poorly understood. What is the role of Spc7 at the Hip1 and Slm9. This analysis identified We previously identified the essential MT-kinetochore interface? Hip3, a previously uncharacterised 187 kinetochore protein Spc7 as a suppres- The Mad2 and Mph1 branches of the sor of the MT-plus-end localized spindle checkpoint are both activated protein Mal3, which belongs to the in spc7 mutant strains indicating that highly conserved EB1 family. Spc7 is a Spc7 is required for the attachment of conserved protein as homologues were kinetochores with MTs as well as bipo- identified from yeast to human lar attachment of sister kinetochores to (Cheeseman et al., 2004, Genes Dev, the spindle. Further analysis of various 18:2255-68; Kerres et al., 2004, Mol spc7 mutants indicates that the forma- Biol Cell, 15:5255-67; Nekrasov et al., tion and function of the mitotic spindle 2003, Mol Biol Cell, 14:4931-46; Obuse is severely affected in these strains. et al., 2004, Nat Cell Biol, 6:1135-41). Interestingly some of the spc7 mutant phenotypes can be suppressed partially We showed that Spc7 and Mal3 inter- by expression of extra tubulin possibly act genetically as well as physically and pointing to a role of Spc7 in MT that expression of a dominant-negative dynamics. Full-length Spc7 interacts variant of Spc7 results in severe with the plus-end MT protein Mal3, defects in the association of kineto- while a truncated version of Spc7 chores with MTs. appears to associate with the mitotic Spc7 associates constitutively with the spindle in a Mal3 independent way. inner centromeric region in S. pombe

T22 T23 Microtubule organisation via Therefore, localisation of Alp7-Alp14 Interactions between the conformational change or by regulation of microtubule- complex must be regulated in order to spindle checkpoint proteins competing with its substrates. execute multiple functions at each There are however many other possible associated proteins location. Since yeast cells undergo and the Anaphase mechanisms, such as binding and TACC/Alp7 and TOG/Alp14 closed mitosis in which the nuclear Promoting Complex in blocking of the APC activator Cdc20 throughout the cell cycle envelope does not break down, Alp7- Schizosaccharomyces pombe which has been shown to form a Alp14 complex needs to translocate complex with checkpoint proteins such Masamitsu Sato and Takashi Toda from the cytoplasm to the nucleus upon Matylda Sczaniecka as Mad2 and Mad3. Changes in the mitotic entry and vice versa upon and Kevin Hardwick status of Cdc20/APC phosphorylation Laboratory of Cell Regulation, Cancer mitotic exit. may also be important. Research UK, 44 Lincoln’s Inn Fields, Wellcome Centre for Cell Biolgy, London, WC2A 3PX, United Kingdom Here we show that Alp7/TACC pos- University of Edinburgh, We are currently looking at one of those sesses an ability to actively transport Kings Buildings, EH9 3JR mechanisms: the direct binding of Mad Centrosomal protein TACC (transform- Alp7-Alp14 complex into the nucleus. and Bub proteins to the APC in fission ing acidic coiled-coil protein) and TOG Surprisingly, nuclear import of Alp7- The spindle checkpoint monitors the yeast Schizosaccharomyces pombe. (tumor overexpressed gene) are con- Alp14 occurs not only during mitosis metaphase – anaphase transition by We have found that Mad2 and Mad3 served MAPs that regulate microtubule but also even during interphase, in ensuring correct microtubule – kineto- proteins coimmunoprecipitate with dynamics in many aspects of cellular which Alp7-Alp14 complex is exported chore attachment. In the case of an Lid1-TAP subunit of the APC in a phenomena. TACC and TOG form a to the cytoplasm. During mitosis, cells unattached kinetochore or if the tension mitotic arrest. This interaction is complex and the localisation of TOG to are utilising molecular schemes which between microtubule and kinetochore partially dependent on the APC the centrosome is dependent upon accumulate Alp7-Alp14 in the nucleus is not sufficient, the kinetochore emits a activator protein – Slp1 (Cdc20 TACC. effectively. Nucleocytoplasmic shuttling signal which is recognised by the com- homologue). Moreover it seems to be In fission yeast, the orthologs of TACC of microtubule-associated proteins ponents of the spindle checkpoint. dependent on specific motifs found in (Alp7) and TOG (Alp14) function TACC-TOG via the Ran GTPase system This results in a series of interactions Mad3 protein, the KEN-boxes. throughout the cell cycle. During inter- is an efficient way to regulate both between the checkpoint proteins KEN-boxes are known to serve as phase, Alp7 and Alp14 are required to interphase and mitotic microtubules in (Mad1, Mad2, Mad3, Bub1, Bub3, recognition signals for APC – mediated organise cytoplasmic microtubule a spatial and temporal manner. Mph1) and several complexes are degradation, similar to D-boxes. We are structure, and in mitosis, they play key Regulatory circuit of TACC-TOG as a formed. This, in turn leads to the investigating the possible role of these roles in formation of bipolar spindle and possible cargo of Ran might be con- inhibition of the Anaphase Promoting motifs in mediating the inhibitory establishment of amphitelic micro- served as a mechanism underlying Complex (APC), accumulation of protein binding. Our results suggest tubule-kinetochore attachment in the microtubule dynamics and spindle mitotic proteins and anaphase delay. that one of the Mad3 KEN-boxes is not nucleus. formation. APC is a multisubunit ubiquitin ligase, only important for the interaction of the which acts as an E3 enzyme in the spindle checkpoint proteins with the ubiquitin pathway. By attaching APC, but it is also required to form the polyubiquitin chains onto its substrates mitotic checkpoint complex (MCC), it targets them for degradation by the which is necessary for proper 26S proteasome. In mitosis APC is checkpoint function. In fact, both of the responsible for targeting for KEN motifs in Mad3 seem to be degradation anaphase inhibitors: required for inducing a checkpoint – securin and cyclin B. dependent metaphase arrest. Further studies in this area will lead to a It is not clear how the checkpoint better understanding of the ways in proteins inhibit APC from degrading its which this important mechanism substrates. One possibility is that they (the spindle checkpoint) prevents bind directly to the APC and block its cells from missegregating their active sites, either by inducing a chromosomes.

T24 T25 Dissociation of mitotic spindle an inhibitor of actin polymerisation, and Mechanism Controlling zone (AMIZ). Simultaneous contact of position from the timing of requires a subset of spindle assembly Perpendicular Alignment of astral microtubules from both poles checkpoint proteins including Bub1, with the AMIZ directs spindle rotation anaphase onset in fission Bub3, Mad3 and Mph1. We find that the Spindle to the Axis of Cell and this requires both actin and two yeast cells lacking Mto1, a centrosomin-like Division in Fission Yeast type V myosins, Myo51 and Myo52. protein, have mis-positioned spindles Finally, we show that disruption of the but are not delayed in the timing of Gachet Y1, Reyes C1, Goldstone S1, actin cytoskeleton is monitored by a John C. Meadows 2 1 and Jonathan B.A. Millar sister chromatid separation. Instead we Hyams J , Tournier S . checkpoint that regulates the timing of find that Latrunculin A delays the onset sister chromatid separation. We find 1 Division of Yeast Genetics, of anaphase and causes mitotic LBCMCP-CNRS UMR5088, that whereas sister kinetochore pairs National Institute for Medical Research, spindles to collapse. This effect is Institut d'Exploration Fonctionelle des normally congress to the spindle The Ridgeway, Mill Hill, exacerbated in cells lacking Ase1, a Génomes (IFR109), Université Paul midzone before anaphase onset, this London NW7 1AA, UK microtubule associated protein (MAP), Sabatier, 118 route de Narbonne, congression is disrupted when the actin which stabilises anti-parallel spindle 31062 Toulouse, France cytoskeleton is disturbed. By analyzing 2 It has previously been proposed that in midzone microtubules. These results Institute of Molecular Biosciences, the timing of kinetochore separation, fission yeast mitotic spindle position suggest that Latrunculin A delays the Massey University, Private Bag 11 222, we find that this anaphase delay is monitored by a checkpoint that onset of anaphase by destabilising Palmerston North, New Zealand. requires the Bub3, Mad3, and Bub1 but controls the timing of anaphase onset. formation of a bipolar mitotic spindle not the Mad1 or Mad2 spindle This checkpoint is activated by rather than by causing spindle In animal cells, the mitotic spindle is assembly checkpoint proteins. In treatment of cells with Latrunculin A, mis-orientation. aligned perpendicular to the axis of cell agreement with this, we find that Bub1 division. This ensures that sister remains associated with kinetochores chromatids are separated to opposite when actin is disrupted. These data sides of the cytokinetic actomyosin ring indicate that, in fission yeast, the (CAR). We show that, in fission yeast, integrity of the actin cytoskeleton is spindle rotation is dependent on the monitored by a subset of spindle interaction of astral microtubules with assembly checkpoint proteins. the cortical actin cytoskeleton. Interaction initially occurs with a region surrounding the nucleus, which we term the astral microtubule interaction

T26 T27 Optical tweezers in fission the role of microtubules in nuclear Generating cell form in yeast cell. The positioning of growth yeast: Mechanism of nuclear positioning, as well as the role of the fission yeast zones is regulated by the polarity nucleus in septum positioning, by marker Tea1 delivered by microtubules and septum positioning displacing the nucleus with optical to cell ends. In the absence of Stefania Castagnetti1, Béla Novák1,2 1 2 tweezers. A displaced nucleus returned 1,3 microtubules, a new growth zone is Isabel Raabe , Leonardo Sacconi , to the cell center by the pushing force and Paul Nurse activated near the nucleus in the Francesco Pavone2, 1 exerted by microtubules against the cell 1 middle of the cell. This only occurs Iva Tolic-Nørrelykke tips. Nuclear displacement during Cancer Research UK, 44 Lincoln’s Inn when the nucleus is a sufficient Fields, London, WC2A 3PX, UK 1 interphase or early prophase resulted in 2 distance from growing ends because Max Planck Institute of Molecular Cell asymmetric cell division, whereas Molecular Network Dynamics Research these ends exert lateral inhibition over Biology and Genetics, Pfotenhauerstr. displacement during prometaphase Group of Hungarian Academy of new growth zone activation. A two 108, 01307 Dresden, Germany Sciences and Budapest University of 2 resulted in symmetric division as in component morphogenetic mechanism European Laboratory for Non-linear unmanipulated cells. These results Technology and Economics, 1111 for activating a new growth zone is Spectroscopy, Via Nello Carrara 1, Budapest, Gellert ter 4, Hungary suggest that the division plane is 3 proposed: a Tea1-microtubular delivery 50019 Sesto Fiorentino (Florence), Italy specified by the pre-dividing nucleus. Rockefeller University, 1230 York component that positions a second Since the yeast nucleus is centered by Avenue, New York, NY 10021, USA spontaneous symmetry breaking Cells of Schizosaccharomyces pombe microtubules during interphase but not module which includes the lateral have a centrally placed nucleus and in mitosis, we propose that the Understanding how cellular form is inhibitory component acting nearby divide by fission at the cell center. establishment of the division plane at generated and maintained requires the existing sites of growth. Microtubules are required for the the beginning of mitosis is an optimal identification of mechanisms acting central position of the nucleus. Genetic mechanism for accurate symmetric beyond direct intermolecular studies suggested that the position of division in these cells. interactions. We have investigated the the nucleus may determine the position activation of a new growth zone in the of the septum. Alternatively, the septum middle of a normally rod-shaped fission may be positioned by the spindle, or by morphogen gradients or reaction diffusion mechanisms. We investigated

T28 T29 Control of growth However, the mechanism underlying Rgf1p is a specific binary switch by cycling between an polarity upon perturbed NETO remains to be understood. Rho1-GEF that coordinates inactive GDP-bound and an active Here we show that S-phase checkpoint GTP-bound conformational state and DNA replication pathway is required for the mainte- cell polarization with cell wall stimulates GS in its GTP-bound nance of the monopolar growth in the biogenesis in fission yeast. prenylated form. Rho1p regulates cell Muneyoshi Kanai1, Takashi Toda2 pol1/mon7 mutant. Indeed, the integrity by controlling the actin and Dai Hirata1 over-expression of either Rad3 or Cds1 Patricia García, Virginia Tajadura cytoskeleton and cell wall synthesis. kinase was able to inhibit the switch to and Yolanda Sánchez 1Department of Molecular bipolar under the normal growth We identified a new GEF (Guanine Biotechnology, Graduate School of condition. Further, the activity of the Instituto de Microbiología Bioquímica, nucleotide Exchange Factor), named Advanced Sciences of Matter, Dyrk family Pom1, essential for the CSIC/Universidad de Salamanca and Rgf1p, specifically regulating Rho1p Hiroshima University, Higashi Hiroshima switch to bipolar, in the pol1 mutant Departamento de Microbiología y during polarized growth. 739-8530, JAPAN was higher than that in the G1-arrested Genética, Universidad de Salamanca. 2Laboratory of Cell Regulation, Cancer cdc10 mutant with monopolar growing Campus Miguel de Unamuno. 37007, rgf1A cells are defective in cell integrity Research UK, London Research manner. These results suggest that the Salamanca, Spain. E-mail: [email protected] and lyse with a phenotype similar to Institute, PO Box 123, 44 Lincoln’s Inn Rad3-Cds1 kinases coordinate NETO cells devoid of Rho1 or Pck1/2 activity. Fields, London WC2A 3PX, UK with completion of DNA replication by In order to maintain intracellular In addition, cells deleted for rgf1+ regulating a molecule(s) that acts osmolarity and to produce cell shapes display a defect in actin organization In fission yeast, at a specific point in downstream of Pom1. other than spheres, cell wall expansion during bipolar growth. the G2 phase, growth polarity switches must be focused on particular regions. drastically from monopolar to bipolar. Fission yeast is a useful model system Overexpression of rho1+ suppressed This phenomenon is called NETO for studying cell wall biosynthesis and the rgf1A phenotypes while deletion of (New End Take Off). For NETO to take how this fits in the complex rgf1+ suppressed the severe growth place, two requirements have to be morphogenetic processes required defect in a Rho1-GAP null mutant fulfilled, a critical cell size and for the cell shape to be attained. (rgf1A). Rgf1p interacts functionally completion of DNA replication. with, and acts as a positive regulator of Schizosaccharomyces pombe cell wall Rho1 increasing the amount of GTP- is an extracellular matrix consisting of bound Rho1p and the a-1,3-glucan an outer layer of glycoproteins and an synthase activity. Rgf1p localized to the inner layer of carbohydrate polymers. It growing ends and the septum, where has been strongly suggested that (1,3)- Rho1p is known to function. Our results a-glucan polymer is synthesized first, suggest that Rgf1p probably activates followed by the addition of other the Rho functions necessary for components. The enzymatic system coordinating actin deposition with cell that catalyzes the synthesis of this wall biosynthesis during bipolar growth, polysaccharide is a(1,3)-glucan allowing the cells to remodel their wall synthase (GS). GS is composed of at without risk of rupture. least two fractions: the catalytic moiety of the enzyme (encoded by the bgs family of genes) and the regulatory component (Rho1p). Rho1p acts as a

T30 T31 Spatial regulation of mid1p medial cortex but redistributes towards Characterization of the fission peg1.1, identified some novel and cortical distribution by one pole of the cell. Coupling between yeast CLASP, Peg1 important properties. First Peg1 flipped mid1p localization and nuclear position from stabilizing mitotic microtubules to pom1 kinase is abolished. The asymetric cortical Agnes Grallert, Christoph Beuter1, de-stabilising interphase microtubules. distribution of mid1p negatively 1 Second, Peg1 was required to slow the a Inga Karig , Deepti Wilks, Séverine Morizur correlates with the monopolar growth 2 3 polymerization of interphase micro- and Anne Paolettia Steve Bagley , Rachel Craven , pattern of pom1 cells. It accounts for Ursula Fleig1, Iain M. Hagan tubules that had established end on a the displacement of the contractile ring contact with the cortex at cell tips. UMR144 du CNRS, Institut Curie, towards the non-growing tip during Third, S. pombe CLASP antagonized Cell Division, Paterson Institute for 26 rue d’Ulm 75248 PARIS Cedex 05 mitosis, while mid1p dissociation from CLIP170 (Tip1) and EB1 (Mal3) because France. Cancer Research, Wilmslow Road, the contractile ring and region of Manchester M20 4BX, 1Heinrich-Heine- microtubule bundles were longer and [email protected] septum formation at mitosis exit is not brighter in peg1.1 and peg1A cells, Universität, Lehrstuhl für funktionelle perturbed. Similar defects in mid1p while they are unusually short and In fission yeast, a major determinant for Genomforschung der Mikroorganismen, distribution were not found in other Universitätsstrasse 1, 40225 unstable in cells that lack Mal3 or Tip1 the division plane placement is mid1p, NETO mutants like tea1A, tea3A or 2 function. Fourth, although Peg1 resem- Düsseldorf, Germany, Advanced which defines a medial cortical com- bud6A. We conclude that pom1 kinase bled higher eukaryotic CLASPs by partment in interphase and recruits imaging facility, Paterson Institute for has a specific role in restricting mid1 Cancer Research, Wilmslow Road, physically associating with both Mal3 myosin II heavy chain myo2p at the distribution from the non growing cell and Tip1, neither of these +TIPs were Manchester M20 4BX onset of mitosis. This event initiates tips. Our data also identify a required for Peg1 to de-stabilise inter- contractile ring assembly in a medial pom1-independent mechanism The +TIPs CLIP170, CLASP and EB1 phase microtubules. Nor did Peg1 position and ensures the production of that prevents mid1p association with require Mal3 or Tip1 to bind accumulate at the plus end of micro- equally sized daughter cells after the growing tips. microtubules. Further, Mal3 and Tip1 cytokinesis. How mid1p anchoring is tubules where they control microtubule dynamics and mediate interactions with do not require Peg1 to associate with restricted to the medial cortex is not microtubules or cell tips. Consistently, specific targets. We identified fission understood. We report that in pom1 while deletion of either mal3 or tip1 mutants, mid1p is not restricted to the yeast CLASP in a screen for mutations that compromised spindle formation disrupts the linear cell growth, ablating peg1 has no effect. We are currently and another for molecules that antago- investigating the mechanism by which nised EB1 function. As expected Peg1 associated with kinetochores, however, Peg1 affects interphase microtubules. analysis of the interphase function of Peg1 with the conditional mutant,

T32 T33 Identification of an MTOC- created in the mto1A mutant. We have Cell growth and cell cycle: subunit and to the 5.8S and 25S rRNA localisation signal domain now identified a small domain that is Cdc2 meets ribosome component of the 60S subunit. necessary to target Mto1 to potential This pathway turns out to be very in Mto1 sites for microtubule organising centres biogenesis. complex in eukaryotic cells and (MTOCs). Unlike the wild-type protein, requires large number of trans-acting Itaru Samejima, Sergio Rincon*, which is present at all MTOCs, a Peter Stansfield, Margaret Harley, factors. In the budding yeast and Ken Sawin mutant lacking the domain was absent Fredrik Berklund, Jerome Wuarin. Saccharomyces cerevisiae, around 170 from the equatorial MTOC (eMTOC) proteins and around 70 small nucleolar Wellcome Trust Centre for Cell Biology, and the SPB. Fine mapping of the Biomedical Research Centre, Ninewells RNAs have been shown to participate University of Edinburgh, domain suggests that the localisation Hospital, University of Dundee, Dundee in the post-transcriptional steps of United Kingdom signals to the eMTOC and SPB involve DD1 9SY ribosome subunit synthesis, in addition two distinct but adjacent subdomains, to the 80 ribosomal proteins and the *visiting from Instituto de Microbiologia and that different mechanisms operate In a growing population of cells, cell rRNAs themselves. Bioquimica, Universidad de Salamanca, during interphase and mitosis to growth and cell cycle have to be Spain establish SPB localisation. Imaging of coordinated to guarantee the We have isolated and analysed GFP-tubulin and wild-type and mutant maintenance of cell size. How this complexes containing the major CDK in The microtubule cytoskeleton is a Mto1-GFP revealed a strong correlation coordination is regulated is still not fission yeast, Cdc2. We have identified dynamic system that creates various between mto1p (mis)localisation and clear. Recent discoveries have the proteins associated with cdc2 using types of microtubule arrays under sites available for microtubule indicated that ribosome biosynthesis a new proteomics technology termed different cellular states. One of the nucleation. It is known that many plays a critical role in establishing iTRAQ. We identified cdc2 and strategies employed to define a proteins at the SPB constitute the critical cell size thresholds at which cell associated cyclins, the ORC complex particular shape of microtubule septation initiation network (SIN), cycle transitions can occur. and also many proteins controlling cytoskeleton is to restrict microtubule a signal transduction pathway that ribosome biogenesis. Mutations in nucleation to specific intracellular sites. organizes events necessary to The synthesis of ribosomes uses up some of these proteins cause cell cycle mto1+ (microtubule organizer 1) is complete cell division. We will discuss vast amounts of the resources in defect, indicating that in addition to central to all cytoplasmic microtubule regulation of Mto1 localisation in rapidly growing cells, and is emerging controlling ribosome biogenesis, these nucleation, from both spindle pole body relation to the SIN pathway, which is as a key control point for the regulation proteins play a critical role in cell cycle (SPB) and non-SPB sites. mto1+ has also required for eMTOC formation. of cell growth and division, in yeast and regulation. We propose that cdc2 been shown to recruit gamma tubulin human cells. interaction with ribosome biogenesis complex to these sites, and new proteins is a key control point in the cytoplasmic microtubules are no longer In all organisms, during ribosome coordination of cell growth and biosynthesis, a polycictronic cell cycle. pre-ribosomal RNA (pre-rRNA) transcript is spliced to the mature 18SrRNA component of the 40S

T34 T35 A novel checkpoint reveals a is inhibited and preparation for DNA Mechanisms of the APC/C- APC/C recognises substrates and coupling between general cell replication (formation of the dependent ubiquitylation ubiquitylates them for degradation. pre-replication complex) is delayed. We have set up APC/C-Fizzy-depend- growth and the cell cycle The checkpoint is totally dependent on and proteolysis ent and APC-Fizzy-related dependent Gcn2. Surprisingly, all components destruction assays in frog egg extracts Tonje Tvegard, Esben A. Nilssen, Hiro Yamano, Michelle Trickey, 1 required for formation of the and using them we have identified Héla Soltani, Stephen Kearsey , pre-replication complex are synthesized Margaret Hanwell and Yuu Kimata several new APC/C substrates in fission Marit Krohn, Cathrine A Bøe, Beáta on time even in UV-irradiated cells, yeast. To investigate receptor(s) that Grallert and Erik Boye. suggesting that an active mechanism is Marie Curie Research Institute, specifically bind to the substrates, inhibiting entry into S phase. In a gcn2 The Chart, Oxted, Surrey, we use a site-specific photo-crosslink- Department of Cell Biology, mutant, there is no reduction in the RH8 0TL, U.K. ing technique. We show that the APC/C Institute for Cancer Research, translation rate and the UV-induced cell or Fizzy family protein binds to the Rikshospitalet-Radiumhospitalet HF, cycle delay is abolished. Possibly, The ubiquitin pathway is an ATP- substrate, but the receptor is not Montebello, 0310 Oslo, Norway dependent tagging system for protein 1 phosphorylation of eIF2`, and not exclusive to either the APC/C or Fizzy, and Department of Zoology, necessarily the reduced translation, is degradation. The anaphase-promoting suggesting that cells use different University of Oxford, UK. required for the UV-induced G1 delay. complex/cyclosome (APC/C) is an receptors depending on the substrates. In a non-phosphorylatable eIF2` essential E3 ubiquitin ligase that We will discuss how the APC/C We have shown that fission yeast cells mutant, the formation of pre-RC is not ubiquitylates numerous proteins at recognises different substrates with delay entry into S phase after UV delayed. However, UV irradiation does specific times in the cell cycle. latest data. irradiation in G1 (1). The G1 delay does repress translation in this mutant, Although the broad outlines of the not depend on any of the classical suggesting an eIF2·-independent APC/C’s regulation are understood, checkpoint proteins Rad3, Cds1 and pathway for Gcn2-dependent regulation many important questions about this Chk1. Consistently, and in contrast to of translation. We are investigating large ubiquitin ligase complex remain other classical checkpoints, Cdc2 is not further how the translation machinery unanswered. We are studying the phosphorylated during the course of generates a signal to regulate cell molecular mechanisms by which the the delay. Our recent data suggest that cycle progression. the G1 delay is caused by a novel checkpoint mechanism linking 1. Nilssen EA, Synnes M, Kleckner N, translation and cell cycle progression. Grallert B, Boye E, (2003) Intra-G1 arrest We report here that UV irradiation in response to UV irradiation in fission activates the kinase Gcn2, which yeast, Proc. Natl. Acad. Sci. U S A.100, phosphorylates eIF2a, a key protein in 10758-63. the regulation of translation. When Gcn2 phosphorylates eIF2·, translation

T36 T37 The Domain of Securin The fragment inhibits separase, while The mechanism of cell cycle cycle arrest in mei4 cells is unknown at separase is recruited normally and progression through meiosis I present. We have recently found that Required for Stabilization and Cdc2p on tyrosine-15 phosphorylation securin is destructed. It may interfere by the meiosis-specific fork- Inhibition of Separase Can the separase activation after securin was maintained in mei4 cells. Have a Separase Cleavage destruction. If the 127DIE129 stretch in head transcription factor Site by Substitutions Cut2 is substituted to AIA, the fragment Mei4p in fission yeast We show here that dephosphorylation toxicity and the full-length function are of Cdc2p tyrosine-15 by overexpres- sion of cdc25+ or inactivation of wee1+ Koji Nagao and Mitsuhiro Yanagida abolished. The Cut2 fragment is Tonami, Y., Yamada-Namikawa, physically interacted with separase. C., Tochigi, A., Nakanishi, M. induced meiosis I in mei4 deleted cells. The level of cdc25+ mRNA was high Initial Research Project, Okinawa Interestingly, Cut2 is cleaved in a and Murakami, H. during meiosis I whereas it was low in Institute of Science and Technology, separase dependent manner if the cleavage consensus by separase is Department of Biochemistry mei4 cells. In contrast, the level of and Graduate School of Biostudies, wee1+ mRNA was low during meiosis I, Kyoto University, Japan introduced following the DIE sequence. and Cell biology These finding is consistent with our Nagoya City University Graduate whereas it was maintained in mei4 proposed model that the DIE region in School of Medicine cells. There are four FLEXs around Securin-separase complex is required cdc25+, while there are five FLEXs near for sister chromatid separation. Cut2 may mimic the cleavage site of wee1+. Electro mobility shift assays Securin degrades in an APC/cyclosome separase and inhibit the activation of In most eukaryotes, the Cdc2p kinase separase as a pseudo substrate. complexed with the B-type cyclin plays showed that Mei4p bound all of FLEX dependent manner at anaphase. around cdc25+ and wee1+ in vitro. Separase is activated upon the A temperature sensitive mutation a central role in the control of entry and Chromatin immunoprecipitation assays destruction of securin and cleaves cut1-K73/separase isolated by its progression through mitosis and specific resistance to the fragment meiosis. The Cdc2p protein kinase is also showed that Mei4p bound most of cohesin. Fission yeast securin/Cut2 the FLEXs around cdc25+ and wee1+ required for proper separase toxicity resides in the superhelical fully activated at the onset of mitosis in vivo. Finally, in cells deleted four localization has the motifs for region of separase, suggesting that and meiosis by dephosphorylation of the catalytic site and the helical Cdc2p tyrosine-15. Phosphorylation of FLEXs around cdc25+, entry into the destruction and separase-binding at meiotic nuclear division was severely the N- and C-termini, respectively. region in separase may cooperate Cdc2p on tyrosine-15 is catalysed delayed and cdc25+ mRNA level was Here we show the third essential for activation. mainly by the Wee1p kinase, and dephosphorylation is carried out mainly low. In addition, overexpression of domain, which becomes toxic when the mei4+ induced meiotic nuclear division 76-amino acid fragment (81-156) in the by the Cdc25p phosphatase. The earlier by inducing cdc25+ mRNA and middle of secrin/Cut2 is overproduced. meiosis-specific forkhead-type tran- scription factor Mei4p is required for reducing wee1+ mRNA. expression of many genes which are necessary for recombination and These results suggest that Mei4p is a sporulation. Mei4p can bind heptamer rate-limiting regulator of meiosis I by sequence (GTAAAYA) designed FLEX. activating cdc25+ and inhibiting wee1+ mei4 deleted cells completely arrest transcriptions. before meiosis I after meiotic DNA replication. The mechanism of cell

T38 T39 Suppressor mutants of the we have found that this dot structure, Poster Abstracts Cytoskeleton and Cell meiosis I arrest caused by composed of Mei2p and meiRNA, Morphogenesis sequesters another RNA-binding Pages 65-83 loss of the Mei2p dot protein named Mmi1p. Mmi1p binds to Bioinformatics, Genomics, and Tools a group of meiosis-specific transcripts Pages 1-18 Akira Yamashita1, Ryo Iwata2 and renders them unstable during the Mitotic Spindle and Masayuki Yamamoto1,2 mitotic cell cycle. The meiotic arrest Pages 84-105 due to the loss of Mei2p dot can be Environmental Responses 1Molecular Genetics Research rescued by a reduction in Mmi1p & Signalling Laboratory, 2Department of Biophysics activity (Harigaya, Y. et al., submitted). Pages 19-32 DNA Metabolism and Biochemistry, Graduate School of However, the molecular mechanism of Pages 106-142 Science, University of Tokyo, Japan mRNA destabilization by Mmi1p remains elusive. To isolate factors that Gene Expression Control: In fission yeast, an RNA-binding protein may function in the same regulatory From Chromatin to Proteins Mei2p is crucial for induction and pro- pathway as Mmi1p, we set out to Pages 33-47 motion of the meiotic cell cycle. Mei2p screen for suppressor mutants of the forms a dot structure in the horse-tail meiotic arrest of the sme2A mutant, nucleus during meiotic prophase. The which cannot form the Mei2p dot. Mitotic and Meiotic Cell-Division Mei2p dot is located at the sme2 locus We obtained several candidates, cloned Cycle on chromosome II. The sme2 gene the responsible genes, and identified Pages 48-64 encodes a non-coding RNA, meiRNA, factors implicated in the regulation of which binds to Mei2p and is essential poly(A) tail generation. for progression of meiosis. If cells lack meiRNA, they cannot form the Mei2 dot and arrest prior to meiosis I. Recently,

Poster Session: Fri 17th March, 1600-1900

Odd numbers: 1600-1730 Even numbers: 1730-1900

T40 00 Poster Abstracts Bioinformatic prediction of on genome-wide peptide sets. Maximum functional linkage applied to likelihood models of trait evolution (Pagel 1994) are then fitted to each pair B i o i n f o r matics, fission yeast proteins of genes, to detect whether there is statistically significant Genomics and To o l s Daniel Barker1*, Valerie Wood2, correlation in gene gain/loss on the Andrew Meade3 and Mark Pagel3 species’ phylogenetic tree. Existing functional annotation is not Phenotype and Gene Ontology is specific to mutant yeast phenotypes, 1 Sir Harold Mitchell Building, required by the method, which derives in Fission Yeast so allowing the capture of more granular data and thus allowing more School of Biology, pairs of functionally associated genes specific database searches. During the University of St Andrews, on the basis of gene presence/absence Tim Beck and John Armstrong St Andrews, Fife, KY16 9TH, U.K. and the phylogenetic tree only. Thus ongoing process of phenotype 2 classification it has been decided to The Wellcome Trust Sanger Institute, the method is complementary to School of Life Sciences, draw a distinction between phenotypes The Wellcome Trust Genome Campus, bioinformatic approaches that use University of Sussex, Falmer, Brighton, observable at the colony level and Hinxton, Cambridge, CB10 1SA, UK. sequence analysis or phylogenomics to BN1 9QG, UK. 3 those at the cellular level. This has AMS Building, School of Biological s h a re functional annotation of a known tentatively formed the basis for two Sciences, University of Reading, p rotein among its homologues or Traditionally, most biological data is not separate ontologies that are used to Whiteknights,Reading, RG6 6AJ, UK. o rt h o l o g u e s . recorded within any particular frame- describe structural phenotypes within *Corresponding author, The method holds promise for work or ontology. An ontology is a each domain. Colony phenotypes email [email protected] improved functional annotation of the structured vocabulary consisting of a contain high-level categories relating to Schizosaccharomyces pombe genome. hierarchy of terms where the terms are We describe a recent bioinform a t i c differentiation events (e.g. ability to We present a case study using S. precisely defined and relate to each method to predict functional linkage mate or form hyphae) and sensitivity pombe, 14 other ascomycetes, three other in meaningful ways. The most among proteins, using patterns of gene events (e.g. growth after exposure to other fungi and three animals, in which prevalent biological ontology is the p resence and absence across several differing environmental conditions). a novel functional association is pre- Gene Ontology (GO). GO has been species’ genomes (Barker & Pagel 2005). Cellular and organelle morphologies dicted between the products of S. utilized to unify the terminology used The method detects independent form the highest order categories for pombe gene sen34 (coding for proba- between model organism databases in instances of the correlated gain or loss cellular phenotypes. The process of ble tRNA-splicing endonuclease sub- three domains of knowledge; molecular of pairs of genes. Once one gene defining lower level terms and unit Swiss-Prot O60156, containing function, biological process and cellular coding for a component of a functional relationships is in progress using PFAM domain PF01974) and an S. component. The University of Sussex is unit (e.g. structural complex or biochemi- phenotype data available from FYSSION pombe gene of unknown function (pro- host to FYSSION, a community cal pathway) has been lost from a and from published work. It is tein O13890, containing domain of resource that makes available genome, we assume the remaining com- expected that during the compilation of unknown function PF02696). The strong Schizosaccharomyces pombe mutant ponents of the unit may be under the phenotype ontology there will be an correlation in losses of these two genes libraries. At present mutant reduced selective pre s s u r e and may also overlap with terms defined in GO. It is (p < 0.005) is strong evidence of func- phenotypes are classified by free-text be rapidly lost during evolution. The intended that where possible the GO tional linkage between their products, keywords. This hinders database data method achieves 100% specificity (i.e. terms will be incorporated to avoid and more generally between protein entry and querying since the z e ro false positives) at stringent cut-off s terminology redundancy. domains PF01974 and PF02696. terminology used is ambiguous. (Barker & Pagel 2005). There is therefore the need to develop The method requires a summary of Barker D, Pagel M 2005. Predicting an ontology where phenotypes can be functional gene links from phylogenetic- gene presence and absence in the statistical analyses of whole genomes. precisely described without the chance genomes of several species PLoS Comput Biol 1: 24–31. of misinterpretation. Work is underway (‘phylogenetic profiles’ for the genes in Pagel M 1994. Detecting correlated at Sussex to develop an ontology that the sense of Pellegrini et al. 1999), and evolution on phylogenies: A general method a phylogenetic tree relating the species for the comparative analysis of discrete (e.g. derived from nucleotide orprotein characters. Proc R Soc Lond B Biol Sci multiple alignments). For fully 255: 37–45. sequenced genomes, gene Pellegrini M et al. 1999. Assigning protein presence/absence data may be functions by comparative genome analysis: Protein phylogenetic profiles. Proc Natl Acad obtained bioinformatically, for example Sci U S A 96: 4285–4288 by sequence similarity searches

P1 P2 Does your species show prokaryotes. To improve annotation S a c c h a romyces pombe genes, our approach to probe design in GO? Gene Ontology coverage further, the GO Consortium 70-mer Genome Set was to consider exons within 1000 has recently begun an effort to actively bases of the 3’ end of each gene as annotation outreach. support new groups seeking to use GO primary design targets. If exons were for gene product annotation, and to Malcolm Cook, Madelaine Marchin, not available, or if they were shorter Jennifer I. Clark and Midori Harris make the resulting data available to the and Chris Seidel than 80 bases, then the terminal 1 kb of public. GO Consortium members offer the gene was used as a design target. GO, EMBL-EBI, WT Genome Campus, annotation tutorials at Consortium-wide Stowers Institute for Medical Research The result is 6918 longmer oligonu- Hinxton, CB10 1SD, United Kingdom courses ('Annotation Camps'), courses 1000 E 50th St cleotides for detecting coding run by individual member databases, Kansas City, Mo 64110 sequences or exons, in which many The best known goal of the Gene and conferences; several databases corresponding author: genes are assayed by more than one Ontology (GO) project is the develop- make annotation tools publicly [email protected] probe. The purpose of the 3’ bias is to ment of controlled vocabularies for the available. Individual database curators compensate for the inefficiency of description of attributes --molecular may also learn directly from 'mentors' Abstract: We have designed a publicly reverse transcriptase labeling of cDNA. function, biological process, and cellu- with extensive experience using the GO available genomic reagent for pro d u c i n g In addition, the set contains probes to lar component – of genes and gene system. In the future, the GO DNA microarrays to measure gene detect 333 3’ UTRs and 1521 introns. products. The usage of GO terms in Consortium plans to develop tools and expression in S. pombe. The set The sequences are publicly available. database annotations is an communication routes to enable indi- currently contains 8785 probes and The genome set will make genomic indispensable aspect of the project; GO vidual researchers to contribute an consists of sequence optimized 70-mer investigations into S. pombe gene annotations are now available for over notations for their area of expertise oligonucleotides which can be expression more accessible to the 30 genomes, with recent additions synthesized and printed on glass slides community. including chicken and several using conventional techniques. While S. pombe has approximately 5000

P3 P4 Imaging and Functional sensitive strains stained with the ProbeExplorer: (S. cerevisiaeand S. pombe). Alignment Analysis of 4,100 vacuolar stain CDCFDA and the a transcriptomic web-tool that tools are used to built the associations endocytic marker FM 4-64. A range of between the Affymetrixmicroarrays Schizosaccharomyces pombe morphological mutants were identified includes S. pombe together probe sequences and the transcrip- Temperature Sensitive including strains affected in vacuole with other genomes and the tomes for human, mouse, rat and Mutants biogenesis and in cell shape; in addition mapping of microarrays oligo yeasts. Search by keywords is available a strain that forms invasive hyphae on and user can also do searches and rich medium was discovered. Further probes to transcripts alignments on the genomes introducing James Dodgson and John Armstrong functional analysis of the morphological any DNA or protein sequence query mutants has centred on describing the Alberto De Luis using an incorporated BLAST2 tool. Department of Biochemistry, mutant phenotypes in greater detail and Javier De Las Rivas ProbeExplorer uses ENSEMBL (v.36) University of Sussex, and in complementing the strains with data for the metazoa genomes, GeneDB Brighton, Sussex, BN1 9QG a plasmid based genomic library. A Bioinformatics and Functional data for the S. pombegenome and SGD classical genetic approach involving Genomics Research Group. data for the S. cerevisiaegenome. In The FYSSION resource incorporates mapping mutations of interest to Cancer Research Center (CIC, USAL- this way the tool integrates in a single two libraries of Schizosaccharomyces markers is underway. CSIC), E37007 Salamanca, Spain. database the fission yeast genomic and pombe mutant strains, one affecting ([email protected], [email protected]) transcriptomic data to allow search and temperature-sensitive essential genes comparative analysis with the other the other non-essential genes. A ProbeExplorer is an open access genomes. Also specific to yeasts, microscopy platform was developed for web-based bioinformatic tool designed ProbeExplorer includes the mapping of the imaging of 4,100 temperature- to show the association between all probe sequences from YEAST2 and Affymetrixmicroarray oligonucleotide Ygs08 Affymetrixexpression GeneChips probes and transcripts in a genomic on S. cerevisiaeand S. pombeloci context, but flexible enough to serve See Web page: also as a simplified genome and http://probeexplorer.cicancer.org/ transcriptome browser. Coordinates and sequences of the genomic entities (loci, exons, transcripts), including vector graphics outputs, are provided for fifteen metazoa organisms and for two yeasts

P5 P6 Comparing haploinsufficient Institute of Bioscience & Biotechnology Where do we GO next? ontology development process. Several mutants between (KRIBB) has constructed genome- Involving biologists in Gene novel mechanisms of soliciting and wide heterozygous mutants in incorporating community input have Saccromyces cereviseae and Schizosaccharomyces pombe. For the Ontology development guided the most recent changes in the Schizosaccharomyces pombe comparative genomics study between ontology content. To promote commu- through Gene Ontology (GO) S. pombe and S. cerevisiae, they Midori Harris nication among contributors and ensure performed the time series experiments consistency within the ontology, the GO functional analysis of 96 well plate-based O.D measure- GO, EMBL-EBI, WT Genome Campus, Consortium has established Curator ments for ~4,000 strains. We applied Hinxton, CB10 1SD, United Kingdom Interest Groups, which are formed of Sangjo Han, Minho Lee logistic growth model to their data and Consortium members and community and Dongsup Kim* estimated growth rates of each strains The Gene Ontology (GO) project experts, and focus on specific areas in each experiment. Then, using nested http://www.geneontology.org/ within the ontologies. In addition, GO * To whom correspondence should be ANOVA model considering the effects constructs and uses ontologies to curators and biologists come together addressed at: Department of among experiments and plates, we facilitate the biologically meaningful to consider specific biological topics at BioSystems, Korea Advanced Institute selected HI mutants which seemed to annotation of genes and their products meetings devoted to ontology content. of Science and Technology, 373-1, show distinctively slow growth rates in a wide variety of organisms. The GO Many recent improvements in GO stem Guseong-dong, Yuseong-gu, Daejeon, when tested at the significance level of ontologies describe gene product from the first content meeting, in which 305-701, Republic of Korea. 0.2. Despite the preliminary nature of attributes in three key biological members of the GO group and domain E-mail: [email protected] the experimental data, we performed domains: molecular function, biological experts in plant pathogens, the cell the GO functional analysis on the process, and cellular component. cycle, and metabolism participated. Haploinsufficiency is defined as a selected HI mutants. We will discuss Within GO, terms and relationships can dominant phenotype in diploid the comparative study of two important be added, refined, and reorganized as organisms that are heterozygous for a models in terms of haploinsufficiency biological knowledge advances. loss-of-function allele. Using complete based on GO functional analysis Curators who use GO terms for gene set of Saccaromyces cerevisiane product annotation play a key role in heterozyous deletion strains, Adam et. [1] Adam M. et al. Genetics 169: 1915-1925 the development of GO. To comple- al revealed that haploinsufficient (HI) ment their input, the GO Consortium mutants are functionally enriched for strives to involve members of the bio- metabolic processes carried out by logical research community in the molecular complexes such as ribosome, and mostly due to insufficient protein production[1]. Recently, our collaborating group at Korea Research

P7 P8 Analysis of systematic genes in fission yeast. Fission yeast The UniProt Knowledgebase, to other databases. genome wide deletions has an estimated 4973 protein coding the Fungal Proteome For example, there are cross-references genes. KanR heterozygous deletion to the initial data source when derived of the fission yeast diploids have been constructed from a Annotation Program (FPAP) from translation of coding sequences Schizosaccharomyces pombe h+/h+ ade6-M210/ade6-M216, and the status of the (EMBL). There are also cross-refer- leu-32/leu1-32, ura4-D18/ura4-D18 Schizosaccharomyces pombe ences to PDB when sequence and 3D- 1 3 3 strain and these diploids used to structure information are available. Hayles, J. , Hoe, K-L. , Kim, DU. , genome data within this Park, H.4, Won. M.3, Yoo, H-S.3, generate haploids spores, by a high Sequence pattern and profile matches Palmer, G.1, Duhig, T.1, Peat, N.1, throughput transformation of pON177 framework are cross-referenced to InterPro. 2 2 containing Mat1-M. The subsequent InterPro is a database of protein fami- Mandeville, R. , Wexler, S. , 1 2 2 spores have been germinated and Vivien Junker , Kati K. M. Laiho , lies, domains and functional sites in and Nurse,P, 1 1 screened for cdc and cut phenotypes, Marc Feuermann , Ivo Pedruzzi , which identifiable features found in 2 1 1 Cell Cycle Laboratory, Cancer cell shape defects and for cell viability. Rolf Apweiler , Amos Bairoch known proteins can be applied to We have found that about 28% of unknown protein sequences. It does Research UK, The London Research 1 Institute, 44, Lincoln's Inn Fields, fission yeast genes are essential Swiss Institute of Bioinformatics, CMU, not create signatures itself but amalga- London WC2A 3PX, UK compared to about 18-20% in budding 1 rue Michel-Servet, CH-1211 Geneva mates the efforts of the member data- 2 yeast and a comparison between 4, Switzerland bases. These currently include The Rockefeller University, 1230 York 2 Avenue, New York, New York 10021 fission yeast and budding yeast The EMBL Outstation - The European PROSITE, Pfam, PRINTS, ProDom, USA suggests that only about 60% of Bioinformatics Institute, Wellcome Trust SMART, TIGRFAMs, PIRSF, SUPER- 3 Korea Research Institute of essential genes in fission yeast are Genome Campus, Hinxton, Cambridge, FAMILY, Gene3D and PANTHER. Bioscience and Biotechnology (KRIBB), also essential in budding yeast. The CB10 1SD, United Kingdom Interaction data and biological informa- Laboratory of Human Genomics P.O. current estimate is that there will be tion, when available, are given via Box115, Yusong, Daejon 305-333 180 new cell cycle genes and 166 new The UniProt Knowledgebase IntAct and GO cross-references, Korea morphology genes from a total set of (UniProtKB) provides a central data- respectively. There can also be cross- 4 Bioneer Corporation 322 cell cycle genes and 244 base of protein sequence and function references to organism-specific data- 49-3 Munpyeong-dong, Daedeok-gu, morphology genes. data. UniProtKB consists of two sepa- bases. Daejeon 306-220, South Korea rate datasets, namely TrEMBL and Swiss-Prot. TrEMBL contains transla- The Fungal Proteome Annotation We are using a genome wide set of tions of coding sequences in the Program (FPAP) was set up to deal deletion mutants to identify the major EMBL/DDBJ/GenBank nucleotide data- with the increasing amount of sets of cell cycle and cell morphology bases and directly determined protein sequence data, resulting from complete sequences extracted from the literature, sequencing of fungal genomes. submitted directly to UniProtKB and Schizosaccharomyces pombe is one of sequences of Protein Data Bank (PDB) the model yeasts within FPAP. structures. Swiss-Prot is the manually The current status of S. pombe entries annotated section of UniProtKB. It as of 24-JAN-2006 in UniProt release strives to provide complete, high quali- 6.9, which consists of Swiss-Prot ty annotation of protein sequences via release 48.9 and TrEMBL release 31.9 reading and analysing corresponding is 2944 and 2251, respectively. articles published about the particular sequence. There is also minimal redun- dancy and extensive cross-referencing

P9 P10 The More the Merrier: regard to the identity and numbers of Comparative tandem repeat repeats in non-coding sequences Comparative Analysis of periodically expressed genes. We pres- analysis of the S. pombe typically have a range of repeat unit ent benchmark and reproducibility lengths up to 35 bp, whereas those in Microarray Studies on Cell analyses showing that the main genome protein coding regions have repeat unit Cycle-Regulated Genes in discrepancies do not reflect differences lengths that are predominantly Fission Yeast in the data themselves, microarray or Ann-Marie Patch and Stephen J. Av e s multiples of three and include many synchronization methods seem to lead greater than 100 bp. Despite the long

1+ only to minor biases, but rather in the School of Biosciences, University of evolutionary distances that separate Samuel Marguerat , Thomas S. 2+ 2 interpretation of the data. Our reanaly- Exeter, Exeter EX4 4QD, UK the three species, tandem repeat Jensen , Ulrik de Lichtenberg , Brian 1 3 sis of the three data sets reveals that [email protected] characteristics are conserved, but the T. Wilhelm , Lars J. Jensen and Jürg 1* combining all independent information patterns of the most commonly Bähler leads to an improved identification of Tandem repeats are approximate or identified consensus sequences are

1 periodically expressed genes. These identical copies of contiguous DNA species-specific. Tandem repeats occur Cancer Research UK Fission Yeast evaluations suggest that the available sequences. They are often more frequently than expected in genes Functional Genomics Group, Wellcome microarray data do not allow reliable polymorphic, undergo copy number encoding proteins involved in the bio- Trust Sanger Institute, Hinxton, identification of more than about 500 mutation, are commonly exploited as genesis and organisation of cell Cambridge CB10 1SA, UK 2 cell cycle-regulated genes. The tempo- genetic markers, have been identified organelles, the cytoskeleton and Center for Biological Sequence ral expression pattern of the as causal agents for several human ribosomes. Analysis of yeast ortholo- Analysis, BioCentrum-DTU, Technical top-500 periodically expressed genes diseases, and increasing evidence gous genes showed over half (53.3%) University of Denmark, DK-2800 is generally consistent across suggests that they play a variety of of S. pombe tandem repeat-containing Lyngby, Denmark 3 experiments, and the three studies regulatory and evolutionary roles. We genes have a similar sequence in a EMBL Heidelberg, Meyerhofstrasse 1, together with our integrated analysis have performed a comprehensive and conserved position in the S. cerevisiae D-69117 Heidelberg, Germany + provide a coherent and rich source of comparative analysis of tandem repeats orthologue. Tandem repeats within pro- These authors equally contributed to information on cell cycle-regulated in the genomes of S c h i z o s a c c h a ro m y c e s tein coding regions of S. pombe non- this study gene expression in S. pombe. We hope pombe, Saccharomyces cerevisiae and orthologues have patterns that point to that this report will resolve the apparent a representative human chromosome. expansion and contraction of encoded The last two years saw the publication discrepancies between the previous Tandem repeats are widespread in alpha helix regions. A DNA fingerprint- of three genome-wide gene expression studies and be useful both for wet-lab S. pombe, despite its small genome ing analysis of six independent S. studies of the fission yeast cell cycle. biologists and for theoretical scientists size. Excluding centromere, telomere pombe wild-type isolates using select- While these microarray papers largely who wish to take advantage of the data and dispersed repeat sequences we ed tandem repeats identified polymor- agree on the main patterns of cell for follow-up work. detected 819 non-overlapping tandem phic loci for 11 out of 15 tandem cycle-regulated transcription and its repeats in S. pombe and 1615 in repeats within protein coding control, there are discrepancies with S. cerevisiae, representing 0.61% and sequences and 10 out of 11 in 0.77% of these genomes respectively; non-coding sequences. Two loci have this compares with 2.08% for human been identified as polymorphic in chromosome 21. Tandem repeats are laboratory strains. widely dispersed in a non-random, clustered distribution, frequently occurring in protein coding as well as non-coding sequences. Tandem

P11 P12 A large-scale screen in pairing, and DNA replication is followed Analysis of 5’ and 3’ the genomic sequencing project (Wood S. pombe identifies seven by high levels of recombination Untranslated Regions et al. Nature, 415, 871- 880, 2002) it is between homologous chromosomes known that the distances between novel genes required for (homologs). This recombination is in S.pombe cDNAs. tandem, convergent and divergent critical meiotic events important for the reductional segrega- genes in S.pombe are greater than tion of homologs at the first meiotic Nigel Peat*, Trevor Duhig*, those in S.cerevisiae. ‡ Cristina Martín-Castellanos1, division; without further replication, a Aengus Stewart , Jacky Hayles* There are several possible explanations # Miguel Blanco1, Ana E. Rozalén1, second meiotic division yields haploid and Paul Nurse for these results; 5’ mRNA regions may Livia Pérez-Hidalgo1, Ana I.García1, nuclei. In the fission yeast be systematically longer in S.pombe; Francisco Conde1, Juan Mata2, Schizosaccharomyces pombe, we have *Cell Cycle Laboratory, Cancer Promoter regions may be of greater Chad Ellermeier3, Luther Davis3, deleted 175 meiotically upregulated Research UK, London UK. complexity in S.pombe and therefore ‡ Pedro San-Segundo1, genes and found seven genes not pre- Bioinformatics & Biostatistics, Cancer require longer 5’ UTRs; S.pombe may Gerald R. Smith3, and Sergio Moreno1 viously reported to be critical for meiot- Research UK, London UK. have genuine intergene regions. ic events. Three mutants (rec24, rec25, #Rockefeller University, New York, USA. So far we have sequenced over 2,790 1Instituto de Biología Molecular y and rec27) had strongly reduced meio- cDNAs and have identified over 500 Celular del Cáncer, CSIC/Universidad sis-specific DNA double-strand break- Using a cDNA library we are unique genes; results from this study de Salamanca, Campus Miguel de age and recombination. One mutant sequencing clones at random to are presented here. Unamuno, 37007 Salamanca, Spain (tht2) was deficient in karyogamy, and analyse the 5’ & 3’ un-translated 2The Sanger Institute, The Wellcome two (bqt1 and bqt2) were deficient in regions (UTRs). We are also using this Trust Genome Campus, Hinxton, telomere clustering, explaining their data to look for the existence of true Cambridge, CB10 1SA, UK defectsin recombination and inter-genic regions in S.pombe. From 3Division of Basic Sciences, Fred segregation. The moa1 mutant was Hutchinson Cancer Research Center, delayed in premeiotic S phase Seattle, WA, U.S.A. progression and nuclear divisions. Further analysis of these mutants will Meiosis is a specialized form of cell help elucidate the complex machinery division by which sexually reproducing governing the special behavior of diploid organisms generate haploid meiotic chromosomes. gametes. During a long prophase, telomeres cluster into the bouquet configuration to aid chromosome

P13 P14 YO G Y: a web-based integrated database provides rich, combined Pfam and the Pombe clan (protein families) have been database to retrieve protein information on orthologs in various Proteome:Comparisions and identified using a variety of methods, other species using data from four including sequence, structure and orthologs from fission and independent resources: KOGs, Complexes profile-profile comparisons. We have budding yeast Inparanoid, Homologene, and a table of made tools available for understanding curated yeast orthologs. Associated Benjamin Schuster-Böckler, the relationship of the families within Christopher J. Penkett, Gene Ontology (GO) terms of orthologs Jaina Mistry, John Tate, Valerie a clan. Clans have enabled the James A. Morris, Valerie Wood, can be retrieved for functional Wood, Alex Bateman, Rob Finn association of many more families to and Jürg Bähler inference. Integrating these different a known structure and enriched and complementary datasets provides Wellcome Trust Sanger Institute, annotation. For example, several Cancer Research UK Fission Yeast a straightforward resource to identify Hinxton, UK domains of unknown function (DUFs) Functional Genomics Group, Wellcome known and predicted orthologs of yeast can be assigned a putative function. Trust Sanger Institute, Hinxton, proteins. Pfam is a protein family database. Each Cambridge CB10 1SA, UK family is represented by curated In addition to domain annotation, YOGY is accessible online at: multiple sequence alignments, profile Pfam allows the exploration of protein http://www.sanger.ac.uk/cgi-bin/ We present YOGY (Yeast OrtholoGY), Hidden Markov Models (HMMs) and interactions at the domain and PostGenomics/S_pombe/YOGY/yogy- annotation. Pfam has over 75% sequence levels using iPfam. iPfam a web based resource for orthologous search.pl proteins from the two yeasts coverage of the S. pombe proteome, is a database of interacting Pfam Schizosaccharomyces pombe and and thus provides a rich resource for domains. These interactions are identi- Saccharomyces cerevisiae. Using a understanding the domain repertoire in fied by mapping Pfam domains on to yeast gene or protein as a query, this S. pombe. Similar levels of coverage known 3D structures and calculating have been achieved for other organ- the physical bonds that form the isms making Pfam an excellent tool for interaction. From these calculations, comparative proteomics. The Pfam multiple sequence alignments with website provides simple tools for markups of contacting residues, analysing the distribution of a domain graphs showing the domain interaction within a single proteome and between networks and details of residue-residue proteomes. Tools for comparing domain bonding are available via a series of repertoire between species are web tools and downloadable files. also available.

Recently, Pfam has introduced a hierarchical classification of protein families, termed ‘clans’. Members of a

P15 P16 Regulatable promoter with After uracil wash out, the gene is Fission yeast, Johnston 2005). However, the fission short induction time ~8-fold repressed within 30 minutes the canonical eukaryote yeast displays a number of biological and ~100-fold within 120 minutes. We features and characteristics (including a * cloned a 675bp fragment taken from higher level of conservation and closer Stephen Watt, Juan Mata, Valerie Wood Gavin Burns, and Jürg Bähler the promoter region of SPAC1002.19 correspondence of many biological into the KanMX6 and NatMX6 processes with higher eukaryotes, cassettes of PCR targeting plasmids, The Wellcome Trust Sanger Institute, lower redundancy, and fewer genes) Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1SA Hinxton, Cambridge, CB10 1SA, UK. enabling the control of transcription of which are making it a strong contender a selected gene at its normal chromo- in this particular race. Moreover, recent *email: [email protected]; somal locus. We demonstrate that this One of the broader aims of biology is to reviews suggest that many S. cere v i s i a e tel: +44 (0)1223-494862 system allows altering transcription of provide a complete description of a genes have produced no data in large the pom1 gene within 15 minutes. eukaryotic cell and ultimately to dissect scale functional genomics experiments, A regulatable promoter that can be Comparisons with different nmt1 completely the molecular mechanisms and that previously known genes are switched on and off within short times which form the basis of eukaryotic life. would be a useful tool for the fission constructs suggest that the amount of highy overrepresented in these regulation is between nmt41 and nmt81 Many core eukaryotic processes are datasets (Hughes et al 2004). yeast community. Microarrays are ideal and the overexpression is much weaker conserved and it is generally accepted Functional genomics is therefore not to screen for genes that are strongly that these are often carried out by regulated under selected conditions, than with the strongest nmt1 promoter. providing the expected clues to the The new system may be more suitable orthologous proteins (Chervitz et al function of these experimentally which otherwise have little effect on to provide a pulse of transcription with- 1998). This conservation has allowed intractable genes. The rate at which global expression profiles. We identified the description of numerous "canoni- three genes whose expression levels in a short time (e.g. during a particular novel genes are characterised, and cell-cycle stage) rather than completely cal" parts and mechanisms to describe current annotation statistics suggest are uracil-dependent. The gene switching off gene expression. More the consensus of shared sequence, that it is not inconceivable that the regulated the most strongly in response parts, features and functions. These to uracil is SPAC1002.19. After uracil experience with other genes is required fission yeast will be the first organism to know how useful this new regulat- consensi can be merged and extended to achieve the landmark status where addition, it is induced ~20-fold within able promoter will ultimately be. into the concept of a 'canonical we have some information about the 15 minutes and ~100-fold within 60 eukaryote' to describe universal parts minutes. The basal expression level basic molecular function, biological and mechansms necessary to define a p rocess and cellular component of every seems low (background signal level). eukaryotic cell. The yeasts S. cerevisiae p rotein coding gene product. This is a and S. pombe are both making major p re requisite step towards a more com- contributions to our fundamental under- plete understanding of a eukaryotic cell. standing of the biology of a 'canonical Here I present the current view of the eukaryote'. This is primarily because proteome based on annotation to gene the majority of the gene products in ontology (GO) terms, from both S. cerevisiae and S. pombe have some literature curation and annotation functional similarities with genes transfer from orthologous proteins, and present in higher eukaryotes, making a comparison with S. cerevisiae. I will them both ideal models for eukaryotic also present the current level of processes (Wood et al 2002). coverage based on membership of The budding yeast S. cerevisiae is identified protein families and a universally considered likely to be the comparison with S. cerevisiae. first eukaryotic organism to be fully understood in terms of its basic biology Current data indicates: (where we know, or can infer, some- i) S. pombe has a lower percentage, thing about the molecular function, and absolute number, of genes which are biological process and cellular not assigned to a single GO term. component of each and every protein ii) S. pombe has fewer orphans coding gene) because of its long (sequences with no identified similarity distinguished history and intensity of and no published data). iii) S. pombe has a higher percentage of study (Hughes et al 2004, Fields and proteins covered by protein families in

P17 P18 the Pfam protein family database, and a I will also present data which identifies the Environmental higher proportion of these families are members of the set of 'core' eukaryotic shared with metazoans. genes conserved from yeast to metazoa, for Responses iv) S. pombe has fewer conserved genes which experimentation has provided no where nothing is known about the clues to molecular function or biological and Signalling biological process or molecular function, process in any organism studied so far, and and a greater number of these ‘unknown are therefore ideal targets for intensive conserved genes’ are shared with higher study. Hyphal Growth in S. Pombe The large amount of information already eukaryotes. available on cell growth and signalling Evelyn Amoah-Buahin, Klara Enczi, in S. pombe provides an excellent Monica Pacurar and opportunity to investigate this process *John Armstrong of differentiation and morphogenesis at the molecular level. We are investigat- Department of Biochemistry, School of ing the differences in RNA and protein Life Sciences, University of Sussex, composition between single cells and Falmer, Brighton, BN1 9QG, UK hyphae. By growing the cells in micro- scope chambers it is possible to image *Presenting author. GFP fusion proteins within hyphae. Email: [email protected] 1Amoah-Buahin, E., Bone N. and S. pombe grows in a single-celled form, Armstrong, J. (2005) Hyphal growth in or can mate and undergo meioisis and Schizosaccharomcyes pombe. Euk. Cell sporulation. We found that wild-type S. 4, 1287-1297. pombe can also differentiate to form elaborate branched hyphae which invade deep into solid medium1. Nitrogen deprivation in the presence of an abundant carbon soruce appears to be the main stimulus for hyphal growth. No MAP kinase is necessary for the response, but components of the cyclic AMP signalling pathway are required, in a reciprocal fashion to their requirement in mating. Deletions of those components not required gives a ‘hyperhyphal’ phenotype in which hyphal growth is more efficient than normal.

P18 P19 Cisplatin-induced stress some sensitive and resistant cells. Ish1p-Ish1p interactions (except budding yeast), localizes to the response of human Cisplatin up-regulated Hsp70, Hsp90, in vivo by FRET nucleus and does not have a definitive Rad51 and the alfa proteasome subunit function associated with it. Bis1p over- drug-sensitive and-resistant in some of the resistant cells. cDNA Finan, K1, Rajagopalan, R., expression causes a cell cycle delay tumor cells array studies in cisplatin-sensitive Theis, S. and P. G. Young. but the cells will still form colonies. and–resistant cervix squamous cell Giovanni Luca Beretta§, Laura Gatti§, carcinoma cells indicated that the We have investigated Ish1p dimeriza- § § Dept. of Biology, Queen’s University, Elisabetta Corna , Franco Zunino , expression of components of the tion/oligomerization in vivo using ‡ § Kingston, ON K7L 3N6, Canada recombinational repair pathway was 1 CFP/YFP FRET. We have shown that Jürg Bähler and Paola Perego current address: Sir William Dunn activated in sensitive cells, whereas Ish1-CFP and Ish1-YFP produce a § School of Pathology, Oxford University, Istituto Nazionale Tumori, resistant cells up-regulated DNA Oxford, OX1 3RE, U.K. FRET signal in all of the membrane via Venezian 1, 20133 Milan ITALY damage recognization/repair proteins. systems to which they are localized ‡ Modulation of genes involved in mitotic during log phase, heat shock and Wellcome Trust Ranger Institute, Ish1p is a protein of largely unknown Hinxton, Cambridge, CB10 1SA, UK progression was shown in resistant function with orthologues in many other osmotic stress. We do not detect cells, similarly to what observed in differences in the relative amount of fungal systems. It is expressed strongly resistant cells. Our results indicate that apparent dimers under any condition Global gene expression studies in in a MAPK dependent fashion during fission yeast have shown that cisplatin some pathways activated by cisplatin stress response and localizes to tested. We have succeeded in partially exposure are conserved between yeast titrating out the Ish1p FRET signal in activates a stress response, including membranes of the nuclear envelope and human cells, thereby supporting the nucleus by overexpressing the glutathione-S-transferase, heat shock, and cell periphery. By two hybrid and repair genes. Transcriptional the interest of yeast as a model system analysis we have shown that Ish1p can Ish1p-Ish1p interaction domain to study selected aspects of cellular targeted to the nucleus. response could explain increased ability bind to itself and presumably it forms response to platinum compounds. to repair or tolerate DNA damage in dimers or oligomers in vivo. resistant cells. In the present study, The interaction domain has been Although we also investigated the Ish1p-Bis1p using CFP/YFP FRET, we investigated whether human genes, partially defined. Strong Ish1p we were unable to detect a FRET signal orthologous to those found in yeast, overexpression is lethal. were modulated by cisplatin in pairs of between Ish1-CFP and Bis1-YFP, even during strong overexpression of cisplatin –sensitive and –resistant By two hybrid and co-immunoprecipita- both proteins. We also found that over- human tumor cell. Molecular and tion, we have shown that Ish1p inter- biochemical approaches were used to acts with Bis1p. Bis1p binds to the expression of Bis1p had no effect on the fraction of Ish1p forming dimers, assess the cell response to cisplatin. same Ish1p domain that is involved in suggesting that Bis1p may bind to an Glutathione-S-transferase activity was Ish1p-Ish1p dimerization. Bis1p is increased upon drug exposure both in highly conserved in all eukaryotes Ish1p dimer/oligomer.

P20 P21 Homocysteine and Cysteine Transcriptional profiling of yeast Global transcriptional changes. We are studying mediated growth defect is not exposed to cysteine and homocysteine responses of fission yeast genome-wide gene expression of revealed that genes coding for fission yeast in response to limiting associated with induction of antioxidant enzymes like glutathione entering stationary phase nutrients and after refeeding. We have oxidative stress response peroxidase, catalase and superoxide followed cells over a period of 11 days genes in yeast. dismutase were down-regulated. Christopher J. Penkett, as they went from exponential growth Furthermore, transcriptional response Luis López-Maury, and Jürg Bähler to stationary phase in EMM medium. Arun Kumar, Lijo John, Md. to homocysteine did not show any Most of the transcriptional and similarity to the response to hydrogen Cancer Research UK Fission Yeast physiological changes occur during a Mahmood Alam, Ankit Gupta, Functional Genomics Group, Gayatri Sharma, Beena Pillai*, peroxide. We also failed to detect short physiological window of 2-3 induction of reactive oxygen species in Wellcome Trust Sanger Institute, hours at the end of exponential growth and Shantanu Sengupta* Hinxton, Cambridge CB10 1SA, UK From Institute of Genomics homocysteine and cysteine treated (ODs between 1.5 and 2.5). cells, using fluorogenic probes. These and Integrative Biology, Mall Road, Cell growth and proliferation are Delhi-110007, India results indicate that homocysteine and Preliminary data on this ongoing project cysteine induced growth defect is not controlled by the availability of nutri- will be presented. ents. When cells exhaust available Intracellular thiols like cysteine, due to the oxidative stress. However, we found an increase in the expression nutrients, they enter a quiescent sta- homocysteine and glutathione play a tionary phase characterized by cell critical role in the regulation of of KAR2 gene, a well known marker of endoplasmic reticulum (ER) stress and cycle arrest and distinct physiological, important cellular processes. Alteration biochemical, and morphological of intracellular thiol concentration also observed HAC1 cleavage in results in many diseased states, for homocysteine and cysteine treated instance, elevated levels of cells which indicates that homocysteine homocysteine is considered to be and cysteine mediated growth defect an independent risk factor for may probably be attributed to ER cardiovascular disease. Budding Yeast stress. Transcriptional profiling also (Saccharomyces cerevisae) has proved revealed that genes involved in one- to be an excellent model system for carbon metabolism, glycolysis and studying many human diseases since it serine biosynthesis were up-regulated carries homologues of nearly 40% of on exogenous addition of cysteine and human disease genes and many homocysteine suggesting that cells try fundamental pathways are highly to reduce the intracellular concentration conserved between the two organisms. of thiols by upregulating the genes Here we demonstrate that cysteine and involved in their metabolism. homocysteine, but not glutathione, inhibits yeast growth in a concentration dependent manner. Using deletion strains (str2A and str4A) we show that cysteine and homocysteine independ- ently inhibit yeast growth.

P22 P23 Mechanism of action of tolerant mutant chr1-66T and the Metazoan RBP-JÎ/Suppressor DNA binding are conserved in the + chromium compounds parental strain 6chr exhibited of Hairless homologs S. pombe proteins (3). In vitro gel-shift significantly lower uptake. (Folia assays proved that both recombinant on Schizosaccharomyces Microbiol. 49, 31-36.2004). Fast trans- in S. pombe and endogenous Cbf1-1p recognize 51 2- pombe cells port of CrO4 and increased and bind to DNA probes. Both Cbf1-1p bioaccumulation was detected in a Martin Pfievorovsk y ˘, Jan Rynes,˘ and Cbf1-2p fusions with EGFP localize M. Pesti,1* Z. Gazdag,1 G. Papp,1 J. sensitive mutant, chr-515, in contrast to Tomás˘ Grousl,˘ Petr Folk, to the nucleus of living S. pombe cells. Antal,1 T. Stromájer-Rácz,1 Zs. the tolerant one, chr-1-66T (FEMS Frantisek˘ Puta˘ Single deletions of each gene appear to Koósz,1 K., Takács,1 Microbiol. Lett. 178:109-115. 1999). be viable (4, 5). I. Pócsi,2 J. Belágyi,1 P. Raspor3 Cr(VI) tolerarance and the oxidative Department of Physiology and stress sensitivity of chr-1-66T mutant Developmental Biology, Faculty of Our data strongly suggest that there are 1University of Pécs, Faculty of were attributed to a decreased specific Science, Charles University in Prague, bona fide homologs of the metazoan Sciences, Department of General and gluthatione reductase (GR) and mito- Vinicná 7, Prague 2, 128 00, Czech CSL transcription factors in S. pombe. Environmental Microbiology, Hungary, chondrial MnSOD activity. (J. Basic Republic We are working on detailed 2University of Debrecen, Faculty of Microbiol. 43:96-103. 2003). Cr(VI) characterization of the cbf1-1 and Sciences, Department of Microbiology sensitivity of the chr-51S mutant The CSL (CBF1/Su(H)/LAG-1) proteins cbf1-2 genes and we hope to solve the and Biotechnology, Hungary, 3University accompanied with significantly are highly evolutionarily conserved tran- mystery of proteins regulating of Ljubjana, Food Science and increased GR and glucose-6-phos- scription factors that mediate the effec- multicellular development appearing Technology Department, Slovenia phate dehydrogenase activities and tor step of the transmembrane receptor in a unicellular organism. high intracellular superoxide and perox- Notch signal transduction pathway. Stable chromium(VI)-sensitive and -tol- ide concentrations in spite of its Upon activation and proteolytic We acknowledge the financial support erant mutants were obtained by decreased glutathione (GSH) content. processing the intracellular part of of GACR˘ (204/03/H066) and GAUK induced mutagenesis of fission yeast These data strongly suggested that Notch enters the nucleus and binds to (B157/2005/B-BIO/PrF). heterothallic strains 6chr+lys1-131h+ and instead of GSH, the NADPH/GR system a CSL protein, converting it from a 9chr+leu1-32h-. The segregation of was the major one-electron Cr(VI) repressor to a potent activator of the References tetrades of selected tolerant mutants, reductant in vivo ( J. Basic. Microbiol. responsive genes. This pathway is 1. S. Artavanis-Tsakonas, M. D. Rand, chr1-66T, chr1-14T, indicated that 42:410-421. 2002). However, transfor- essential for metazoan development R. J. Lake, Science 284, 770 (1999). tolerance was determined by single mation of chr-1-66T mutants with and its misregulation results in severe 2. A. P. Weng, J. C. Aster, Curr. Opin. mutation of non allelic origin. Both vector carrying GR gene did not alter abnormalities, lethality or cancer (1, 2). Genet. Dev. 14, 48 (2004). chromium(III) and (IV) induced decreas- the Cr(VI) sensitivity. But sequence 3. R. A. Kovall, W. A. Hendrickson, es in the phase transition temperatures analysis of the transcription factor We have identified and cloned two EMBO J. 23, 3441 (2004). of the 5- and 14-doxyl stearic acid and Pap1/Chr1/Caf3 (Mol.Gen. Genomics putative CSL family member genes in 4. A. Decottignies, I. Sanchez-Perez, 3-doxylbutyric spin probes labelled 271:161-170. 2004) proved that Pap1 S. pombe, provisionally designated P. Nurse, Genome Res. 13, plasma membranes. Cr(III) treatment plays a central role in the determination cbf1-1 and cbf1-2. Their protein 399 (2003). caused a strong fluidizing effect and of chromium (VI) resistance via down- products share 35% and 23% similarity 5. J. Gregan et al., Curr. Biol. 15, the loss of metabolites adsorbity at 260 regulation of GR enzyme (unpublished). to human CBF1, respectively. Manual 1663 (2005). nm which process might be the main Pro-oxidative vs. antioxidative proper- inspection of their sequences revealed cause of growth inhibition and cell ties of ascorbic acid in chromium(VI)- that all amino acid residues required for killing by these impermeable ions. The induced damage was studied. Ascorbic changes in local structure produced by acid influenced Cr(VI) toxicity both as a Cr(VI) and its unstable intermediates, reducing agent, by decreasing Cr(V) the presence of Cr(III) in the outher and persistence, and as an antioxidant, by inner headgroup regions of the plasma decreasing intracellular superoxide membrane, and the changes in the anion and hydrogene peroxide electric charge of the cell surface could formation and by quenching free radi- all affect the physiological function of cals formed during Cr(VI) to Cr(V) the cells.(BBA 1421:175-182.1999; reduction (J. Appl. Toxicol. 25: 2005. FEMS Microbiol. Lett. 182:375-380. in press). 2000; BBA 1611:217-222. 2003). Fast 51 2- 1* transport of CrO4 was detected in a Corresponding author: sensitive mutant, chr-51S, while the [email protected]

P24 P25 Investigating the mechanism of Int6 have identified a 70 amino acid Multiple roles of the Ran and Importin-alpha proteins also of Int6-induced drug fragment sufficient to induce the drug RAN GTPASE and the influence nuclear envelope structure in resistance phenotype. Mutation to both higher and lower eukaryotes. resistance alanine of a single conserved IMPORTIN-ALPHA transport When the Ran GTPase is mis-regulated phenylalanine residue within this region receptors in fisson yeast in fission yeast, the nuclear pore com- Emma Rawson, Caroline Jenkins, abrogated resistance. His-tag fusions plexes are not uniformly distributed in Chris Norbury of this 70aa minimal fragment and the Shelley Sazer, Makoto Umeda, the nuclear envelope and cells undergo inactive mutant form have been Shahed Izaddoost, Gerald Lim H.W., unequal nuclear division followed by Sir William Dunn School of Pathology, constructed with a view to structural Yoshihiro Torii and Jonathan Miller nuclear envelope fragmentation. University of Oxford, UK studies and identification of interacting Deletion of the nuclear pore complex factors responsible for Pap1 activation. Department of Biochemistry and component nup133 or overexpression The translation initiation factor Other work currently being undertaken Molecular Biology, Baylor College of of either cut15 or imp1 exacerbates component Int6 (eIF3e) was previously indicates that the Sty1/Wis1 stress Medicine, Houston, Texas 77030 U.S.A. these nuclear envelope defects. identified in an overexpression screen signalling cascade may have a role in through its ability to cause multidrug the Int6/Pap1 drug resistance pathway. During the closed mitosis of S. pombe, resistance in fission yeast. Further The nuclear import of classical Nuclear the nucleus undergoes dramatic investigation showed this resistance to Localization Signal-containing proteins changes in shape from round, to oval, be dependent on Int6-mediated (cNLS) depends on the Ran GTPase to peanut, to dumbbell, before resolv- activation of the AP-1 transcription system and the importin-alpha family of ing itself into two discrete daughter factor Pap1, but not on previously transport receptors. Higher eukaryotes nuclei. In order to determine the bio- described protein-protein interactions have multiple importin-alpha family physical properties governing these of endogenous Int6. Serial truncations members, budding yeast has just one, changes in size and shape, we are but fission yeast have two, making it a developing a physical model based on good model system in which to current understanding of the fission characterize the specialized roles of the yeast nuclear architecture, nuclear importin-alpha proteins. shapes found during normal and Our investigations into the abnormal mitoses in fission yeast and isoform-specific functions of Imp1p and the physical characteristics of lipid Cut15p, the two importin-alpha pro- bilayers. teins in fission yeast, show that they have both unique and common roles (Umeda et al. Genetics (2005) 171: 7- 21). We are currently examining the specialized in vivo roles of Imp1p and Cut15p in fission yeast.

P26 P27 Nuclear transport in the putative Gag-binding region; nsp1 : A novel shock being the only exception. fission yeast as a useful a C-terminal large domain with 11 stress-responsive protein in We have named it nsp1(novel stress FXFG repeats and a C-terminal bipartite responsive protein 1). Our studies model for the nuclear import NLS-like motif. S. pombe demonstrate that the Spc1-Atf1 SAPK and replication of retroviruses: pathway in S.pombe regulates the Functional characterization of In this presentation we show that while Geetanjali Sundaram, Santanu Pal expression of this ORF. Computational the FXFG-repeat region of Nup124p is Chaudhuri, K.Sheelarani and sequence analysis further revealed a nucleoporin involved in absolutely required for Tf1 activity, the Dhrubajyoti Chattopadhyay interesting features about this ORF, retrotransposition FXFG repeats themselves are not. Dr. B. C. Guha including a major potential for Swapping this region with similar phosphorylation and glycosylation. Srivani Sistla regions from the Nup124p orthologues, Centre for Genetic Engineering and This coupled with the observation that and David Balasundaram Nup153 (human) or Nup1p Biotechnology, Department of this small protein has an unusually high (S. cerevisiae) allowed complete activity Biochemistry, University of Calcutta, 35 serine content (16%), indicates that Laboratory of Nucleopore Biology, of the protein to be restored. A knock- Ballygunje Circular Road, Kolkata- phosphorylation may play a major role Institute of Molecular and Cell Biology, down of Tf1 transposition was 700019, INDIA in regulating the function of the protein 61 Biopolis Drive, Proteos, Singapore observed upon overexpression of the encoded by this novel ORF. We have 138673 FXFG region from Nup124p as well as Our lab has been investigating the investigated the possibility of any Nup153 and Nup1p alone and not upon cellular responses towards Cigarette growth advantage or disadvantage pro- The propagation of Tf1, a long terminal expressing a FG rich region or a non- smoke using S.pombe as a model vided to the cells during various stress repeat (LTR)-containing retrotransposon specific region of similar size from system (Chaudhuri et al ,Yeast 2005; conditions upon overexpression nsp1. in the fission yeast nucleoporins of S. pombe. The FxFG 22: 1223–1238.). Our transcript level We have also studied its sub-cellular Schizosaccharomyces pombe is known repeats are known to interact with differential display studies identified a localization by overexpressing an to require many of the same processes a,importin transport factors. Results of 321 bp ORF to be highly upregulated N-terminal EGFP tagged version of the used by retroviruses to complete its life our work, exploring the function of the upon exposure to Cigarette smoke protein in S.pombe. Flow Cytometry cycle within the host cell and can there- FXFG region will be presented. Extract. Subsequent cloning and analysis of cells overexpressing this fore serve as a very useful model for sequencing of this ORF revealed it to protein indicates that nsp1 may directly the replication of retroviruses. Critical residues in the C-terminal be a sequence orphan coding for a or indirectly affect the cell cycle Nucleoporins are proteins at the bipartite NLS-like motif that are 11.4kDa Hypothetical protein. We have checkpoint machinery , especially when nuclear pore that play a role in traffick- required for Tf1 function have been confirmed the translation of this S.pombe is subjected to heat shock ing proteins between the nucleus and identified. The motif itself is well differentially expressed transcript by (42 deg C), a condition where the the cytoplasm. Extensive studies have conserved among the aforementioned demonstrating its association with transcript level of this ORF exhibit shown the organization of these orthologues of Nup124p. Interacting polysomal fractions of fission yeast. maximum induction. proteins at the nuclear pore complex in partners to this region are being Studies on the expression profile of this yeast. Nup124p is a non-essential dissected to understand its specific role gene show that it’s expression is nucleoporin found in S. pombe and in the Tf1 life-cycle and its phylogenic upregulated under most stress required for the nuclear import and conservation. Elucidation the conditions (Oxidative stress, Nucleotide activity of the retrotransposon Tf1. mechanism of Tf1 transposition would depletion, heat chock etc), osmotic Architecturally, it has 3 major domains, help in understanding retroviral entry the N-terminal region encoding a into the nucleus.

P28 P29 Repression of ergosterol indicating that Pof14 is degraded by SR-Protein Specific Kinase not another SRPK, Kic1p, phosphory- synthesis during adaptation to an autocatalytic mechanism. Pof14 Dsk1 Regulates the Cellular lates Srp1p in vivo. In contrast, Srp2 is interacts with the squalene synthase phosphorylated by not only Dsk1p but oxidative stress requires the Erg9, a key enzyme in ergosterol Localizations of SR Proteins also Kic1p when Dsk1p is not present new F-box protein Pof14 metabolism, in a membrane bound by Phosphorylation in Fission in the cell. Furthermore, although the independently of SCF complex that does not contain the core Yeast Schizosaccharomyces cellular distribution of the three SR- SCF. pof14 transcription is induced related proteins varies within the cell, 1 1 by hydrogen peroxide in a Pap1- pombe their localization patterns all depend on Lionel Tafforeau , Sophie Bamps , + + Monique Dewez1, Jean Vandenhaute1 dependent manner and a pof14 dsk1 ; whereas deletion in kic1 exhibits # Zhaohua Irene Tang, Amy Tsurumi, and Damien Hermand1, deletion strain quickly loses viability in little effect on their localizations. the presence of hydrogen peroxide due Sarah Alaei, Christopher Wilson, Therefore, the in vivo phosphorylation to its inability to repress ergosterol syn- Cathleen Chiu, of the SR proteins by Dsk1p correlates 1Laboratoire de Génétique Moléculaire Jessica Oya, Benson Ngo + (GEMO), Facultés Universitaires Notre- thesis. A pof14 mutant lacking the with their dsk1 -dependent localization. F-box behaves as wild type showing The data prove that the SR proteins Dame de la Paix, Rue de Bruxelles 61, W.M. Keck Science Center, Claremont 5000 Namur, BELGIUM that this function of Pof14 is independ- are the in vivo substrates of Dsk1p and ent of SCF. This indicates that repres- Colleges Claremont, demonstrate that Dsk1p is the major # CA 91711, USA corresponding author: sion of ergosterol metabolism by Pof14, kinase responsible for the modulation [email protected] and consequently modulation of of the SR protein localization in membranes composition plays a key Dsk1p is the orthologues of human SR- S. pombe. As Srp2p and Prp2p are specific protein kinase1 (SRPK1) in We describe a new member of the role in adaptation to oxidative stress. known to be involved in exonic splicing Schizosaccharomyces pombe. Dsk1p enhancers (ESEs) and spliceosome F-box family, Pof14 that forms a specifically phosphorylates fission canonical, F-box dependent SCF assembly, respectively, our results yeast SR (serine/arginine-rich) proteins, provide strong in vivo evidence for a ubiquitin ligase complex. The Pof14 Srp1p and Srp2p, and also Prp2p, the protein has intrinsic instability that is mechanism which regulates the cellular orthologue of human U2AF large sub- localization of the SR-related proteins abolished by inactivation of its F-box unit in vitro. However, little is known motif, Skp1 or the proteasome, by Dsk1p-mediated phosphorylation, about the in vivo targets of Dsk1p and thereby governing their activities in the biological consequences of the pre-mRNA splicing and/or in other Dsk1p-mediated phosphorylation. In cellular processes in S. pombe. this study we investigated the in vivo phosphorylation of the SR proteins and the effect of Dsk1p function on the cel- lular localizations of these SR-related proteins by ectopical expression of the corresponding GFP fusion proteins in wild-type and dsk1-deletion fission yeast strains. Interestingly, Dsk1p, but

P30 P31 S.pombe importin-alpha temperature-sensitive lethality. Gene Expression proteins, Imp1p and Cut15, Differences in the cellular functions that depend on Imp1p and Cut15p indicate Control: have common and unique- that they each have unique physiologi- functions. cal roles. They also have common roles From Chromatin to because the imp1null and the cut15-85 Proteins Makoto Umeda, Shahed Izaddoost temperature-sensitive mutations are and Shelley Sazer synthetically lethal; overexpression of separation. Here we have identified by cut15 partially suppresses the tempera- ACE2P controls the microarray analysis a group of genes Department of Biochemistry and ture sensitivity, but not the mitotic delay + + + + + expression of several genes (adg1 , adg2 , adg3 , cfh4 , agn1 , Molecular Biology, Baylor College of in imp1null cells; and overexpression of + + involved in cell separation in eng1 , and mid2 ) whose expression is Medicine, Houston, Texas 77030, USA. imp1 partially suppresses the mitotic dependent on the transcription factor defect in cut15-85 cells but not the loss Schizozaccharomyces pombe Ace2p and hence they were named The nuclear import of classical nuclear of viability. Both Imp1p and Cut15p are and constitutes a part of a Ace2-dependent genes (adg). Northern localization signal-containing proteins required for the efficient nuclear import transcriptional cascade at the analyses revealed that the expression depends on importin-alpha transport of both an SV40 nuclear localization end of mitosis. of the adg genes is dependent on receptors. In budding yeast there is a signal-containing reporter protein and + Ace2p and that ace2 transcription single importin-alpha gene and in high- the Pap1p component of the stress Maria Luisa Alonso-Núñez, requires Sep1p. The expression of all of er eukaryotes there are multiple response MAP kinase pathway. Imp1p Ana Belén Martín-Cuadrado, these genes varied during the cell importin-alpha-like genes, but in fission and Cut15p are essential for efficient Francisco del Rey and cycle, maximum transcription being yeast there are two: the previously nuclear protein import in S. pombe. Carlos R. Vázquez de Aldana observed during septation. Mutants characterized cut15 and the more lacking the identified genes showed recently identified imp1. Like other Departamento de Microbiología y defects or delays in cell separation importin-alpha family members, Imp1p Genética, Instituto de Microbiología to different extents, but a double supports nuclear protein import in vitro. Bioquímica, CSIC/Universidad de mutant devoid of the Eng1p In contrast to cut15, imp1 is not essen- Salamanca, 37007 Salamanca, Spain endo-a-1,3-glucanase and the Agn1p tial for viability, but imp1 null mutant endo-1,3-`-glucanase showed a cells exhibit a telophase delay and mild Schizosaccharomyces pombe cells phenotype very similar to that of ace2A divide by medial fission through mutants. According to these contraction of an actomyosin ring and observations, these two enzymes are deposition of a multilayered division likely to be the major enzymatic septum that must be cleaved to release activities involved in the dissolution of the two daughter cells. During the last the septum during cell separation. few years, the isolation of mutants On the other hand, it has been showed affected to different extents in cell that other two proteins, Fkh2p separation has provided some insight (a forkhead transcription factor) and into the mechanistic details of this Mbx1p (a MADS box protein) are process. Some of these mutants are involved, with Sep1p, in the expression an endo-`-1,3-glucanase (agn1+), during M phase of several genes like + an endo-a-1,3-glucanase (eng1+), ace2 . Because of that, we have an anillin homologue (mid2+), and two checked and showed that the transcription factors (sep1+ and ace2+). expression of Ace2p targets is The fact that the phenotype of sep1A modified to diferent extends in or ace2A mutants is more severe than these mutants. that of cells lacking eng1+, agn1+ or mid2+ suggests that these transcription factors also control the expression of other genes involved in cell-cell

P32 P33 Characterisation of Histone Protein domain prediction produces 12 Jmj1 is a member of Swr1 include a JmjC domain protein(2). Posttranslational Modification SET domain proteins in the S. pombe complex in S.pombe and has When an immunoprecipitation genome. The S. pombe genome also experiment with Pht1,(the homolog of in Schizosaccharomyces contains homologues of recently an activity at subtelomeric H2A.z in S.pombe) as a bait was pombe using LC-MS/MS Mass identified human histone demethylases, region performed, the Swr1 complex and Spectrometry. namely Jumonji (JmjC) domain proteins additionally nuclear assembly proteins (7) and SWIRM domain/Amine Authors: Sakalar M.Cagri1, were recovered. Chromatin oxidase proteins (2). Roguev Assen1, Wilhelm Brian2, Immunoprecipitation (ChIP) Buchanan, L., Roguev, A., 3 2 Shevchenko, A. and Stewart, A, F. Shevchenko Andrej , Bähler Jürg , experiments showed that in swr1 and Which of these proteins and domains Stewart A.Francis1 jmj1 deletion strains, Pht1 incorporation are responsible for methylation and into chromatin decreased. Deletion BIOTEC, Technische Universität 1 Dresden, Dresden, Germany. demethylation on different histone Affiliations: BioInnovationZentrum, strains of jmj1,pht1 and swr1 all Max Planck Institute of Molecular Cell residues? To investigate this question The University of Technology Dresden, showed sensitivity to TSA (a histone we are using mass spectrometry Dresden, Germany deacetylase inhibitor). Microarray Biology and Genetics, Dresden, 2 Germany. (LC LTQ MS/MS) to detect and quantify The Wellcome Trust Sanger Institute, expression analysis showed that the global levels of histone PTM in various Cambridge, UK sets of genes affected by jmj1, pht1 3 Country of Origin: New Zealand. genetic backgrounds of S. pombe, MPI of Molecular Cell Biology and swr1 deletions overlap significantly in combination with traditional and Genetics, Dresden,Germany and the upregulated genes show a Histone posttranslational modification molecular biology. Ultimately we want localisation bias towards the (PTM) is a fundamental feature of to comprehensively describe the Covalent histone modifications such as chromosome ends. Hence we propose higher order chromatin and is important histone code in S. pombe and methylation, acetylation as well as that Jmj1, in collaboration with Swr1C , in regulating chromatin structure and characterise the responsible proteins. differential incorporation of histone functions in incorporation of Pht1 and transcription. The combinatorial variants are shown to coincide with has an activity at subtelomeric region complexity of histone PTM Preliminary results from this study different chromatin compartments of chromosome ends in S.pombe. (the “histone code”) defines another indicate that S. pombe has a (eu- and heterochromatin) and mark layer of information responsible for remarkably similar chromatin active or repressed genes. Proteins References: epigenetic and cell-memory environment to higher eukaryotes and having JumonjiC (JmjC) domain were phenomena. A good example of such shares common features with shown to be involved in the 1. Ayoub N, Noma K, Isaac S, Kahan T, PTM is histone H3 methylation, Saccharomyces cerevisiae. maintenance of heterochromatin- Grewal SI, Cohen A. A novel jmjC domain protein modulates a modification that essentially defines euchromatin boundary and While retaining the fundamental manipulation of histone heterochromatization in fission yeast. different chromatin states throughout Mol Cell Biol. 2003 Jun;23(12):4356-70. the genome and provides markers for features of higher eukaryotic chromatin, modifications(1). In S.pombe, Jmj1 is silent chromatin (H3 K9 me) or actively the complexity of the system in one of the seven JmjC domain proteins. 2. Krogan, N.J., Keogh, M.C., Datta, transcribed genes (H3K4 and K36 me). S. pombe appears to be reduced by a In order to identify its interaction N., Sawa, C., Ryan, O.W., Ding, H., et al. smaller genome size and fewer partners, Jmj1(Msc1) was TAP-tagged A Snf2 family ATPase complex required Bioinformatics reveals a multitude of potential histone-modifying enzymes. and immunoprecipitated. We found that for recruitment of the histone H2A genes in the Schizosaccharomyces This should increase the ease of Jmj1 is a part of a multi-protein variant Htz1. pombe genome encoding specific exploring the histone PTM landscape, complex that resembles S. cerevisae Mol. Cell, 2003 12: 1565–1576. protein domains implicated in histone allowing greater resolution of the Swr1 complex (Swr1C). In S. cerevisae, H3 methylation. The SET domain is one modifications and a better Swr1C incorporates a H2A variant, of the catalytic domains responsible for understanding of their inter-relatedness. H2A.z, into nucleosomes and does not histone methylation on lysine residues.

P34 P35 RNA pol II subunit Rpb7 In many eukaryotes RNAi mediates Role of the APC/C E3 oligomerization, establishes the promotes centromeric DNA and chromatin modifications Ubiquityl ligase in heterochromatin structure. A further resulting in transcriptional silencing. stabilization of heterochromatin transcription and In fission yeast centromeric repeats are heterochromatin assembly involves recruitment of Cohesin by RNAi-directed chromatin transcribed into siRNA precursors in fission yeast Swi6. Degradation of cohesin subunit silencing (pre-siRNAs), which are processed by Rad21 via the pathway involving Dicer to direct assembly of centromeric Rudra N. Dubey1, Nandni Nakwal3, the APC/C E3 ubiquityl ligase helps 2 1# 2# heterochromatin. It is not known how Mitsuhiro Yanagida to bring about sister chromatid Ingela Djupedal , Manuela Portoso , 3 Henrik Spåhr3, Carolina Bonilla1, pre-siRNAs are structured and which and Jagmohan Singh separation. 3 RNA polymerase is responsible for their Claes M. Gustafsson , 3 Robin C. Allshire2 and Karl Ekwall1,4 synthesis. Here we show that Institute of Microbial Technology, A surprising new result indicates the pre-siRNAs are synthesized by PolII. Sector 39A, Chandigarh-160036. India involvement of the Anaphase 1 1Karolinska Institutet, Dept of We define the centromeric pre-siRNA Department of Pharmacology, Robert Promoting Complex (APC) in silencing Biosciences/Dept of Natural Sciences, promoter from which initiation is Wood Johnson Medical School, at mat, cen and rDNA loci in fission Univ. College Sodertorn, Alfred Nobel’s exquisitely sensitive to a conditional University of Medicine and Dentistry of yeast. Results show that APC subunits mutation in the Rpb7 subunit of PolII. New Jersey, Piscataway, NJ, USA. Cut4 and Cut9 interact with Swi6/HP1 Allé 7, S-141 89, Huddinge, Sweden. 2 2Wellcome Trust Centre for Cell Biology, Defective Rpb7 causes reduced siRNA Department of Gene Mechanisms, and help in stable inheritance of Inst. of Cell and Molecular Biology, production and inefficient assembly of Graduate School of Biostudies, Kyoto Swi6/HP1 (and possibly H3-Lys9- Univ. Edinburgh, Edinburgh centromeric heterochromatin. Rpb7 University, Kitashirakawa-Oiwakecho, methyl) at the heterochromatin loci. EH9 3JR, UK. thus provides a functional link Sakyo-ku, Kyoto 606-8502, Japan Surprisingly, Swi6, in turn, is required 3Karolinska Institutet, between PolII transcription and for recruitment of APC subunits to Dept of Medical Nutrition, Novum, chromatin silencing. Gene silencing is mediated by mainly heterochromatin, suggesting a mutually S-141 86 Huddinge, Sweden. two pathways in fission yeast: orchestrated role of Swi6 and Cut4 in 4Corresponding author post-transcriptional (PTGS) and facilitating the stabilization of transcriptional (TGS). Recent advances heterochromatin and sister chromatid #These authors contributed equally have revealed that components of separation. Finally, proteolysis of the to this work. PTGS initiate silencing by recruiting the Rad21subunit of Cohesin is required machinery that sets the heterochro- for stable binding of Swi6 to matin-specific histone code, heterochromatin. i.e., methylation of H3 at Lys9. The latter is recognized by the This work was supported by funding from evolutionarily conserved heterochro- the Department of Science and Technology, matin protein Swi6/HP1 (a component New Delhi, India of TGS), which, through

P36 P37 Analysis of the SAGA composition of S. pombe SAGA by Genetic analysis of fission nucleotide and the 3’splice-site, differ in tandem affinity purification (TAP) on two their dependence on ScPrp18 or transcriptional coactivator yeast step pre-mRNA splicing + independent core SAGA subunits (Spt7 ScSlu7. Genetic depletion of spprp18 complex from S. pombe and Ada1). Mass spectrometry factors- SpPrp18 and SpSlu7 and spslu7+, or repression of their

1 1 analysis revealed that the 19 subunits expression, cause splicing defects for Dominique Helmlinger , Judit Villén , + 2 1 forming SAGA in S. cerevisiae are Piyush Khandelia sptfIID pre-mRNAs. By examining the Steven P. Gygi and Fred Winston conserved in S. pombe and co-purify and Usha Vijayraghavan splicing status for several intron-con-

1 2 with both Spt7 and Ada1. Interestingly, taining transcripts we are investigating Department of Genetics, Department S. pombe Spt7 is present in two or Department of Microbiology and Cell pre-mRNAs dependent on these fission of Cell Biology, Harvard Medical more isoforms that could result from an Biology, Indian Institute of Science, yeast factors. Our analyses of fission School, Boston, MA 02115, USA N-terminal proteolytic processing, Bangalore 560012, India yeast Prp18 and Slu7 reveal certain splicing, or post-translational distinct differences. They do not The SAGA (Spt-Ada-Gcn5 acetylase) modifications. Preliminary results Pre-mRNA splicing reactions and interact in the two-hybrid assay; unlike complex is a multifunctional coactivator indicated that S. pombe Spt7 harbors a factors are evolutionarily conserved and the stable association between their that regulates transcription by RNA highly acetylated and phosphorylated predicted to function similarly in diverse budding yeast counterparts. Further, polymerase II. SAGA was originally region, adjacent to its bromodomain. metazoans. Six S. cerevisiae protein while ScPRP18 and ScSLU7 display discovered in Saccharomyces Deciphering the role of posttranslational factors, including ScPrp18 and ScSlu7, functional overlap the fission yeast cerevisiae and homologous complexes modifications on SAGA structure and are needed for 3’splice-site recognition proteins have distinct non-overlapping have been identified in higher function, and their possible involvement and cleavage during the second-step of functions. Comparative homology eukaryotes. We have started to in mating and cell division might splicing. Direct interactions between modeling of the predicted Spprp18 characterize SAGA composition and illuminate how the specific activities of Slu7- an essential factor, and Prp18 - a protein based on the crystal structure functions in Schizosaccharomyces multi-protein coactivator complexes are non-essential factor, underlie their of ScPrp18 provides a structural basis pombe to characterize its roles in a controlled to regulate the expression spliceosome recruitment and functions for the lack of its interaction with yeast in which chromatin structure and of developmental genes. therein. The intron-rich S. pombe SpSlu7. Using epitope-tagged strains modifications are similar to higher genome, with multiple predominantly for both SpPrp18 and SpSlu7 we are eukaryotes. Deletion of the genes short introns per transcript, is an investigating indirect protein encoding two SAGA core members, + + interesting model system to probe interactions and association with spt7 and ada1 , has shown that SAGA splicing mechanisms in an organism spliceosomal snRNAs. Together our is critically required for growth, mating, evolutionarily distant from budding current data implicate unique functions and septation. We have also deter- yeast. We report our studies on two and spliceosomal interactions for mined the biochemical putative S. pombe splicing factors: SpPrp18 and SpSlu7 and suggest spprp18+ and spslu7+. In contrast to functional divergence of these factors the viable but temperature-sensitive with genome evolution. growth of ScPRP18::HIS3 we find spprp18+ is essential. Slu7 is essential in both yeasts. Splicing of some budding yeast pre-mRNAs, with varied spacing between the intron branch

P38 P39 Genome-wide identification RBPs. Several pioneer studies have PLO1P regulates M/G1-phase that we named PBF (Pombe cell cycle targets of RNA-binding used microarray-based approaches to specfic transcription in box factor), composed of at least three achieve this goal. Their results have transcription factors (Fkh2p, Sep1p and proteins revealed the existence of complex fission yeast Mbx1p), that binds to a DNA promoter networks of RBP-RNA interactions, motif PCB (Pombe cell cycle box). Juan Mata and Jürg Bähler comparable in complexity to the Christopher J. McInerny*, This motif is found in the promoters of transcription factors networks that Szu Shien Ng*, a number of genes required for Cancer Research UK Fission Yeast Kyriaki Papadopoulou* regulate transcription. § cytokinesis, specifically expressed at Functional Genomic Group and Stephen Sedgwick the M/G1 transition. Wellcome Trust Sanger Institute, The first step to identify RBP targets at Wellcome Trust Genome Campus a genome-wide scale is to isolate an *Division of Biochemistry and Molecular We are interested to understand further Hinxton CB10 1SA, UK RBP together with any RNAs Biology, Institute of Biomedical and Life how the PBF-PCB transcription system associated with it; the RNAs are then Sciences, University of Glasgow, controls M/G1 gene expression. We RNA-binding proteins (RBPs) recognise Glasgow G12 8QQ, UK identified using DNA microarrays. § previously showed that the PBF-PCB specific RNA sequences and regulate This technique is often referred to as Division of Yeast Genetics, transcription system is necessary for every stage in the life of RNA RIp-chip (for RBP Immunoprecipitation National Institute for Medical Research, the periodic mRNA accumulation of molecules, including splicing, nuclear followed by analysis with DNA chips). The Ridgeway, Mill Hill, M/G1 transcribed genes, possibly export, subcellular localisation, London NW7 1AA, UK through auto-regulation of fkh2+. degradation and translation. To study the global role of RBPs in We also provided evidence of a stable fission yeast we have set up the Cytokinesis, the process at the end of PBF-PCB complex throughout the cell Although there are many examples of technique of Rip-chip and we have the cell division cycle that results in cycle, suggesting potential post-trans- how specific RBPs regulate single started to apply this approach to a separation of the two daughter cells, lational activation. Plo1p kinase is a transcripts, little is known about the variety of RBPs. We will present results requires a series of precisely prominent multi-functional mitotic extent of this control, and about how that validate the method and coordinated events. We study this regulator found in many eukaryotic cells co-ordinately regulate groups of preliminary data on its application process in the fission yeast systems, and we present data that genes at the posttranscriptional level. to several RBPs. Schizosaccharomyces pombe, as this components of PBF are substrates of organism shows significant similarities Plo1p, thus suggesting a mechanism To get a global view of posttranscrip- in cell separation to higher eukaryotes, by which this protein kinase regulates tional regulation it is crucial to identify including humans. M/G1-specific gene expression. all the RNAs that are bound by specific Previously, we identified a mechanism that regulates the expression of a group of genes required for cytokinesis. We characterised a protein complex

P40 P41 Comparative proteomic Through this approach a number of Genome wide patterns of shows a distinct peak of histone analysis of chromatin related proteins were found to be shared histone modifications in acetylation around the ATG and between two or more different protein gradually decreased acetylation levels complexes in S. cerevisiae complexes and were termed ‘proteomic fission yeast in the coding region. The patterns are and S. pombe hyperlinks’. Eight of these serve as independent of gene length but links in a network of protein complexes Indranil Sinha, Marianna Wire´n dependent on the gene expression Assen Roguev1, Daniel Schaft1,2, including the major yeast histone and Karl Ekwall* levels. H3K9Ac shows a stronger peak Anna Shevchenko3, Rein Aasland4, deacetylase complex (Rpd3C), the near the ATG and is more reduced in Andrej Shevchenko3, major H4 acetyltransferase complex Karolinska Institutet, Dept. of the coding regions of genes with high A. Francis Stewart1 (Esa1/NuA4C) and two chromatin Biosciences/School of Life Sciences, expression compared with genes with remodeling complexes, one of which University College Sodertorn, low expression levels. H4K16Ac is 1BIOTEC TU-Dresden, Tatzberg 47-51, (Swr1C) is responsible for incorporation Alfred Nobel’s Alle´ 7, S-141 89, strongly reduced in coding regions of 01307 Dresden, Germany of H2A.Z into chromatin. Huddinge, Sweden; highly expressed genes. A second 2present address: The Victor Chang Tel: +46-8-6084713; microarray platform was used to Cardiac Research Institute, Level 6, 384 A similar approach was then applied Fax: +46-8-6084510; confirm the 5’ to 3’ polarity effects Victoria Street, Darlinghurst NSW 2010, then in S. pombe. S. pombe was E-mail: [email protected] observed with tiling microarrays. Australia chosen mainly because of its evolution- By comparing coding region histone 3Max Planck Institute for Molecular Cell ary distance from budding yeast and We have used oligonucleotide tiling acetylation data in HDAC mutants and Biology and Genetics, vertebrates, its simplicity and ease of arrays to construct genome-wide high- wild type, we found that hos2 affects Pfotenhauerstrasse 108, 01307 handling. The S. pombe homologs of resolution histone acetylation maps for primarily the 5’ regions, sir2 and clr6 Dresden, Germany the proteomic hyperlinks from fission yeast. The maps are corrected affect middle regions, and clr6 affects 4Department of Molecular Biology, S. cerevisiae were TAP-tagged, purified for nucleosome density and reveal 3’ regions. Thus, mechanisms involving University of Bergen, HiB, P.O. box and protein complex members surprisingly uniform patterns of different HDACs modulate histone 7800, N-5020 Bergen, Norway identified. While this analysis revealed modifications for five different histone acetylation levels to maintain a 5’ to 3’ remarkably conserved proteomic cores acetylation sites. We found that histone polarity within the coding regions. We have applied sequential epitope of the complexes in the two yeasts, acetylation and methylation patterns tagging and mass spectrometry (SEAM) important aspects of the network’s are generally polar, i.e. they change as approach in Saccharomyces cerevisiae topology differed. The network in a function of distance from the ATG and Schizosaccharomyces pombe to S. pombe appears to be more complex codon. A typical fission yeast gene determine and compare the proteomic and more closely related to partial environments of chromatin related networks derived from higher protein complexes. organisms. To our knowledge this is the first systematic comparative proteomic Initially 29 proteins in S. cerevisiae were attempt between different species. selected bioinformaticaly on the basis The nature of the proteomic hyperlinks of presence of chromatin related connecting complexes of related but domains (SET, SANT, chromo, HDACs seemingly antagonizing functions raises and Sir2-homology deacetylase many questions in regards to the proteins). They were then TAP-tagged targeting and regulation of such and purified. Of the initial 29, 14 important gene expression machinery proteins were found to exist in especially in the light of the recent multi-protein complexes. findings on the role of Eaf3 in Co-immunoprecipitating proteins H3-K36Me recognition. were identified by mass-spectrometry, TAP-tagged and purified themselves.

P42 P43 Comparative analysis the role analysis of the S.pombe genome Genome-wide transcript sity Affymetrix tiling arrays for fission on RNAi mechanisms of the identified a putative homologue of the analyses in fission yeast yeast, our goal is to clearly define the D.discoideum HelF gene. Experimental transcribed portion of the fission yeast Dictyostelium discoideum results show that HelF is a natural Brian Wilhelm, Samuel Marguerat, genome (including transcript lengths, HelF gene and its putative nuclear suppressor of RNAi in Chris Penkett, Stephen Watt, splicing structure, non-coding RNAs, D.discoideum begging the questions: and anti-sense transcripts). The arrays Schizosaccharomyces pombe Jürg Bähler 6 Do these two enzymes have have approximately 1.2x10 probes homologue overlapping functions, and does the Fission Yeast Functional Genomics (25mers) which cover the three putative S.pombe HelF homologue chromosomes at 20 bp resolution on Group, Wellcome Trust Sanger Institute, Huan Wang, Brendan Curran also negatively regulate RNAi in each strand or 10 bp using probes from Hinxton, Cambridge CB10 1SA, U.K. and Conrad Lichtenstein S.pombe? both strands. RNA from fission yeast growing under various conditions/per- Although the complete genome School of Biological and Chemical Here using heterologous protein turbations has been hybridized to Sciences, Queen Mary, University of sequence for Schizosaccharomyces expression and fluorescence pombe has been available for several provide as complete a view as possible London, London, E1 4NS, UK microscopy we have revealed that of transcript structure and regulation. years, its full transcriptome remains although the D.discoideum HelF protein Preliminary data on this project will RNA-induced gene silencing, first unknown. While much of the genome localizes to the nuclei of S.pombe cells, content has been characterized through be presented. discovered in plant cells, appears to the product of the S.pombe HelF similarity to genes in other organisms, occur in the majority of eukaryotic homologue does not localize to the organisms. One form of such gene many other genes remain poorly nuclei of D.discoideum cells. described or are simply hypothetical regulation is post-transcriptional gene This suggests that despite their open reading frames. Using high-den- silencing (PTGS). In PTGS, dsRNAs are similarities these proteins have different digested by the RNaseIII-related cellular functions. Moreover, although enzyme Dicer into 21–23bp small HelF is a non-essential gene in interfering RNAs (siRNAs), which D.discoideum, attempts to isolate a assemble with cellular proteins to bind haploid S.pombe strain carrying a HelF the target messenger RNA (mRNA) by homologue knock-out have so far sequence complementary. This leads failed. We are currently attempting this to cleavage of the target mRNA thereby knock-out in a diploid strain to test the preventing protein synthesis. hypothesis that the putative HelF protein is encoded by an essential gene It has already been established that the in S.pombe. We can also use our Dicer enzyme of the soil amoeba, already developed RNAi selective D.discoideum lacks a highly conserved system to investigate the role of this helicase domain. Intriguingly, a genome HelF homologue on RNAi mechanisms search in D.discoideum revealed a new in S.pombe. gene, HelF, with high homology to the helicase domain usually found in the [email protected] Dicer proteins. Moreover subsequent

P44 P45 Comparative analysis of of both organisms upon exposure to Genome-wide Translational transcripts seem to be associated with mechanisms that program KCl and CaCl2 under identical culture Control in Fission Yeast many more ribosomes than less conditions. Mutant strains of either abundant transcripts, although gene expression: yeast that lack Gcn5 grow poorly under ribosome density seems to only Daniel H. Lackner1, Traude H. role of the Gcn5 histone these conditions. We have used DNA 2 1 correlate weakly with transcript levels. microarray analysis to compare Beilharz , Samuel Marguerat , We also found a strong inverse acetyltransferase in 2 1 changes in the expression of over 2000 Thomas Preiss and Jürg Bähler correlation between ribosome density S. cerevisiae and S. pombe. orthologous pairs of genes during KCl and gene length: shorter genes tend to 1Wellcome Trust Sanger Institute, 1,2 adaptation in the two yeasts. be much tighter packed with ribosomes Yongtao Xue-Franzén , Fission Yeast Functional Genomics 1,2 3 While there is a group of orthologous than longer genes. Anna Johnsson , Valerie Wood Group, Hinxton, Cambridge, 1,2 genes that exhibit a conserved We are now testing whether these and Anthony Wright adaptation response, both yeasts also CB10 1SA, U.K. trends depend on other mRNA features 2Molecular Genetics Program, 1 show species-specific responses to such as stability or polyA length. To School of Life Science, KCl-induced stress at the transcription- Victor Chang Cardiac Research obtain global data on polyA length, Södertörns Högskola, Sweden Institute, Darlinghurst (Sydney) 2 al level. We identified a subset of KCl transcripts are fractionated using polyU Department of Biosciences, adaptation genes that are Gcn5 NSW 2010, Australia columns and fractions of different tail Karolinska Institute, Sweden 3 dependent. Interestingly, these genes lengths are quantified with DNA The Sanger Institute, are almost exclusively genes that We are interested in global roles of microarrays. Transcript stability is exhibit species-specific KCl adaptation translational regulation and its determined by measuring mRNA The budding yeast Saccharomyces responses. We conclude that the coordination with other levels of levels at different times after cerevisiae (S. cerevisiae) and the fission differential role of Gcn5 during KCL gene expression control. blocking transcription. yeast Schizosaccharomyces pombe adaptation in the two yeasts is either To obtain translational profiles for all Integration of these varied data sets will (S. pombe) are thought to have (i) an example of independent evolution mRNAs, polysome preparations are provide insight into global mechanisms diverged from one another about one or (ii) divergent evolution. Both models separated according to their size using of post-transcriptional regulation. billion years ago. It is therefore possible suggest that co-regulator proteins like a sucrose gradient and the mRNAs in Moreover, combining translational and to study the conservation/divergence of Gcn5 are variable in their functional role each fraction (or pools of fractions) are expression profiling of cells in different mechanisms that reprogram the even though they are structurally identified and quantified with DNA cell-cycle stages or subjected to genome in response to changes in the conserved. microarrays. Starting with exponentially various genetic and environmental external environment over a long growing cells, we analyzed 12 perturbations will provide a genome- evolutionary distance. We have shown polysome fractions using DNA wide view of translational regulation that the Gcn5 histone acetyltransferase microarrays containing elements for all in fission yeast, complementing our (HAT) is required for efficient adaptation known and predicted genes of fission expression profiling data. yeast. This approach provided data on average numbers of associated ribosomes for most transcripts. Integration with data on mRNA steady-state levels revealed an interesting bias: the most abundant

P46 P47 Mitotic and Meiotic Myo51, a fission yeast type and the organisation of the mitotic V myosin with multiple roles spindle, whereas the function of Myo51 Cell-Division Cycle has remained elusive (Cell Motil in meiosis. Cytoskeleton. 51:53-56). Analysis of negative inhibited by mitotic kinase activity and a two-component GTPase activating A. Doyle and D. P. Mulvihill. Myo51 expression increases regulators of the septation protein (GAP) composed of byr4p and dramatically upon entry to meiosis initiation network cdc16p. A number of other negative Department of Biosciences, (Nature Genetics 32:143-147), regulators of the SIN have been identi- University of Kent, Canterbury Kent, and we have been focusing upon iden- *Philippe Collin and Viesturs Simanis fied, including fin1, dma1, zfs1, scw1 CT2 7NJ, UK. tifying Myo51’s function during this cel- and par1-PP2A. We will present our lular process. Swiss Institute for Experimental Cancer work studying the function and cell The fission yeast Schizosaccharomyces Research (ISREC), 155 Chemin des cycle regulation of inhibitors of the SIN. pombe, is a non-motile unicellular Myo51 has a distinct localisation Boveresses, Epalinges, 1066, eukaryote, and has proven to be during meiosis and appears to have Switzerland. extremely useful in the study of cellular multiple functions during this event. E-mail: [email protected]. processes such as mitosis, meiosis and We will describe our latest findings on Phone: +41-21-692-58-58 regulation of the cell cycle, due to its this motors role in meiosis. FAX: +41-21-652-69-33. similarity to mammalian cells.

The SIN is a signal transduction S. pombe expresses three classes of network that triggers the initiation of the motor protein myosin (types I, II septum formation. It is composed of and V) to maintain basic cellular four protein kinases and their regula- function. Of the two type V myosins, tory subunits (plo1, cdc7 – spg1, sid1 - Myo52 is involved in vacuole cdc14 and sid2 - mob1). SIN proteins distribution, cytokinesis, cell growth are anchored to the poles of the mitotic spindle by a scaffold comprising sid4 and cdc11. Signalling is mediated by activation of the ras-superfamily GTPase spg1p. Signalling by the SIN is

P48 P49 Neck domain independent The genome of the fission yeast, A role for the septation interphase, though how this is achieved movement of the fission yeast Schizosaccharomyces pombe, encodes initiation network in the remains unclear. We are making use of only 5 myosins, thus providing the spg1 overexpression to evaluate the type V myosin, Myo52 perfect model system in which to study formation of the cytokinetic involvement of the SIN in CAR the regulation and function of individual ring in fission yeast assembly. Agnes Grallert1 Suzanne R. Edwards2 myosins. Using fluorescent labelling, 2 and Daniel P. Mulvihill the S. pombe type V myosin, Olivier Hachet and Viesturs Simanis We characterized the properties of Myo52, can be visualised as vesicle like rings induced by spg1 overexpression 1 Cancer Research UK Cell Division dots, which move rapidly along actin Swiss Institute for Experimental Cancer in G2 arrested cells. Using ring markers Group, Paterson Institute for Cancer cables throughout the fission yeast Research (ISREC) such as cdc15-GFP, rlc1-GFP and Research, Wilmslow Road, Manchester, cytoplasm. We will present data from myp2-YFP, we found that interphasic 2 Chemin des Boveresses 155 M20 4BX. Department of Biosciences, our mutant analysis of the Myo52 CH-1066 Epalinges Switzerland spg1 induced rings are functional in University of Kent, Canterbury, protein. Removing the long IQ motif that they can contract, however most Kent, CT2 7NJ. containing neck region had little affect In most eukaryotes, cytokinesis is fail to be correctly positioned. This on Myo52 velocity, demonstrating that mediated by a contractile actomyosin observation show that interphasic spg1 Intra-cellular movement is a a property other than step size ring (CAR). The formation of the CAR induced CAR lack some spatial fundamental property of all cell types, determines this motor protein’s cellular depends on the reorganization of the information suggesting a bypass of the as the precise sub-cellular localisation velocity. In contrast mutations within actin cytoskeleton at the onset of mito- CAR positioning mechanism or a failure of organelles is essential for their the motor domain affecting the motor sis. In Schizosaccharomyces pombe, to maintain CAR positioning. Using correct function and therefore a basic protein’s affinity for nucleotide and the onset of septation is temporally different genetic backgrounds, requirement for cell viability. actin fail to complement the growth coordinated with the end of mitosis we addressed the requirements for the Many organelles and molecules are defects associated with the myo52A through the combined action of the formation of interphasic spg1 induced actively transported throughout the allele. Models will be presented which septation initiation network (SIN) and CAR. Our results suggest a requirement cytoplasm by molecular motors. attempt to explain these findings. the polo kinase Plo1p. The function of of the SIN pathway for the formation of Myosins Vs are one such motor protein. the SIN is to trigger the formation of the such rings. These observations They are dimeric actin based motor division septum and the contraction of suggest that, although the SIN appears proteins, which transport organelle the CAR after completion of chromo- not required for ring assembly, cargoes throughout a cell. They some partitioning. CAR assembly the SIN pathway may participate in possess a long neck region that has occurs at the onset of mitosis, this process. previously been hypothesized to allow independently of SIN signaling. the protein to move rapidly by take However, ectopic activation of spg1 is large steps along actin cables. sufficient to trigger CAR assembly in As yet very little is known about their regulation or precise cellular function.

P50 P51 Lsk1p and Lsc1p form a CDK- perturbation of the cell division Fission yeast MES1 is an fission yeast arrests cells at metaphase cyclin complex that positively machinery. We demonstrate that Lsk1p APC/C substrate and is coincident with accumulation of Cdc13 – a member of the cyclin dependent and Cut2, whereas expression of D-box regulates the Septation kinase (CDK) family – forms a physical directly recognized by WD40 and KEN-box double mutant does not. Initiation Network and is complex with the previously activator proteins To ensure the idea that Mes1 is an required for Ser-2 uncharacterized cyclin, Lsc1p, and that APC/C substrate, we show that Mes1 both deletion mutants display indistin- Yuu Kimata and Hiro Yamano is destroyed in an APC/C- and Fizzy- phosphorylation of the guishable phenotypes. In addition, dependent manner in Xenopus egg regulatory heptad repeat genetic and physiological analysis Marie Curie Research Institute, Oxted, extracts, which requires intact D-box found in the carboxy terminal demonstrate that Lsk1p-Lsc1p acts in Surrey, RH8 0TLUnited Kingdom and KEN-box. Furthermore Mes1 is domain of RNA polymerase II parallel to Clp1p to promote acto- efficiently polyubiquitinated by APC/C myosin ring stability upon checkpoint In meiosis, to ensure successive in vitro. These data strongly suggest activation. Remarkably, deletion of chromosome segregation without that Mes1 inhibits APC/C-dependent Karagiannis J, either gene is capable of rescuing the intervening DNA replication, the destruction of Cdc13 as a competitive and Balasubramanian MK. lethal, multi-septate phenotype prevention of complete inactivation of substrate. We also show by using site- conferred by constitutive the Cdc2 activity is required between specific photo-crosslinking techniques Laboratory of Cell Division, hyper-activation of the SIN, strongly meiosis I and meiosis II. We have that Mes1 directly binds to the Fizzy Temasek Life Sciences Laboratory, suggesting that the Lsk1p-Lsc1p recently shown that the fission yeast family of proteins from various species, Singapore 117604 complex acts to stabilize the acto- Mes1 protein has a role in maintaining suggesting that APC/C activator myosin ring through its action as a a certain amount of Cdc13 in this proteins are able to recognize D-box In Schizosaccharomyces pombe positive regulator of this module. period by inhibiting the APC/C activity. and KEN-box independently of APC/C cytokinesis is monitored by a check- Intriguingly, Lsk1p is most closely Here we investigate the molecular and this ability is conserved in WD40 point system capable of delaying cell related to the family of CDKs that mechanism of APC/C inhibition by domains of activators. cycle progression and promoting function, at least in part, by modulating Mes1. Mes1 has putative D-box and actomyosin ring stability. the phosphorylation status of a KEN-box in its sequence. Key components of the checkpoint are repeated regulatory heptad found in the Over-expression of wildtype Mes1 in the phosphatase, Clp1p, and the carboxy terminal domain of RNA Septation Initiation Network (SIN), polymerase II. Consistent with such a which function in a positive feedback role we are able to demonstrate that loop to lengthen the duration in which Ser-2 phosphorylation of the heptad cells are competent for cytokinesis. is abolished in both lsk1A and To identify novel regulators of this lsc1A mutants. system we screened a genome-wide bank of kinase deletion mutants and identified one knockout strain, lsk1A, that displayed a striking fragmentation of the actomyosin ring upon

P52 P53 Role of the Septation Initiation cells due to a failure of cytokinesis. Functional characterization of rescued with the cds1 deletion, which Network during meiosis When starved, S. pombe cells of Nap1 protein in is a kinase involved in the activation of opposite mating type will fuse to form a the replication checkpoint. In contrast, diploid zygote that undergoes meiosis cell cycle progression of the mitotic delay due to nap1 deletion A. Krapp, P. Collin, A. Cokoja, S. producing four spores. No septa or Shizosaccharomyces pombe is not rescued with chk1 deletion, Dischinger, E. Cano and V. Simanis contractile rings are formed during this which responds to the DNA damage process, but SIN proteins are Eva Lambea, Maribel Grande, Sandra checkpoint. Therefore, it indicates that ISREC, Epalinges, Switzerland expressed and localise to the SPB. López-Avilés and Rosa Aligué. the deletion of nap1 gene activates Moreover, the localisation pattern of specifically the replication checkpoint. When nutrients are abundant, cells of SIN activators and inhibitors suggests Department of Cell Biology, University These results suggest a model in which the fission yeast Schizosaccharomyces that the SIN is activated during the of Barcelona. C/ Casanova 143. 08036- the role for Nap1 during DNA synthesis pombe grow as rods, dividing by second meiotic division. In agreement, Barcelona. Spain. is linked with the onset of mitosis. medial fission after formation of a SIN mutants cannot form spores, while medially placed cell wall or division all the other steps of meiosis appear Nap1, a member of the Nap/Set family, In order to further characterize the role septum. Septum formation is triggered normal. This defect in spore formation has been described in human cells as a of Nap1 during cell cycle progression by a group of proteins called the is due to a failure in the proper histone chaperone protein that pro- we have purified Nap1 binding proteins septation initiation network, or SIN. development of the forespore motes nucleosome assembly in vitro. by affinity chromatography and we Ectopic activation of the SIN can membrane. In budding yeas, Nap1 has also been have identified Sda1 as a protein that uncouple septum formation from other described as a member of mitotic binds directly Nap1. In Saccharomyces cell cycle events, while failure of SIN signalling network that controls cell cerevisiae It has been described that signalling gives rise to multinucleated growth during mitosis and appears to Sda1 is involved in the synthesis of require shuttling of Nap1 between cyclins during G1 progression. nucleus and cytoplasm. In order to establish this mitotic signalling network Altogether, these results suggest a we have studied the role of the homo- model in which the role of Nap1 during logue Nap1 protein in DNA synthesis is linked with the onset Schizosaccharomyces pombe. We have of mitosis. found that Nap1 is required for the B- type cyclin Cdc13 activity. Deletion of nap1 leads to a delay in the onset of mitosis and we show that this delay is

P54 P55 The dual role of the mitotic Cds1(Chk2) kinases, and that Characterization of the addition to the F-box two independent kinase Srk1 in fission yeast phosphorylation of Cdc25 on these cellular function of the protein interaction domains, an sites is necessary for Srk1 to impose a N-terminal TPR and a C-terminal LRR G2 arrest. Furthermore we show that ubiquitin ligase motif. Pof3 is part of the SCF complex, Sandra Lopez-Aviles, Pof3 Maribel Grande, Eva Lambea overexpression of Srk1 causes Cdc25 complex SCF as it interacts with Pcu1 and Rosa Aligue to accumulate in the cytoplasm, an (the fission yeast Cullin-1) and Skp1. effect which is dependent on Rad24, a Yasmine Mamnun, Satoshi Katayama Department of Cell Biology, University member of the14-3-3 protein family. and Takashi Toda In the absence of Pof3 cells exhibit a Given these observations, we propose number of phenotypes including of Barcelona. C/ Casanova 143. 08036- a model in which Srk1 inhibits Cdc25, appearance of lagging chromosomes Barcelona. Spain. Laboratory of Cell Regulation, London avoiding a premature onset of mitosis. Research Institute, and a high rate of chromosome loss. pof3 cells are highly sensitive to UV In fission yeast, the Chk1 and Cancer Research UK In addition to this role during a normal and show G2 delay due to constitutive Cds1(Chk2) checkpoint kinases block 44 Lincoln’s Inn Fields, London WC2A mitotic entry by inhibiting the Cdc25 cell cycle, we show that Srk1 regulates 3PX, UK activation of the DNA damage Cdc25 in response to environmental checkpoint, which is required to phosphatase in response to replication stress. Treatment of wild type cells prevent lethal mitosis. Loss of Pof3 arrest or DNA damage. In this work we Ubiquitination and subsequent show that the stress-activated kinase, with osmotic stress leads to the degradation of proteins regulate many additionally causes substantially accumulation of Cdc25 in the cyto- shortened telomers and desilencing of Srk1, also inhibits mitotic entry by important cellular processes. Key plasm and to an increase of the Cdc25- heterochromatin regions. Taken phosphorylating Cdc25. We find that regulators of cell cycle and division are Rad24 binding, whereas this behaviour together these phenotypes hint a over-expression of Srk1 kinase causes targeted for degradation by a family of Pof3 is not observed in srk1-deleted cells. central role of SCF in protection cell cycle arrest in late G2 phase, an E3 ubiquitin ligases, termed Skp1- This effect is mirrored in vivo by the of genome integrity. effect which is dependent on the Cdc53-F-box (SCF) complexes. In this catalytic activity of Srk1. Conversely, stabilization of Cdc25 in the wild type enzyme complex the F-box proteins act strain compared to the strain lacking This work is supported by fellowships of the cells lacking srk1 enter mitosis as receptors to bind and recruit Srk1. Moreover, the activity of Srk1 FWF (J-2398) and EMBO (ALTF-53-2004). prematurely. The G2/M arrest caused substrates thereby conferring the by overexpression of Srk1 is due to increases dramatically following the specificity of the ubiquitin transfer. exposure of cells to osmotic stress. hyperphosphorylation of Cdc2 on Tyr15 We have characterized the S. pombe Taken together, these results suggest and can be attributed, primarily, to inhi- F-box protein Pof3, which contains in bition of Cdc25 activity. Consistent with that, in a similar manner to Chk1 and Cds1 in response to genotoxic stress, this we find that Srk1 interacts with Srk1 is responsible for the cell cycle Cdc25 in vivo. Most importantly, we find that Srk1 phosphorylates the arrest and Cdc25 stabilization following a non-genotoxic environmental insult. N-terminal non-catalytic region of Cdc25 by Srk1 in vitro, at the same sites phosphorylated by the Chk1 and

P56 P57 The fission yeast Chs2p activity. Chs2p localisation and Putative identification of the gas1p, gas2p, protein interacts with the assembly into a ring that contracts -1,3-glucanosyltransferases gas4p and gas5p proteins during division requires the general a (the products of the SPAC19B12.02c, type-II myosin Myo3p and is system for polarised secretion and in S. pombe SPBC29A10.08, SPBC342.3 and required for the integrity of some components of the actomyosin C11E3.13c ORF, respectively). Three of + + the actomyosin ring. ring. Chs2p interacts physically with the María de Medina-Redondo, these genes (Sp gas1 , Sp gas2 and type-II myosin Myo3p showing a Carlos R. Vázquez de Aldana Sp gas5+) are expressed periodically physical link between the plasma and Francisco del Rey. during vegetative growth. Sp gas4+ has Rebeca Martín-García* membrane and the ring. In chs2A a basal expression during vegetative and M.-Henar Valdivieso. mutants the actomyosin ring integrity Instituto de Microbiología Bioquímica, growth, but its mRNA levels increase is compromised during the last stages Departamento de Microbiología y during sporulation. gas2A and gas4A Departamento de Microbiologia y of contraction and it remains longer in Genética, CSIC/Universidad de cells showed no apparent growth Genetica/Instituto de Microbiologia the midzone. Salamanca, Campus Miguel de defect in either rich or minimal medium, Bioquimica. Universidad de Unamuno, 37007 Salamanca, Spain. neither at 32ºC nor at 37ºC. However, Salamanca/CSIC. Edificio Additionally, chs2A cells are gas5A cells showed a slower division Departamental. Laboratorio 231. hypersensitive to a minor perturbation The cell wall of Schizosaccharomyces rate at 37ºC and thicker cell walls at Campus Miguel de Unamuno. 37007 of the actomyosin ring. In a pombe is a dense network of polysac- this restrictive temperature. gas4A Salamanca. Spain. synchronous culture, chs2A cells charides, composed of `-galactoman- diploids cells divide normally in rich Ph: +34 923 121589. Fax: +34 923 exhibit a delay in septation with respect nans, `-1,3-glucan and a-glucan (linear medium and undergo meiosis correctly 224876. to the control strain. All these results a-1,3-glucan, a-1,6-glucan and a-1,6- in media lacking nutrients. In contrast, show that Chs2p participates in the glucan branched a-1,3-glucan). a-1,3- this mutant cannot complete sporula- In Schizosaccharomyces pombe correct function of the medial ring. glucan chains are synthesized by a tion, and forms aberrant spores which cytokinesis requires the function of a glucan synthase complex, and remain are not totally individualized. To deter- contractile actomyosin ring. Fission unorganized and alkali-soluble until mine the subcellular localization of yeast Chs2p is a transmembrane covalent linkages occur between a-1,3- these proteins, fusions with the yellow protein structurally similar to chitin glucans and other cell wall compo- fluorescent protein (YFP) were synthases that lacks such enzymatic nents. In Saccharomyces cerevisiae constructed, under the control of the and in Candida albicans, proteins native promoter of each gene. So far, encoded by the GAS and PHR genes we have been able to determine the have a-1,3-glucanosyltransferase activi- localization of gas1p protein in the cell ty, belonging to the GHF72 family. All of wall all around the cells, and also in the them are attached to the membrane septa. gas4p protein is located in the through a glycosylphosphatidylinositol spore wall. (GPI).

A search of the S. pombe database for proteins with sequence similarity to the S. cerevisiae Gas1p resulted in the

P58 P59 Biochemical analysis of the tion and therefore for cytokinesis to Rgf3p is a specific Rho1-GEF unit of the 1,3-a-glucan synthase is the fission yeast tropomyosin, occur in S. pombe (Nature 360: 84-7). that regulates septum small GTPase Rho1p which acts as a Facilitated by the use of a novel anti- molecular switch varying from an inac- Cdc8. body (raised against the full-length synthesis during cytokinesis tive state bound to GDP to an active Cdc8 protein), we have characterised a in fission yeast. state bound to GTP. Kalomoira Skoumpla, number of cell biological and Sheran Attanapola, Arthur C. biochemical properties of Cdc8. Virginia Tajadura, Patricia García and We identified rgf3+ as the gene affected Coulton, Michael A. Geeves, Both Immunofluorescence and GFP Yolanda Sánchez in the ehs2-1 (equinocandin hypersen- and Daniel P. Mulvihill tagging methodologies confirm that sitive) mutant. ehs2-1 is defective in Cdc8 associates with the actin ring Instituto de Microbiología Bioquímica, 1,3-a-glucan synthesis and is ther- Department of Biosciences, University during mitosis, as reported previously. CSIC/Universidad de Salamanca and mosensitive, at the restrictive tempera- of Kent, Canterbury, Kent, CT2 7NJ. In addition these reagents reveal that Departamento de Microbiología y ture cells lyse as doublets. Mutant cells the fission yeast tropomyosin Genética, Universidad de Salamanca. lacking rgf3+ are unviable. Cytokinesis, the division of a cell associates with actin filaments during Campus Miguel de Unamuno. 37007, brought about by the contraction of an interphase. Consistent with this, Salamanca, Spain. E-mail: [email protected] Rgf3p is a specific Rho1p GEF actin-based contractile ring, is an western blot analysis demonstrate that (Guanine nucleotide Exchange Factor). essential process, conserved in all Cdc8 protein levels do not vary through Schizosaccharomyces pombe cells Rgf3p activates Rho1p promoting the eukaryotes. Much of what we under- the cell cycle, and suggests that the divide by binary fission through the change of the GDP bound to the stand today about the process of protein is subjected to constant post- formation of a septum that divides the GTPase by GTP and hence increasing cytokinesis and its regulation comes translational modifications. Biochemical mother cell into two equal daughter the 1,3-a-glucan synthase activity. rgf3+ from studies using the fission yeast, and kinetic analysis of purified endoge- cells. At the onset of mitosis a expression is cyclic with a peak in M- Schizosaccharomyces pombe. nous Cdc8 revealed that the protein is contractile actomyosin ring (CAR), phase of the cell cycle and it exclusive- Tropomyosin, an essential actin-binding acetylated in vivo, and this modification essential for cell division, is assembled ly localizes at the medial region of the protein is required for the maintenance modulates the protein’s ability to in the medial region of the cell but it cell where the contractile ring is located and the stabilisation of actin cables associate with actin, thus regulating only contracts at the end of mitosis and the division septum is assembled. throughout the cell cycle, including Cdc8 function in vivo. when the two nuclei have separated Early in mitosis, Rgf3p forms a ring-like those incorporated into the cytokinetic and the mitotic spindle has structure that contracts to a dot during actomyosin ring (CAR). disassembled. At the same time of ring the latest stages of mitosis and disap- contraction, the synthesis of the pears when the septum is completely We present data from a cross-discipline primary septum, mainly composed of formed. rgf3A spores arrest with two study of the fission yeast tropomyosin, linear 1,3-a-glucan, begins and it fol- nuclei and an actomyosin ring but Cdc8, which has previously been lows as the CAR contraction proceeds they are unable to form any septum shown to be essential for CAR forma- to form a plate at the division site. and thus are unable to divide and The secondary septa, with a propagate. composition similar to the cell wall, are then deposited to each side of the Flanking the RhoGEF catalytic domain, primary septum. The cleavage of the Rgf3p contain two regulatory domains, primary septum at the end of a Serine-Threonine rich region at the cytokinesis liberates the two daughter amino terminus and a CNH (Citron cells. Thus proper formation and kinase Homology) domain at the cleavage of the division septum is carboxy terminus; this one is essential essential for cell division and to main- for Rgf3p activity. The presence of tain cell integrity during cell separation. these domains suggests that Rgf3p and thus Rho1p functions could be The synthesis of the 1,3-a-glucan, the regulated by interaction with other main component of the primary and proteins to switch on 1,3-a-glucan secondary septa, in the medial region is synthase activity. carried out by at least two different 1,3- a-glucan synthases whose catalytic subunits are coded by the cps1+/bgs1+ and bgs4+ genes. The regulatory sub-

P60 P61 Opportune retirement of complex (x-TuC) as well as with a Timely destruction of S.pombe which carry destruction boxes (D-box), Hrs1p, the organiser of astral meiotic SPB component, suggesting Rad54 by the Anaphase a nine-residue motif RXXLXXXN/D/E that Hrs1p facilitates formation of the and/or KEN boxes. Using an in vitro microtubule arrays for the HAA, responsible for the vigorous Promoting Complex assay utilising Xenopus laevis egg horsetail nuclear movement, HNM, by stabilising connection extracts we have identified Rhp54, the ensures smooth spindle between the SPB and minus ends Michelle Trickey and Hiro Yamano S.pombe homolog of the human and of microtubules. S.cerevisiae homologous recombination formation at the onset Marie Curie Research Institute, Oxted, protein Rad54, as a target of the of Meiosis I Expression of Hrs1p is restricted to Surrey, RH8 0TL, United Kingdom APC/C. However, no other members of meiotic prophase. Upon the onset of the Rad52 recombination family in Kayoko Tanaka, Shinya Okamoto, meiosis I, Hrs1p abruptly disappeared The Anaphase Promoting Complex S.pombe, or human and S.cerevisiae Ayano Kagami from the SPB even when it was forcibly (APC)/cyclosome is a large multi homologs of Rhp54 are targets. In vitro and Masayuki Yamamoto expressed under the regulation of a subunit E3 ligase which is known to Rhp54 destruction is dependent upon a strong nmt1 promoter. To see the effect orchestrate the cell cycle transition KEN box within its far N-terminus and Departament of Biophysics of ectopic Hrs1p expression during from mitosis to G1. Originally identified Fizzy-related activated APC/C with and Biochemistry, Graduate School meiosis, we isolated a few hrs1 as the factor required for cyclin B which it interacts in an APC/C of Science, University of Tokyo, mutants in which mutated Hrs1 degradation, the APC/C is now known independent manner. Studies in Tokyo 113-0033, Japan. proteins were stabilised at the SPB to be required for the timely S.pombe have found that Rhp54 e-mail: [email protected] during meiosis I and II. In one of the degradation of numerous proteins, destruction is restricted to the G1 tokyo.ac.jp mutants ( hrs1.d10), relative timing of which include securin, Cyclin A, Cdc6, phase of the cell cycle and is the spindle formation after the Xkid, UbcH10, geminin and Plk1. dependent upon the S.pombe Fizzy- Upon commitment to sexual cessation of HNM was delayed and The APC/C functions with the aid of related homolog Ste9. However, muta- differentiation, cells exchange the malformed spindles were observed. co-activator proteins known as tion of the KEN box to AAA is not mating factor signal which induces These results indicate a possible Fizzy/Cdc20/Slp1 and Fizzy- sufficient in S.pombe to stabilise dramatic remodelling of microtubule mechanism to down-regulate Hrs1p related/Cdh1/Ste9, these interact with Rhp54, and an addition factor resides architecture to form an astral micro- at the onset of meiosis I to ensure a APC/C from metaphase to anaphase within its N-terminus. tubule array emanating from the spindle smooth spindle formation. and during G1 respectively. It has been pole body (SPB)1, 2. During meiotic shown that both Fizzy and Fizzy-related prophase, an oscillatory nuclear References are destroyed in an APC/C dependent movement termed horsetail nuclear 1. Petersen, J., Heitz, M. J. & Hagan, manner. The APC/C degrades proteins, movement (HNM) takes place3, 4. HNM I. M. Curr Biol 8, 963-6 (1998). is led by the astral microtubule array 2. Ding, D. Q., Chikashige, Y., Haraguchi, aided by the dynein-dynactin complex T. & Hiraoka, Y. J Cell Sci 111 (Pt 6), 701-12 (1998). and is proposed to facilitate the 3. Robinow, C. F. Genetics 87, alignment of homologous chromo- 491-97 (1977). somes necessary for efficient meiotic 4. Chikashige, Y. et al. Science 264, 2, 5, 6 recombination . 270-3 (1994). 5. Yamamoto, A., West, R. R., McIntosh, We have shown that a meiosis-specific J. R. & Hiraoka, Y. J Cell Biol 145, SPB component Hrs1p (also known as 1233-49 (1999). Mcp67) is a key molecule to remodel 6. Ding, D. Q., Yamamoto, A., Haraguchi, microtubules into the horsetail-astral T. & Hiraoka, Y. Dev Cell 6, 8 329-41 (2004). array (HAA) . Deletion of Hrs1p 7. Saito, T. T., Tougan, T., Okuzaki, impaired HAA formation, leading to D., Kasama, T. & Nojima, H. compromised HNM. Ectopic J Cell Sci 118, 447-59 (2005). expression of Hrs1p during the mitotic 8. Tanaka, K., Kohda, T., Yamashita, cell cycle resulted in the formation of a A., Nonaka, N. & Yamamoto, HAA-like astral microtubule array, which M. Curr Biol 15, 1479-86 (2005). drove an oscillatory nuclear movement in interphase cells. Hrs1p interacted with components of the x-tubulin

P62 P63 Cellular roles of the Cdc37 cells, Cdc2 kinase activity is greatly Cytoskeleton and Cell molecular chaperone in reduced, as is the level of Cdc2 associated with its mitotic cyclin Morphogenesis fission yeast partner Cdc13, indicating that Cdc37 is required for assembly or maintenance qualitatively normal but quantitatively Emma Turnbull, Jun Liang, Ina Martin of the active complex. On the other Non-core components of and Peter Fantes the fission yeast x-tubulin reduced numbers of active interphase hand, the tyrosine 15 phosphorylation microtubule organizing centers in vivo, status of Cdc2 is not affected. complex Institute of Cell Biology, University of and this is exacerbated by mutations in mod22+. Simultaneous deletion of Edinburgh, Mayfield Road, Edinburgh In a separate study, we have been EH9 3JR, UK Andreas Anders gfh1p, mod21p and alp16p, a third carrying out a genetic screen with the and Kenneth E. Sawin non-essential x-TuC protein, does not aim of identifying novel clients of lead to additive defects, suggesting Cdc37 is a molecular chaperone found Cdc37, and have identified candidates. Wellcome Trust Centre for Cell Biology, that all three proteins contribute to a in fungi and animals. In mammalian Several of these are the products of Edinburgh University,Swann Building, single function. Co-immunoprecipitation cells it interacts strongly with the genes with known roles in cell cycle Mayfield Road, Edinburgh, EH9 3JR, experiments indicate that gfh1p and heat-shock protein Hsp90, another progress and cell morphogenesis: chaperone, but direct interaction United Kingdom alp16p are codependent for association we are testing whether they interact with a small “core” x-TuC, while appears to be much weaker in directly with Cdc37. fungal systems (1,2). Relatively little is known about the in mod21p is more peripherally associat- vivo function of individual components ed, and that gfh1p and mod21p can 1. Turnbull, E.L., Martin, I.V. and Fantes, Most client (substrate) proteins of P.A. (2005) FEBS J, 272, 4129-4140. of the eukaryotic x-tubulin complex (x- form a sub-complex independent of the Cdc37 are protein kinases. We have 2. Farrell, A. and Morgan, D.O. (2000) TuC). We identified three genes, gfh1+, small x-TuC. Interestingly, sucrose gra- shown that the cell cycle regulatory Mol Cell Biol, 20, 749-754. mod21+ and mod22+, in a screen for dient analysis suggests that the major kinase Cdc2 is a major client of Cdc37, 3. Turnbull, E.L., Martin, I.V. and Fantes, fission yeast mutants affecting micro- form of the x-TuC in fission yeast may by a combination of genetics, cell P.A. (2006) J Cell Sci, 119, 292-302. tubule organization. gfh1+ is a previ- be the small complex. We propose that physiology and biochemistry (3). ously characterized x-TuC protein gfh1p, mod21p and alp16 act as facul- When Cdc37 function is impaired in weakly similar to human x-TuC subunit tative “non-core” components of the cdc37ts mutants, cells arrest in G2 GCP4, while mod21+ is novel and fission yeast x-TuC and enhance its with a phenotype very similar to that of shows limited similarity to human x-TuC microtubulenucleating ability. cdc37ts mutants. In arrested cdc37ts subunit GCP5. We show that mod21p is a bona fide x-TuC protein and mod21A mutants are viable. We find that gfh1A and mod21A mutants have

P64 P65 Self-organization of dynamical (motor protein–mediated) btn1, the orthologue of the response pathway was severely cytoplasmic microtubule self-organization. We show that inter- Batten disease gene CLN3, is delayed, and following subsequent phase microtubule arrays in the fission founds of mitosis, F-actin patches were arrays in fission yeast yeast also self-organize. This organiza- involved in the maintenance either loosely polarised and monopolar tional process requires the microtubule- of cylindrical rod shape or exhibited random cortical localisa- 1 Rafael E. Carazo-Salas interacting genes mod20, tip1, ase1 morphology and polarised tion. Subsequent cell cycles resulted in and Paul Nurse1,2 and klp2, but not the spindle pole body. the formation of pear-shaped cells, with Moreover, this process allows the cell growth daughter cells showing total rounded, 1Cell Cycle Laboratory, 44 Lincoln’s Inn dynamical generation of a central posi- depolarised cell growth. Cell swelling 1 1 2 Fields, Cancer Research UK, London tion in the cell independently of pre- S Codlin and S E Mole , occurred first at the non-growing end, Research Institute, WC2A 1PX, defined spatial cues. We suggest that suggesting a defect in the correct posi- United Kingdom microtubule self-organization may be 1MRC Laboratory for Molecular Cell tioning of the polarisome machinery in 2Rockefeller University, 1230 York essential for the robust generation of Biology, University College London, an attempt to pass through NETO. A Avenue, NY10021, USA intracellular coordinates throughout Gower Street, London WC1E 6BT, genetic interaction was established eukaryotes. United Kingdom. with for3, supporting a role for Btn1 in Animal cells can acquire highly ordered 2Department of Paediatrics & Child F-actin polarisation. Cell lysis was microtubule patterns via a combination Health, and Biology, UCL. found to be due to defective cell wall of structurally enforced assembly and composition - btn1A cells were highly We have previously reported btn1, the sensitive to zymolase and swelling and Schizosaccharomyces pombe homo- lysis, but not cell curving and monopo- logue of the human Batten disease larity, were rescued by the addition of 1 gene, CLN3, as being involved in vac- M sorbitol to osmotically stabilise cells. uolar homeostasis. Btn1 is a transmem- The localisation of Btn1 became more brane protein that traffics slowly to the endosomal and bipolar upon a temper- vacuolar membrane via the endomem- ature shift to 37ºC, and this localisation brane system. Here we show that was actin dependent and essential for btn1A is temperature sensitive for the rescue of the polarity defects in growth at 37ºC. Cell death is due to cell btn1A . lysis, which occurs within two to three Btn1p therefore, links the endomem- cell cycles and 18 h following heat brane system with both the actin and stress. Early temperature sensitive microtubule cytoskeleton, in a heat growth defects include curving and stress response pathway. Since Batten bundling of interphase microtubules disease is a lysosomal storage disorder leading to curved shaped cells within with progressive loss of neurons, spe- 15 mins of the temperature shift, and cialised and highly polarised cell types, by 7h, all cells were curved. Also, cells fission yeast is proving to be a good exhibited defects in NETO transition model system to understand the after completion of the first round of molecular basis of this disorder. cytokinesis at 37ºC, resulting in monopolar growth. The loss of rod- shaped morphology was accompanied by a progressive failure in F-actin patch movement. First, F-actin re-polarisation following activation of the heat stress

P66 P67 Role of Phosphorylation in EB1 homologue, mal3p, is a general Force Provides a Local Cue Langevin equations we regulating CLIP-170 and EB1 promoter of microtubule growth. for the Organization of built a 3-dimensional model of the cell It localizes all along the microtubules containing the nucleus together with its Fission Yeast Homolgues, and accumulates on their tips, from Microtubules in Fission Yeast associated microtubules within a fixed TIP1 and MAL3 where it disappears preceding shape. Microtubule properties such as catastrophes. Dietrich Foethke,1 Damian Brunner,1 mechanic elasticity and production 1 Andréia Feijão and Damian Brunner The mechanisms that allow tip1p and Francois Nédélec of force by polimerization are consid- mal3p to influence and regulate ered together with different models of 1 European Molecular Biology microtubule dynamics are not yet Cell Biology and Biophysics how microtubule dynamics might be Laboratory, EMBL, Heidelberg understood and itís also not known European Molecular Biology Laboratory regulated. what regulates their activities. This may Meyerhofstr. 1, 69117 Heidelberg, With the help of the simulation we CLIP-170 and EB1 protein family mem- involve phosphorylation because both Germany compared the influence of these mod- bers are plus end-tracking proteins that tip1p and mal3p are phosphorylated. In els on the geometry of the microtubule accumulate to growing microtubule tips this context, we are investigating the To understand how the complex layout network and on the positioning of the being implicated in the local control of role of tip1p and mal3p phosphory l a t i o n . of the microtubule network in inter- nucleus. We find that force dependent microtubule dynamics and attachment We show that both mal3p and tip1p are phase arises from the properties of sin- microtubule dynamics can explain most of microtubules with the cell membrane phosphoproteins and that the gle dynamic microtubules, we created a of the features observed in wild type or kinetochores. Tip1p, the fission yeast phosphorylation state of malp3 clearly computer simulation of an interphase cells. CLIP-170 homologue is required for changes along the cell cycle. We are cell and compared it with experimental targeting of microtubules to cell ends. It currently investigating which sites are observations. Using overdamped spatially regulates of microtubule phosphorylated in these proteins and dynamics suppressing catastrophe in which kinases could be responsible the middle regions of the cell, but not for their phosphorylation. at the cell ends. On the other hand the

P68 P69 Mapping functional regions The most common disease-causing Network from spindle pole constitute a morphogenesis network of btn1, the mutation in CLN3 is a 1 kb internal body for cell morphogenesis that is important for polarity control and deletion that occurs on 81% of disease cell separation. These proteins are Schizosaccharomyces pombe chromosomes and results in the loss of essential for cell viability and mutations orthologue of the human a large region of homology between the Dai Hirata result in failure to establish rod shape Batten disease gene CLN3 two proteins. When overexpressed in upon septum formation. Pmo25 is btn1A cells, GFP.Btn1p containing an Department of Molecular localized at mitotic SPBs and septum, Biotechnology, Graduate School of 1 1 internal deletion that models this muta- and act upstream of Orb6. We have R. Haines , S. Codlin , 102-176del Advanced Sciences of Matter, 1 2 tion (GFP.Btn1p ), is localised to found that localization patterns of and S. E. Mole , the endoplasmic reticulum and the Hiroshima University, Higashi Hiroshima Pmo25 to the SPB are asymmetrical, 739-8530, JAPAN 1 golgi complex, but not to early endo- reminiscent of components of the sep- MRC Laboratory for Molecular Cell cytic compartments unlike full length tation initiation network (SIN). Further Biology, University College London, GFP.Btn1p. Furthermore it was unable Cell morphogenesis and the cell cycle analysis has indicated that the SIN is Gower Street, London, WC1E 6BT, to rescue the cell curving defect are coordinately regulated. In fission required for Pmo25 localization, in United Kingdom yeast, the growth polarity dynamically 2 observed in btn1A cells after 4 hours at which Nak1-Orb6 kinase activities are Departments of Paediatrics & Child 37°C. However this protein was able to changes during 3 stages of the cell under the control of the SIN. We pro- Health and Biology, University College partially rescue the vacuole size defect cycle, initiation of growth upon cell divi- pose that Pmo25 plays a connecting London in btn1A cells, suggesting that it retains sion, NETO (new end take off), and role between the SIN and the morpho- partial function. We have also designed septum formation. However, the coordi- genesis network. In this presentation, btn1 has been reported by our labora- a set of deletion and truncation con- nated mechanism remains elusive. we will present a new molecule tory to be involved in vacuole home- structs to map regions of the protein Recently, we have found that the con- involved in the network. ostasis, and to be a functional homo- required for controlling its intracellular served proteins, MO25-like Pmo25, GC logue of the human gene CLN3. location and its varied functions (curv- kinase Nak1, Drosophila Furry-like Mutations in this gene cause juvenile ing, polarised growth and vacuole Mor2, NDR kinase Orb6, and Mob2 neuronal ceroid lipofuscinosis (JNCL) or homeostasis). In addition we have used Batten disease, a neurodegenerative site directed mutagenesis to model all lysosomal storage disease. Since disease causing missense mutations of S. pombe cells have over 50 vacuoles CLN3 in btn1 and expressed these in (the equivalent of the mammalian lyso- our btn1A strain. Since all of these some) and exhibit striking polarised mutations occur at residues that are growth, as do neurons, we have con- conserved in the S. pombe protein they cluded that the fission yeast system are likely to be essential for function. provides an excellent tool for the study These studies have revealed residues of JNCL. We have shown that btn1A that are required for correct trafficking cells have larger vacuoles that are also of the protein, as well has those that less acidic, and that this can be res- have impacts, both positive and nega- cued by overexpression of GFP.Btn1p. tive, on vacuole size and pH. btn1A cells also lose rod-shaped mor- The results from this study will help to phology upon a temperature shift to define the complex mechanism of 37°C, with defects including cell curv- Btn1p action and its apparent role in ing, inability to repolarise actin patches cytoskeletal-related functions and vac- and failure to undergo NETO. In addi- uole homeostasis, and will add consid- tion we have shown that the ability of erably to our understanding of the GFP.Btn1p to rescue these defects is molecular basis of JNCL. dependent on its location, implying that Btn1p has more than one function and that controlling the location of the pro- tein may be functionally important.

P70 P71 Tomography shows the way of pendently of each other. Cell Polarity Determinants ed Mid1p is required for selection of interphase microtubules in Electron tomography is ideally suited Tea1p, Tea4p and Pom1p the division site in fission yeast. for a detailed study of microtubule Intriguingly, we find that, although mid1 fission yeast organisation, as it enables the investi- Inhibit Cell Division at Cell mutants misplace the division septa, gation and reconstitution of a volume Ends in Fission Yeast the misplaced septa are occluded from J.L. Höög*, C. Schwartz§, A.T. Noon?, with optimal resolution. Using this the cell ends. This process, referred to E.T. O’Toole§, D.N. Mastronarde§, J.R. method, we have investigated the exact as tip-occlusion, is essential for viability § Yinyi Huang, Ting Gang Chew, McIntosh , C. Antony* range of the number of microtubules in Wanzhong Ge, Hongyan Wang, of Mid1p-defective cells and requires a single bundle and the details of tip-localized kelch repeat protein Tea1p § Naweed I. Naqvi and Mohan K. *EMBL, Heidelberg, Germany; The microtubule organization at both the Balasubramanian and associated factors, Tea4p / Wsh3p, Boulder Laboratory of 3D EM of Cells, cell tips and in the nuclear area, where and Pom1p (tip-complex). In a genetic Boulder Colorado, USA microtubule nucleation occurs. Cell Division Laboratory, Temasek Life screen aimed at identifying molecular Solving the microtubule organization Sciences Laboratory and the mechanism(s) of tip occlusion we iden- Cellular organisation and the spatial within these cells requires the 3-D Department of Biological Sciences, tified Cyk3p, an SH3 domain containing positioning of protein complexes within reconstruction of volumes larger than National University of Singapore, protein that localizes to the actomyosin a cell are critical for the maintenance of those represented by a single tomo- Singapore e-mail: [email protected] ring and cell ends in a tip-complex cellular architecture and directed cell gram. Thus, we have used montaging independent manner. The tip complex growth. Fission yeast to cover a greater volume of cells, and The mechanisms positioning the plane appears to negatively regulate cell end (Schizosaccharomyces pombe) has in some cases we gained an entire cell of cell division are crucial to all cells. localized actomyosin ring proteins such proven to be an excellent model for volume. This is, to our knowledge, the While bacteria appear to choose their as Cyk3p in order to achieve tip occlu- studying cell division and cell morpho- first time a whole eukaryotic cell has division plane by negative regulatory sion. Our studies reveal interesting genesis. Maintaining the polarity of been reconstructed to this resolution. mechanisms that prevent cell division analogies between the tip-complex and these rod shaped cells as well as tar- Large volumes provide further informa- at inappropriate sites, most eukaryotic the bacterial Min-proteins, both of geting polarity factors to the right loca- tion about the structure of microtubule cells do so by signals that positively which prevent cell division at inappro- tion requires an intact microtubule net- bundles as well as their respective ori- influence this process. Cells of the fis- priate sites. work. Interphase microtubules are entation and interactions with other sion yeast Schizosaccharomyces arranged into 4-5 cytoplasmic bundles, organelles i.e. mitochondria. pombe are cylindrically shaped with oriented along the long cell axis. Based hemispherical ends and divide through on fluorescence microscopy data, the the use of an actomyosin based con- individual microtubules within a bundle tractile ring. The metazoan anillin-relat- are believed to grow and shrink inde-

P72 P73 Microtubules, MAPs, and The fission yeast Schizosaccharomyces Control of growth polarity critical cell size and completion of DNA motors: How to organize the pombe is an ideal organism to study upon perturbed DNA replication. However, the mechanism linear arrays of microtubules and underlying NETO remains to be microtubule cytoskeleton. MTOCs because they are genetically replication understood. Here we show that tractable, optically convenient for high S-phase checkpoint pathway is Marcel Janson, Isabelle Loiodice, spatial-temporal resolution imaging and Muneyoshi Kanai1, Takashi Toda2 required for the maintenance of the and Phong Tran analysis, and have MTOCs which and Dai Hirata1 monopolar growth in the pol1/mon7 organize four linear arrays of micro- mutant. Indeed, the over-expression of University of Pennsylvania, tubules. There are three classes of 1Department of Molecular either Rad3 or Cds1 kinase was able to Philadelphia, PA, USA MTOCs in the fission yeast: the centro- Biotechnology, Graduate School of inhibit the switch to bipolar under the some-equivalent spindle pole body Advanced Sciences of Matter, normal growth condition. Further, the The microtubule cytoskeleton is essen- (SPB), the equatorial MTOC (eMTOC) Hiroshima University, Higashi Hiroshima activity of the Dyrk family Pom1, tial for cellular processes such as mito- which forms during cytokinesis, and 739-8530, JAPAN essential for the switch to bipolar, in the sis, organelle transport, and cell polari- multiple interphase MTOCs (iMTOCs). 2Laboratory of Cell Regulation, Cancer pol1 mutant was higher than that in the ty. The key organizer of microtubules is Works from ours and several other labs Research UK, London Research G1-arrested cdc10 mutant with the microtubule organizing center have identified mto1p/mto2p, ase1p, Institute, PO Box 123, 44 Lincoln’s Inn monopolar growing manner. These (MTOC). All MTOCs are composed of and klp2p as playing key roles in the Fields, London WC2A 3PX, UK results suggest that the Rad3-Cds1 multi-protein complexes and share organization of the interphase micro- kinases coordinate NETO with three general properties: a) They nucle- tubule arrays. In fission yeast, at a specific point in completion of DNA replication by ate microtubules. b) They arrange the Using a combination of in vivo live cell the G2 phase, growth polarity switches regulating a molecule(s) that acts microtubules into functional patterns. imaging and in vitro motility assays with drastically from monopolar to bipolar. downstream of Pom1. And c) They attach the microtubules to purified proteins, we are investigating This phenomenon is called NETO (New their proper organelle targets. In recent the molecular mechanism of how End Take Off). For NETO to take place, years the molecules that are localized antiparallel linear arrays of microtubules two requirements have to be fulfilled, a to the centrosome, the primary MTOC are formed. We discovered that: 1) in animal cells, have been catalogued mto2p recruits x-TuRCs to pre-existing and much progress has been made in microtubules and activates de novo understanding the mechanism by which nucleation of a new microtubule on the the x-tubulin ring complex (x-TuRC) pre-existing microtubule, 2) ase1p pref- located at the centrosome nucleate erentially bundles the new and old microtubules. However, most cytoplas- microtubules into an antiparallel array, mic x-TuRCs are not located at the and 3) this new antiparallel microtubule centrosome, and their cellular functions array is then pulled to the site of the are unknown. Furthermore, while the iMTOC by the minus end kinesin klp2p. canonical centrosome arranges radial Our results suggest a model where arrays of microtubules which are microtubules, MAPs, and motors inter- attached to the nucleus, many highly act in a coordinated manner to organize differentiated cell types – neurons, linear and dynamic microtubule struc- myotubes, and polarized epithelial cells tures. – have linear arrays of microtubules which are not attached to the nucleus. The mechanisms which generate linear arrays of microtubules are unknown.

P74 P75 Screening for the mutants that induces a Wee1-dependent G2-delay, Fission yeast MO25 protein is Pmo25, GC kinase Nakl, Mor2, and have the defect in cell size indicating that the mutation activates essential for cell Orb6, constitute a morphogenesis net- the mechanism coordinating growth work that is important for polarity con- control polarity with cell cycle progression. morphogenesis trol (establishment and maintenance) However, the mechanism inducing the and cell separation. Pmo25 was local- Shunsuke Kubota, Muneyoshi Kanai, Wee1-dependent G2-delay remains elu- Kazunori Kume1, Muneyoshi Kanai1, ized at mitotic SPBs and then under- Kazunori Kume, Daisuke Urata, sive. Takashi Toda2 and Dai Hirata1 went translocation to the dividing medi- Tetsuya Goshima, and Dai Hirata To identify a molecule(s) involved in the al region upon cytokinesis. Our results regulation of Wee1, we tried to isolate 1Department of Molecular indicated that Pmo25 interacts with Department of Molecular the mutants that have the defect in cell Biotechnology, Graduate School of Nak1 transiently at SPBs and is Biotechnology, Graduate School of size control like wee1 mutant (wel: Advanced Sciences of Matter, required for both the localization and Advanced Sciences of Matter, WEe1-Like mutant). Wild-type cells Hiroshima University, Higashi Hiroshima kinase activity of Nak1. Pmo25 and Hiroshima University, Higashi Hiroshima were mutagenized with NTG and cul- 739-8530, JAPAN Nak1 were essential for Orb6 kinase 739-8530, JAPAN tured at 36°C for 5 h. Using this culture, 2Laboratory of Cell Regulation, Cancer activity. Further, the Pmo25 localization we isolated the small cells by elutria- Research UK, London Research at the SPBs and the Nak1-Orb6 kinase In fission yeast, Drosophila Furry-like tion. From this collection, we screened Institute, PO Box 123, 44 Lincoln’s Inn activities during interphase were under Mor2, in concert with the for the temperature-sensitive and small Fields, London WC2A 3PX, UK the control of the Cdc7 and Sid1 kinas- NDR/Tricornered kinase Orb6, becomes cell size mutants. In this screening, we es in septation initiation network (SIN), localized at the polarized growing isolated 33 wel mutants and classified In fission yeast, Drosophila Furry-like suggesting a functional linkage regions and plays an important role in into at least 3 loci including the wee1 Mor2 plays an essential role in cell mor- between SIN on SPBs and the network the restriction of growth zone(s) to mutation. phogenesis in concert with the for cell morphogenesis following cytoki- which CLIP170-like protein Tip1 is tar- NDR/Tricornered kinase Orb6. nesis. geted. Further the mor2 mutation Mutations of these genes result in the loss of cell polarity. Here we show that the conserved proteins, MO25-like

P76 P77 Characterisation of Tip1p- Although we now have a good under- Pob1 functions in the 664 cells, presumed secretory vesicles interacting proteins in standing of how Tip1p regulates micro- secretory pathway, interacting (~90 nm in diameter) were accumulated tubule dynamics, the mechanisms and along F-actin cables at the restrictive Schizosaccharomyces pombe proteins that modulate the activity of with Rho3 and the exocyst temperature. This indicates that Pob1 Tip1p are not yet understood: Tip1p complex in fission yeast may play an important role in vesicle Lindsay Murrells1, Mika Toya1,2 could be a component of many differ- transport associated with actin cables. and Damian Brunner1 ent protein complexes and such addi- Kentaro Nakano1, Mika Toya2, Rho3, a small-GTPase, was obtained tional roles for Tip1p, involving interac- Naomi Kamasawa3, Aki Yoneda3, as a multi-copy suppressor of p o b 1 - 6 6 4 1European Molecular Biology tions with different cellular structures, Masako Osumi3 mutation. Rho3 has been shown to Laboratory, Meyerhofstrasse 1, 69117 are expected. and Masayuki Yamamoto2 have functions at least in two path- Heidelberg, Germany. To identify additional roles of Tip1p, ways; controlling localization of For3 2Present Address: Laboratory of Cell screens have been carried out in the 1Department of Structural Biosciences, (Nakano et al., 2002) and modulating Regulation, Cancer Research UK, lab to identify proteins that interact with Graduate School of Life and exocyst complex including Sec8 (Wang London Research Institute, Lincoln's Tip1p in vitro. Initially, four Environmental Sciences, University of et al. 2003). Given that the pob1-664 Inn Fields Laboratories, London WC2A Tip one-interacting (Toi) proteins were Tsukuba, Tsukuba, Ibaraki 305-8577, cells showed a vesicle accumulation 3PX, United Kingdom identified from a pulldown using bacte- 2Department of Biophysics and phenotype similar to sec8-1 mutant rially-expressed GST-Tip1p, chemically Biochemistry, Graduate School of cells, we studied the functional interac- In fission yeast, growth is restricted to cross-linked to ProteinA beads using Science, University of Tokyo, Hongo, tion of these genes. pob1-664sec8-1 the cell ends and, as a consequence, an anti-GST antibody, as bait. Proteins Tokyo 113-0033, double mutant cells showed a severe cells are rod-shaped cylinders. Correct isolated from an SDS gel were identi- 3Depertment of Chemical and Biological growth defect at the permissive tem- positioning of the growth zones and the fied using mass spectrometry, however Sciences, Faculty of Science, Japan perature. At the restrictive temperature, maintenance of polarity requires the only those proteins giving a clear signal Women’s University, Tokyo 112-0015, Sec8p was delocalized in pob1-664 microtubule cytoskeleton, which in turn above background could be detected Japan cells, while Pob1 localized properly at is regulated by microtubule-associated using this method. In order to more the cell tips or division site in sec8-1 proteins (MAPs). One such protein is sensitively detect Tip1p interactors, an Cell morphogenesis is coordinated with background. This indicates that the Tip1p, a microtubule plus-end tracking isotope-coded affinity tag (ICAT) several steps including establishment localization of exocyst complex protein, homologous to human method was employed and six addi- of cell polarity, organization of polarized depends on the localization of Pob1. CLIP170. Tip1p is transported to the tional candidate Tip1p-interactors were cytoskeleton, and trafficking of mem- These results suggest that Pob1 plays plus-ends of microtubules by the identified (collaboration with E Brunner, brane and other cell materials. In fission a role in maintenance of cell morpholo- kinesin Tea2p, where it is important for University of Zurich), which are current- yeast, round, bent or T shape mutants gy determining appropriate sites for the the spatial regulation of microtubule ly under investigation. have been studied as morphological secretory vesicle targeting by the local- dynamics, suppressing catastrophe at defective mutants. We have previously ization of the exocyst complex. the cell cortex in the central regions of shown that pob1-664 mutant cells the cell, but not at the cell ends. The show a lemon-like shape at the restric- presence of Tip1p at the plus-ends of tive temperature (Toya et al., 1999). microtubules therefore ensures that Here we study how Pob1p is involved microtubules efficiently target the cell in the maintenance of cell morphology. ends. Electron microscopy using the high- pressure freezing and freeze-substitu- tion method revealed that, in the pob1-

P78 P79 Schizosaccharomyces pombe rho5+ expression was hardly detected Multi-mode and multi-step different regions of tea1p and tea3p are Rho5, a protein highly during log phase growth but was cortical anchoring of tea1p required for their respective interactions induced under nutritional starvation with an essential central region of homologous to Rho1, is mod5p. We demonstrate that tea3p is conditions. Rho5 overproduction Hilary Snaith and Ken Sawin involved in stationary phase caused a similar, but less penetrant, required for proper cortical localization cell survival and spore phenotype than Rho1 overproduction. of tea1p, specifically at non-growing + Wellcome Trust Centre for Cell Biology, Importantly, overexpression of rho5 but cell tips, and that tea1p and mod5p are morphogenesis School of Biological Sciences, no other rho genes was able to rescue independently required for tea3p local- University of Edinburgh, Edinburgh, A ization. Further, we find that tea3p is the lethality of rho1 cells. EH9 3JR, UK Sergio A. Rincón, Beatriz Santos Rho5 has a role in stationary phase co-transported with tea1p by micro- and Pilar Pérez because the rho1A rho5A strain had tubules to cell tips, but this occurs only Tea1p is the key cell polarity regulator in the absence of mod5p. reduced viability during stationary in fission yeast. It is an 1147 amino- Instituto Microbiología-Bioquímica. phase compared to that of rho1A. Together these results suggest that acid protein which contains six N-termi- Departamento de Microbiología- To get information about the possible independent protein-protein interac- Genética. CSIC/Universidad de nal kelch repeats and a largely alpha- tions among tea1p, tea3p and mod5p function of Rho5 in sporulation, we helical coiled-coil C-terminal domain. Salamanca. 37007 Salamanca. Spain made a dominant-negative (rho5T20N) collectively contribute to tea1p cortical Cells lacking tea1p are unable to estab- and a hyperactive (rho5G15V) allele of anchoring at cell tips, and allow us to Rho GTPases are key molecules that + lish proper bipolar growth and as a propose a multi-step and multi-mode rho5 in a homothallic background and result grow in a monopolar manner, regulate several cellular functions, such both strains showed severe ascospore model for this process. According to often forming additional growth axes. as cell polarity, secretion or cell wall formation defects. The rho5T20N strain the model, tea1p is initially in a trans- biogenesis. Schizosaccharomyces tea1A mutants are also defective in the port complex on microtubule plus ends, produced four ascopores less renfrin- cortical localization of other polarity pombe has six Rho GTPases, Cdc42 gent and very sensitive to glusulase. in association with other plus-end bind- factors, including the membrane protein and Rho1-Rho5. Rho1 is an essential These spores showed a very faint ing proteins. (1) Upon reaching cell tips protein involved in the regulation of the mod5p and the tea1p-related protein tea1p interacts with mod5p. This inter- signal of Mde10-GFP, a protein that tea3p. Tea1p itself is predominantly cell wall biosynthesis since it is the reg- localizes to the ascospore wall, sug- action is important for the anchoring of localised at cell tips, following targeting ulatory subunit of the a-(1,3)glucan syn- gesting weak spores walls. On the tea1p at the cortex but is by itself insuf- thase. Rho1 is also important for the by association with growing micro- ficient for the correct steady-state other hand, the rho5G15V mutant tubule ends. It is subsequently maintenance of actin patches. produced four aberrant spores with localization of tea1p and mod5p. (2) anchored at cell tips via an unknown Rho5 is a highly homologous protein to some wall thickenings, and slightly Tea1p then further interacts with corti- Rho1 (86% identity). We hypothesized mechanism that requires both the cally-localized tea3p. This may occur at sensitive to glusulase. These results tea1p C-terminus and mod5p. Once at that Rho5 might be acting in a similar suggest that Rho5 functions in a both cell tips but is functionally impor- the cell tips tea1p organised a zone of way than Rho1. However, rho5A similar, but less efficient way than tant primarily at non-growing tips. (3) mutant cells show no morphological or polarised filamentous actin which We also suggest that at growing cell Rho1, plays a non-essential role during drives growth. cell wall defects. In order to make sure stationary phase, and participates in tips there may be a parallel, tea3p- + Currently there are three major out- that rho5 is not a pseudogene, we spore wall formation. independent pathway of tea1p reten- checked the expression of the gene. standing issues in relation to how tea1p tion, involving both a tea1p-mod5p functions in microtubule-mediated cell interaction and an interaction of tea1p polarity in fission yeast. Firstly, how with other cortical proteins. (4) Finally, tea1p is associated with, and transport- we propose that in addition to the ed on, microtubule plus ends? mod5p-dependent pathway of cortical Secondly, how does tea1p become tea1p anchoring, a complementary anchored at the cortex at cell tips? mechanism exists whereby tea1p and Thirdly, how does tea1p interact with tea3p functionally interact both in the the actin cytoskeleton? In this work we cytoplasm and at the cortex to address the second of these questions: “bypass” mod5p. This bypass operates tea1p cortical anchoring. We show that only in the absence of mod5p and the tea1p-related protein tea3p, binds therefore may represent a homeostatic independently to both mod5p and mechanism that can maintain polarity tea1p, and that tea1p and mod5p can fidelity irrespective of the precise levels also interact directly, independent of of mod5p. tea3p. Despite their related structures,

P80 P81 Modeling of the regulation of through which the system variables act DYRK-family protein kinase, the adult brain size. Human DYRK1A is fission yeast growth modes on each other were represented by cir- Pom1, interacts with Rho GAP among the genes mapped to the Down cuit linkages expressed as logical oper- syndrome critical region. ations. Different types of mutants were S. pombe Pom1 DYRK localizes at cell Sasa˘ Svetina Hisashi Tatebe1, Kentaro Nakano2, predicted by abolishing a single or 1 1 tips and septation sites in a manner more circuit linkages. The bipolar Attila Glatz and Kaz Shiozaki dependent on the cell-end marker Tea1 Institute of Biophysics, Faculty of growth mutants obtained in this manner and Tea4/Wsh3. The Apom1 mutant is 1 Medicine, University of Ljubljana, could be essentially grouped into those Section of Microbiology, University of defective in NETO and shows monopo- Ljubljana, Slovenia that arise due to the loss of cell ability California, Davis, California, USA lar growth with cytoskeletal actin con- 2 to inhibit the activators of the monopo- Department of Structural Biosciences, centrated to the growing end. We have Fission yeast growth machinery acts lar growth mode, and those that arise Graduate School of Life and found that, like other DYRKs, Pom1 is within localized growth zones which are due to the loss of cell ability to activate Environmental Sciences, University of indeed Tyr-phosphorylated. A yeast 2- during interphase at the rounded ends the bipolar growth mode. In order to Tsukuba, Tsukuba, Ibaraki, JAPAN hybrid screen for proteins that interact and during the division process in the relate the predictions of the proposed with Pom1 has identified Rga4, one of middle of this rod-like cell. A wild type regulatory circuit to underlying molecu- The DYRK (dual-specificity tyrosine the nine Rho GTPase-activating pro- cell first grows in a monopolar growth lar mechanisms, different known gene regulated kinase) family members have teins (GAPs) in S. pombe. Rga4-GFP is mode at the preexisting cell end. Later products that act as polarity factors a Tyr-X-Tyr motif in the activation loop localized to cortical regions except cell on in the cell cycle a bipolar growth have been assigned to different circuit and are structurally related to MAP tips, showing “corset”-like staining mode is established, by the growth linkages. In the present work we kinases that have a Thr-X-Tyr motif around the cell. Interestingly, Apom1 being induced also at the new cell end. include into the assignment analysis instead. DYRKs autophosphorylate the cells cannot maintain this characteristic Several gene products and their mutual the literature data on growth initiation Tyr-X-Tyr motif, resulting in full activa- localization pattern of Rga4. The Arga4 interactions have already been revealed sites of different bipolar growth tion to phosphorylate Ser/Thr residues mutant is viable but actin patches are that are involved in the positioning of mutants, and also take into considera- in substrate proteins. DYRK genes have less polarized and ~50% of the mutant the growth machinery and thus in the tion recently described polarity factors been identified ubiquitously among cells fail NETO. We will discuss the role regulation of fission yeast growth Sla2p and Tea4p. eukaryotes and implicated in the con- of Rga4 and Rho-family GTPase in cell 1 modes. Therefore the attempts to inte- trol of neuronal cell growth. A mutation polarity, together with our latest results. grate the existing information in terms 1Martin SG and Chang F: New end take in a DYRK gene in Drosophila, mini- of a model seem to be justified. In a off. Regulating cell polarity during the brain, brings about marked reduction in switch-like model introduced recently it fission yeast cell cycle, Cell Cycle 8 was assumed that different growth (2005) 1046-1049. modes correspond to different stable 2Bähler J and Svetina S: A logical cir- steady states of an underlying regulato- cuit for the regulation of fission yeast ry circuit which was described in terms growth modes. J. Theor. Biol. 237 of a logical circuit based on three bina- (2005) 210-218. ry variables.2The signaling pathways

P82 P83 Mitotic Spindle Characterisation of the fission deletions of the kinesin-like molecules yeast DASH-kinetochore Klp5/6 (kinesin-8). Klp5 and Klp6 are known to play a role in promoting Cdc2 phosphorylation of here report that Dis1 is hyper phospho- complex bipolar attachment of the spindle to the fission yeast Dis1 similar to rylated by Cdc2 in mitotic metaphase kinetochore. Thus DASH may regulate and rapidly dephosphorylated in Karen Crawley, Takashi Toda microtubule attachment at the kineto- XMAP215/TOG improves anaphase. Whilst Dis1-6A mutant cells chore in concert with Klp5/6, thereby segregation accuracy via that substitute all of the six Cdc2 sites Cell Regulation Laboratory, generating tension. We have screened metaphase kinetochore can produce colonies at 22-36°C, they London Research Institute, for DASH mutants in a klp5 null back- lose minichromosome at a high fre- Cancer Research UK localization ground by selecting survivors that are quency, become hypersensitive to a functional at the permissive tubulin poison and synthetic lethal with Bipolar attachment of the kinetochore temperature but lose DASH function Keita Aoki, Yukinobu Nakaseko, mis12-537, a centromere defective to the mitotic spindle is a crucial step # + when shifted to the restrictive Kazuhisa Kinoshita , Gohta Goshima mutant. Cdc2 phosphorylation of Dis1 for sister chromatid separation during tempe-rature. We are characterising the and Mitsuhiro Yanagida* improves the fidelity of chromosome mitosis. The kinetochore is a spe- mitotic phenotypes of these mutants to segregation through conferring on cialised proteinaceous structure formed gain insight into DASH function Department of Gene Mechanisms, Dis1 the ability to locate at metaphase on centromeres that is required for during mitosis. Graduate School of Biostudies, Kyoto kinetochores. The signals of tethering the spindle. It consists of University, Sakyo-ku, Kyoto 606-8502, GFP-tagged unphosphorylatable many sub complexes including inner This work is partly performed under Japan Dis1-6A are greatly diminished in the core platform proteins and outer pro- # collaboration with Dr. Jonathan Millar’s Present address: Max Planck Institute metaphase kinetochores, but instead teins. In the budding yeast group (Sanchez-Perez et al., 2005). of Molecular Cell Biology and Genetics, present along the short spindle. The Saccharomyces cerevisiae, the DASH D-01307 Dresden, Germany. wild type Dis1 is enriched at the plus complex is an essential complex con- + Sanchez-Perez, I., Renwick, S.J., Present address: Department of and minus ends of microtubules, stituting of 10 components and plays a Crawley, K., Karig, I., Buck, V., Cellular and Molecular Pharmacology, respectively, in metaphase and vital role in chromosome segregation. Meadows, J.C., Franco-Sanchez, A., University of California-San Francisco, anaphase. The transition is regulated All 10 homologues of DASH have been Fleig, U., Toda, T., and Millar, J.B. San Francisco, CA 94107 by Cdc2 phosphorylation and dephos- recently identified in the fission yeast, (2005). The DASH complex and phorylation. Dis1-6A resembles and Schizosaccharomyces pombe, an Klp5/Klp6 kinesin coordinate bipolar Spindle formation and microtubule partly substitutes Alp14/Mtc1, another organism that contains a larger and chromosome attachment in fission dynamics are vital for chromosome XMAP215-like protein in fission yeast. more complex centromere structure. In yeast. EMBO J. 24, 2931-2943. segregation. Kinetochore microtubules this organism all the DASH genes are are shortened in anaphase to bring not essential but nonetheless play an separated chromatids toward the aop- important role in chromosome segrega- posite spindle poles. Fission yeast tion. Deletion mutants of the DASH Dis1, similar to frog XMAP215, human genes display chromosome instability Tog, fly mini spindles, nematode Zyg-9 and are synthetically lethal with and budding yeast Stu2 that are required for proper microtubule dynam- ics, is immuno-coprecipitated with the central centromere DNAs in mitosis. We

P84 P85 Analysis of Spindle Pole Body localised coiled-coil protein that is The DASH complex and is not essential), one that requires Protein Pcp1 in fission yeast recently identified to be the orthologue Klp5/Klp6 kinesin co-ord i n a t e l y DASH and one that does not. of budding yeast Spc110p and human We carry out a genetic analysis to control chromosome Chii Shyang Fong, Masamitsu Sato kendrin, has been shown to play an establish any link between DASH com- and Takashi Toda essential role in bipolar spindle forma- bi-orientation. plex and kinetochore associated pro- tion and chromosome segregation. Also teins as Mal2p, Mis6p or Mis12p and it is known that overproduction of Pcp1 Kinesins as Klp5p or Klp6p. We were Laboratory of Cell Regulation, Cancer Franco-Sanchez A., Sanchez-Perez Research UK, London Research results in SPB abnormalities. With the unable to construct a DASH complex aim to unravel the role for Pcp1 in spin- deleted strain an either Klp5 or Klp6 Institute, Lincoln’s Inn Fields M.I., Meadows J.C., Buck V. and Millar dle nucleation and its regulation during deleted, indicating that DASH complex Laboratories, 44 Lincoln’s Inn Fields, J.B.A. (2006). London WC2A 3PX, UK the cell cycle, we are currently examin- and kinesins Klp5/Klp6 perform an ing mitotic phenotypes of conditional In order for each set of sister chro m a t i d s essential over-lapping function. On the mutants of pcp1 that were isolated in other hand double mutants between Bipolar mitotic spindle, which emanates to be separated to opposite spindle from the spindle pole bodies (SPBs), is the lab. Preliminary observations of the poles, each kinetochore must be bound deleted DASH complex and either mutants indicated multiple mitotic others kinesins (Klp1, Klp2, Klp3 or essential for proper chromosome seg- to microtubules from one pole and its defects including chromosome misseg- Klp4), are viable indicating that the regation. Whilst many aspects of SPB’s sister kinetochore to microtubules from function have been extensively studied, regation, deformed mitotic spindles and opposite pole, a phenomenon known interaction between the DASH and delayed mitotic progression. Klp5/Klp6 is specific. mechanisms underlying microtubule as spindle bi-orientation. nucleation from this structure has The mechanism governing the estab- remained elusive. Pcp1, an SPB lishment of spindle bi-polarity has been To examine the nature of the essential examined in some detail in budding role shared by DASH and Klp5, spores yeast, in which each kinetochore is lacking both genes could germinate. bound by only a single microtubule (2-4 The cells had an elongated mitotic in fission yeast). In this mechanism are spindle and separated spindle poles involved a number of microtubule bind- indicating that the cells had entered but ing proteins and kinetochore associat- failed to exit mitosis. ed proteins. These proteins are phos- In conclusion, we find that mitotic cen- phorilated by the Aurora like Ipl1 tromere associated Klp5/Klp6 kinesin kinase, which triggers microtubule complex is essential in cells lacking detachment. One of these complexes is components of the DASH complex. DASH, which is essential in budding Cells lacking both complexes undergo yeast and forms both rings and spiral a first cell cycle arrest in mitosis due to structures on microtubules in vitro. a failure to establish chromosome bi- However in fission yeast posses at orientation. least two pathways to establish a correct bi-polar mitotic spindle (DASH

P86 P87 Characterisation of a new the commitment to mitosis as it geneti- Mad3 and its regulation the activity of Slp1-APC/C and thereby allele of CUT12: A Spindle cally interacts with cdc25 and the Polo of the APC/C the metaphase to anaphase transition. kinase, plo1. In this study, a novel However, there may be other mecha- Pole Body component cut12 allele, cut12.2, is being charac- Kevin G. Hardwick, Matylda nisms whereby spindle checkpoint pro- implcated in the control of terised. It is a thermo-sensitive loss-of- Sczanieka, Emma King, Julie Blyth teins inhibit Slp1-APC/C. commitment to Mitosis function allele of cut12 : cut12.2 cells and Sjaak van der Sar enter mitosis but nucleate microtubules Here we describe multiple mechanisms Daphné Garcin, Agnes Grallert, from only one of their two SPBs. The The Wellcome Trust Centre for Cell whereby Mad3 regulates the activity of lower penetrance of cut12.2 compared Slp1-APC/C. Mad3 (and BubR1) con- Anna Poziemba-Niedbala, Biology, University of Edinburgh to the one of cut12.1 has enabled us to tain two conserved KEN boxes. Iain Hagan King’s Buildings, Mayfield Road, clone multi-copy suppressors of cut12, Edinburgh, EH9 3JR, UK Mutation of either of these KEN boxes Paterson Institute for Cancer Research such as CyclinB. New genetic interac- abrogates the fission yeast spindle tions between Cut12 and proteins checkpoint. We will provide evidence (CRUK), Christie Hospital, Wilmslow The spindle checkpoint prevents cells Road, Manchester M20 4BX, United involved in cell division have also been with spindle or kinetochore defects that these signals are crucial for MCC uncovered: for example the dominant formation, and for the interaction Kingdom. [email protected] + from initiating chromosome segrega- activating mutation in cdc2 , cdc2-3w, between the Mad2/Mad3 checkpoint tion. Mutations in the Mad (mitotic In eukaryotic cells, entry into mitosis is suppresses the conditional lethality of arrest defective) or Bub (budding unin- proteins and mitotic APC/C. cut12.2. In addition, we find that phosphory l a t i o n tightly occurs once a trigger level of hibited by benomyl) genes inactivate of Mad3 by aurora kinase is a key Cdc2-CyclinB activity is reached. This the checkpoint and allow cells with trigger is then amplified by a positive defective spindles to proceed through requirement for the spindle checkpoint response to the lack of tension at yeast feedback loop to promote complete mitosis. Such cell divisions lead to kinetochores. entry into mitosis. Dissecting the mech- chromosome loss, aneuploidy anisms that control the activation of and death. this complex remains a challenge. Cut12/Stf1 is a spindle pole body (SPB) Mad3, Mad2 and Slp1(Cdc20) co- component that has been involved in immunoprecipitate in the mitotic check- point complex (MCC), and it is thought that formation of this complex inhibits

P88 P89 Identification of the Alp14- domain contains 2 TOG domains that Dynamics of Spindle lapse microscopy and FRAP analysis to interacting protein using yeast by themselves consist of 5 HEAT Checkpoint Proteins study the dynamic localisation of these (Huntingtin-Elongation A-TOR) repeats, proteins during the cell cycle and on two-hybrid screening which are believed to be involved in activation of the spindle checkpoint. protein-protein interaction. On the other K. May and K. Hardwick Chiho Ikebe and Takashi Toda hand, the C-terminal region possesses Bub1 and 3, and Mad1, 2 and 3 a microtubule-binding activity that is The Wellcome Trust Centre for Cell localise to the kinetochore early in an Laboratory of Cell Regulation, Cancer required for targeting of this protein to Biology, University of Edinburgh, Kings unperturbed mitosis. As mitosis Research UK, London Research the SPB via binding to Alp7, the TACC Buildings, Mayfield Rd., Edinburgh EH9 progresses we observe differences in Institute, Lincoln’s Inn Fields (transforming acidic coiled-coil-contain- 3JR. Scotland. localisation between these proteins. Laboratories, 44 Lincoln’s Inn Fields, ing) homolg. We previously showed that Mad1 and 2, which interact with each London WC2A 3PX, UK Alp14 is required for bipolar spindle for- The mitotic spindle checkpoint prevents other constitutively, become localised mation. In its absence, spindle-kineto- progression of mitosis unless all chro- to the spindle and spindle pole body Bipolar spindle formation during chore interaction is not established, mosomes are correctly attached to the (SPB) while the Bub1, Bub3 and Mad3 prometaphase is vital for accurate chro- resulting in chromosome mis-segrega- spindle and tension is generated. Bub1 signals are lost as anaphase mosome segregation in anaphase. The tion. That means Alp14 acts as a struc- and Bub3, Mad1-3 and Mph1 are progresses. Unlike the other checkpoint Dis1/TOG microtubule-associated pro- tural bridge between the kinetochore essential components of the spindle proteins Mad1 and 2 localise to the tein (MAP) family is evolutionally con- and the spindle, thereby facilitating checkpoint. On activation of the check- nuclear periphery during interphase. served in all eukaryotes from yeasts to microtubule-kinetochore attachment. In point they localise to the site of We show that this interaction is humans. All this family proteins localize order to identify more Alp14-interacting chromosome/spindle microtubule mediated by the N-terminal coiled coil to microtubule-organizing centers proteins, we undertook yeast two- attachment, the kinetochore. Although domain of Mad1 and the S.pombe Mlp (MTOCs) that include the spindle pole hybrid screening, using Alp14 as a bait. these proteins have been well homologue Alm1. We are investigating bodies (SPBs) in yeast and the centro- We isolated several proteins that inter- characterised it is still unclear how they the significance of these interactions some in animals. This family plays a act with Alp14. We have started to function to generate the signal which with respect to the spindle checkpoint. pivotal role in microtubule functions, characterize these integrators and the ultimately leads to APC inhibition and, including mitotic spindle formation, results of the functional analysis will be once generated, how this signal is kinetochore function and cell polarity presented. turned off. To gain insight into the roles and morphogenesis. of these proteins we have used time- In the fission yeast, the two Dis1/TOG MAPs homologs, Alp14 and Dis1 have been identified. Alp14 was originally identified in our laboratory as one of morphological mutants (alp stand for altered polarity). Alp14 consists of two separable domains. The N-terminal

P90 P91 Dissociation of mitotic checkpoint proteins including Bub1, Characterisation of the Mph1 is phosphorylated throughout the spindle position from the Bub3, Mad3 and Mph1. We find that spindle checkpoint kinase cell cycle and the levels of this do not cells lacking Mto1, a centrosomin-like change except during checkpoint timing of anaphase onset protein, have mis-positioned spindles Mph1 in fission yeast activation, for example in response to in fission yeast but are not delayed in the timing of spindle damage, when it becomes sister chromatid separation. Instead we Laura Milne and Kevin Hardwick hyperphosphorylated. This modification John C. Meadows find that Latrunculin A delays the onset is Bub1-independen and we are and Jonathan B.A. Millar of anaphase and causes mitotic Wellcome Trust Centre for Cell Biology, currently testing other kinases for their spindles to collapse. This effect is University of Edinburgh, EH9 3JR involvement in Mph1 phosphorylation. Division of Yeast Genetics, National exacerbated in cells lacking Ase1, a Institute for Medical Research, microtubule associated protein (MAP), The spindle checkpoint monitors the In addition to a role in the spindle The Ridgeway, Mill Hill, which stabilises anti-parallel spindle kinetochore-microtubule interactions to checkpoint Mph1, as well as Bub1 London NW7 1AA, UK midzone microtubules. These results ensure accurate segregation of sister (another spindle checkpoint kinase) suggest that Latrunculin A delays the chromatids during mitosis to prevent also has a role in chromosome It has previously been proposed that in onset of anaphase by destabilising aneuploidy. It arrests cells at the segregation. These are the only spindle fission yeast mitotic spindle position is formation of a bipolar mitotic spindle metaphase to anaphase transition in checkpoint components in fission yeast monitored by a checkpoint that rather than by causing spindle response to lack of tension and/or kine- involved in chromosome segregation. controls the timing of anaphase onset. mis-orientation. tochore-microtubule attachment until In a deletion mutant a high percentage This checkpoint is activated by the problem can be corrected and of lagging chromosomes and chromo- treatment of cells with Latrunculin A, anaphase can progress. some loss is observed, much the same an inhibitor of actin polymerisation, and as in a Bub1 deletion. Bub1 is also requires a subset of spindle assembly Mph1, the fission yeast homologue of known to have a role in chromosome Mps1, is one of the kinases involved in segregation. When a double deletion of the spindle checkpoint. Deletion of Mph1 Mph1 and Bub1 is made an additive results in cells with a compromised effect is seen with respect to spindle checkpoint. They do not arrest chromosome segregation. in response to spindle damage by The percentage of lagging drugs such as Benomyl or mutations chromosomes and chromosome loss like the cold sensitive tubulin mutant almost doubles with respect to the nda3, but attempt to continue through single mutants. We are currently anaphase with the result being cells using live cell imaging to study these showing cut phenotypes and ultimately chromosome segregation defects in cell death due to chromosome loss. more detail.

P92 P93 Regulation of exchanging the formation with latrunculin B or heat- Optical tweezers in fission role of microtubules in nuclear position- daughter nuclei and co-sym- inactivation of the temperature-sensi- yeast: Mechanism of nuclear ing, as well as the role of the nucleus in tive cdc7-24 gene product induced the septum positioning, by displacing the metric localization of Cdc7 on precocious onset of nuclear exchange. and septum positioning nucleus with optical tweezers. A dis- the SPBs in the dikaryotic cell The Cdc7-GFP signal persisted placed nucleus returned to the cell cen- 1 2 division of S. pombe. preferentially on both of the outer SPBs Isabel Raabe , Leonardo Sacconi , ter by the pushing force exerted by but disappeared from both of the inner Francesco Pavone2, microtubules against the cell tips. 1 Koei Okazaki and Osami Niwa SPBs. Like haploid cell division, the Iva Tolic-Nørrelykke Nuclear displacement during interphase Cdc7-GFP positive SPBs were in most or early prophase resulted in asymmet- 1 (Kazusa DNA Research Institute, Japan) cases “new” SPBs in dikaryotic cell Max Planck Institute of Molecular Cell ric cell division, whereas displacement division. The head-to-head spindle Biology and Genetics, Pfotenhauerstr. during prometaphase resulted in sym- orientation was not compromised in the 108, 01307 Dresden, Germany metric division as in unmanipulated We found that, if a temperature-sensi- 2 tive SIN mutant is returned to permis- presence of latrunculin B. European Laboratory for Non-linear cells. These results suggest that the sive temperature after binucleation at This orientation appeared to be Spectroscopy, Via Nello Carrara 1, division plane is specified by the pre- the non-permissive temperature, cells established at an early phase of SPB 50019 Sesto Fiorentino (Florence), Italy dividing nucleus. Since the yeast nucle- restart propagation with the two nuclei. separation and maintained throughout us is centered by microtubules during The two nuclei that had been associat- the most part of mitosis. Transient Cells of Schizosaccharomyces pombe interphase but not in mitosis, we pro- ed each other during interphase with a disruption of cyoplasmic microtubules have a centrally placed nucleus and pose that the establishment of the divi- Klp2-dependent mechanism began by a low temperature treatment in G2 divide by fission at the cell center. sion plane at the beginning of mitosis is synchronized back-to-back mitoses. phase compromised partially the head- Microtubules are required for the cen- an optimal mechanism for accurate Each spindle appeared to only extend to-head spindle orientation in tral position of the nucleus. Genetic symmetric division in these cells. outward during early anaphase B. subsequent dikaryotic cell division. studies suggested that the position of However, at a certain point in anaphase We propose a model that the new the nucleus may determine the position B, the spindles began to extend inward SPBs are built opposite to the nuclear of the septum. Alternatively, the septum to facilitate the exchange of the inner partner, and the new-and-old SPB may be positioned by the spindle, or by daughter nuclei. The onset of inner polarity is maintained by the SPB-asso- morphogen gradients or reaction diffu- nuclei exchange coincided with the ciated cytoplasmic microtubules during sion mechanisms. We investigated the completion of bundling the split cytoki- G2 phase. netic actomyosin fibers into a fine ring. It also coincided with the time point when the localization of Cdc7-GFP on the SPB becomes asymmetric, sug- gesting mechanisms coupling these three events. In accordance with this notion, inhibition of actomyosin ring

P94 P95 Precise spindle cutting using exposures resulted in partial or Dynamic Studies of allows direct testing whether two pro- a picosecond laser complete cutting of the spindle. Checkpoint Protein teins (one donor, eg. Mad2-CFP and The bleached and partially cut spindles one acceptor, eg. Mad1-YFP) come in typically continued elongating. Interactions in Fission Yeast very close proximity and can form com- Isabel Raabe, Sven K. Vogel, The completely cut spindles broke into S. pombe plexes. New tags, with pair fluorescent Iva Tolic-Norrelykke two segments, which crossed each fluorophores (CFP/YFP; other or moved away from each other. Cerulean/Citrine) for donor/acceptor MPI of Molecular Cell Biology and Patricia E. Rischitor The segments later reconnected to and Kevin Hardwick checkpoint proteins were constructed, Genetics Dresden, Germany form a functional elongating spindle in and consequently several methods for ~50% cases. Mitosis and cytokinesis measuring FRET – acceptor photo- Laser cutting experiments have led to Institute of Cell Biology, University of proceeded normally in most irradiated Edinburgh, UK bleaching and sensitized fluorescence – important discoveries in cell biology cells, though the separation of the are being used. (Berns et al., Int. Rev. Cytol. 1991). With daughter cells was delayed in Our other strategy is to study the this technique it is possible to ablate Cells must accurately replicate their comparison to non-irradiated cells. chromosomes during S phase and then “molecular output” of the interactions specific structures inside a cell locally We conclude that a picosecond pulsed between kinetochores and spindle and with rapid time resolution. Here we segregate the genetic material equally laser diode can be used for precise during mitosis. Failure to correctly repli- checkpoint proteins. Bub1 protein is use a low-cost and easy to use ablation of fluorescently labeled thought to act as a “kinetochore scaf- picosecond pulsed 405 nm laser diode cate and segregate chromosomes structures in the smallest and most results in the gain or loss of genetic fold”, as its presence at kinetochores is to dissect GFP-labeled spindle micro- genetically tractable model system required for targeting other checkpoint tubules in fission yeast. Laser irradia- information, which may cause lethality for eukaryotic cell division. or promote the genetic changes that proteins (eg. Bub3 and Mad3). Using an tion at short exposure times induced ectopically targeted Bub1 scaffold to spindle bleaching, whereas longer predispose higher eukaryotic cells to cancer. The spindle assembly check- telomeres (Bub1-GFP-Taz1Myb), one point is an elegant regulatory system can test whether Bub1 is sufficient to that delays the onset of anaphase until recruit other proteins to these sites. each and every chromosome has This will give more insights into deci- established a bipolar orientation. The phering the actual role of kinetochores components of this system, Mad and in checkpoint signalling. Bub proteins are known to form dynamic protein complexes, which par- tially are activated through their interac- tion at and with the kinetochores. This poster focuses on two strategies of analyzing such interactions in S. pombe, one strategy being based on FRET technique (Fluorescence Resonance Energy Transfer), which

P96 P97 Regulation of chromosome kinetochores are structurally and Microtubule organisation via location. Since yeast cells undergo bi-orientation in fission yeast functionally conserved. regulation of microtubule- closed mitosis in which the nuclear We have sought factors that are envelope does not break down, important for the establishment of associated proteins Alp7-Alp14 complex needs to Isabel Sanchez-Perez1, Steven J. 1 2 chromosome bi-orientation in fission TACC/Alp7 and TOG/Alp14 translocate from the cytoplasm to the Renwick , Karen Crawley , Inga yeast. We have identified a truncated nucleus upon mitotic entry and vice Karig3, Vicky Buck1, John C. throughout the cell cycle 1 allele of dam1 as a multicopy versa upon mitotic exit. Meadows , Alejandro Franco- suppressor of the sensitivity of cdc13- Sanchez1, Ursula Fleig3, Takashi Masamitsu Sato and Takashi Toda 2 1 117 (cyclin B) and mal3-1 (EB-1) cells Here we show that Alp7/TACC Toda and Jonathan B.A. Millar to thiabendazole, a microtubule poison. possesses an ability to actively Laboratory of Cell Regulation, Cancer 1 Dam1 bind to the plus end of spindle transport Alp7-Alp14 complex into the Division of Yeast Genetics, National microtubules and kinetochores as cells Research UK, 44 Lincoln’s Inn Fields, nucleus. Surprisingly, nuclear import of Institute for Medical Research, enter mitosis. Dam1 is a component of London, WC2A 3PX, United Kingdom Alp7-Alp14 occurs not only during The Ridgeway, Mill Hill, a heterotetrameric complex, variously mitosis but also even during interphase, London NW7 1AA, UK Centrosomal protein TACC (transform- 2 called the Dam1/DASH/DDD complex in which Alp7-Alp14 complex is Laboratory of Cell Regulation, Cancer that forms rings around the microtubule ing acidic coiled-coil protein) and TOG exported to the cytoplasm. During Research UK London Research in vitro. In fission yeast cells lacking (tumor overexpressed gene) are con- mitosis, cells are utilising molecular Institute, Lincoln's Inn Fields Dam1, or other components of the served MAPs that regulate microtubule schemes which accumulate Alp7-Alp14 Laboratories 44 Lincoln's Inn Fields, DASH complex, anaphase is delayed dynamics in many aspects of cellular in the nucleus effectively. London WC2A 3PX, UK phenomena. TACC and TOG form a 3 due to activation of the spindle Nucleocytoplasmic shuttling of micro- Institut für Mikrobiologie, Heinrich- assembly checkpoint and lagging sister complex and the localisation of TOG to tubule-associated proteins TACC-TOG Heine-Universitat Düsseldorf, 40225 chromatids are frequently observed and the centrosome is dependent upon via the Ran GTPase system is an Düsseldorf, Germany. occassionally sister chromatid pairs TACC. efficient way to regulate both inter- segregate to the same spindle pole. In fission yeast, the orthologs of TACC phase and mitotic microtubules in a In order for each set of sister chro- We find that one of the DASH proteins, (Alp7) and TOG (Alp14) function spatial and temporal manner. matids to be separated to opposite Dad1, is also a component of the Mis6 throughout the cell cycle. During inter- Regulatory circuit of TACC-TOG as a spindle poles, each kinetochore must kinetochore sub-complex, suggesting it phase, Alp7 and Alp14 are required to possible cargo of Ran might be be bound to microtubule(s) from one might tether microtubule associated organise cytoplasmic microtubule conserved as a mechanism underlying pole and its sister kinetochore to micro- DASH complex to the kinetochore. structure, and in mitosis, they play key microtubule dynamics and spindle tubule(s) from the opposite pole, a phe- Finally, we show that the DASH roles in formation of bipolar spindle formation. nomenon known as chromosome bi- complex is essential in cells lacking the and establishment of amphitelic orientation or bi-polar chromosome mitotic centromere associated microtubule-kinetochore attachment in attachment. Fission yeast is an excel- Klp5/Klp6 kinesin complex. the nucleus. lent model system in which to study Cells lacking both Dam1 and Klp5 Therefore, localisation of Alp7-Alp14 this process for multiple reasons. undergo a first cell cycle arrest in complex must be regulated in order to Firstly, fission yeast centromeres close- mitosis due to a failure to establish execute multiple functions at each ly resemble those in animal cells in that bipolar chromosome attachment. they are large (~35-110 kb) and contain We will discuss the roles of DASH and multiple inverted repeat units which are Klp5/Klp6 in establishing chromosome important for establishing centromeric bi-orientation. cohesion. Secondly, unlike budding yeast, spindle microtubules are only nucleated during mitosis and each kinetochore is bound to multiple (2-4) microtubules. Finally, many of the pro- teins of fission yeast and animal cell

P98 P99 Msd1p, a novel protein which tubulin mutant, atb2-983. The encoded Identification and protein) domain. Both the UBA and localises to mitotic SPBs and protein localised to mitotic SPBs and characterisation of Soc2, arfGAP domains of Soc2 are necessary spindle MTs, and no specific l o c a l i s a- to suppress thiabendazole sensitivity of spindle MTs, is implicated in tion is evident during interphase, hence a novel regulator of cdc13-117 cells and viability of Asoc2 anchoring of spindle MTs to we named it Msd1p (Mitotic Specific microtubule stability cells. Notably, the soc2 gene was SPB and proper chromosome Dot). The msd1 deletion was viable but previously identified as a multicopy resulted in minichromosome loss phe- suppressor of the cold sensitivity of segregation Nadeem Shaikh, Steven Renwick, notype. In addition, msd1 mutants Vicky Buck, and Jonathan B.A. Millar cdc2-r4 cells (GeneDB, Sanger Centre), showed abnormal spindle MTs that although we have been unable to Mika Toya and Takashi Toda slipped through SPB(s) in ~50% of Division of Yeast Genetics, National repeat this observation. Instead we find mitotic cells. The ends of slipped- Institute for Medical Research, The that soc2 additionally suppresses the Laboratory of Cell Regulation, Cancer through spindle MTs were less dynamic Ridgeway, Mill Hill, London NW7 1AA, sensitivity of Amal3 cells to Research UK, London Research and Alp4p, a component of gamma- UK thiabendazole, suggesting that Soc2 is Institute, 44 Lincoln’s Inn Fields, tubulin complex, was observed at these a general regulator of microtubule London, WC2A 3PX, UK sites, suggesting that they are minus To identify novel regulators of spindle stability. However live analysis of ends of MTs. DAPI staining showed microtubule stability we isolated soc2-gfp cells revealed that Soc2 does Accurate transmission of chromosomes that DNA was unequally segregated in multicopy suppressors of the sensitivity not bind microtubules but appears during mitosis is critical for maintaining a part of these cells. A potential role of of cdc13-117 (cyclin B) cells to as multiple rapidly moving dots genome integrity. In fission yeast, the Msd1p in anchoring spindle MTs to thiabendazole, a microtubule poison. underneath the plasmamembrane spindle pole body (SPB) plays pivotal SPBs and its implication in proper We identified five distinct genes which concentrate at the cell tips and roles in formation of proper bipolar chromosome segregation will be dis- including cdc13+ (5 clones), a truncated division septa. We are currently spindle, thereby ensuring accurate cussed. allele of dis2+ type 1 phosphatase exploring the possibility that Soc2 chromosome segregation. We cloned a (8 clones), a small heat stable inhibitor may act as a GAP for multiple ARFs gene which encodes a novel coiled-coil of type 1 phosphatase (1 clone) and a (ADP-ribosylation factor) including Arf1 protein during the screening to find truncated allele of dam1, which and Arf2, which are involved in vesicle multi-copy suppressors for the alpha- encodes a component of the trafficking, and Alp41, which is required heterodecameric DASH complex that for `-tubulin biogenesis. links the +TIPS of spindle microtubules to kinetochores. In addition we identified soc2 (7 clones), which encodes an essential protein that contains a UBA (Ubiquitin associated) and an arfGAP (GTPase activating

P100 P101 Genetic analysis of the cut12 Cut12 enables cdc25.22 cells to divide. The Mitotic Role and spindle assembly and chromosome gene highlights roles for RNA The most attractive hypothesis to Regulation of Kinesin-8 Klp5 segregation. explain these observations is that the metabolism in fission yeast association between Plo1 and Cut12 and Klp6 in Fission Yeast Klp5 and Klp6 are two such mitotic cell cycle control on the SPB plays a critical role in the microtubule depolymerising kinesins feedback loop that regulates commit- Amy Unsworth and Takashi Toda (kinesin-8) in fission yeast, which are * Victor A.Tallada, Alan J. Bridge, ment to mitosis. We have used a genet- thought to function together as a Patrick Emery and Iain Hagan ic approach to study Cut12 function in London Research Institute, Cancer heterodimer. They localise to greater detail. We have isolated muta- Research UK, 44 Lincoln’s Inn Fields, cytoplasmic microtubules during Paterson Institute for Cancer Research, tions at five loci which can simultane- London, WC2A 3PX, UK interphase and upon mitotic entry, Wilmslow Road, Manchester M20 4BX, ously suppress the cut12.1 mutation translocate to mitotic spindles and UK. [email protected] (Fax +44 and confer a cold sensitive lethal It is vital for cells to segregate their kinetochores, and then to the spindle 161 446 3109) growth defect. All of the identified DNA equally during mitosis. To do this, midzone during anaphase. We aim to genes are involved in RNA production, chromosomes must be captured by explore the role and regulation of these Commitment to mitosis is regulated by processing or translation. Interestingly, microtubules and bipolar attachment proteins during mitosis. We have done MPF. MPF is inhibited by phosphoryla- most of the mutants show cell cycle established. Chromosomes then live microscopy analysis in deletion tion of p34cdc2 by protein kinases related defects, including monopolar spindle congress at the cell equator before mutants and found that chromosome to Wee1. Removal of this phosphate by phenotypes that are strikingly reminis- being segregated to opposite poles. congression prior to segregation does Cdc25 promotes entry into mitosis. cent of the cut12.1 defect, defective These chromosome motions are not occur. We are also studying the Full-scale commitment to mitosis spindle pole structure, altered inter- powered by microtubule dynamics and significance of heterodimerisation of involves the promotion of Cdc25 activi- phase microtubules arrays and nuclear also by motor proteins such as kinesin Klp5 and Klp6 and are analysing ty and down regulation of Wee1 in a morphology defects. These findings and dynein. There are, however, the behaviour of ATPase positive feedback loop that is triggered suggest that enhanced production of intriguing motor proteins belonging to “rigor” mutants. by active MPF and involves the protein cell cycle regulators may underlie the the Kinesin-8, -13 or –14 families that kinase polo. Fission yeast cdc25.22 suppression of cut12.1 by these muta- influence microtubule dynamics by mutants can divide if cdc2, wee1 or tions. Consistently we have found that depolymerisng microtubules. Most of cut12 are also mutated. Cut12 is an elevating the levels of Cdc25 with a those characterised play roles in essential spindle pole body (SPB) com- mutant that lacks sequences in the ponent that associates with the fission 5’UTR, that link Cdc25 production to yeast polo kinase Plo1. Recessive translation competence of the cell, sup- cut12 mutants block spindle formation presses cut12.1. while the dominant cut12.s11 (stf1.1) mutant permits division of cells which lack Cdc25 and promotes the prema- ture association of Plo1 with the SPB and activation of its kinase activity. The recruitment of Plo1 to the interphase SPB, and the suppression of cdc25.22 by cut12.s11 is dependent upon the function of Plo1 itself. Activating Plo1 activity independently of the status of

P102 P103 Regulation of the Pic1/INCENP and Ark1/AuroraB. Pre-anaphase Spindle We observed mitosis in wild type cells Chromosomal Passenger Consistent with this, co-immunoprecip- Positioning expressing GFP-`2-tubulin and meas- itation experiments reveal that the ured the spindle length and angle in proteins by Shugoshin2 in Bir1/Pic1 complex, but not the three dimensions. The mitotic phases fission yeast mitosis Ark1/Pic1 complex, is destabilised in Sven K. Vogel, Isabel Raabe were determined by examine the spin- the absence of Sgo2. This suggests and Iva Tolic-Norrelykke dle elongation rates. We show that the Vincent Vanoosthuyse, that there are multiple chromosomal spindle gets aligned already in pre- Sergey Prykhozhij passenger complexes, subject to dis- Max Planck Institute of Molecular Cell anaphase: The angle at the beginning and Kevin G. Hardwick tinct regulation. Interestingly, the Biology and Genetics (MPI-CBG), of phase II was 37.6 ± 2.2 (mean ± absence of Sgo2 has only a minor Pfotenhauerstrasse 108, 01307 SEM, n=46) which represents a mis- Dresden, Germany. Wellcome Trust Centre for Cell Biology, effect on chromosome segregation in a aligned spindle (by definition >30 University of Edinburgh, Edinburgh normal unperturbed mitosis but very degrees), whereas by the end of phase Correct positioning of the mitotic spin- EH9 3JR, United Kingdom significantly increases the rate of chro- II the angle decreased to 27.4 ± 1.7 mosome loss and chromosome attach- dle in eukaryotic cells is necessary for (mean ± SEM, n=46) which represents an adequate separation of sister chro- Fission yeast has two members of the ment defects (syntely, merotely) follow- an aligned spindle. matids before cytokinesis. It also Shugoshin family, Sgo1 and Sgo2. ing the release from a spindle check- Previous studies have shown that dur- directs symmetric versus asymmetric Although Sgo1 has clearly been estab- point arrest. This suggests that Sgo2 is ing pre-anaphase intra-nuclear astral cell division and thereby influences cell- lished as a protector of centromere an important factor for the successful microtubules nucleate from the spindle fate choice in many developing cells. It cohesion in meiosis I, the roles of Sgo2 recovery from a spindle checkpoint pole bodies. We hypothesize that those is therefore of fundamental interest to remain elusive. Here we show that arrest. We also show that Sgo2 and the astral microtubules may contribute to understand the mechanisms underlying Sgo2 contributes to proper chromo- CPC localise to telomeres. Interestingly the spindle alignment. Alternatively, the positioning of the mitotic spindle. some segregation in mitosis by regulat- their telomere localisation is dependent asymmetric properties of the nuclear Schizosaccharomyces pombe is an ing the Chromosomal Passenger on the spindle checkpoint kinase Bub1. envelope might promote directed excellent model system to study spin- Complex (CPC). Interestingly, whilst We discuss the possible roles of Sgo2 movement of the spindle pole bodies dle positioning because of the small Sgo2 has a pronounced effect on the and the CPC on telomeres at the and hence align the spindle. We are number of microtubules and the repro- localisation of Bir1/Survivin on chromo- metaphase to anaphase transition. currently examining these hypotheses. ducible and highly symmetrical cylindri- somes in early mitosis, it has only a cal cell shape. The cell geometry itself milder effect on the localisation of the most likely contributes to the spindle two other known CPC components alignment during anaphase B when the spindle length exceeds the width of the cell. In order to examine positioning of the spindle that is not confined by the cell geometry, we focused on the spin- dle alignment during pro-metaphase (phase II).

P104 P105 DNA Metabolism TRAX (spTRAX). First, we cloned and Analysis of GINS function in judged by flow cytometry. Inactivation expressed the two yeast proteins in the fission yeast cell cycle of Psf1 and Psf2 in G2 allows normal E. coli. We have shown that, like the execution of mitosis and cytokinesis, Characterization of the previously characterized human followed by arrest in the subsequent Chen Chun Pai1, Ignacio Garcia S. pombe homologues of the Translin, the spTranslin is an octamer of 2 1 S phase. In addition, we also found that Llorente , Xiaowen Yang , Stuart identical subunits that specifically binds 2 1 inactivation of Psf1 in meiosis causes human proteins Translin d(GT)n repeats, though at a lower MacNeill and Stephen Kearsey arrest in pre-meiosis S phase, and TRAX affinity. However, unlike the human 1 suggesting that GINS is also required Translin, spTranslin binds the Department of Zoology, University of for the meiotic cell cycle. Ron Ben Yosef, Orly Laufman corresponding RNA transcripts, (GU)n, Oxford, South Parks Road, Oxford, Levels of the Psf2 subunit of GINS are OX1 3PS, UK and Haim Manor with a much higher affinity. By using the 2 constant during the cell cycle and yeast two-hybrid technique, we showed Institute of Molecular Biology and during G1 arrest by nitrogen starvation. Department of Biology, that the spTranslin and the spTRAX Physiology, University of Copenhagen, Using an in situ chromatin binding Technion-Israel Institute of Technology, specifically interact. We also deleted Sølvgade 83H, 1307 Copenhagen K, assay, we have shown that the binding Haifa 32,000, Israel the Translin and/or TRAX ORFs in Denmark of Psf2 to chromatin is periodic in S. pombe and found that these genes fission yeast and occurs during Translin is a single-stranded DNA and are non-essential. Currently, we are Studies in X e n o p u s and S a c c h a ro m y c e s S phase. To investigate the relevance of RNA binding protein. It is highly searching for proteins that may form cerevisiae have shown that the GINS GINS function to the chromatin conserved in evolution and homologues specific complexes with spTranslin complex plays an essential role in the association of other replication are found in various mammals, aves, and/or spTRAX in the yeast cells. initiation and elongation stages of DNA proteins, we are examining how the insects, plants and in S. pombe, For this purpose, we constructed replication. To analyze this Sld5-Psf1- chromatin binding of DNA polymerase but not in S. cerevisiae. A Translin S. pombe strains in which the Psf2-Psf3 tetramer in alpha and Cdc45 is affected by homologue designated TRAX, which spTranslin ORF was fused to the Schizosaccharomyces pombe, we have Psf1 inactivation. specifically interacts with Translin but Tandem Affinity Purification (TAP) tag constructed conditional Psf1 and Psf2 does not bind DNA or RNA, is also either through its N terminus, mutants where the proteins are fused to found in all these organisms. or through its C terminus. We are an estradiol-binding domain. On estra- The human and the mouse Translins purifying native complexes including diol depletion, both of these strains selectively bind single stranded G rich the tagged spTranslin from these cells show a DNA replication arrest as DNA and RNA sequences, including by affinity chromatography. SDS-PAGE d(GT)n microsatellites, d(TTAGGG)n of these complexes has already telomeric repeats and certain types of revealed the presence of other proteins mRNAs. Based primarily on their in these complexes. The identity of nucleic acid binding characteristics, these proteins will be determined by various functions have been suggested mass spectrometry. We are also for Translin and TRAX. However, constructing similar strains expressing a definition of their functions in precise TAP-tagged spTRAX and will use molecular terms is lacking. We rea- affinity purification for identification of soned that S. pombe would be most proteins that are specifically associated suitable for such functional analysis with the spTRAX. and therefore initiated a study of the S. pombe Translin (spTranslin) and

P106 P107 Dual roles for Homologous translocations involving a homologous Cellular response to UVA damage DNA as well as lipids and recombination in bre a k - i n d u c e d chromosome which resulted from both radiation in fission yeast proteins. Indeed, UVA radiation are allelic crossovers and break-induced known to induce oxidative stress loss of Heterozygosity in replication. Non-reciprocal response pathways and formation of fission yeast translocations were found to require the Delphine Dardalhon, Anne Reynaud, low levels of oxidized bases (mainly 8- homologous recombination (HR) genes Eveline Sage and Stefania oxoguanine) and dipyrimidine photo- rhp51+ (RAD51Sc), rad22+ (RAD52Sc), Francesconi products. While an effort has been Jason Cullen, Sharon Hussey, Carol + Sc + Sc Walker, John Prudden, Boon-Yu Wee, rhp55 (RAD55 ) rhp54 (RAD54 ) and done to understand the role of UVA + (CNRS/ Institut Curie, UMR 2027, Anoushka Davé and Tim Humphrey mus81 , in addition to the MRN radiation in DNA damage and component nbs1+. Surprisingly, break- Centre Universitaire d’Orsay, 91405 cancerogenesis, very little is known on Orsay, France) MRC Radiation and Genome Stability induced LOH resulted predominantly the effects of UVA-induced stress from de novo telomere addition at the response and on the impact of UVA Unit, Harwell, Didcot Oxfordshire, Ultraviolet (UV) radiation comprises OX11 0RD, UK. break-site in these HR-defective radiation on cell cycle progression. strains, although at reduced levels approximately 5.4 % of total solar Thus, we decided to study the cellular when HR was disrupted at later stages. spectrum and, within the UV spectrum, response to UVA radiation in S.pombe Loss of heterozygosity (LOH) is the only UVA (320-400 nm) and UVB most common form of genome As levels of de novo telomere addition and we will present data on the effects were reduced in rad50A, rad50S or wavelengths (280-320 nm) reach Earth’ of UVA on cell cycle progression. instability in cancer cells. Despite its surface. UVA itself comprises more potential initiating role in tumorigenesis, exo1A backgrounds, in which end processing is compromised, these than 90 % of total solar UV radiation. the mechanisms by which such genetic While UVB radiation directly damage alterations arise are poorly understood. results together suggest that de novo telomeres are added preferentially to DNA by inducing formation of We have investigated whether DNA dipyrimidine photoproducts, UVA double-strand breaks (DSBs) can cause resected ends. HR therefore performs dual roles in both facilitating radiations mainly generate reactive LOH by screening for auxotrophic oxygen species (ROS) that may marker loss ~30kb away from an HO- LOH through the generation of endonuclease break-site within a non- non-reciprocal translocations and in essential minichromosome in preventing LOH, possibly through Schizosaccharomyces pombe. competition with telomerase Extensive break-induced LOH was recruitment factors for resected observed in ~2% of cells and was DSB. associated with large non-reciprocal

P108 P109 Multiple distinct mechanisms mutants results in similar LOH levels Capture of extranuclear DNA evidence suggest that DSB regulate de novo telomere but mainly through de novo telomere at fission yeast double-strand repair-driven chromosomal integration addition at the break-site, indicating a of mtDNA may not exclusively occur addition at DNA double-strand key role for HR in preventing de novo breaks under experimental conditions. Firstly, breaks in fission yeast telomere addition at DSBs. recent studies reported the association We further find that disrupting Rqh1, Anabelle Decottignies of human genetic diseases with de Anoushka Davé, Boon-Yu Wee, the S. pombe homologue of the human novo insertions of mtDNA in the nuclear Jason Cullen, Carol Walker, John tumour suppressor, BLM, in an Catholic University of Louvain, Faculty genome including a patient exposed to Prudden and Tim Humphrey HR-deficient background leads to a of Medicine, Cellular Genetics. 74+3, Chernobyl radiations. Secondly, dramatic increase in LOH levels Avenue Hippocrate, 1200 Brussels, systematic sequencing of nuclear MRC Radiation and Genome Stability compared to the corresponding single BELGIUM. genomes from budding yeast, human Unit, Harwell, Didcot, Oxfordshire, mutants, predominantly through de [email protected] and various plant species revealed that OX11 0RD, UK. novo telomere addition. Moreover, integration of mtDNA fragments truncating the minichromosome arm by Proper repair of DNA double-strand occurred during evolution and is Loss of heterozygosity (LOH) is a adding a telomere directly distal to the breaks (DSBs) is necessary for the probably an ongoing process. common form of genetic instability in break-site in HR-deficient strains maintenance of genomic integrity. Accordingly, this study unraveled the cancer cells that is thought to promote results in a striking increase in de novo A new simple assay was used to study presence of mtDNA sequences in the tumorigenesis through functional telomere addition at the break-site. extrachromosomal DSB repair in fission yeast nuclear genome. inactivation of tumour suppressor A further increase in de novo telomere Schizosaccharomyces pombe. In the second part of the work, genes. However, the mechanisms addition was observed when Rqh1 was Strikingly, DSB repair was associated co-transformation experiments revealed leading to LOH and how they are sup- also deleted in this context. We have with the capture of endogenous fission that microsatellite DNA from higher pressed in normal cells are poorly thus identified three distinct factors – yeast mitochondrial DNA (mtDNA) at eukaryotes competed with fission yeast understood. We have investigated disruption of HR, deletion of Rqh1 and high frequency. Capture of mtDNA mtDNA for insertion at DSBs. mechanisms of LOH distal to an location of the break-site proximal to a fragments required the Lig4p/Pku70p Finally, this study revealed that, in HO-endonuclease-induced DNA telomere, which when combined, result non-homologous end-joining (NHEJ) NHEJ-deficient cells, capture of double-strand break (DSB) within a in remarkably high levels of break- machinery. The Mre11 complex extranuclear DNA at DSBs was non-essential minichromosome, Ch16, in induced LOH through de novo telomere Rad32p/Rad50p/Nbs1p was also observed if homologies -as short as 8 Schizosaccharomyces pombe. We find addition. These data identify multiple required for efficient capture of mtDNA, bp- were present between DNA that break-induced LOH in a wild-type distinct mechanisms which function to supporting a role for the complex in substrate and DSB ends. Hence, background is infrequent (~2%) and regulate levels of de novo telomere promoting intermolecular ligation. whether driven by NHEJ, results from homologous recombination addition at a break-site in fission yeast MtDNA capture frequency was highly microhomology-mediated end-joining (HR)-dependent allelic crossovers and and together are likely to play an increased in fission yeast cells grown to or homologous recombination, DNA break-induced replication (BIR). important role in maintaining genome stationary phase, a phenomenon capture associated with DSB repair is In contrast, DSB induction in HR stability in eukaryotes. requiring vacuolar carboxypeptidase a mutagenic process threatening activity. Further genetic requirements genomic stability. for mtDNA fragment production and transfer to the nucleus are currently under investigation. Several pieces of

P110 P111 Regulation of DSC1 by (DNA synthesis control), the The fate of dysfunctional Our studies have shown that Taz1 The Replication Checkpoint transcriptional factor complex telomeres through the (orthologue of the human telomere responsible for normal S-phase binding proteins TRF1 and TRF2) is transcription. cell cycle central to telomere protection in fission Chaitali Dutta, Prasanta Patel, yeast. Surprisingly, cells lacking Taz1 Anna Oliva*, Janet Leatherwood* In order to confirm our suspicion that Miguel Godinho Ferreira* are as viable as wild type cells in and Nicholas Rhind the checkpoint controls the entire and Julia Promisel Cooper optimal growth conditions. However, S-phase transcription machinery we in G1-arrested taz1- cells, University of Massachusetts Medical analyzed the global transcriptional Telomere Biology Laboratory, 44 chromosomes undergo lethal end-to- School, USA induction in response to HU by gene Lincoln's Inn Fields, Cancer Research end fusions via non-homologous end- *State University of New York, array in collaboration with Dr. Janet UK, London WC2A 3PX, joining (NHEJ). In dividing taz1- cells, Stony Brook, USA Leatherwood (Stony Brook NY). United Kingdom. which spend the majority of the cell We find that the activated checkpoint *Present address: Telomere and cycle in S and G2 phases, Transcriptional regulation in response to affects all S-phase transcripts and this Genomic Stability Laboratory, Instituto chromosome ends undergo replication arrest is important for cell response is Cds1 dependent. Gulbenkian de Ciência, Lisboa, homologous recombination (HR) that survival and genomic stability. However Furthermore, our data suggests that Portugal. protects dysfunctional telomeres from the mechanism by which checkpoint checkpoint has no effect of S-phase end-fusions. The profile of DNA repair regulates transcription is not well transcription when DSC1 activity is Telomeres, the natural chromosome at taz1- telomeres reflects the reciprocal understood. The exception is in compromised, corroborating the ends of eukaryotes, have unique regulation of NHEJ and HR through the Saccharomyces cerevisiae, where a hypothesis that DSC1 is the properties that distinguish them from cell cycle; so that NHEJ is higher in G1 checkpoint specific transcription factor checkpoint target. damage-induced DNA ends. Most than in other stages of the cell cycle up-regulates the enzymes that are human somatic cells lack telomerase, and, conversely, HR repair is higher in needed to produce more Currently, our study is focused on the enzyme responsible for generating G2 than in G1. deoxynucleotides. This transcriptional regulation of DSC1 activity by the telomeres. Proliferation of cells lacking In conclusion, Taz1 protects chromo- strategy does not appear to be checkpoint. Studies have shown that telomerase leads to telomere depletion some-ends from being recognized as conserved. We are using C-terminal 61 amino acids of Cdc10, and ultimately to telomere loss. When deleterious breaks and used as Schizosaccharomyces pombe to study a major component of the DSC1, are telomere function is lost, chromosome substrates of DNA repair. The outcome checkpoint dependent transcriptional crucial for its regulation. We have ends are treated as DNA breaks that, in of these processes at telomeres varies regulation. shown that bacterially expressed most cases, leads to cell death. If the greatly through the cell cycle, leading to GST-tagged Cdc10 is phosphorylated cell survives, chromosomes will be chromosome-end fusions and lethality In fission yeast the checkpoint not only in vitro by Cds1 and have mapped the joined by their ends yielding dicentric in G1 and chromosomal rearrange- up-regulates the genes that are needed phosphorylation of Cdc10 to C-terminal entities that can break upon mitosis. ments in G2. We propose to investigate to produce more deoxynucleotides but serines. We are currently investigating This, in turn, causes unequal the mechanisms underlying also up-regulates many genes normally the checkpoint dependent in vivo distribution of genetic information to chromosome-end protection and to expressed during S phase with no phosphorylation of Cdc10 by western daughter cells as well as to the understand the outcomes of its failure. obvious checkpoint function. This blotting and isoelectric focusing. formation of new unprotected ends. observation suggests the checkpoint In addition we are examining the The ensuing genomic instability is regulates the entire S-phase requirement of in vivo Cdc10 thought to be involved in the transcription program. We hypothesize phosphorylation in checkpoint development of cancer. that in response to HU, the checkpoint response. kinase Cds1 maintains high level of S phase transcripts by regulating DSC1

P112 P113 Stalled replication forks absence of DNA replication checkpoint. The role of Rad9 We focused on this Thr. 225 because process in absence of Cds1 We observed that the recombination phosphorylation in recovery the phosphorylation become protein Rad22 is recruited on early and dependent on Rad9-Cut5/Rad4 late replication origins in response to from stressed replication fork complex only at replication fork. B. Froget, S. Lambert, P. Meister, HU treatment in cds1 deleted strain. in fission yeast The mutant which Thr225 is no longer and G. Baldacci Moreover, replication factories phosphorylated rescues post replicative containing PCNA foci and Pol` foci are Kanji Furuya, Francesca Paderi, repair (PRR) pathway mutant rhp18-d. UMR2027, Institut Curie, Orsay less abundant after HU treatment in Antony Carr Although the mutant showed a weak University, France. cds1 deleted cells compare to wild type Camptothecin sensitivity, it showed strain. These results suggest that in DNA checkpoint is the one of the hyper recombination phenotype and During replication, cells have to absence of DNA replication checkpoint, mechanism to prevent the genomic hyper-mutator phenotype, and showed coordinate DNA synthesis with cell a replication complex destabilization instability, and the defect causes cell fragmented chromosomes after cycle progression, checkpoints and occurs at stalled forks, which could be death, scenescence, neural camptothecin treatment. Surprisingly, recombination. A defect in these targeted by nucleases. In line with this, degeneration or tumorigenesis. DNA the threonine 225 mutant failed to processes may result in genetic using PFGE analysis, we observed that replication is the one of the event which activate chk1 upon replicative stress, instability. Hydroxyurea (HU) treatment cds1 deleted cells experience DNA causes genomic instability when things but manage to survive by activating slows down replication forks fragmentation in response to HU go wrong and DNA checkpoint pathway Mad2 checkpoint which probably is progression by deregulating the treatment. Moreover, this DNA is shown to monitor the process mainly activated by abnormal chromosome nucleotide pool, and results in DNA fragmentation depends on the by stabilising the replication fork structure. These phenotype is seen only replication checkpoint activation, endonuclease Mus81 and is structure. under replicative DNA damage but not mediated by the Cds1 kinase, which independent of Rhp51. Altogether, on G2 DNA damage. We hypothesize maintains replication forks integrity. In our data are consistent with a role for Here we focus on the Rad9 protein and Rad9 phosphorylation somehow absence of this checkpoint (cds1-d Cds1 in replication forks integrity show its phosphorylation contribute to modulate the way the replication fork is strain), we have previously shown that protection by preventing 1 the replication fork stability possibly by repaired thus ensures proper activation in response to HU treatment : 1) cells aberrant stalled forks processing by modulating post-replicative repair path- of PRR and replication checkpoint are unable to complete DNA DNA repair enzymes leading to way. Rad9, which forms PCNA-like pathway and thus, we are currently replication, 2) aberrant DNA structures unscheduled recombination events. trying to see the difference in stalled accumulate at collapsed replication structure together with Hus1 and Rad1, is phosphorylated on Thr225 (inside replication fork between mutant and forks and 3) the formation of aberrant 1. Meister P, Taddei A, Vernis L, Poidevin M, wild type. DNA structures depends on Gasser SM, Baldacci G. Temporal PCNA domain) by Rad3 kinase. homologous recombination. These separation of replication and results suggested that Cds1 prevents recombination requires the intra-S unscheduled recombination events at checkpoint. J Cell Biol. HU-induced stalled replication forks. 2005 Feb 14;168(4):537-44. In this context, we used Chromatin Immunoprecipitation (ChIP) to determine whether recombination proteins are recruited at stalled forks in

P114 P115 Rad4/Cut5 interact with events are dependent upon the Rad3 A novel Double Strand with an HO recognition site and the Cdc2/Cdc13 in response to kinase. We observed that the Break system in hygromycin resistance gene. The two phosphorylation of Rad4 is also halves of the LEU2 gene contain 500 replication stress partially dependent upon the Cdc2 Schizosaccharomyces pombe, bp of homology between them which and DNA damage kinase. Cdc2 is the sole cyclin- specific for Single Strand can utilised for SSA. The HO dependant kinase in S.pombe and has Annealing endonuclease is expressed from a Valerie Garcia and Antony Carr been found to activate recombination plasmid under the control of the pathways (Caspari et al, 2002). Stephen Gill, Petra Werler, Anthony inducible nmt promoter. Once Fission yeast Cut5/Rad4 and its We have shown that Rad4 is M Carr and Johanne M Murray expressed, the HO endonuclease budding Yeast, Xenopus and Human phosphorylated in vitro by Cdc2, generates a single DSB within the ura4 homologues are required for both the and identified phosphorylation sites by Genome Damage & Stability Centre, gene. Following break formation and DNA replication process and for mass-spectrometry. The function of University of Sussex, BN1 9RQ, UK strand resection, SSA is used to repair S phase checkpoints. Cut5/Rad4 is an these sites in vivo is under investigation the break, leading to the formation of a essential protein containing 4 BRCT and will be presented. Furthermore, The repair of double strand breaks fully functional LEU2 gene. Thus cells domains which are common to many Cdc13 was identified as a Rad4 partner (DSBs) is essential for genomic stability switch from ura4+/LEU2- to ura4- DNA repair/checkpoint proteins. in TAP purification of Rad4 complex and cell survival. Experimentally, DSBs /LEU2+. Although Rad4 interacts with several from extracts prepared from HU treated can be introduced non-specifically proteins implicated in DNA replication cells. This HU induced interaction was through exposure to irradiation and The requirement for proteins known to and checkpoint functions (Drc1, Rad9, confimed by co-immunoprecipitation. genotoxic chemicals, whereas be involved in SSA in S. cerevisiae was Crb2 and Chk1), its molecular function Altogether, these results position the utilisation of an endonucleases allows investigated using this system. While is still not understood. We show that Rad4-Cdc13/Cdc2 complex as a key introduction of DSBs at specific sites. broadly in agreement with data from Rad4 is phosphorylated after treatment intermediate in the stabilisation or In Saccharomyces cerevisiae a number S. cerevisiae (Rad22 (RAD52) is of cells with HU or DNA damaging processing of stalled replication forks. of assays have been developed which required but Rhp51 (RAD51) is not) agents, and these phosphorylation utilise HO endonuclease recognition there were a number of differences. sites to induce a single DSB at a S. pombe Rad16/Swi10, homologues specific location within the genome, of the RAD1/RAD10 flap endonuclease, allowing the kinetics of DSB repair to which is essential for SSA in be studied. However, this technique S. cerevisiae, were shown to be impor- is not widely used in tant, but not essential for SSA in S. Schizosaccharomyces pombe. We have pombe. Interestingly, the checkpoint designed an HO system specifically to protein Rad17, like S. cerevisiae the study the single strand annealing homologue RAD24, was found to be (SSA) pathway, about which little is essential for SSA. Furthermore, we known about in S. pombe. found that, unlike its S. cerevisiae homologue SRS2, Srs2 was not The system comprises two halves of required for checkpoint recovery the LEU2 marker gene, integrated in following DSB induction. chromosome II, separated by non- essential DNA containing the ura4 gene

P116 P117 Bypass of replication arresting the rare disorder Fanconi Anaemia, Smc5/6 complex is required clear. All components are essential and as the human homologue. loss of Smc5/6 complex function DNA lesions: Involvement of - - for recombination repair at + + We constructed both mhp1 and mhp2 results in chromosomal fragmentation S. pombe mhp1 and mhp2 ? mutants (mph1 homologue pombe) in collapsed replication forks during S phase. Hypermorphic mutants S. pombe and have started to are defective in HR repair and Ralph Gruber1, Jonathan Framton3, characterise the phenotypes of both Anja Irmisch, Eleni Ampatzidou segregation of the rDNA (Torres-Rosell Wilfried Kramer2 single and double mutants. We found and Jo Murray et al., 2005; Lehmann et al., 1995). and K. Anke Schürer1 similar phenotypes as shown for mph1 In S. pombe Smc6 is nuclear and from S. cerevisiae supporting the Genome Damage and Stability Centre, predominantly localises to the 1Leibniz Institute for Age Research-Fritz hypothesis of mhp1+ and mhp2+ being University of Sussex, Brighton, nucleolus. However, Chromatin Lipmann Institute, Beutenbergstr. 11, orthologues of bakers yeast Mph1. BN1 9RQ, UK immunoprecipitation (ChIP) experi- D-07745 Jena, Germany, Email: We got evidence for an apparent ments show that it associates with DNA [email protected] separation of function diversification Structural Maintenance of throughout the genome. Epistasis 2University of Göttingen, Institute for during evolution. However, none of the Chromosome (SMC) proteins are highly analysis, ChIP and visualisation of Microbiology and Genetics, Dept. of S. pombe genes was able to conserved proteins involved in higher- replication intermediates by 2-D gels Molecular Genetics and Preparative complement the S. cerevisiae mph1 order chromosome organization. suggest that Smc6 is required at a late Biology, Grisebachstr. 8, D-37077 phenotypes (sensitivity to DNA In eukaryotes they exist in three stage in homologous recombination at Göttingen, Germany damaging agents and spontaneous conserved multi-subunit protein collapsed replication forks. The role of 3Genome Damage and Stability Centre, mutator phenotype). Further complexes. The Smc1/3 complex the Smc6 complex in regulation of University of Sussex, Falmer, Brighton characterisations including genetic (cohesin) mediates sister chromatid recombination at stalled forks will BN1 9RQ, Great Britain interaction studies with genes from cohesion, while the Smc2/4 complex be discussed. post-replicative repair, from (condensin) facilitates chromosome In S. pombe two sequence homologues homologous recombination and other condensation at mitosis (Hirano, 2002). Hirano, T. Genes Dev 16, 399-414 (2002). of S. cerevisiae MPH1, mhp1+ and genes involved in reinitiation of stalled The function of the third complex, Lehmann, A. R. et al. Mol Cell Biol 15, 7067- mhp2+, can be identified. replication forks are presently in which comprises Smc5, Smc6, and 80 (1995). S. cerevisiae MPH1 (mutator progress. These analysis may also four stochiometric non-Smc core Torres-Rosell, J. et al. Nat Cell Biol 7, 412-9 (2005). phenotype) constitutes a new pathway open new perspectives on the complex components (Nse1-4), is less for error-free bypass of DNA lesions function of FancM, the human during replication that requires MPH1/mhp1+/mhp2+ homologue. homologous recombination but is distinct from post-replicative repair. Mph1 is conserved in archaea and eukaryotes with FancM, defective in

P118 P119 The role of rrp1 and rrp2 complex that is involved in Rhp51- Premeiotic G1 to S transition chromosome pairing, double-strand genes in the repair of DNA dependent but Rhp55/57 independent is affected by recombination break (DSB) formation, and repair of recombination repair and it is DSBs, showed that the start of damage in suggested that this complex is a proteins, and mutual inhibition premeiotic S phase (and of subsequent Schizosaccharomyces pombe second mediator for Rhp51 is observed for two events) is advanced for up to two hours recombinase. Here, we report an recombination hotspots in several mutants (eg rec11, rec12, Pawel Karpinski*, analysis of a novel DNA repair protein, rec14), like wild-type in a few mutants Rrp1 that has SNF2N, zf-C3HC4 and (mde2, dmc1), and delayed in one case Agnieszka Dyjankiewicz Jürg Kohli, Eveline Doll, Monika and Dorota Dziadkowiec helicases C domains. The strain devoid (rec8). A preliminary model on the of the rrp1 gene is slightly sensitive to Molnar, Benjamin Sakem, regulation of the preimeiotic G1 to Emil Parvanov Institute of Genetics and Microbiology, high doses of UV, but not to MMS. S transition will be presented. Arrp1Arhp51 double mutant shows the Wroclaw University, Przybyszewskiego Institute of Cell Biology, 63/77, 51148 Wroclaw, Poland. same UV sensitivity as a Arhp51 single The meiotic recombination hotspot at mutant, indicating that Rrp1 functions University of Berne, Baltzer-Strasse 4, ura4A was correlated with a double- * present address: Department of Genetics, in the Rhp51-dependent repair process CH-3012 Berne, Switzerland strand break (DSB) 500 bp upstream of Wroclaw Medical University, for the UV-induced DNA damage. e-mail: [email protected] the gene. The dependence of ura4A Marcinkowskiego 1, 50368 Wroclaw, While a Arrp1 mutant is less sensitive hotspot activity and DSB formation on Poland to UV than a Asfr1 mutant, the Three types of fission yeast meiosis are proteins modifiying chromatin structure Arrp1Asfr1 double mutant shows the used for experimental analysis. The and enhancing the M26 hotspot was Homologous recombination is an A shortest and most synchronous meiosis important process assisting in the same sensitivity as sfr1 single mutant. investigated. In particular, it was found On the other hand, rrp1 mutation has is induced by temperature shift of that ura4A hotspot and DSB was not repair of DNA damage in all studied an additive effect on DNA repair pheno- strains carrying the pat1-114 mutation. dependent on intact Pcr1, which is organisms. In Schizosaccharomyces A A longer prophase and poorer pombe a Rhp51 recombinase type of a rhp57 mutant. These results required for hotspot activity at ade6- indicate that Rrp1 functions synchrony are displayed by the M26. Nevertheless, recombination (functionally homologous to RecA + - independently of Rhp55/57 and azygotic h /h meiosis of diploids analysis indicated that the two hotspots protein in E.coli and Rad51 in induced by nitrogen starvation. S.cerevisiae) and mediators, Rhp55 and possibly takes part in a sub-pathway of (distance 15 kb) mutually inhibit each Sfr1-dependent recombiantion repair. The natural zygotic meiosis used for other to a significant extent for Rhp57 (Rad55 and Rad57 in The phenotypes of a deletion mutant of recombination analysis in crosses of inter- and intragenic recombination. S.cerevisiae), take part in homologous haploid strains is not suitable for the pairing and strand exchange, essential a rrp1 paralogue, rrp2, are also In addition the hotpots showed compe- investigated in order to determine if study of the timing of prophase events. tition (deficit of double events at both to the homologous recombination this gene is involved in DNA A systematic analysis of strains hotspots) which was a minor part of repair process. It was previously shown replication/repair. carrying mutations in genes that are the overall reduction. that Sfr1 and Swi5 form a protein coding for proteins involved in meiotic sister chromatid cohesion,

P120 P121 Streptomyces 'phage iC31 includes members that promote A bioinformatics approach to (Patel et al, 2006). Due to this and iBT1 integrases in fission unidirectional reactions between two predict origin efficiencies as a stochasticity as well as the limitations different attachment sites of about of the experimental techniques used, it yeast 40bp in the absence of additional function of the genomic is strongly speculated that more origins proteins and are thus potentially ideal context exist. Observed efficiencies are a Nicholas CO Lee for exactly the type of genome function of the intrinsic probability of a and William RA Brown manipulation to which Cre and Flp are Legouras Ioannis1, given region of the genome to act as an unsuited. The Streptomyces 'phage Kouretas Panagiotis2, Nurse Paul3, origin and by the rate at which it is Institute of Genetics, Queens Medical iC31 and iBT1 integrases are Lygeros John2, Lygerou Zoi1 converted to the post-replicative state Centre, Nottingham University, examples of such proteins. We have by passive replication from nearby NG7 2UH, UK shown that these integrases promote 1Departement of Medicine, origins. To detect a possible greater both excision and insertion reactions in University of Patras, Greece number of origins than the ones cur- Site specific recombinases belong to fission yeast but the reactions do not 2Depertment of Electrical Engineering, rently mapped and to define intrinsic two main families; the tyrosine and go to completion. Incomplete reaction University of Patras, Greece firing efficiencies of known origins, serine recombinases. The tyrosine due to attachment site damage is a 3Rockefeller University, New York, we are implementing a bioinformatics recombinase family include the feature of the reactions promoted by NY, USA approach that correlates the following enzymes Cre and Flp which are widely the iC31 integrase in vertebrate cells in a genome-wide level: known origin used in genome manipulation in and probably arises because the Following the publication of the fission data (positions and efficiencies), AT metazoans. These enzymes promote reaction intermediate is recognized as yeast genome (Wood V. et al, 2002), richness and the locations of tran- reversible reactions between identical DNA damage. We are investigating high throughput approaches to scribed regions. This approach will be sites and are thus unsuited to whether this is also true in fission yeast investigate origin function became insightful in terms of the possible promoting insertions, translocations and the nature of the nucleases that are feasible. Recent analyses of DNA effects of AT content and nearby or inversions. We are trying to develop required for the inappropriate repair of replication across the fission yeast transcribed DNA on origin efficiencies. techniques for manipulating the the reaction intermediate. genome have mapped origin positions AT richness is crucial for origin centromeric DNA of fission yeast and estimated their firing efficiencies. establishment, but it remains elusive if in vivo. The serine recombinases family Such analyses have shown that in order it can also determine efficiency. to function, origins must satisfy certain Furthermore disturbance of the criteria; they must have an AT content replication machinery due to the nearby above average (>69%) for a sufficiently transcription machineries has long been large segment of DNA (~1kb). However, considered detrimental to origin not all potential origins will fire in every establishment. Current progress in this cell cycle: origin efficiencies vary from area will be presented with emphasis below 10% (the experimental detection on the various aspects of genomic limit) to around 80% and origin context that allegedly affect intrinsic selection in each individual cell is origin efficiencies, namely AT content, stochastic. The stochastic nature of genes and chromatin structure. origin selection in fission yeast has long been speculated and was recently proved with single molecule analysis

P122 P123 Control of RNR by the Re-localisation of Suc22 does not Interactions of SpMcm10 with Rad4/Cut5. In addition to the physical Signalosome (CSN) and occur in these null mutants. DNA replication factors interactions with Mcm4-6 genetic Upon irradiation of G2 cells, Suc22 is interactions have also been demon- checkpoints also exported from the nucleus to the strated with mcm2 as well as mcm4, cytoplasm in wildtype cells. Again, this Karen Moore, Simone Contini mcm5 and mcm6. Recombinant Cong Liu, Thomas Caspari does not occur in the null mutants. and Stephen J. Aves Mcm10 has single stranded DNA and Antony M. Carr The inability to relocalise Suc22 binding activity in vitro and activates explains the DNA damage sensitive School of Biosciences, University of DNA polymerase ` via the p180 Genome Damage and Stability Centre, phenotypes. Exeter, Exeter EX4 4QD, UK catalytic subunit. In vivo we find no University of Sussex, Falmer Brighton [email protected] genetic or yeast 2-hybrid interactions BN1 9RQ. Deletion of spd1, that encodes a small between Mcm10 and DNA polymerase protein with similarity to S. cerevisiae S. pombe mcm10 (cdc23) encodes an ` catalytic subunit, or either of the The COP9/signalosome complex (CSN) Sml1, results in constitutive essential protein of 593 amino acids primase subunits. Chromatin binding is highly conserved multi component cytoplasmic localisation of Suc22 and that binds to chromatin. Mcm10 assays have shown that SpMcm10 is protein complex with homology to suppresses the slow S phase and homologues are found in all eukaryotes required to load Cdc45 onto chromatin the19S regulatory lid complex of the radiation sensitivity phenotypes of where they function in DNA replication after pre-replicative complex formation. proteosome. The signalosome is csn1, csn2, pcu4, ddb1 and cdt2 but their precise roles remains elusive. In vitro Mcm10 is required to stimulate required for the removal of covalently mutants. Spd1 degradation is ubiquitin- Temperature sensitive mcm10 mutants phosphorylation of Mcm4 by attached Nedd8 from cullins. In S. dependent and CSN-Pcu4-Ddb1-Cdt2 display a typical cell division cycle Dfp1/Hsk1 kinase. In Saccharomyces pombe CSN co-purifies with the Cullin dependent in S phase, but checkpoint arrest phenotype at 36°C with cerevisiae interactions with Dna2, DNA 4 homolog (Pcu4 ), the Ddb1 and Cdt2 pathway-independent. In contrast, elongated cell morphology indicative of polymerase d and PCNA indicate that it (a WD-repeat protein). Null mutations in in response to irradiation of G2 cells, an intact checkpoint. SpMcm10 is is involved in the elongation phase. csn1, csn2, pcu4, ddb1 and cdt2 genes Spd1 degradation is ubiquitin-depend- required for both the initiation and Intriguingly, interactions with a variety in S. pombe are DNA damage sensitive ent, CSN-Pcu4-Ddb1-Cdt2-dependent elongation phases of DNA replication, of other proteins have been reported and exhibit slow DNA replication and checkpoint pathway-dependent. but it is not necessary for pre-replica- including Sir2 and Sir3 suggesting a because of misregulation of RNR. In We have shown that Cdt2 acts as an tive complex formation; it is, however, role in gene silencing. The S. pombe wild type cells in G1, G2 and mitosis adaptor protein, bringing Spd1 to the essential for activation of the replication Mcm10 amino acid sequence contains the small subunit of RNR (Suc22) is CSNPcu4-Ddb1 complex, and that the complex. Consistent with a role at motifs associated with some sequestered in the nucleus, while the key regulatory step in Spd1 initiation SpMcm10 interacts physically bacteriophage primases and this has large subunit (Cdc22) is distributed degradation is the presence of the with four subunits of the origin led to the hypothesis that Mcm10 is a throughout the cell. In S phase, Suc22 Cdt2 adaptor. recognition complex (Orc1, Orc2, Orc5 primase. However, both mcm10 and relocalises to the cytoplasm. and Orc6). We have used yeast 2- spp1 are essential genes and hybrid analysis to demonstrate a over-expression of mcm10 is physical interaction with three subunits insufficient to rescue spp1-4 mutants in of the MCM2-7 replicative helicase which the primase catalytic subunit (Mcm4, Mcm5 and Mcm6), two GINS is defective. subunits (Psf2 and Sld5) and

P124 P125 Aberrant chromosome RuraR system, the viability of the Sap1 associates with (FPC) that plays a central role in the rearrangement induced by palindrome system is dramatically replication origins and is activation of the replication checkpoint reduced when replication fork stalling is and the stabilization of stalled forks. replication fork stalling at a induced. Southern blot analysis and required for the replication To further understand the mechanisms palindrome PFGE show that RFS at the palindrome stress response pathway of the replication checkpoint, we have leads to rapid chromosomal carried out screens to identify genes Ken’ichi Mizuno, Johanne Murray rearrangement and the formation of Chiaki Noguchi and Eishi Noguchi that genetically interact with FPC. Here and Antony Carr dicentric and acentric sister we describe one of FPC interacting chromatids. The cells progress through Department of Biochemistry factors, Sap1, a protein bound to DNA Genome Damage and Stability Centre, G2 and enter mitosis with normal and Molecular Biology, sequences required for switching of University of Sussex, Brighton, kinetics suggesting that the Drexel University College of Medicine, sexual identity in the fission yeast BN1 9RQ, UK. rearrangements occur during S phase Philadelphia, PA 19102 Schizosccharomyces pombe. We show and the products do not activate the that Sap1 is required for replication Genomic rearrangements linked to G2/M DNA damage checkpoint. The To achieve faithful DNA replication, checkpoint signaling. Inactivation of aberrant recombination are associated dicentrics align correctly at metaphase eukaryotic cells are equipped with a Sap1 causes DNA damage represented with cancer and human genetic with bipolar attachment to the spindle dedicated sensor-response system, by Rad22 DNA repair foci during diseases. Such recombination has but are then torn apart at anaphase termed the replication checkpoint. S-phase, indicative of fork damage. indirectly been linked to replication fork leading to a catastrophic mitosis. This checkpoint is activated by stalled A pulsed-field gel electrophoresis stalling. We have previously established The generation of the non-equivalent replication forks and required for analysis shows that the sap1 mutants a system, designated RuraR, to induce sister chromatids is dependent on the accurate transmission of genetic have a defect in resumption of DNA replication fork stalling (RFS) at a RTS1-Rtf1 system and a subset of information. Stalled forks are inherently replication after fork arrest. We also specific site in a controllable manner, recombination and checkpoint proteins. unstable structures that are prone to provide evidence by chromatin using a defined replication fork barrier These results lead us to conclude collapse, regression and recombination, immunoprecipitation that Sap1 sequence RTS1 and trans-acting 1) RFS at the palindrome triggers the posing serious threats to genomic associates with a replication origin. replication fork block protein Rtf1 chromosomal rearrangements through integrity. In humans, defects in this Our blast search reveals that Sap1 is (Lambert et al, 2005). In the RuraR homologous recombination; 2) the checkpoint cause genetic instability, conserved throughout evolution. system, RFS is overcome by generation of non-equivalent sister leading developmental and neurological We propose that Sap1 is required for homologous recombination between chromatids is dependent upon the defects and a predisposition to cancer. the maintenance of replication forks RTS1 sites leading to inversion of the palindromic sequences, which are able Recently, our investigations revealed in eukaryotic cells. intervening ura4 marker and rarely to to self-pair, forming intrastrand hairpin that two proteins, Swi1 and Swi3, form site-specific gross chromosome structures; and 3) the palindrome a replication fork protection complex rearrangements. system provides an inducible system We have constructed a derivative with which to analyse the behaviour palindrome system, Rura-aruR, by of dicentric chromosomes. inserting another copy of the ura4 gene in reverse orientation. In contrast to the

P126 P127 Associates with a replication a replication fork protection complex Checkpoint pathway regulates replication fork. We here show that rad9 origin and is required for the (FPC) that plays a central role in the Post Replicative Repair Thr 225 interacts with rhp18 post activation of the replication checkpoint replication repair pathway (PRR) replication stress response and the stabilization of stalled forks. thought Damage Dependent protein. pathway To further understand the mechanisms Rad9t225 phosphorylation After CPT (campthotecin) treatment, of the replication checkpoint, we have which causes replicative DNA damage, Chiaki Noguchi and Eishi Noguchi carried out screens to identify genes Francesca Paderi we observe that rad9 T225 mutation that genetically interact with FPC. rescues rhp18-d. Synchronous culture Department of Biochemistry Here we describe one of FPC Genome Damage and Stability Centre experiment shows cell cycle delay and Molecular Biology, interacting factors, Sap1, a protein Science Park Road induced by CPT in rhp18-d mutant is Drexel University College of Medicine, bound to DNA sequences required for University of Sussex Falmer, Brighton compromised by rad9 T225 mutation. Philadelphia, PA 19102 switching of sexual identity in the East Sussex BN1 9RQ UK Consistently Chk1 phosphorylation is fission yeast Schizosccharomyces reduced in rhp18-d - rad9 t225 To achieve faithful DNA replication, pombe. We show that Sap1 is required Checkpoint pathways respond to background. eukaryotic cells are equipped with a for replication checkpoint signaling. changes in DNA structure. Its activation Our data suggest that Thr225 residue dedicated sensor-response system, Inactivation of Sap1 causes DNA ensures the integrity of DNA by specifically responds to S phase termed the replication checkpoint. damage represented by Rad22 DNA delaying cell cycle upon DNA damage. damage to generate checkpoint signal. This checkpoint is activated by stalled repair foci during S-phase, indicative of We focus on Thr 225 phosphorylation The function of Thr 225 may involves replication forks and required for fork damage. A pulsed-field gel site on checkpoint protein rad9. DNA processing on damaged accurate transmission of genetic electrophoresis analysis shows that the Thr 225 phosphorylation does not replication fork; the phosphorylation at information. Stalled forks are inherently sap1 mutants have a defect in involve checkpoint activation after Thr 225 site may acts as a coordinator unstable structures that are prone to resumption of DNA replication after fork general DNA damage, but it is for polimerase network at damaged collapse, regression and recombination, arrest. We also provide evidence by suggested that this phosphorylation is replication fork. posing serious threats to genomic chromatin immunoprecipitation that required for the repair at the damaged integrity. In humans, defects in this Sap1 associates with a replication checkpoint cause genetic instability, origin. Our blast search reveals that leading developmental and neurological Sap1 is conserved throughout defects and a predisposition to cancer. evolution. We propose that Sap1 is Recently, our investigations revealed required for the maintenance of that two proteins, Swi1 and Swi3, form replication forks in eukaryotic cells.

P128 P129 Mrn comlex and the spindle and the complex is thought to be Involvement of Cid1 poly(A) disorder Bloom’s syndrome. The rqh2 checkpoint involved in the early stages of DNA polymerase and the sub- gene is thought to be present in four damage repair. In this study, S. pombe + sub-telomeric copies, at each arm of mrn mutants are shown to be sensitive telomeric rqh2 DNA helicase chromosomes I and II; at least one of Hayatu Raji, Edgar Harstuiker to spindle depolymerisation drugs. gene in a common pathway these copies is expressed after 3 When observed under the microscope following inhibition of DNA telomere erosion or replication stress. UK the mutants show defective septum The open reading frame itself contains formation and sister chromatid replication sequences encoding the RecQ helicase Cells have functional checkpoints that mis-segregation after treatment with 1 domain and sequences similar to ensure critical steps are not initiated Olivia S Rissland , Abigail thiabendazole. This indicates that the 1 1 centromeric dh repeats. Unlike rqh1, until prior steps have been accurately Stevenson , Li Phing Liew , Shao-Win cells are unable to arrest at mitosis 2 1 3 the other RecQ helicase family member completed. DNA damage and spindle Wang , Daniel Lackner , Juan Mata , + following thiabendazole treatment. 3 1 in S. pombe, rqh2 is normally silenced defects would go undetected in the The results of this study suggest that Jürg Bähler and Chris J Norbury in sub-telomeric heterochromatin. absence of these checkpoints, which DNA repair proteins are involved in the 1 This silencing occurs by both the RNAi- can lead to cancer and aneuploidy, Sir William Dunn School of Pathology spindle checkpoint in S. pombe. 2 RITS pathway, which is dependent on both of which can be deleterious to the and Department of Zoology, University 3 transcription of rqh2-associated dh survival of the cell. The Mrn complex is of Oxford and Wellcome Trust Sanger sequences1, 2, and an RNAi-independent involved in double strand break repair Institute, Hinxton, UK. pathway, which itself is dependent on 2 + the telomere-associated protein Taz1 . S. pombe cid1 was identified through However, upon expression, rqh2 a screen for S-phase cell cycle 5 transcripts undergo a distinctive pattern checkpoint genes . Surprisingly, the of the 3’ end processing, which Cid1 protein is a cytoplasmic non- 4 suggests that this may be a direct Cid1 canonical poly(A) polymerase . We target. We propose that after replication hypothesized that, upon S phase stress Cid1-mediated cytoplasmic arrest, Cid1 might regulate the poly(A) polyadenylation of rqh2 mRNA occurs tail length of checkpoint gene mRNAs. and allows Rqh2 translation. Rqh2 itself Using microarray hybridisation, we may then stabilise and/or resolve identified putative targets as those stalled DNA replication forks, RNAs whose levels changed upon Cid1 particularly those that are telomere deletion. One of the most striking proximal, and may also maintain the changes occurred in the levels of S-M checkpoint signal. rqh2+/SPAC212.11: these transcripts were dependent on Cid1 cds1A 1. Cam et al. Nat Genet 37, 809-19 mutants arrested in S phase. Rqh2 is (2005). the second member of the RecQ DNA 2. Kanoh et al. Curr Biol 15, 1808-19 (2005). helicase family to be identified in S. Mandell et al. J Biol Chem 280, 5249-57 pombe. RecQ helicases are associated (2005). with several human disorders. For 3. Read et al. Proc Natl Acad Sci USA 99, instance, mutations of the BLM heli- 12079-84 (2002). case, which is the closest relative of 4. Wang et al. Mol Cell Biol 20, 3234-44 (2000). Rqh2, cause the cancer predisposition

P130 P131 The telomere protein Taz1 is efficient replication through internally High-resolution analysis of In budding yeast, the transcription required for semi-conservative positioned telomeric sequences, cohesin association along machinery has been shown to locate regardless of their orientation in relation cohesin to sites between convergently DNA replication through to the direction of replication. fission yeast chromosomes transcribed genes. Likewise, in fission telomeres Therefore, telomere sequences lacking II and III yeast, cohesin peaks are preferentially Taz1 constitute an obstacle to the but less exclusively localised to these Ofer Rog, Kyle Miller replication machinery. Christine Schmidt1, Yuki Katou2, intergenic regions forming cluster-like and Julia Promisel Cooper Despite the impairment of telomere Katsu Shirahige2 and Frank Uhlmann1 arrangements. Less than half the replication in the absence of Taz1, convergence sites are occupied by Telomere Biology Laboratory, Cancer telomeres in taz1A cells are very long, 1Chromosome Segregation Laboratory, cohesin whereas in budding yeast Research UK, 44 Lincoln's Inn Fields, suggesting that in taz1A cells, the very Cancer Research UK, London Research almost all of these sites are used. London WC2 3PX, UK long telomeres are re-synthesized in Institute, Lincoln’s Inn Fields Interestingly, although cohesin peaks every S-phase by telomerase. Laboratory, 44 Lincoln’s Inn Fields, appear less confined to places between Telomeres are bound by a complex of To test this idea, we monitored London WC2A 3PX, United Kingdom convergently transcribed genes they proteins that regulates both telomeres following loss of telomerase 2Center for Biological Resources and seem to be highly restricted to chromosome end-protection and the activity. Strikingly, taz1A cells lose their Informatics, Division of Gene Research, intergenic regions in general. activity of telomerase, which telomeres immediately upon telomerase and Graduate School of Bioscience and We have monitored cohesin dynamics synthesizes the terminal repeats. loss, in contrast to wild type cells, Biotechnology, Tokyo Institute of during mitotic chromosome segregation Here we show that a telomeric DNA which exhibit gradual attrition of Technology, 4259 Nagatsuta, Midori-ku, in a highly synchronous culture binding protein is also required for telomeres. This observation supports Yokohama, 226-8501, Japan released from G2. Surprisingly, the efficient semi-conservative replication the idea that taz1A telomeres break number of cohesin association sites of the bulk of the telomeres, and during S-phase and erode abruptly in After replication, sister chromatids are increases towards anaphase, possibly explore possible causes and the absence of telomerase. held together by the cohesin complex due to the fragmentation of single consequences of fork-stalling We will present our progress in which persists on chromosomes until cohesin peaks observed in G2. at telomeres. understanding why Taz1 is required to mitosis when the dissolution of sister It will be interesting to determine the Fission yeast Taz1, an ortholog of the orchestrate passage of replication forks chromatid cohesion at anaphase-onset sites at which cohesin is loaded onto human telomere binding proteins through telomere sequences, and allows the spindle to pull sister chromosomes. This might help to hTRF1 and hTRF2, binds double- whether specialized helicases are chromatids apart. We have performed explain the cluster-like arrangement of stranded telomeric repeats and required for this process. We will also analysis of cohesin association along cohesin association sites and could regulates diverse telomere functions. address possible genetic interactions fission yeast chromosomes II and III also be a crucial step for identification Using two-dimensional gel elec- linking stalled forks to the chromosome using different cohesin subunit of possible cohesin removal sites trophoresis, we find that loss of Taz1 mis-segregation exhibited by taz1A chromatin immunoprecipitates for during mitosis. results in stalled replication forks both cells at cold temperatures. hybridisation to high-density within and directly adjacent to the oligonucleotide microarrays telomere. Taz1 is also required for (ChIP on chip).

P132 P133 Fission yeast Cid12 has dual Cid12 had an increased chromosome What is the functional form between taz1A telomeres; these functions in chromosome segregation failure rate due to significance of the bouquet fusions are mediated by Lig4-depend- precocious loss of sister chromatid ent nonhomologous end-joining. segregation and checkpoint cohesion at the centromere but not structure? Thus, taz1A mutants have not only control along the chromosome arms. impaired telomere clustering but also In keeping with a recently described Kazunori Tomita, chromosome fusions through meiotic Abigail Stevenson, Thein Z. Win, function for Cid12 in RNAi-mediated and Julia Promisel Cooper cell-cycle. and Shao-Win Wang heterochromatin assembly, this was To assess the importance of telomere accompanied by an accumulation of Telomere Biology Laboratory, Cancer clustering without worrying about Department of Zoology, University of polyadenylated transcripts Research UK, London, UK chromosome fusions, we characterized Oxford, South Parks Road, Oxford, corresponding to naturally silenced e-mail: [email protected] meiotic phenotypes in lig4A taz1A United Kingdom repeat elements within heterochromatic cells. 50% of lig4A taz1A asci are domains, with consequent defects in Telomeres are nucleoprotein structures abnormal. This defect is partially Fission yeast Cid12 is a member of the centromeric gene silencing. These cells that form and protect chromosome suppressed by the concomitant Cid1 family of specialised poly(A) also suffered increased meiotic defects ends and play a critical but poorly deletion of mad2, suggesting that polymerases. Like cells lacking cid1, and their viability was dependent on understood role in meiosis. Telomere defects in telomere clustering trigger a cid12A mutants were shown to have the spindle checkpoint protein Bub1. function changes profoundly as cells checkpoint-mediated block to meiotic checkpoint defects when DNA To account for the effects of Cid12 on proceed from the mitotic to meiotic cell progression. We monitored the SPB replication was inhibited. Here, we various aspects of DNA metabolism, cycle. In early meiosis, telomeres gath- and microtubules during meiosis to show that Cid12 is also required for including chromosome segregation and er at the centrosome to form the so- investigate the basis for the spore faithful chromosome segregation and the checkpoint control, we suggest that called ‘bouquet’ structure observed in formation problem in lig4A taz1A cells. that mutation of amino acid residues Cid12 has dual functions in RNAi diverse eukaryotes. This is particularly We found defective SPB separation and predicted to be essential for poly(A) silencing and regulating mRNA stability. striking in fission yeast, as the segregation, as well as severe spindle polymerase activity resulted in loss of telomeres localize to the spindle pole defects in some lig4A taz1A zygotes. Cid12 function in vivo. Cells lacking body (SPB) during meiotic prophase, in Because Taz1 is a component of the contrast to the mitotic interphase meiotic SPB, we checked the timing of configuration in which the centromeres telomere dissociation from SPB. localize to the SPB. The S. pombe Telomeres dissociate from the SPB telomere binding protein Taz1 is almost simultaneously with SPB required for telomere clustering at the division. We propose that telomere SPB. Taz1 also protects telomeres from dissociation from the SPB is an being recognized as DNA breaks and initiating event of meiosis I and controls subjected to inappropriate DNA repair the timing of SPB division through reactions. During the G1 arrest that Mad2 inactivation. precedes meiosis, end-to-end fusions

P134 P135 Role of intra-S phase a Replication Termination Sequence Characterisation of the role of is sensitive to DDA. In contrast, checkpoint proteins at stalled from the mat locus (Lambert et al., Cell sumoylation in the DNA deletion of pli1 is not lethal: cells are 2005). The role of the present study is not sensitive to DDS, but are sensitive replication forks to examine the interplay between damage response and to TBZ and display loss of silencing at checkpoint proteins and recombination genome stability the centromere (3). Null mutants of ulp1 Ellen Tsang at stalled forks in RuraR. We present (required for the majority of the data showing an elevated level of Watts FZ, Andrews A, Ho JCY, processing of SUMO to the mature Genome Damage and Stability Centre Rad22 (Rad52 homologue) recruitment Taylor DL and Zhou L form) display aberrant cell and nuclear University of Sussex to stalled forks in the absence of the morphologies, suggesting that multiple Rad3 checkpoint protein, but not in the Genome Damage and Stability Centre, processes are affected in this strain (2). The intra-S phase checkpoint absence of Rad9, Rad17, Cds1 or School of Life Sciences, University of ulp2 null mutants resemble pli1.d null coordinates a variety of cellular Chk1. Rad22 recruitment also spreads Sussex, Falmer, Brighton, E Sussex mutants, but with subtle differences. responses to replicative stress. Lambert further along the chromosome away The various roles of these proteins will et al. have recently shown that several from the site of fork stalling in the SUMO (small ubiquitin-like modifier) is be discussed. recombinogenic pathways occur to absence of Rad3. This suggests an covalently attached to proteins. bypass stalled replication forks in the involvement of Rad3 in fork processing Unlike ubiquitin, SUMO does not target 1. Andrews, E. A., J. Palecek, site-specific fork stalling system that is independent of the J. Sergeant, E. Taylor, A. R. Lehmann, proteins for degradation. In contrast, and F. Z. Watts. 2005. Nse2, “RuraR”. Here the S. pombe ura4 gene Rad9/Rad17/Cds1/Chk1 pathways. it affects protein localisation, protein was flanked by two copies of RTS1, a component of the Smc5-6 –protein interactions and protein complex, is a SUMO ligase required for activity. S. pombe mutants defective in the response to DNA damage. Mol Cell sumoylation are sensitive to DNA Biol 25:185-96. damaging agents (DDA) and the 2. Taylor, D. L., J. C. Ho, A. Oliver, microtubule inhibitor, thiabendazole and F. Z. Watts. 2002. Cell-cycle- (TBZ). This implies that sumoylation has dependent localisation of Ulp1, a roles in the DNA damage response and Schizosaccharomyces pombe Pmt3 (SUMO)-specific protease. J Cell Sci genome stability. In order to more 115:1113-22. precisely identify the roles of sumoyla- 3. Xhemalce, B., J. S. Seeler, G. Thon, A. tion we have been characterising the Dejean, and B. Arcangioli. 2004. Role of S. pombe SUMO ligases (Nse2 and the fission yeast SUMO E3 ligase Pli1p in Pli1) and the SUMO proteases centromere and telomere maintenance. (Ulp1 and Ulp2). Embo J 23:3844-53. Epub 2004 Sep 09.

Nse2 is essential for viability and is a member of the Smc5-6 complex (1). It is required for sumoylation of Smc6, and a SUMO ligase null mutant of Nse2

P136 P137 Analysis of Cohesin Function repair in S.pombe. Increase in cell A Mathematical Model of the can explain not only wild type behavior, in Fission Yeast survival during DNA replication has Initiation of DNA Replication but many other situations as well (e.g. been attributed to the formation of two HU block caused S phase block, OP 1 Saeeda Bhatti, Tracy Riddell identical sister chromatids, the physical Orsolya Kapuy*, Attila Csikász-Nagy cdc18 and cdc18TA mutants). and Christopher J. McInerny linkage of sister chromatids by the and Béla Novák Our model reveals two “oscillators” cohesin complex is vital for double centered around the two cell cycle strand break (dsb) repair. This central regulating Cdk/cyclin complexes: Division of Biochemistry and Molecular Molecular Network Dynamics Research role of cohesin in DNA dsb repair Cdc2/Cig2 and Cdc2/Cdc13. Biology, Institute of Biological and Life + Group of the Hungarian Academy became clear when mutants of rad21 The Cdc2/Cig2 “oscillator” is not Sciences, University of Glasgow, of Sciences had decreased survival after exposure essential for mitotic cycle, because the Glasgow G12 8QQ, Scotland, UK. and Budapest University of Technology to ionizing radiation. and Economics, Hungary Cdc2/Cdc13 “oscillator” can initiate both S and M phases. The Cdc2/Cig2 Survival of an organism is dependant Here we analyse the effects of inducing “oscillator” can work in the absence of on the accurate transmission of genetic *email: [email protected] information from one cell to its double strand. The alkylating agent phone: + 36-1-463-2910 Cdc2/Cdc13, but it drives only methly methane sulfonate (MMS) was periodic S phases. Both “oscillators” daughters. Such faithful transmission used to mimic the effects of ionizing can work in the absence of Cdc18, requires accuracy in the replication of DNA replicates once, and only once per DNA, precision in chromosome radiation. Increasing concentrations cell cycle. After exit from mitosis but their interaction is sensitive to of MMS were administered to three Cdc18 level. We propose that Cdc18 distribution and the ability to survive prereplictive complexes are formed on S. pombe strains, plays a role of coordinating their action: spontaneous and induced DNA the origins of replication. During S damage. wild-type with functional Rad21p, a phase these prereplictive complexes up-regulation of Cdc18 level might rad21+ mutant strain and a strain in block Cdc2/Cdc13 “oscillator” and let + are transformed to postreplicative which the meiotic cohesin rec8 was the Cdc2/Cig2 “oscillator” periodically Cohesin is a chromosomal protein complexes. Proper initiation of S phase expressed in the mitotic cycle, replac- initiate S phase. complex holding replicated sister DNA + in fission yeast requires Cdc10 mediat- ing the Rad21p. Expression of rec8 in strands together after synthesis. ed transcription. Cdc10 transcribes this manner serves as a useful tool for 1 D. Hermand, N. Fersht and P. Nurse: t plays a critical role in the successful Cig2 cyclin (the regulatory subunit of Cdc18/Cdc6 plays a central checkpoint separation of sister chromatids in both investigating the meiotic specific role of the S phase Cdk/cyclin complex), Rec8p and allows us to determine if role in restraining mitosis during DNA mitosis and meiosis for which there are Cdc18 (the licensing factor of the there is an overlap in cohesin function. replication (submitted) specific cohesin proteins. The fission replication), and Mik1 tyrosine-kinase yeast Schizosaccharomyces pombe (the inhibitor of Cdk). We incorporated These experiments revealed mitotic (S.pombe), has two cohesin genes the major regulations of this pathway + + and meiotic specific roles for the two rec8 and rad21 that are specific into a simple mathematical model of to meiosis and mitosis cohesins. When Rec8p was expressed the fission yeast cell cycle. The model in the mitotic cycle replacing the respectively.Expression of both genes includes the effect of the unreplicated Rad21p cell survival decreased, is regulated during their respective cell DNA checkpoint (Rad3 system), and cycles, with maximum transcript and thus indicating that Rec8p does not have a role in DNA repair. protein being present during DNA replication.

Long before the essential role of cohesin in chromosome segregation was recognized investigators found that one of the sub-units was involved in

P138 P139 Gross chromosomal In human cells, chromosomal fragile Permanently ubiquitinated suppressed the UV and 4-NQO rearrangements and elevated sites are thought to be the signature of PCNA and Translesion sensitivity of rhp18-d and K164R stalled forks and are prone to lead to mutants that are unable to recombination at an inducible GCR in cancer cells and to synthesis mono-ubiquitinate PCNA endogenous- site-specific replication chromosomal breakage in response to ly. This shows that our fusion construct fork barrier. replication inhibitor treatment. Sharada Ramasubramanyan, is able to substitute partially for PCNA To investigate the consequences of Dr. Catherine Green, ubiquitinated on K164 in repair 1 2 Prof. Alan Lehmann S. Lambert , A. Carr , stalled forks on GCR, we have processes after DNA damage. and G. Baldacci1. developed various site-specific fork However, the fusion construct did not stalling substrates: 1) Rura and uraR Genome Damage and Stability Centre, suppress the UV sensitivity of rad8-d University of Sussex 1UMR2027, Institut Curie, Orsay which stall the telomere or the mutants that are able to University, France. centromere proximal replication fork, mono-ubiquitinate but not 2 respectively, 2) RuraR which stall both During Translesion Synthesis (TLS), poly-ubiquitinate PCNA, suggesting Genome Damage and Stability Centre, DNA is synthesized past unrepaired University of Sussex, Brighton UK. replication forks leading potentially to that the fusion construct may not be a ura4 locus unreplicated, and 3) _ura_ damage in the template DNA by substrate for poly-ubiquitination. specialized error-prone polymerases Genomic rearrangements linked to which stall both replication forks with- out leading to ura4 locus unreplicated. that insert correct or incorrect To determine whether the Ub-PCNA aberrant recombination are associated nucleotides opposite the damaged site. with cancer and human genetic Using PFGE following Southern-blot fusion can substitute for PCNA during analysis, we observed that a one-sided However, the mechanism by which the replication, the fusion construct was diseases. Such recombination has indi- cells switch from replicative polymeras- rectly been linked to replication fork replication fork breakage occurs only at integrated into the genomic Pcn1 locus RuraR-specific fork stalling. Moreover, es to translesion polymerases at the of diploid wild type S. pombe cells. stall. Using fission yeast, we have stalled forks is still unknown. It has developed a genetic system to induce by scoring the lost of ura4 marker and When the cells were sporulated, only by PCR-based molecular analysis, been shown recently that after DNA two out of four spores from each ascus a site-specific fork barrier at a damage, PCNA (Proliferating cell nonhistone/DNA complex. We used the translocations between chromosome II were viable, suggesting that Ub-PCNA and chromosome III are observed only nuclear antigen), the sliding clamp is not able to substitute for endogenous RTS1-dependent fork stalling, previous- required for DNA replication, is ly identified at the mat locus1, which is at RuraR-specific fork stalling. These PCNA in its essential function and that results suggest that stalled forks mono-ubiquitinated and this modified unmodified PCNA may be necessary able to stall replication fork in a polar form has higher affinity for pol_ (TLS way. To create a region of DNA difficult leading to unreplicated DNA regions are for cell viability. prone to fork breakage and to lead polymerase), suggesting a role for this to replicate, we integrated RTS1 post-translational modification of PCNA sequences on either side of the ura4 to GCR. We are currently analyzing further the in mediating the polymerase switch. phenotype of cells expressing PCNA- gene on chromosome III, to create To gain further insights into the role of RuraR. By controlling the expression of 1. Dalgaard JZ, Klar AJ. A DNA replication- Ub fusions along with localization and arrest site RTS1 regulates imprinting by PCNA modification, we have attempted interaction studies of the TLS genes required for RTS1 activity, fork determining the direction of replication at to mimic permanently ubiquitinated stalling could be efficiently induced at polymerases in association with mat1 in S. pombe. Genes Dev. 2001 Aug PCNA with a Ubiquitin-PCNA fusion mono-ubiquitinated PCNA. ura4. We previously reported that2: 1) 15;15(16):2060-8. protein, which we have expressed in Recombination is required for cell different Schizosaccharmomyces viability in response to RuraR-specific 2. Lambert S, Watson A, Sheedy DM, pombe strains. Expressing the fusions Martin B, Carr AM. Gross chromosomal fork stalling, 2) Recombination proteins did not have any dominant negative are recruited at such stalled fork and 3) rearrangements and elevated recombination at an inducible effect in wild type cells and expressing RuraR-specific fork stalling leads to the fusion constructs could partially elevated recombination and Gross site-specific replication fork barrier. Cell. 2005 Jun 3;121(5):689-702. Chromosomal Rearrangements (GCR).

P140 P141 Requirement of fission yeast Schizosaccharomyces pombe. Unlike Author index Finan, Kieran P21 Cid14 in polyadenylation of trf4 trf5 double mutants, cells lacking Finn, Rob P16 Cid14 are viable, though they suffer an Foethke, Dietrich P69 ribosomal RNAs Aligué, Rosa P55, P56 increased frequency of chromosome Alonso-Nunez, Maria P33 Fong, Chii-Shyang P86 mis-segregation. The Cid14 protein is Francesconi, Stefania P109 1 2 Alvarez-Tallada, Victor P102 Thein Z. Win , Chris J. Norbury , constitutively nucleolar, and is required Franco-Sanchez, Alejandro P85, P87 and Shao-Win Wang1 Anders, Andreas P65 for normal nucleolar structure. A minor Aoki, Keita P84 Froget, Benoît P114 population of polyadenylated rRNAs Furuya, Kanji P115 1 Arcangioli, Benoît T3, P137 Department of Zoology and was identified. These RNAs Garcia, Valerie P116 2Sir William Dunn School of Pathology, Armstrong, John T11 P1, P5, P19 accumulated in an exosome mutant Attanapola, Sheran P60 García-Rodriguez, Patricia T31, P61 University of Oxford, South Parks and their presence was largely depend- Garcin, Daphne P88 Road, Oxford, United Kingdom Aves, Stephen P12, P125 ent on Cid14, in line with a role for Ayté, José T20 Gill, Stephen P117 Cid14 in rRNA degradation. Bähler, Jürg T2, T16, P11, P15, P17, Goldstone, Sherilyn T27 Polyadenylation in eukaryotes is Surprisingly, both fully processed 25S Grallert, Agnes T33, P50, P88 conventionally associated with P20, P23, P35, P40, P45, P47, rRNA and rRNA processing P82, P131 Grallert, Beata T36 increased nuclear export, translation intermediates appear to be channelled Greenall, Amanda T22 and stability of mRNAs. In contrast, Balasubramanian, Mohan P52, P73 into this pathway. Our data suggest Barker, Daniel P2 Hachet, Olivier P51 recent studies suggest that the Trf4 and that additional substrates may include Hagan, Iain T15, T33, P62, P88, P102 Trf5 proteins, members of a widespread Beck Tim P1 the mRNAs of genes involved in Ben-Yosef, Ron P106 Haines, Rebecca P70 family of non-canonical poly(A) meiotic regulation. Polyadenylation- Han, Sangjo P7 polymerases, share an essential Bhatti, Saeeda P138 assisted nuclear RNA turnover is Bone, Neil T11, P19 Hardwick, Kevin T25 P89, P91, P93, function in Saccharomyces cerevisiae therefore likely to be a common eukary- P97, P104 that involves polyadenylation of nuclear Bonilla, Carolina T17, P36 otic mechanism affecting diverse Boye, Erik T36 Harley, Margaret T35 RNAs as part of a pathway of biological processes. Harris, Midori P3, P8 exosome-mediated RNA turnover. Bridge, Wendy P102 Brown, William P122 Hartsuiker, Edgar T8, P130 Substrates for this pathway include Hayles, Jacqueline P9, P14 aberrantly modified tRNAs and Carazo-Salas, Rafael P66 Carr, Anthony T4, T8, P115, P116, Hebden, Anna T7 precursors of snoRNAs and rRNAs. Helmlinger, Dom P38 Here we show that Cid14 is a Trf4/5 P117, P124, P126, P140 Castagnetti, Stefania T29 Hermand, Damien P30, P139 functional homolog in the distantly Hirata, Dai T30, P71, P75, P76, P77 related fission yeast Codlin, Sandra P67, P70 Collin, Phillippe P48, P54 Hoog, Johanna P72 Cooper, Julia T7, P113, P132, P135 Humphrey, Tim P108, P110 Crawley, Karen P85, P98 Ikebe, Chiho P90 Csikasz-Nagy, Attila Irmisch, Anja P119 Cullen, Jason P108, P110 Jaendling, Alessa Dafydd, Heledd T9 Jones, Nic T13 Dave, Anoushka P108, 110 Joseph, Nimesh Day, Alison T14 Junker, Viv P10 Decottignies, Anabelle P25, P111 Kanai, Muneyoshi T30, P75, P76, P77 de Medina Redondo, Maria P59 Kapuy, Orsolya P139 Djupedal, Ingela T17, P36 Karagiannis, Jim P52 Dodgson, James P5 Kerres, Anne T23 Doyle, Alexander P49 Kim, Dongsup P7, P9 Duhig, Trevor P9, P14 Kimata, Yuu T37, P53 Dutta, Chaitali P112 Kjaerulff, Søren T10 Dziadkowiec, Dorota P120 Kohli, Jürg P121 Ekwall, Karl T17, T21, P36, P43 Krapp, Andrea P54 Fantes, Peter T15, P64 Krohn, Marit T36 Feijao, Andreia P68 Kubota, Shunsuke T76 Ferreira, Miguel Godinho, P113 Kumar, Arun P22 Kume, Kazunori P76, P77

P142 143 Lackner, Daniel T2, T16, P47, P131 Pérez-Hidalgo, Livia P13 Tournier, Hyams T27 Watts, Felicity P137 Lambea, Eva P55, P56 Pesti, Miklos P24 Toya, Mika P78, P79, P100 Wee, Boon-Yu P108, P110 Lambert, Sarah P114, P140 Petersen, Janni T15, P62 Tran, Phong P74 Whitehall, Simon T22 Lawrence, Clare T13 Pfievorovsky, Martin P25 Trickey, Michelle T37 P63 Wilhelm, Brian P11, P35, P45 Lee, Minho P7 Raabe, Isabel T28, P95, P96, P105 Tsang, Ellen P136 Wilkinson, Caroline T13 Legouras, Ioannis T1, P123 Raji, Hayatu P130 Tvegaard, Tonje T36 Wood, Valerie P2, P14, P15, P16, P18, Liew, Li Phing T2, P131 Ramasubramanyan, Sharada P141 Uhlmann, Frank P133 P46, P123 López, Luis P23 Rawson, Emma P26 Umeda, Makoto P27, P32 Wright, Anthony T18, P46 López-Avilés, Sandra P55, P56 de Medina-Redondo, Mara P59 Unsworth, Amy P103 Wuarin, Jérome T35 Lygerou, Zoi T1, P123 Reiter, Wolfgang T13 Valdivieso, M.-Henar P58 Xue-Franzen, Yongtao T18 P46 Mamnun, Yasmine P57 Reyes, Gachet T27 Vanoosthuyse, Vincent P104 Yamano, Hiro T37, P53, P63 Mandeville, Ryoko P9 Rincon, Sergio T34, P80 Vijayraghavan, Usha P39 Yamashita, Akira T40, P62 Manor, Haim P106 Rischitor, Patricia P97 Vogel, Sven P96, P105 Yanagida, Mitsuhiro T38, P37, P84 Marchin, Madelaine P4 Rissland, Olivia T2, P131 Wang, Huan P44 Young, Paul T12, P21 Marguerat, Samuel T16 P11 P45 P47 Rog, Ofer P132 Wang, Hongyan P73, P79 Zhu, Xuefeng T21 Martín, Rebeca P58 Roguev, Assen T19, P34, P35, P42 Wang, Shao-Win T2, P131, P134, P142 Martín-Castellanos, Cristina P13 Roseaulin, Laura T3 Watt, Stephen P17, P45 Mata, Juan T2, P13, P17, P40, P131 Rozalen, Ana P13 May, Karen P91 Russell, Paul T6, T15 McFarlane, Ramsay T9 Sakalar, Cagri P35 Meadows, John T26, P85, P92, P98 Samejima, Itaru T34 Millar, Jonathan T26, P27, P85, P87, Franco-Sanchez, Alejandro P85, P92, P98, P101 P87, P98 Milne, Laura P93 Sanchez-Perez, Isabel P25, P85, Mizuno, Kenichi T4, T8, P126 P87, P98 Mole, Sara P67, P70 Santos, Beatriz P80 Moore, Karen P125 Sato, Masamitsu T24, P86, P99 Moreno, Sergio P13 Sawin, Ken T34, P65, P81 Mulvihill, Dan P49, P50, P60 Sazer, Shelley P27, P32 Murakami, Hiroshi T39 Schmidt, Christine P133 Murray, Jo T4, P117, P119, P126 Schürer, Anke P118 Murrells, Lindsay P78 Sczaniecka, Matylda T25, P89 Nagao, Koji T38 Shaikh, Nadeem P101 Nakano, Kentaro P79, P83 Shiozaki, Kaz T15, P83 Ng, Szu-Shien P41 Sinha, Indranil T17, T21, P43 Nielsen, Olaf T10 Sistla, Srivani P28 Niwa, Osami T5, P94 Snaith, Hilary P81 Noguchi, Chiaki P127, P128 Soltani, Héla T36 Noguchi, Eishi P127, P128 Stansfield, Peter T35 Norbury, Chris T2, P26, P131, P142 Stevenson, Abigail T2, P131, P134 Novák, Béla T29, P139 Sundaram, Geetanjali P29 Nurse, Paul T1, T11, T15, T20, T29, P9, Svetina, Sa_a P82 P14, P25, P66, P123, P139 Tajadura, Virginia T31, P61 Okazaki, Koei P94 Tanaka, Kayoko T62 Pacurar, Monica P19 Tang, Zhaohua P31 Paderi, Francesca P115, P129 Toda, Takashi T24, T30, P57, P75, P77, Pai, Chen Chun P107 P85, P86, P90, P98, P99, Paoletti, Anne T32 P100, P103 Peat, Nigel P9, P14 Tolic-Nørrelykke, Iva T28, P96, P105 Penkett, Christopher P15, P23, P45 Tomita, Kazunori P135 Perego, Paola P20 Tonami, Yuko T39

144 145 Delegate List Mr Keita Aoki Dr Jose Ayte Mrs Samia Ben Hassine Kyoto University Universitat Pompeu Fabra Pasteur Institute Yoshida-Honmachi, Sakyo-ku Doctor Aiguader, 80 25-28 rue du docteur Roux Dr Rosa Aligue Kyoto Barcelona Paris University of Barcelona 606-8317 08003 75015 Casanova 143 JPN ESP FRA Barcelona +075-753-4206 +34-93-542-2891 +33 140613686 08036 [email protected] [email protected] [email protected] ESP +34934037252 Dr Benoit Arcangiolo Dr Jurg Bahler Mr Ron Ben-Yosef [email protected] Pasteur Institute Wellcome Trust Sanger Institute Technion-Israel Institute of technology 25-28 rue du Dr Roux Wellcome Trust Genome Campus Faculty of Biology Miss Maria Luisa Alonso Nunez Paris Hinxton Technion In Instituto de Microbiología Bioquímica 75015 Cambridge Haifa Edificio Departamental. Campus FRA CB10 1SA 32000 Unamuno +33 1 45 68 8454 GBR ISR Lab 236 [email protected] +44 (0)1223 494861 +972-8293456 Salamanca [email protected] [email protected] 7 007 Dr John Armstrong ESP University of Sussex Dr Mohan Balasubramanian Mr Fredrik Berglund +34923294400 Falmer Temasek Lifesciences Laboratory University of Dundee [email protected] Brighton 1 Research Link Level 5 BN1 9QG The National University of Singapore Ninewells Hospital and Medical School Dr Beatriz Alvarez GBR Singapore Dundee Instituto de Biologica Molecular y +44 (0)1273 678576 117604 DD1 9SY Celular del Cancer, CSICUniversidad de [email protected] SGP GBR Salamanca +65-6872-7000 +44 (0)1382 660 111 ext 32566 Campus Miguel de Unamuno Mr Sheran Attanapola [email protected] [email protected] Salamanca University of Kent 37007 Mulvihill Lab Dr Daniel Barker Ms Saeeda Bhatti ESP Department of Biosciences University of St Andrews University of Glasgow +34923294810 Canterbury School of Biology University Avenue [email protected] Kent University of St Andrews Glasgow CT2 7NJ Sir Harold Mitchell Building G12 8QQ Dr Victor Alvarez Tallada GBR St Andrews GBR Paterson Institute +44 (0)1227 764 000 Fife +44 (0)141 339 8855 x0417 Wilmslow Road [email protected] KY16 9TH [email protected] Withington GBR Manchester M20 3DT Dr Stephen Aves +44 (0)1334 476161 Dr Hasanuzzaman Bhuiyan GBR University of Exeter [email protected] University College Sodertorns +44 (0)161 446 3167 The Henry Wellcome Building for Alfred Nobel s Alle 3 [email protected] Biocatalysis Mr Tim Beck Huddinge Stocker Road University of Sussex S-14152 Dr Andreas Anders Exeter Falmer SWE University of Edinburgh EX4 4QD Brighton +4686084000 Swann Building, Mayfield Road GBR BN1 9QG [email protected] Edinburgh +44 (0)1392 264675 GBR EH9 3JR [email protected] +44 (0)1273 674341 GBR [email protected] +44 (0)131 650 7074 [email protected]

146 147 Ms Claudia Bicho Miss Katalin Boros Dr Rafael Edgardo Carazo Salas Dr Julia Cooper National Institute for Medical Research CRUK Paterson Institute for Cancer Cancer Research UK Cancer Research UK The Ridgeway Research 44 Lincolns Inn Fields 44 Lincolns Inn Field Mill Hill Wilmslow Road London London London Manchester WC2A 3PX WC2A NW7 1AA M20 4BX GBR GBR GBR GBR +44 (0) 207 242 0200 +44 (0)207 2693415 +44 (0)20 8959 3666 +44 (0)1614463156 [email protected] [email protected] [email protected] [email protected] Prof Antony Carr Miss Karen Crawley Dr Perry Blackshear Prof Erik Boye University of Sussex London Research Institute, Cancer NIHNIEHS Rikshospitalet-Radiumhospitalet HF Falmer Research UK 111 T.W. Alexander Dr. Montebello Brighton Cell Regulation lab, Room 329 P.O. Box 12233 MD A2-05 Oslo Sussex Cancer Research UK Research Triangle Park 0310 BN1 9RQ 44 Lincolns Inn Fields North Carolina NOR GBR London 27709 +47-22934256 +44 (0)1273 678122 WC2A 3PX USA [email protected] [email protected] GBR +1 919-541-4899 +44 (0)20 7242 0200 [email protected] Dr Wendy Bridge Dr Stefania Castagnetti [email protected] Radiation and Genome Stability Unit Cancer Research UK Ms Cathrine Arnason Boee Medical Research Council PO 123 Dr. Attila Csikasz-Nagy The Norwegian Radium Hospital Oxford 44 Lincolns Inn Field Budapest University of Technology and Ullernchausseen 70 OX11 0RD London Economics Montebello GBR WC2A 3PX Gellert ter 4 Oslo +44 (0)1235 841000 GBR Budapest 0310 [email protected] +44 (0)207 2693235 1521 NOR [email protected] HUN +4722935945 Dr William Brown +3614632910 [email protected] Nottingham University Dr Sandra Codlin [email protected] Institute of Genetics University College London Neil Bone School of Biology Gower Street Mr Jason Cullen University of Sussex Queens Medical Centre London Medical Research Council Falmer Nottingham WC1E 6BT Harwell Brighton NG7 2UH GBR Dicot BN1 9QG GBR +44 (0)2076792122 OX11 0RD GBR +44 (0)115 849 3263 [email protected] GBR +44 (0)1273 678308 [email protected] +44 (0)1235 841000 [email protected] Mr Philippe Collin [email protected] Mr Juan Antonio Canete ISREC Ms Carolina Bonilla Centro de Investigacion del Cancer Ch. des Boveresses 155 University College Sodertorns Avda. universidad de Coimbra, SN. Epalinges Miss Heledd Dafydd Alfred Nobel s Alle 7 Campus Miguel de Unamuno Vaud Prifysgol Cymru Bangor Huddinge Salamanca 1066 Lab G30 S-14152 37007 CHE Adeilad Coffa SWE ESP +41216925858 Ffordd Deiniol +4686084000 +34923294805 [email protected] Bangor [email protected] [email protected] LL57 2UW GBR +44 (0)1248 382362 [email protected]

148 149 Dr Anoushka Dave Mrs Helena Diaz-Cuervo Dr Thomas Duke Mrs Andreia Feijao Medical Research Council Centro de Investigacion del Cancer University of Cambridge EMBL Harwell Avda. universidad de coimbra, SN. Cavendish Laboratory Meyerhofstrasse 1 Didcot Campus Miguel de Unamuno Madingley Road Heidelberg OX11 0RD Salamanca JJ Thompson Avenue 60117 GBR 37007 Cambridge DEU +44 (0)1235 841000 ESP CB3 0HE +49 387336 [email protected] +34923294805 GBR [email protected] [email protected] +44 (0)1223 337200 Dr Alison Day [email protected] Dr Miguel Godinho Ferreira University of Newcastle upon Tyne Mrs Ingela Djupedal Cancer Research UK Catherine Cookson Building Karolinska Institute Mrs Chaitali Dutta 44 Lincoln’s Inn Fields Framlington Place Huddinge University of Massachusetts Medical London University of Newcastle upon Tyne Sweden School, USA WC2A 3PX Newcastle upon Tyne 14140 364 Plantation Street, LRB 940E GBR Tyne and Wear NE2 4HH SWE Worcester +44 (0)2073212 GBR +4686084737 Massachusetts [email protected] +44 (0)191 222 7444 [email protected] 01605 [email protected] USA Mr. Kieran Finan Mr James Dodgson +1 5088568317 Queen’s University Miss Maria de Medina Redondo University of Sussex [email protected] Barrie Street Instituto de Microbiologa Bioqumica Falmer Kingston Departamento de Microbiologa y Brighton Dr Dorota Dziadkowiec ON Geneica, CSICUniversidad de BN1 9QG Wroclaw University K7L 3N6 Salamanca GBR Przybyszewskiego 6377 CAN Campus Miguel de Unamuno +44 (0)1273 678308 Wroclaw +1-613-533-6148 Salamanca [email protected] 51 148 [email protected] 37007 POL ESP Mr Alexander Doyle +48 71 3756250 Mr Dietrich Foethke +34 923 294 462 University of Kent [email protected] European Molecular Biology Laboratory [email protected] Giles Lane Meyerhofstr. 1 Dr Anabelle Decottignies Canterbury Dr Karl Ekwall Heidelberg Catholic University of Louvain Kent Karolinska Institute 69117 Avenue Hippocrate, 74+3 CT2 7NJ Alfred Nobels Alle 7 DEU Brussels GBR Huddinge +49 6221 3878813 1200 +44 (0)1227 764000 ext 7930 S-141 89 [email protected] BEL [email protected] SWE +32-2-7647474 +46 8 6084713 Mr Chii Shyang Fong [email protected] Mr Trevor Duhig [email protected] Cancer Research UK London Research Cancer Research UK Institute Mrs Ayguel Dereli 44, Lincolns Inn Fields Dr Peter Fantes 44 Lincoln’s Inn Fields MPI-CBG Dresden London University of Edinburgh London Pfotenhauer st. 108 WC2A 3PX Mayfield Road WC2A 3PX Dresden GBR Edinburgh GBR 01307 +44 (0)20-7269-3492 EH9 3JR +44 (0)20 7269 2945 DEU [email protected] GBR [email protected] + 351 210 20 +44 (0)131-650-5669 direct [email protected] [email protected]

150 151 Dr Stefania Francesconi Mrs Ana Isabel Garcia Dr Sherilyn Goldstone Prof Iain Hagan CNRS Institut Curie Instituto de Biología molecular y Celular Universite Paul Sabatier Paterson Institute for Cancer Research Centre Universitaire d Orsay del Cáncer CSIC- USAL Batiment 4R3 Porte 1 Wilmslow Road Orsay Campus Miguel de Unamuno 118 Route de Narbonne Manchester 91405 Salamanca Toulouse M20 4BX FRA 37007 31062 GBR +33-0169863131 ESP FRA +44 (0)161 446 8193 [email protected] + 34 923 29 48 10 +33 05 61 55 64 34 [email protected] [email protected] [email protected] Dr Alejandro Franco-Sanchez Miss Rebecca Haines MRC National Institute for Medical Miss Patricia Garcia Rodriguez Dr Agnes Grallert MRC LMCB Research Universidad de SalamancaCSIC Paterson Institute for Cancer Research University College London NIMR Edificio Departamental, lab 231 Wilmslow Road Gower Street The Ridgeway Campus Miguel de Unamuno Manchester London Mill Hill Salamanca M20 4BX W1E 6BT London 37007 GBR GBR NW7 1AA ESP +44 (0)1614463167 +44 (0)2076797806 GBR +34-923121589 [email protected] [email protected] +44 (0)2088162367 [email protected] [email protected] Dr Beata Grallert Mr Sangjo Han Ms Daphne Garcin Institute for Cancer Research, DNR Korea Advanced Institute of Science Mr Benoit Froget Paterson Institute for Cancer Research Ullernchausseen 70 Montebello and Technology Institut Curie, CNRS. Christie Hospital Oslo 373-1, Guseong-Dong, Yuseong-gu Bat 110, Centre Universitaire d Orsay Wilmslow Road 0310 Daejeon Orsay Withington NOR 305-701 91405 Manchester +47 22 93 47 29 KOR FRA M21 0AS [email protected] +82-42-869-4357 +1 69 86 30 13 GBR [email protected] [email protected] 01614463167 Dr Amanda Greenall [email protected] University of Newcastle upon Tyne Dr Kevin Hardwick Dr Kanji Furuya The Medical School University of Edinburgh Genome Damage and Stability Centre Dr Thomas Germe Framlington Place Kings Buildings University of Sussex Cancer Research UK Newcastle upon Tyne Mayfield Road Brighton 44 Lincolns Inn Fields NE2 4HH Edinburgh BN1 9RQ London GBR EH93JR JPN WC2A 3PX +44 (0)191 2227444 GBR +44 (0)1273873118 GBR [email protected] +44 (0)131 650 7091 [email protected] +44 (0)2072420200 [email protected] [email protected] Dr Olivier Hachet Dr Valerie Garcia ISREC Miss Margaret Harley University of Sussex Dr Stephen Gill Ch. des Boveresses 155 University of Dundee Falmer University of Sussex Epalinges Ninewells Hospital Brighton Science Park Road CH-1066 Level 5 BN1 9RQ Falmer CHE Dundee GBR BN1 9RQ +41 021 692 58 58 DD1 9SY +44 (0)1273873118 GBR [email protected] GBR [email protected] [email protected] +44 (0)1382 660111 ext 33520 [email protected]

152 153 Miss Sonya Hartmuth Dr Damien Hermand Dr Tim Humphrey Dr Nimesh Joseph University of Manchester FUNDP-GEMO MRC Radiation and Genome Stability Paterson Institute for Cancer Research Michael Smith Building rue de Bruxelles, 61 Unit Wilmslow Road Oxford Road Namur Harwell Withington Manchester 5000 Oxford Manchester M13 9PT BEL OX11 0RD Greater Manchester GBR +3281724241 GBR M20 4BX +44 (0)161 275 5512 [email protected] +44 (0)1235 841114 GBR sonya.hartmuth@postgrad. [email protected] +44 (0)161-4463156 manchester.ac.uk Dr Ana B Herrero [email protected] Center for Cancer research Dr Chiho Ikebe Dr Edgar Hartsuiker Campus Miguel de Unamuno Cancer Research UK, London Mrs Viv Junker GDSC Salamanca Research Institute Swiss Institute of Bioinformatics University of Sussex 37007 44 Lincolns Inn Fields CMU, 1 Michel-Servet Falmer ESP London Geneva Brighton +34923294810 WC2A 3PX CH-1211 BN1 9RQ [email protected] GBR CHE GBR +44 (0)20-269-3330 +41-22-379 58 22 +44 (0)1273 873118 Prof Dai Hirata [email protected] [email protected] [email protected] Hiroshima University 1-3-1 Kagamiyama Ms Anja Irmisch Mr Muneyoshi Kanai Dr Jacqueline Hayles Higashi-Hiroshima University of Sussex Hiroshima University Cancer Research UK, London Research 739-8530 Science Park Road Kagamiyama1-3-1 Institute JPN East Sussex Higashihiroshima 44, Lincolns Inn Fields +81 82 424 7764 Falmer, Brighton Hiroshima London [email protected] BN1 9RQ 739-8530 WC2A PX GBR JPN GBR Miss Cassandra Hogan +44 (0)1273 678123 +81-82-424-7764 +44 (0)20 7269 2072 The Babraham Institute [email protected] [email protected] [email protected] Babraham Research Campus Cambridge CB2 4AT Miss Alessa Jaendling Mrs Orsolya Kapuy Miss Anna Hebden GBR University of Wales, Bangor Budapest University of Technology and Cancer Research UK +44 (0)1223 496000 Memorial Building Economics 44 Lincolns Inn Fields [email protected] Deiniol Rd Budapest London Bangor H-1111 WC2A 3PX Dr Christian Holmberg Gwynedd HUN GBR Department of Genetics, LL57 2UW +3614632910 +44 (0)20 7269 3212 University of Copenhagen GBR [email protected] [email protected] Oester Farimagsgade 2A +44 (0)1248 382369 DK-1353 Copenhagen K [email protected] Dr Jim Karagiannis Mr Dom Helmlinger DNK Temasek Life Sciences Laboratory Harvard Medical School [email protected] Professor Nic Jones 1 Research Link 77 avenue Louis Pasteur Paterson Institute for Cancer Research National University of Singapore New Research Building Mrs Johanna Hoog Christie Hospital NHS Trust Singapore room 239 EMBL Wilmslow Road 117604 Boston Meyerhofstrasse 1 Manchester SGP MA Heidelberg M20 4BX +65-6872-7000 02215 69117 GBR [email protected] USA DEU +44 (0)161 446 3101 +1-617-432-7557 +49-6221-387620 [email protected] [email protected] [email protected]

154 155 Mrs Anne Kerres Dr Andrea Krapp Mr Daniel Lackner Mr Minho Lee Heinrich-Heine-Universite ISREC Wellcome Trust Sanger Institute Korea Advanced Institute of Science Lehrstuhl funktionelle Genomforschung Boveresses 155 Wellcome Trust Genome Campus and Technology Dusseldorf Epalinges Hinxton Protein BioInformatics Laboratory 40225 1066 Cambridge Department of BioSystems DEU CHE CB10 1SA Korea Advanced Institute of Science +49 211 8114581 +41 21 692 58 58 GBR and 373-1, Guseong-dong, [email protected] [email protected] +44 (0)1223-494862 Yuseong-gu, Daejeon [email protected] 305-701 Dr. Dongsup Kim Ms Marit Krohn KOR Korea Advanced Institute of Science The Norwegian Radium Hospital Mr Ronak Lakhani +82-42-869-4357 and Technology Montebello University of Sussex [email protected] Guseong-dong, Yuseong-gu Oslo Falmer Daejeon 0310 Brighton Mr Ioannis Legouras 305-701 NOR BN1 9QG University of Patras KOR +47 22935945 GBR Rio +82-42-869-4317 [email protected] +44 (0)1273 678308 Patra [email protected] [email protected] Achaia Mr Shunsuke Kubota 26500 Dr Yuu Kimata Hiroshima University Mrs Eva Lambea GRC Marie Curie Research Institute Kagamiyama 1-3-1 University of Barcelona +30-2610-991822 The Chart Higashi Hiroshima C Casanova 143 [email protected] Oxted Hiroshima Barcelona Surrey 739-8530 08036 Miss Li Phing Liew RH8 0TL JPN ESP University of Oxford GBR +81 82 424 7764 +34934037252 South Parks Road +44 (0)1883 722306 [email protected] [email protected] Oxford [email protected] OX1 3RE Mr Arun Kumar Mrs Sarah Lambert GBR Mr Soeren Kjaerulff Insitute of Genomics and Integrative Institut Curie, CNRS +44 (0)1865 285510 University of Copenhagen Biology Institut Curie, UMR2027 [email protected] 216. Farimagsgade 2A Opposite Jublee Hall , bat 110 Centre universitaire Dr Luis Lopez Copenhagen Delhi university North campus Orsay Wellcone Trust Sanger Institute DK-1353 Kbh K Mall Road 91405 Wellcome Trust Genome Campus DNK Delhi FRA Hinxton +45 35 32 21 03 India +1 69 86 71 91 CB10 1SA [email protected] 1100007 [email protected] GBR IND +44 (0)1223494862 Prof Juerg Kohli +91-011-27666156, 27666157, 276 Dr Clare Lawrence [email protected] Institute of Cell Biology [email protected], Paterson Institute for Cancer Research University of Berne [email protected] Christie Hospital NHS Trust Mrs Sandra Lopez-Aviles Baltzer-Strasse 4 Wilmslow Road University of Barcelona Bern Mr Kazunori Kume Manchester C Casanova 143 CH-3012 Hiroshima University M20 4BX Barcelona CHE Kagamiyama 1-3-1 GBR 08036 +41-31-631-4615 Higashi Hiroshima +44 (0)161 446 3171 ESP [email protected] Hiroshima [email protected] +34934037252 739-8530 [email protected] JPN +81 82 424 7764 [email protected]

156 157 Dr Zoi Lygerou Dr Samuel Marguerat Mr Orestis Mavroudis-Chocholis Dr Jonathan Millar University of Patras, School of Wellcome Trust Sanger Institute Paterson Institute for Cancer Research National Institute for Medical Research Medicine Wellcome Trust Genome Campus Christie Hospital The Ridgeway University Campus Hinxton Wilmslow Road Mill Hill Rio, Patras Cambs Withington London NW7 1AA 26500 CB10 1SA Manchester GBR GRC GBR M20 4BX +44 (0)208 959 3666 + 30 2610 997621 +44 (0)1223 834244 GBR [email protected] [email protected] [email protected] +44 (0)161 446 3171 [email protected] Ms Vicky Miller Dr Yasmine Mamnun Prof Ian Marshall University of Edinburgh Cancer Research UK, LRI University of Kent Dr Karen May The Wellcome Trust Centre for Cell 44 Lincolns Inn Fields Computing Laboratory Edinburgh University Biology London Canterbury Kings Buildings Swann Building WC2A 3PX CT2 7NF Mayfield Road Mayfield Road GBR GBR Edinburgh Edinburgh EH9 3JR +44(0)207 269 3330 +44 (0)1227 827753 EH9 3JR GBR [email protected] [email protected] GBR +44 (0131 650 7063 +44 (0)131 650 8087 [email protected] Ms Ryoko Mandeville Miss Rebeca Martin [email protected] The Rockefeller University Universidad de SalamancaCSIC Miss Laura Milne 1230 York Avenue Edificio Departamental Ramsey McFarlane University of Edinburgh New York, NY Campus Miguel de Unamuno NWCRFI, UWB Michael Swann Building NY Salamanca Memorial Building King’s Building USA 37007 Deiniol Road Mayfield Road +1-212-327-8472 ESP Bangor Edinburgh EH9 3JR [email protected] +34923121589 Gwynedd GBR [email protected] LL57 2UW 0131 650 7083 Prof Haim Manor +44 (0)1248 382360 [email protected] Technion-Israel Institute of Technology Dra Cristina Martín-Castellanos GBR Dr Izumi Miyabe Technion City Instituto de Biología Molecular y Celular [email protected] Genome Damage and Stability Centre Haifa del Cáncer University of Sussex 32,000 Campus Miguel de Unamuno Mr John Meadows Brighton ISR Salamanca National Institute for Medical Research BN1 9RQ +972-4-8293456 37007 The Ridgeway GBR [email protected] ESP Mill Hill +44 (0)1273 873118 +34 923 29 48 10 London NW7 1AA [email protected] Ms Madelaine Marchin [email protected] GBR Stowers Institute for Medical Research +44 (0)20 8959 3666 Dr Kenichi Mizuno 1000 E 50th St Dr Juan Mata [email protected] Genome Damage and Stability Centre Kansas City The Wellcome Trust Sanger Institute University of Sussex MO Wellcome Trust Genome campus Dr Margaret Migocki Falmer 64110 Hinxton MRC National Institute of Medical East Sussex USA CB10 1SA Research BN19RQ +1 816 926 4454 GBR The Ridgeway GBR [email protected] +44 (0)1223 494862 Mill Hill +44 (0)1273 877521 [email protected] London [email protected] NW7 1AA GBR +44 (0)20 8959 3666 [email protected]

158 159 Dr Sara Mole Dr Hiroshi Murakami Miss Szu Shien Ng Dr Chris Norbury University College London Nagoya City University, University of Glasgow University of Oxford MRC Laboratory for Molecular Cell 1 Kawasumi, Mizuho-cho, Mizuho-ku, Davidson Building, IBLS South Parks Road Biology Nagoya Glasgow Oxford University College London 467-8601 G12 8QQ OX1 3RE Gower Street JPN GBR GBR London +81-52-853-8145 [email protected] +44 (0)1865 275500 WC1E 6BT [email protected] [email protected] GBR Prof Olaf Nielsen +44 (0)20 7679 37806 Dr Jo Murray University of Copenhagen Prof Bela Novak [email protected] Sussex University Farimagsgade 2A Budapest University of Technology and Falmer Copenhagen K Economics Dr Karen Moore Brighton DK-1353 Gellert ter 4 University of Exeter BN1 9RQ DNK Budapest The Henry Wellcome Building for GBR +45 3532 2000 1111 Biocatalysis +44 (0)1273 678123 [email protected] HUN Stocker Road [email protected] +3614631364 Exeter EX4 4QD Dr Osami Niwa [email protected] GBR Ms Lindsay Murrells Kazusa DNA Research Institute +44 (0)1392 262085 European Molecular Biology Laboratory 2-6-7 Kazusa-kamatari Paul Nurse [email protected] Meyerhofstrasse 1 Kisarazu The Rockefeller University Heidelberg Chiba 1230 York Avenue, Box 257 Professor Sergio Moreno D-69117 292-0818 New York Instituto de Biologica Molecular y DEU JPN NY 10021 Celular del Cancer. CSICUniv. +49 0 6221 387-0 +81-438-52-3923 USA Salamanca [email protected] [email protected] +1 212-327-8080 Campus Miguel de Unamuno [email protected] Salamanca Dr Koji Nagao Mrs Chiaki Noguchi 37007 Initial Research Project, Okinawa Drexel University College of Medicine Mr Yung-Chin Oei ESP Institute of Science and Technology 245 N 15th Street University of Cambridge +34923294810 Suzaki 12-22 MS497 Cavendish Laboratory [email protected] Uruma Philadelphia Madingley Road Okinawa PA JJ Thompson Avenue Dr Dan Mulvihill 904-2234 19102 Cambridge University of Kent JPN USA CB3 0HE Department of Biosciences +81 98 921 3985 +1 215-762-4424 GBR University of Kent [email protected] [email protected] +44 (0)1223 337200 Department of Biosciences, University [email protected] of Kent Dr Kentaro Nakano Dr Eishi Noguchi Canterbury University of Tsukuba Drexel University College of Medicine Dr Koei Okazaki Kent 1-1-1 Tennohdai 245 N 15th Street Kazusa DNA Research Institute CT2 7NJ Tsukuba MS497 2-6-7 Kazusa-Kamatari GBR Ibaraki Philadelphia Kisarazu +44 (0)1227 764000 305-8572 PA Chiba [email protected] JPN 19101 292-0818 +81-29-853-6642 USA JPN [email protected] +1 215-762-4424 +81-438-52-3925 [email protected] [email protected]

160 161 Ms Aslihan Ors Dr Christopher Penkett Mr Martin Pfievorovsky Miss Emma Rawson Marie Curie Research Institute Wellcome Trust Sanger Institute Faculty of Science, Charles University University of Oxford The Chart Wellcome Trust Genome Campus in Prague South Parks Road Oxted, Surrey Hinxton Vinicna 7 Oxford RH8 0TL Cambridge Prague 2 OX13RE GBR Cambridgeshire 12843 GBR +44 (0)1883-722-306 CB10 1SA CZE +44 (0)1865285510 [email protected] GBR +420221951785 [email protected] +44 01223 834244 [email protected] Ms Monica Pacurar [email protected] Dr Wolfgang Reiter University of Sussex Dr Bernd Pulverer Paterson Institute for Cancer Research Falmer Dr Paola Perego Nature Cell Biology Wilsmlow Road Brighton BN1 9QG Istituto Nazionale Tumori London Manchester GBR via Venezian 1 GBR M20 4BX +44 (0)1273 678308 Milan +44 (0)20 7843 4892 GBR [email protected] 20133 [email protected] +44 (0)161 446 3156 ITA [email protected] Miss Francesca Paderi +39-0223901 Mrs Isabel Raabe University of Sussex [email protected] MPI-CBG Dresden Miss Caroline Reyes Genome Damage & Stability Centre Pfotenhauerstr. 108 CNRSUMR5088 Brighton BN1 9RQ Dr Livia Perez-Hidalgo Dresden 118 route de Narbonne GBR Instituto de Biología Molecular y Celular 01307 TOULOUSE [email protected] del Cáncer CSIC-USAL DEU 31062 Campus Miguel de Unamuno +0351 21020 FRA Miss Chen-Chun Pai Salamanca [email protected] +05 61 55 64 34 University of Oxford 37007 [email protected] South Parks Road ESP Mr Hayatu Raji Oxford +34 923 29 48 10 University of Sussex Mr Sergio Rincon OX1 3PS [email protected] Science Park Road Instituto de Microbiologica Bioqumica GBR Brighton BN1 9QR CSICUSAL +44 (0)1865271230 Prof Miklos Pesti GBR Edificio Departamental, Lab. 223 [email protected] Pecs University, Faculty of Sciences +44 (0)1273 678123 Campus Miguel de Unamuno Ifj&250s&225g u. 6. [email protected] Salamanca Dr Anne Paoletti P&233cs 37007 Institut Curie 7624 Sharada Ramasubramyan ESP 26 rue d Ulm HUN University of Sussex +34-923-121644 PARIS +3672501573 Brighton [email protected] 75005 [email protected] East Sussex BN1 FRA GBR Dr Patricia Elena Rischitor +33 1 42346413 Dr Janni Petersen [email protected] University of Edinburgh [email protected] University of Manchester Kings Buildings Michael Smith Building Ms Soshila Ramayah Mayfield Road Mr Nigel Peat Oxford Road University of Wales Bangor Edinburgh Cancer Research UK Manchester Memorial Building EH9 3JR 44 Lincolns Inn Fields M13 9PT Deiniol Road GBR London GBR Bangor +44 0131 650 5366 WC2A 3PX +44 (0)0161 275 5512 Wales [email protected] GBR [email protected] LL57 2UW +44 (0)207269-3476 GBR [email protected] +44 (0)1248 382369 [email protected]

162 163 Ms Olivia Rissland Prof Paul Russell Dr Masamitsu Sato Miss Matylda Sczaniecka University of Oxford The Scripps Research Institute Cancer Research UK, London Research University of Edinburgh South Parks Road 10550 North Torrey Pines Road Institute Swann Building S5.08 Oxford OX1 3RE MB3 44 Lincolns Inn Fields Kings Buildings GBR La Jolla London Mayfield Road +44 (0)1865 275500 CA WC2A 3PX Edinburgh [email protected] 92037 GBR EH9 3JR USA +44 (0)2072693330 GBR Mr Ofer Rog +1 858 784-8273 [email protected] +44 (0)131 650 7083 Cancer Research UK [email protected] [email protected] 44 Lincolns Inn Fields Dr Kenneth Sawin London WC2A 3PX Mr Cagri Sakalar University of Edinburgh Mr Nadeem Shaikh GBR BIOTEC TU Dresden Swann Building, Mayfield Road National Institute for Medical Research +44 (0)2072693212 Biotechnologisches Zentrum Edinburgh The Ridgeway [email protected] TU Dresden EH9 3JR Mill Hill Dresden GBR London Mr Assen Roguev 01062 + (0)131-650-706 NW7 1AA BIOTEC TU-Dresden DEU [email protected] GBR Tatzberg 47-51 +4935146340101 +44 (0)20 8959 3666 Dresden [email protected] Dr Shelley Sazer [email protected] 01307 Baylor College of Medicine DEU Dr Itaru Samejima One Baylor Plaza Professor Kazuhiro Shiozaki +4935146340107 University of Edinburgh Houston University of California, Davis [email protected] Michael Swann Building 77030 One Shields Ave. Mayfield Road 7+1 13 798-4531 Davis Mrs Laura Roseaulin Edinburgh [email protected] 95616 Pasteur Institute EH9 3JR USA 25-28 rue du docteur Roux GBR Ms Christine Schmidt +1530752-7467 Paris +44 (0)131 650 7063 CRUK - London Research Institute [email protected] 75015 [email protected] Lincoln s Inn Fields Laboratories FRA 44 Lincolns Inn Fields Mr Indranil Sinha +33 140613686 Dr Yolanda Sanchez London Karolinska Institutet [email protected] Universidad de SalamancaCSIC WC2A 3PX S&246dert&246rns H&246gskola, Alfred Edificio Departamental, lab 231 GBR Nobels Alle 7, Mrs Maja Rothenberg Campus Miguel de Unamuno +44 (0)2072693229 Huddinge University of Bern Salamanca [email protected] 14189 Baltzer-Strasse 4 37007 SWE Bern ESP Dr K. Anke Schürer +4686084000 CH-3012 Bern +34-923-121589 Leibniz Institute for Age Research - [email protected] +41-31-631-4654 [email protected] Fritz Lipmann Institute [email protected] Beutenbergstrasse 11 Dr Srivani Sistla Dr Beatriz Santos Jena Institute for Molecular and Cell Biology Mrs Ana Elisa Rozalén CSIC University of Salamanca D-07745 61, Biopolis Drive Instituto de Biología Molecular y Celular Avd. Campo Charro sn Hab.223 DEU 05-01, PROTEOS de Cáncer CSIC-USAL Salamanca +49-3641-656137 Singapore Campus Miguel de Unamuno 37007 [email protected] 138673 Salamanca ESP SGP 37007 +34-923-121644 +65- 6586 95 57-58 ESP [email protected] [email protected] +34 923 29 48 10 [email protected]

164 165 Miss Kate Sloan Miss Abigail Stevenson Dr. Zhaohua Irene Tang Dr Yuko Tonami CRUK Paterson Institute for Cancer University of Oxford Claremont Colleges Nagoya City University Graduate Research South Parks Road 925 N. Mills Ave. School of Medicine Christie Hospital NHS Trust Oxford Claremont Colleges 1 Kawasumi, Mizuho-cho, Mizuho-ku Wilmslow Road OX1 3PS Claremont Nagoya Manchester GBR California Aichi M20 4BX +44 (0)1865 271234 CA 91711 467-8601 GBR [email protected] USA JPN +44 (0)1614463156 +1 909-607-9067 +81-52-853-8146 [email protected] Ms Geetanjali Sundaram [email protected] [email protected] Guha Centre for Genetic Engg. Miss Helen Tinline-Purvis Dr Hilary Snaith and Biotechnology Medical Research Council Dr Sylvie Tournier Wellcome Trust Centre for Cell Biology 35 Ballygunje Circular Road Harwell UMR5088 University of Edinburgh Ballygunje Science College Didcot 118 route de Narbonne Swann Building University of Calcutta OX11 0RD TOULOUSE Mayfield Road Kolkata GBR 31062 Edinburgh 700019 +44 (0)1235 841000 FRA EH9 3JR IND [email protected] +33 05 61 55 69 10 GBR +91-033- 24614983 [email protected] +44 (0)131 650 7063 [email protected] Dr Takashi Toda [email protected] Cancer Research UK Dr Mika Toya Prof Sasa Svetina London Research Institute Cancer Research UK, London Research Mrs Hela Soltani Faculty of Medicine, 44 Lincoln’s Inn Fields Institute Institute of Cancer Research, DNR University of Ljubljana London 44 Lincolns Inn Fields Ullernch. 70 Lipiceva 2 Ljubljana WC2A 3PX London Oslo 1000 GBR WC2A 3PX 0310 SVN +44 (0)207269 3535 GBR NOR +386 1 5437600 [email protected] +44 (0)207 269 3330 +47 22 93 40 00 [email protected] [email protected] [email protected] Dr Iva Tolic-Norrelykke Miss Virginia Tajadura MPI-CBG Max Planck Institute of Dr Phong Tran Dr Nathalie Spielewoy Universidad de SalamancaCSIC Molecular Cell Biology and Genetics University of Pennsylvania Cancer Research UK Edificio Departamental, lab. 231 Pfotenhauerstr. 108 421 Curie Blvd, Room 1009 44 Lincolns Inn Field Institute Campus Miguel de Unamuno Dresden Philadelphia London Salamanca37007 01307 PA WC2A 3PX ESP DEU 19104 GBR +34-923-121589 +493512102020 USA +44 (0)207 061 8080 [email protected] [email protected] +1 215 746 2755 [email protected] [email protected] Dr Kayoko Tanaka Dr Kazunori Tomita Mr Peter Stansfield Graduate School of Science, University Cancer Research UK Dr Michelle Trickey University of Dundee of Tokyo 44 Lincolns Inn Fields Marie Curie Research Institute Level 5 7-3-1 Hongo, Bunkyo-ku London Limpsfield Chart Ninewells Hospital and Medical School Tokyo WC2A 3PX Oxted Dundee 113-0033 GBR Surrey DD1 9SY JPN +44 (0)207-269-3415 RH8 0TL GBR +81-3-5841-4388 [email protected] GBR +44 (0)1382 660 111 ext 32566 [email protected] +44 (0)1883 722306 [email protected] [email protected]

166 167 Miss Ellen Tsang Dr Noelia Valbuena Mr. Sven K. Vogel Mr Boon-Yu Wee Genome Damage and Stability Centre Instituto de Biología Molecular y Celular Max Planck Institute MRC Radiation and Genome Stability Science Park Road del Cáncer Pfotenhauer Str. 108 Unit University of Sussex Campus Miguel de Unamuno Dresden Harwell Falmer Salamanca D-01307 Oxford BN1 9RQ 37007 DEU OX11 0RD GBR ESP +49-351-210-0 GBR +44 (0)1273 873118 +34923294810 [email protected] +44 (0)1235 841114 [email protected] [email protected] [email protected] Miss Huan Wang Ms. Tonje Tvegaard Miss Henar Valdivieso Queen Mary, University of London Mr Jikai Wen Institute for Cancer Research, DNR Universidad de SalamancaCSIC 333 Mile End Road The University of Birmingham Montebello Edificio Departamental. Campus London Room 601, School of Biosciences Oslo Unamuno E1 4NS The University of Birmingham 0310 Salamanca GBR Edgbaston NOR 37007 +44 (0)02078824784 Birmingham +47 22 93 40 00 ESP [email protected] B15 2TT [email protected] +34923121589 GBR [email protected] Dr Shao-Win Wang +44 (0)121 414 5406 Dr Frank Uhlmann University of Oxford [email protected] Cancer Reserach UK London Research Mr Luis Valente South Parks Road Institute Cancer Research UK Oxford Dr Simon Whitehall 44 Lincolns Inn Fields Telomere Biology Laboratory OX1 3PS University of Newcastle London 44 Lincolns Inn Fields GBR Catherine Cookson Building WC2A 3PX London +44 (0)1865 271212 Framlington Place GBR WC2A 3PX [email protected] Newcastle upon Tyne +44 (0)207 269 3024 GBR NE2 4HH [email protected] +44 (0)20 7242 0200 Mr Stephen Watt GBR [email protected] Wellcome Trust Sanger Institute +44 (0)191 222 6000 Dr Makoto Umeda Hinxton [email protected] Baylor College of Medicine Dr Vincent Vanoosthuyse Cambridge One Baylor Plaza Wellcome Trust Centre for Cell Biology CB101SA Dr Brian Wilhelm Houston University of Edinburgh GBR WTSI 77030 King’s Buildings +44 (0)1223 834244 Wellcome Trust Genome Campus USA Mayfield Road [email protected] Hinxton +1 713-798-5486 Edinburgh CB10 1SA [email protected] Ecosse Dr Felicity Watts GBR EH93JR University of Sussex +44 (0)1223 834244 Mrs Amy Unsworth GBR School of Life Sciences [email protected] Cancer Research UK +44 (0)131 650 7083 lab0131 650 7 Falmer 44 Lincolns Inn Fields [email protected] Brighton Dr Caroline Wilkinson London E Sussex Paterson Institute for Cancer Research WC2A 3PX Dr Usha Vijayraghavan BN1 9QG Wilmslow Road GBR Indian Institute of Science GBR Manchester +44 (0)20 72693330 C. V. Raman Avenue +44 (0)1273 678257 M20 4BZ [email protected] Bangalore [email protected] GBR Karnataka +44 (0)161 446 3171 560012 [email protected] IND + 91 80 23600168 [email protected]

168 169 Dr Jo Wixon Dr Hiro Yamano Notes John Wiley and Sons Ltd Marie Curie Research Institute 4 Pieces Court The Chart Waterbeach Oxted, Surrey Cambridgeshire CB5 9QL RH8 0TL GBR GBR +44 (0)1223 479052 +44 (0)1883 722306 [email protected] [email protected]

Ms Valerie Wood Dr Akira Yamashita Wellcome Trust Sanger Institute University of Tokyo Wellcome Trust Genome Campus 7-3-1, Hongo, Bunkyo-ku Hinxton Tokoyo Cambridge CB10 1SA 113-0033 +44 (0)1223 494954 JPN [email protected] +81-3-5841-4387 [email protected] Prof Anthony Wright Sodertorns Prof Mitsuhiro Yanagida Alfred Nobels Alle 7 Kyoto University Huddinge Yoshida-Honmachi SE-141 89 Sakyo-ku SWE Kyoto +4686084708 Kyoto [email protected] 606-8501 JPN Dr Pei-Yun Jenny Wu +81 75 753 4205 The Rockefellere University [email protected] 1230 York Avenue New York NY Dr Paul G. Young 10021 Queen s University USA R.2443 Biosciences Complex +1-212-327-8472 Kingston [email protected] Ontario K7L 3N6 Dr Jerome Wuarin CAN University of Dundee +1 613-533-6148 Level 5, Ninewells Hospital [email protected] Dundee DD1 9SY GBR Dr Xuefeng Zhu +44 (0)1382 632566 Karolinska Institutet [email protected] Stockholm SE-141 86 Mrs Yongtao Xue-Franzén SWE Sodertörns Univerisity College & +46-8-58583761 Karolinska Institute [email protected] Alfred Alle 3 Huddinge 147 70 SWE +6084000 [email protected]

170 171 Notes Notes

172 173 Notes Notes

174 175