DOCSLIB.ORG

Werner Syndrome Protein and DNA Replication

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Werner Syndrome Protein and DNA Replication International Journal of Molecular Sciences Review Werner Syndrome Protein and DNA Replication Shibani Mukherjee, Debapriya Sinha, Souparno Bhattacharya, Kalayarasan Srinivasan, Salim Abdisalaam and Aroumougame Asaithamby * Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; [email protected] (S.M.); [email protected] (D.S.); [email protected] (S.B.); [email protected] (K.S.); [email protected] (S.A.) * Correspondence: [email protected]; Fax: +1-214-648-5995 Received: 17 September 2018; Accepted: 25 October 2018; Published: 2 November 2018 Abstract: Werner Syndrome (WS) is an autosomal recessive disorder characterized by the premature development of aging features. Individuals with WS also have a greater predisposition to rare cancers that are mesenchymal in origin. Werner Syndrome Protein (WRN), the protein mutated in WS, is unique among RecQ family proteins in that it possesses exonuclease and 30 to 50 helicase activities. WRN forms dynamic sub-complexes with different factors involved in DNA replication, recombination and repair. WRN binding partners either facilitate its DNA metabolic activities or utilize it to execute their specific functions. Furthermore, WRN is phosphorylated by multiple kinases, including Ataxia telangiectasia mutated, Ataxia telangiectasia and Rad3 related, c-Abl, Cyclin-dependent kinase 1 and DNA-dependent protein kinase catalytic subunit, in response to genotoxic stress. These post-translational modifications are critical for WRN to function properly in DNA repair, replication and recombination. Accumulating evidence suggests that WRN plays a crucial role in one or more genome stability maintenance pathways, through which it suppresses cancer and premature aging. Among its many functions, WRN helps in replication fork progression, facilitates the repair of stalled replication forks and DNA double-strand breaks associated with replication forks, and blocks nuclease-mediated excessive processing of replication forks. In this review, we specifically focus on human WRN’s contribution to replication fork processing for maintaining genome stability and suppressing premature aging. Understanding WRN’s molecular role in timely and faithful DNA replication will further advance our understanding of the pathophysiology of WS. Keywords: cancer; DNA double-strand repair; premature aging; post-translational modification; protein stability; replication stress; Werner Syndrome; Werner Syndrome Protein 1. Introduction Werner Syndrome (WS) is an autosomal recessive genetic disorder that causes symptoms of premature aging and is accompanied by a higher risk of cancer [1–3]. Individuals with WS show a greater predisposition to diseases usually observed in older age, such as arteriosclerosis, cataracts, osteoporosis, and type II diabetes mellitus [4–6]. In addition, individuals with WS are more susceptible to rare cancers that are mesenchymal in origin [1,2]. Myocardial infarction and cancer are the most common causes of death among patients with WS [2]. Primary cells derived from these patients exhibit elevated levels of chromosomal translocations, inversions, and deletions of large segments of DNA, and they have a high spontaneous mutation rate [7,8]. Additionally, WS fibroblasts have a markedly shorter replicative life span than age-matched controls in culture [4,9]. Most WS cases have been linked to mutations in a single gene, the Werner syndrome gene (WRN), which is located on chromosome 8 [10]. Int. J. Mol. Sci. 2018, 19, 3442; doi:10.3390/ijms19113442 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 2 of 17 Int. J. Mol. Sci. 2018, 19, 3442 2 of 18 WRN, the protein defective in WS, belongs to the RecQ helicase family. The human genome containsWRN, five the RecQ protein genes: defective RecQ1, in Bloom WS, belongssyndrome to protein the RecQ (BLM), helicase WRN, family. RecQ4, The and human RecQ5. genome WRN containsis a 1432 five amino RecQ acid-long genes: RecQ1, multifunctional Bloom syndrome protein protein that comprises (BLM), WRN, four RecQ4, distinct and functional RecQ5. WRN domains is a 1432(Figure amino 1). acid-longWRN has multifunctional an exonuclease protein (E84) that domain comprises (38–236 four distinct aa) and functional a WRN-WRN domains interaction (Figure1). WRN(multimerization has an exonuclease or oligomerization) (E84) domain domain (38–236 (251–333 aa) and aa) a WRN-WRNin the N-terminal interaction region. (multimerization It has adenosine ortriphosphatase oligomerization) (ATPase), domain helicase (251–333 (K577) aa) in (558–724 the N-terminal aa), and region. RecQ ItC-terminal has adenosine (RQC) triphosphatase (749–899 aa) (ATPase),domains helicasein the middle (K577) region (558–724 and aa), a helicase-and-ribonuclease and RecQ C-terminal (RQC) D-C-terminal (749–899 aa) (HRDC) domains domain in the middle (940– region1432 aa) and in athe helicase-and-ribonuclease C-terminal region. Though D-C-terminal the crystal (HRDC) structure domain for full-length (940–1432 WRN aa) in is the not C-terminal available region.yet, crystal Though structures the crystal of the structure exonuclease for full-length and HRDC WRN domains is not have available been solved. yet, crystal The structurescrystal structure of the exonucleaseof the exonuclease and HRDC domain domains (1–333 have aa) been at solved.2.0 angstrom The crystal resolution structure showed of the exonucleasea ring of six domain WRN (1–333exonuclease aa) at 2.0domains, angstrom the resolution perfect size showed to slip a around ring of sixa DNA WRN helix, exonuclease with their domains, binding the and perfect catalytic size tosites slip oriented around ainward DNA helix, toward with the their encircled binding andDNA catalytic [11]. This sites orientedstudy furt inwardher revealed toward thethat encircled WRN’s 2+ 2+ DNAexonuclease [11]. This domain study furtherpossesses revealed Mg thatand WRN’s Mn exonucleasebinding sites, domain which possesses help modulate Mg2+ and WRN’s Mn2+ bindingexonuclease sites, whichactivities help [11]. modulate Additionally, WRN’s exonucleasefull-length activitiesWRN forms [11]. Additionally,a trimer [12], full-length and the WRNWRN formsexonuclease a trimer construct [12], and (1–333 the WRN aa) exonuclease forms a trimer construct when (1–333 purified aa) forms by gel a trimer filtration when analysis purified and by gelhomohexamers filtration analysis upon and interaction homohexamers with DNA upon or interaction with Proliferating with DNA cell or with nuclear Proliferating antigen cell(PCNA), nuclear as antigenexamined (PCNA), by atomic as examined force microscope by atomic [13,14]. force Subsequently, microscope [13 Perry,14]. et Subsequently, al. (2010) identified Perry etthe al. 250–333 (2010) identifiedamino acids the as 250–333 being aminonot only acids responsible as being not for onlyWRN’ responsibles homomultimerization, for WRN’s homomultimerization, but also critical for but its alsoexonuclease critical for processivity its exonuclease [15]. The processivity HRDC domain’s [15]. The cr HRDCystal structure domain’s revealed crystal structurethat this domain revealed exists that thisas a domain monomer exists in assolution a monomer and inhas solution weak DNA and has binding weak DNAability binding in vitro ability [16].in However, vitro [16]. the However, HRDC thedomain HRDC is known domain to is interact known with to interact many differen with manyt proteins, different which proteins, suggests which that suggestsWRN’s DNA that WRN’sbinding DNAspecificity binding is dictated specificity by is another dictated domain. by another Thus, domain. structural Thus, analyses structural of analyses N- and ofC-terminal N- and C-terminal domains domainshave provided have provided a wealth a of wealth information of information about WRN’s about WRN’sexonuclease exonuclease activities activities and its andability its abilityto act on to actdifferent on different DNA DNAstructures. structures. FigureFigure 1. SchematicSchematic showing showing different different functional functional domain domains,s, exonuclease exonuclease (E84), (E84), helicase helicase (K577) (K577) active activesites, and sites, DNA-PKcs and DNA-PKcs (S440 and (S440 S467), and ATM S467), (S1058, ATM S1141 (S1058, and S1141 S1292), and ATR S1292), (S991, ATR S1411, (S991, T1152 S1411, and T1152S1256) and and S1256) CDK1 and(S1133) CDK1 phosphorylation, (S1133) phosphorylation, and acetylation and acetylation(K366, K887, (K366, K1117, K887, K1127, K1117, K1389 K1127, and K1389K1413) and sites K1413) in WRN. sites TDD-Trimerization in WRN. TDD-Trimerization (oligomerization/multimerization) (oligomerization/multimerization) domain (250–333aa); domain A- (250–333aa);acidic repeats A-acidic (2X27; 424–477 repeats aa); (2X27; RQC-RecQ 424–477 C-term aa); RQC-RecQinal (749–899 C-terminal aa); NLS-nuclear (749–899 localization aa); NLS-nuclear signal; localizationaa-amino acid; signal; black aa-amino dotted lines acid; denote black dottedacetylat linesion events; denote solid acetylation red arrows events; indicate solid redDNA-PKcs- arrows indicatemediated DNA-PKcs-mediated phosphorylation sites; phosphorylation solid dark blue sites; lines solid represent dark blue ATM-mediated lines represent phosphorylation ATM-mediated phosphorylationevents; dotted orange events; arrows dotted represent
Load more

Recommended publications
  • Complex Interacts with WRN and Is Crucial to Regulate Its Response to Replication Fork Stalling
    Oncogene (2012) 31, 2809–2823 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE The RAD9–RAD1–HUS1 (9.1.1) complex interacts with WRN and is crucial to regulate its response to replication fork stalling P Pichierri, S Nicolai, L Cignolo1, M Bignami and A Franchitto Department of Environment and Primary Prevention, Istituto Superiore di Sanita`, Rome, Italy The WRN protein belongs to the RecQ family of DNA preference toward substrates that mimic structures helicases and is implicated in replication fork restart, but associated with stalled replication forks (Brosh et al., how its function is regulated remains unknown. We show 2002; Machwe et al., 2006) and WS cells exhibit that WRN interacts with the 9.1.1 complex, one of enhanced instability at common fragile sites, chromo- the central factors of the replication checkpoint. This somal regions especially prone to replication fork interaction is mediated by the binding of the RAD1 stalling (Pirzio et al., 2008). How WRN favors recovery subunit to the N-terminal region of WRN and is of stalled forks and prevents DNA breakage upon instrumental for WRN relocalization in nuclear foci and replication perturbation is not fully understood. It has its phosphorylation in response to replication arrest. We been suggested that WRN might facilitate replication also find that ATR-dependent WRN phosphorylation restart by either promoting recombination or processing depends on TopBP1, which is recruited by the 9.1.1 intermediates at stalled forks in a way that counteracts complex in response to replication arrest. Finally, we unscheduled recombination (Franchitto and Pichierri, provide evidence for a cooperation between WRN and 2004; Pichierri, 2007; Sidorova, 2008).
    [Show full text]
  • Structure and Functional Implications of the Human Rad9-Hus1-Rad1
    Supplemental Material can be found at: http://www.jbc.org/content/suppl/2009/06/17/C109.022384.DC1.html ACCELERATED PUBLICATION This paper is available online at www.jbc.org THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 31, pp. 20457–20461, July 31, 2009 © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Structure and Functional onto DNA lesion sites by Rad17-RFC2–5 (which consists of one large subunit, Rad17, and four small subunits, RFC2–5), where Implications of the Human it triggers the activation of the cell cycle checkpoint (3, 4). Rad9-Hus1-Rad1 Cell Cycle Moreover, the 9-1-1 complex can also directly participate in DNA repair via physical association with many factors involved *□S Checkpoint Complex in base excision repair (BER), translesion synthesis, homolo- Received for publication, May 18, 2009, and in revised form, June 5, 2009 gous recombination, and mismatch repair pathways (5–9). Published, JBC Papers in Press, June 17, 2009, DOI 10.1074/jbc.C109.022384 Although both the 9-1-1 and the PCNA complexes perform Min Xu‡§, Lin Bai‡§, Yong Gong‡, Wei Xie‡§, Haiying Hang‡1, ‡2 critical functions in eukaryotic cells with predicted similar and Tao Jiang structures (10), their specific roles are distinct. First, the 9-1-1 ‡ From the National Laboratory of Biomacromolecules, Institute of complex is a DNA damage sensor in the cell cycle checkpoint Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101 and the §Graduate University of Chinese Academy but does not function as a scaffold for the major DNA replica- of Sciences, 19A Yuquan Road, Shijingshan District, Beijing 100039, China tion factors; however, PCNA plays exactly the opposite role (1, 11).
    [Show full text]
  • Checkpoint-Dependent and Independent Roles of the Werner
    12628–12639 Nucleic Acids Research, 2014, Vol. 42, No. 20 Published online 28 October 2014 doi: 10.1093/nar/gku1022 Checkpoint-dependent and independent roles of the Werner syndrome protein in preserving genome integrity in response to mild replication stress Giorgia Basile1,2,†, Giuseppe Leuzzi1,2,†, Pietro Pichierri2,3 and Annapaola Franchitto1,2,* 1Section of Molecular Epidemiology, Department of Environment and Primary Prevention, Istituto Superiore di Sanita,` Viale Regina Elena, 299-00161 Rome, Italy, 2Genome Stability Group, Istituto Superiore di Sanita,` Viale Regina Elena, 299-00161 Rome, Italy and 3Section of Experimental and Computational Carcinogenesis, Department of Environment and Primary Prevention, Istituto Superiore di Sanita,` Viale Regina Elena, 299-00161 Rome, Italy Received July 22, 2014; Revised October 08, 2014; Accepted October 09, 2014 ABSTRACT agents perturbing DNA replication (1–3). Based on WRN enzymatic activities and substrate preferences in vitro,it Werner syndrome (WS) is a human chromosomal in- is thought that WRN may participate in multiple DNA stability disorder associated with cancer predispo- metabolic pathways in vivo, such as replication, recombi- sition and caused by mutations in the WRN gene. nation and repair (4–6). WRN has been implicated in the WRN helicase activity is crucial in limiting breakage correct and fruitful recovery from replication fork arrest at common fragile sites (CFS), which are the prefer- (1,7–8). Given a coordinate action of WRN and DNA poly- ential targets of genome instability in precancerous merase delta in the replication of DNA substrates contain- lesions. However, the precise function of WRN in re- ing G4 tetraplex structures (9), the crucial requirement of sponse to mild replication stress, like that commonly the WRN helicase activity in maintaining common frag- used to induce breaks at CFS, is still missing.
    [Show full text]
  • Regulation of ATR Substrate Selection by Rad17-Dependent Loading of Rad9 Complexes Onto Chromatin
    Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Regulation of ATR substrate selection by Rad17-dependent loading of Rad9 complexes onto chromatin Lee Zou,1,2 David Cortez,1,2 and Stephen J. Elledge1–4 1Verna and Marrs McLean Department of Biochemistry and Molecular Biology, 2Howard Hughes Medical Institute, and 3Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA Cells respond to DNA damage by activating a network of signaling pathways that control cell cycle progression and DNA repair. Genetic studies in yeast suggested that several checkpoint proteins, including the RFC-related Rad17 protein, and the PCNA-related Rad1–Rad9–Hus1 protein complex might function as sensors of DNA damage. In this study, we show that the human Rad17 protein recruits the Rad9 protein complex onto chromatin after damage. Rad17 binds to chromatin prior to damage and is phosphorylated by ATR on chromatin after damage but Rad17’s phosphorylation is not required for Rad9 loading onto chromatin. The chromatin associations of Rad17 and ATR are largely independent, which suggests that they localize to DNA damage independently. Furthermore, the phosphorylation of Rad17 requires Hus1, suggesting that the Rad1–Rad9–Hus1 complex recruited by Rad17 enables ATR to recognize its substrates. Our data are consistent with a model in which multiple checkpoint protein complexes localize to sites of DNA damage independently and interact to trigger the checkpoint-signaling cascade. [Key Words: Rad17; Rad1–Rad9–Hus1 complex; ATR; Chk1; checkpoint] Received October 2, 2001; revised version accepted November 26, 2001.
    [Show full text]
  • Eukaryotic DNA Replication & Genome Maintenance
    Eukaryotic DNA Replication & Genome Maintenance Session 1 NEW APPROACHES AND PERSPECTIVES MONDAY 9/9/2013, 7:30 PM A. van Oijen / K. Labib # lname Title Talk Length 1 van Oijen Single-molecule studies of DNA replication 12 2 Remus Origin specificity during budding yeast DNA replication in vitro 12 3 Urban Mapping DNA replication origins in the human genome 12 4 Mechali DNA replication origins profiling—Genetic and epigenetic features, and relationships with cell 12 identity 5 Ni Subunits of the origin recognition complex bind BubR1 and are essential for kinetochore function 12 during mitosis in human cells 6 Labib Regulation of the replisome by ubiquitin and SUMO 12 7 Koren Random replication of the inactive X chromosome 12 8 Xie Replication-dependent DNA fragility and adaptive evolution in natural populations 12 9 Raghuraman Replication origins and the generation of palindromic amplifications in yeast 12 10 Dutta MicroDNAs and microdeletions—Genomic instability in vertebrate cells 12 Session 2 ORIGIN SELECTION AND LICENSING TUESDAY 9/10/2013, 9:00 AM M. Aladjem / S. Bell # lname Title Talk Length 11 Aladjem Regulatory interactions governing replication initiation patterns in human cells 12 12 Petryk Sequencing of Okazaki fragments allows determination of replication fork polarity and origin 12 location and efficiency in the human genome 13 Debatisse Chk1 or Rad51 deficiency perturbs replication dynamics via p53R2-mediated modulation of dNTP 12 availability 14 Liachko Dual modes of DNA replication initiation in the methylotrophic yeast
    [Show full text]
  • DNA Distress: Just Ring 9-1-1 [9,11]
    CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector Dispatch R733 DNA Distress: Just Ring 9-1-1 [9,11]. Interestingly, the Rad9 carboxyl terminus plays an important role in activation of the DNA damage The Rad9–Hus1–Rad1 checkpoint clamp (9-1-1) is a central player in the cellular checkpoint, which halts cell cycle response to DNA damage; three groups have determined the crystal structure progression to provide time for of 9-1-1, providing new insight into its loading mechanism and association with DNA repair [1]. Phosphorylation of DNA damage checkpoint and repair enzymes. this domain is required for binding and recruiting TopBP1 to sites of Michael Kemp and Aziz Sancar PCNA and 9-1-1 require the DNA damage [17] in order to activity of heteropentameric clamp activate the DNA damage response The genome is constantly exposed to loading complexes termed replication kinase ATR [18]. Unfortunately, to cellular metabolites and exogenous factor C (RFC) and Rad17-RFC, obtain recombinant 9-1-1 protein agents that induce lesions in DNA respectively, which bind to, open, suitable for crystallization, all capable of causing mutation, cancer, and clamp the complexes around three groups [9–11] used a truncated or cell death. In response to such DNA [13]. Whereas PCNA is loaded form of Rad9 lacking this region, damage, eukaryotic cells activate onto 30-primer–template junctions and hence the structures provide signaling pathways that promote DNA by the canonical RFC made up of no insight into the role of the Rad9 repair, allow bypass of lesions during RFC1–5, 9-1-1 is instead preferentially carboxyl terminus in the checkpoint DNA replication, and halt cell-cycle loaded onto 50-recessed ends by response.
    [Show full text]
  • Cdna Cloning and Gene Mapping of Human Homologs For
    GENOMICS 54, 424–436 (1998) ARTICLE NO. GE985587 cDNA Cloning and Gene Mapping of Human Homologs for Schizosaccharomyces pombe rad17, rad1, and hus1 and Cloning of Homologs from Mouse, Caenorhabditis elegans, and Drosophila melanogaster Frank B. Dean,*,1 Lubing Lian,† and Mike O’Donnell*,‡ *The Rockefeller University and ‡The Howard Hughes Medical Institute, 1230 York Avenue, New York, New York 10021; and †Myriad Genetics, Inc., 390 Wakara Way, Salt Lake City, Utah 84108 Received July 13, 1998; accepted September 17, 1998 INTRODUCTION Mutations in DNA repair/cell cycle checkpoint genes can lead to the development of cancer. The cloning of The progression of a eukaryotic cell through the human homologs of yeast DNA repair/cell cycle check- stages of the cell cycle can be arrested if the events of point genes should yield candidates for human tumor the previous stage of the cell cycle, such as DNA rep- suppressor genes as well as identifying potential tar- lication, have not been completed or if the DNA has gets for cancer therapy. The Schizosaccharomyces sustained some type of damage. The controls on cell pombe genes rad17, rad1, and hus1 have been identi- cycle progression are termed checkpoints (Hartwell fied as playing roles in DNA repair and cell cycle and Weinert, 1989), and they are able to detect checkpoint control pathways. We have cloned the whether the processes of the individual stages of the cDNA for the human homolog of S. pombe rad17, cell cycle have been completed and whether the DNA is RAD17, which localizes to chromosomal location 5q13 intact or in need of repair.
    [Show full text]
  • Opening Pathways of the DNA Clamps Proliferating Cell Nuclear Antigen and Rad9-Rad1-Hus1 Xiaojun Xu, Carlo Guardiani, Chunli Yan and Ivaylo Ivanov*
    10020–10031 Nucleic Acids Research, 2013, Vol. 41, No. 22 Published online 12 September 2013 doi:10.1093/nar/gkt810 Opening pathways of the DNA clamps proliferating cell nuclear antigen and Rad9-Rad1-Hus1 Xiaojun Xu, Carlo Guardiani, Chunli Yan and Ivaylo Ivanov* Department of Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, GA 30302, USA Received July 8, 2013; Revised August 15, 2013; Accepted August 16, 2013 ABSTRACT are DNA clamps, which act as platforms for the assembly of core replisomal components on DNA (6). In this Proliferating cell nuclear antigen and the checkpoint capacity, DNA clamps are essential in cellular activities clamp Rad9-Rad1-Hus1 topologically encircle DNA ranging from DNA replication, repair of DNA damage, and act as mobile platforms in the recruitment of chromatin structure maintenance, chromosome segrega- proteins involved in DNA damage response and tion, cell-cycle progression and apoptosis (2,6–12). cell cycle regulation. To fulfill these vital cellular PCNA is a recognized master coordinator of multiple functions, both clamps need to be opened and pathways controlling replication and DNA damage loaded onto DNA by a clamp loader complex—a response. The clamp has a toroidal shape that wraps process, which involves disruption of the DNA around DNA and topologically links the replicating clamp’s subunit interfaces. Herein, we compare DNA polymerase to its substrate. During lagging strand the relative stabilities of the interfaces using the DNA synthesis, PCNA organizes three core replication proteins—DNA polymerase, Flap endonuclease 1 and molecular mechanics PoissonÀBoltzmann solvent DNA ligase I (13,14). Similarly, the checkpoint clamp accessible surface method.
    [Show full text]
  • Roles of the Checkpoint Sensor Clamp Rad9-Rad1-Hus1 (911)-Complex and the Clamp Loaders Rad17-RFC and Ctf18-RFC in Schizosaccharomyces Pombe Telomere Maintenance
    REPORT REPORT Cell Cycle 9:11, 2237-2248; June 1, 2010; © 2010 Landes Bioscience Roles of the checkpoint sensor clamp Rad9-Rad1-Hus1 (911)-complex and the clamp loaders Rad17-RFC and Ctf18-RFC in Schizosaccharomyces pombe telomere maintenance Lyne Khair,1 Ya-Ting Chang,1 Lakxmi Subramanian,1 Paul Russell2 and Toru M. Nakamura1,* 1Department of Biochemistry and Molecular Genetics; University of Illinois at Chicago; Chicago, IL USA; 2Departments of Molecular Biology and Cell Biology; The Scripps Research Institute; La Jolla, CA USA Key words: telomere, checkpoint, Rad9-Rad1-Hus1, Rad17, Ctf18 Abbreviations: 911, Rad9-Rad1-Hus1; ChIP, chromatin immunoprecipitation; DBD, DNA binding domain; DSBs, double- strand breaks; dsDNA, double-stranded DNA; HU, hydroxyurea; MRN, Mre11-Rad50-Nbs1; NHEJ, non-homologous end- joining; OB fold, oligonucleotide/oligosaccharide-binding fold; PCNA, proliferating cell nuclear antigen; PFGE, pulsed-field gel electrophoresis; PIKK, phosphatidyl inositol 3-kinase-like kinase; RFC, replication factor C; SHREC, Snf2/Hdac-containing repressor complex; TERT, telomerase reverse transcriptase; UV, ultraviolet While telomeres must provide mechanisms to prevent DNA repair and DNA damage checkpoint factors from fusing chromosome ends and causing permanent cell cycle arrest, these factors associate with functional telomeres and play critical roles in the maintenance of telomeres. Previous studies have established that Tel1 (ATM) and Rad3 (ATR) kinases play redundant but essential roles for telomere maintenance in fission yeast. In addition, the Rad9-Rad1-Hus1 (911) and Rad17-RFC complexes work downstream of Rad3 (ATR) in fission yeast telomere maintenance. Here, we investigated how 911, Rad17-RFC and another RFC-like complex Ctf18-RFC contribute to telomere maintenance in fission yeast cells lacking Tel1 and carrying a novel hypomorphic allele of rad3 (DBD-rad3), generated by the fusion between the DNA binding domain (DBD) of the fission yeast telomere capping proteinP ot1 and Rad3.
    [Show full text]
  • Gene Section Review
    Atlas of Genetics and Cytogenetics in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS Gene Section Review HUS1 (HUS1 checkpoint homolog (S. pombe)) Amrita Madabushi, Randall C Gunther, A-Lien Lu Department of Biochemistry and Molecular Biology, School of Medicine, University of Maryland, 108 North Greene Street, Baltimore, Maryland 21201, USA (AM, RCG, ALL) Published in Atlas Database: September 2010 Online updated version : http://AtlasGeneticsOncology.org/Genes/HUS1ID40899ch7p12.html DOI: 10.4267/2042/45024 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology Identity Protein HGNC (Hugo): HUS1 Description Location: 7p12.3 Amino acids: 280. Molecular weight: 31.7 kDa. Hus1 Note: NCBI accession: NM_004507.2; NP_004498.1. is a component of the 9-1-1 cell cycle checkpoint complex that plays a critical role in sensing DNA DNA/RNA damage and maintaining genomic stability. Description Expression Found in all tissues. 15464 bp; 8 exons. Transcription Localisation Nucleus and cytoplasm. In discrete nuclear foci upon The transcribed mRNA has 2143 bp and the coding DNA damage. region is 840 bp, encodes a 280 amino acids, 31691 Da protein. Pseudogene None. Figure 1. HUS1 gene adapted from NCBI database Homo sapiens chromosome 7, GrCh37 primary reference assembly with kilobases from the telomere of p-arm on bottom. The exons 1-8 are represented by boxes with transcribed and untranscribed sequences in pink and orange, respectively. The exon numbers are labeled on top. The grey arrowhead symbolizes the direction of transcription and the arrows show the ATG and the stop codons, respectively.
    [Show full text]
  • An Essential Role for Drosophila Hus1 in Somatic and Meiotic DNA Damage Responses
    1042 Research Article An essential role for Drosophila hus1 in somatic and meiotic DNA damage responses Uri Abdu1,*,‡, Martha Klovstad2,*, Veronika Butin-Israeli1, Anna Bakhrat1 and Trudi Schüpbach2 1Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University, Beer-Sheva, 84105, Israel 2Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA *These authors contributed equally to this work ‡Author for correspondence (e-mail: [email protected]) Accepted 18 January 2007 Journal of Cell Science 120, 1042-1049 Published by The Company of Biologists 2007 doi:10.1242/jcs.03414 Summary The checkpoint proteins Rad9, Rad1 and Hus1 form a We subsequently studied the role of hus1 in activation of clamp-like complex which plays a central role in the DNA- the meiotic checkpoint and found that the hus1 mutation damage-induced checkpoint response. Here we address the suppresses the dorsal-ventral pattering defects caused by function of the 9-1-1 complex in Drosophila. We decided to mutants in DNA repair enzymes. Interestingly, we found focus our analysis on the meiotic and somatic requirements that the hus1 mutant exhibits similar oocyte nuclear defects of hus1. For that purpose, we created a null allele of hus1 as those produced by mutations in DNA repair enzymes. by imprecise excision of a P element found 2 kb from the These results demonstrate that hus1 is essential for the 3Ј of the hus1 gene. We found that hus1 mutant flies are activation of the meiotic checkpoint and that hus1 is also viable, but the females are sterile.
    [Show full text]
  • Hus1 in Responding to Endogenous and Exogenous Stresses in Vivo
    ROLES FOR THE DNA DAMAGE CHECKPOINT GENE HUS1 IN RESPONDING TO ENDOGENOUS AND EXOGENOUS STRESSES IN VIVO A Dissertation Presented to the Faculty of the Graduate School of Cornell University In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Gabriel Balmus, D.V.M. January 2013 © 2013 Gabriel Balmus, D.V.M. ii ROLES FOR THE DNA DAMAGE CHECKPOINT GENE HUS1 IN RESPONDING TO ENDOGENOUS AND EXOGENOUS STRESSES IN VIVO Gabriel Balmus, D.V.M., Ph.D. Cornell University 2013 The DNA damage response (DDR) represents the primary line of defense against exogenous and endogenous genotoxic agents that threaten the stability of our genomes. The ATM and ATR pathways are central to the response to DNA-damage and their understanding can bring important information in the fight against inborn disease and cancer. While the roles for the ATM pathway in DDR are well understood, the lack of a model organism for the ATR pathway has impeded its understanding. Here I show how we use Hus1, a component of the RAD9-RAD1-HUS1 heterotrimeric clamp and a vital member of the ATR pathway, to deregulate the ATR pathway and dissect its importance in development and disease. This was done by engineering in mice a Hus1 allelic series by combining the wild-type (Hus1+) with a hypomorphic (Hus1neo) or null (Hus1Δ1) alleles. As opposed to the germline constitutive deletion that leads to embryonic lethality, Hus1 hypomorphic mice are born at expected frequencies and show no overt phenotype but have increased levels of genomic instability. Thus they have enough Hus1 to deal with physiologic stress and could be used for our research.
    [Show full text]