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A Non-Canonical Function of Topoisomerase II in Disentangling Dysfunctional Telomeres

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A Non-Canonical Function of Topoisomerase II in Disentangling Dysfunctional Telomeres The EMBO Journal (2009) 28, 2803–2811 | & 2009 European Molecular Biology Organization | All Rights Reserved 0261-4189/09 www.embojournal.org TTHEH E EEMBOMBO JJOURNALOURN AL A non-canonical function of topoisomerase II in disentangling dysfunctional telomeres Thomas Germe, Kyle Miller1 and situated at the end of chromosomes and form the telomere Julia Promisel Cooper* (Ferreira et al, 2004; Rog and Cooper, 2008). Telomeres recruit and regulate telomerase, a reverse transcriptase that Telomere Biology Laboratory, Cancer Research, London, UK re-synthesizes the DNA lost due to the end replication problem (Bianchi and Shore, 2008). Telomeres also suppress The decatenation activity of topoisomerase II (Top2), their own non-homologous end joining (NHEJ), homologous which is widely conserved within the eukaryotic domain, recombination (HR) and checkpoint activation, promoting is essential for chromosomal segregation in mitosis. It is chromosome stability. less clear, however, whether Top2 performs the same In fission yeast, telomeric sequences are bound by Taz1, function uniformly across the whole genome, and whether which recruits Rap1. Together, Taz1 and Rap1 negatively all its functions rely on decatenation. In the fission yeast, regulate telomerase activity at telomeres, conferring telomere Schizosaccharomyces pombe, telomeres are bound by Taz1, length homoeostasis (Cooper et al, 1997; Kanoh and which promotes smooth replication fork progression Ishikawa, 2001), and inhibit NHEJ, protecting chromosomes through the repetitive telomeric sequences. Hence, repli- from lethal fusions (Ferreira and Cooper, 2001; Miller et al, cation forks stall at taz1D telomeres. This leads to telo- 2005). Fortunately for taz1D and rap1D cells, growing fission meric entanglements at low temperatures (p201C) that yeast spend little time in G1 and, therefore NHEJ frequencies cause chromosomal segregation defects and loss of viabi- are virtually zero in growing cultures, allowing taz1D and lity. Here, we show that the appearance of entanglements, rap1D cells to be viable (Ferreira and Cooper, 2001, 2004). and the resulting cold sensitivity of taz1D cells, is sup- Taz1 also has a positive effect on telomeric sequence main- pressed by mutated alleles of Top2 that confer slower tenance, as it favours replication fork (RF) progression through catalytic turnover. This suppression does not rely on the the telomere region. Hence, taz1 þ deletion results in stalling of decatenation activity of Top2. Rather, the enhanced pre- the RF at telomeres (Miller et al, 2006). This stalling is thought sence of reaction intermediates in which Top2 is clamped to lead to at least two different outcomes: rapid telomere loss in around DNA, promotes the removal of telomeric entangle- the absence of telomerase, and a high rate of telomere rear- ments in vivo, independently of catalytic cycle completion. rangement (Miller et al,2006;Roget al, 2009). Fission yeast We propose a model for how the clamped enzyme–DNA cells lacking Taz1 also show chromosome entanglements, complex promotes proper chromosomal segregation. distinct from NHEJ-mediated fusions, at cold growing tempera- The EMBO Journal (2009) 28, 2803–2811. doi:10.1038/ tures ( 201C). These entanglements cannot be resolved at emboj.2009.223; Published online 13 August 2009 p mitosis and therefore induce lethality; hence, taz1D cells are Subject Categories: genome stability & dynamics cold sensitive (Miller and Cooper, 2003). Such mitotic defects Keywords: DNA replication; Schizosaccharomyces pombe; are only observed in taz1D cells that have undergone the telomeres; topoisomerase II preceding S-phase at p201C (Miller and Cooper, 2003). Furthermore, although Rap1 shares many functions with Taz1, it is dispensable for the prevention of both entanglements and stalled telomeric RFs. Hence, the entanglements seem to Introduction arise as a by-product of stalled RF processing. Interestingly, chromosome entanglements and the cold sen- In proliferating eukaryotic cells, linear chromosomes face two sitivity of taz1D cells are suppressed by a mutation of the Top2 important problems. First, as the semi-conservative DNA gene (top2 þ ), top2-191 (Miller and Cooper, 2003). Top2 is a replication machinery is unable to completely replicate the homodimeric enzyme, widely conserved within the eukaryotic terminal DNA sequences, chromosome ends erode with each domain, able to create a transient DSB in a DNA duplex and to cell cycle. Second, free DNA ends can be recognized as promote the passage of another duplex through this ‘DNA gate’. double-strand breaks (DSBs) by the specialized DNA repair This strand passage activity confers the ability of Top2 to machineries whose activities can be deleterious at natural modify the topology of DNA molecules in vitro and in vivo.It chromosome ends. These problems are solved by various should be noted that Top2 is able to relax supercoiled DNA, and specialized proteins that bind the G-rich repetitive sequences to resolve or promote catenation between two DNA circles (Baldi et al, 1980; Hsieh and Brutlag, 1980; Liu et al,1980; *Corresponding author. Telomere Biology Laboratory, Cancer Research, Goto and Wang, 1982) (Figure 1A). 44 Lincoln’s Inn Fields, London WC2A 3PX, UK. Top2 is essential in eukaryotes for chromosomal conden- Tel.: þ 44 20 7269 3415; Fax: þ 44 20 7269 3258; E-mail: [email protected] sation and segregation (Holm et al, 1985; Uemura et al, 1987; 1Present address: The Wellcome Trust and Cancer Research Gurdon Adachi et al, 1991; Shamu and Murray, 1992). It is generally Institute and the Department of Zoology, University of Cambridge, accepted that its strand passage activity is required during Tennis Court Road, Cambridge CB2 1QN, UK mitosis to decatenate residual intertwining that persists be- Received: 2 March 2009; accepted: 13 July 2009; published online: tween sister chromatids after the completion of semi-conser- 13 August 2009 vative DNA replication. As a consequence, yeast cells bearing &2009 European Molecular Biology Organization The EMBO Journal VOL 28 | NO 18 | 2009 2803 Topoisomerase II and dysfunctional telomeres T Germe et al A Transported DNA duplex “Gate” DNA duplex 2× ADP + 2Pi Clamp closure Gate duplex cleavage Clamp re-opening 2× ATP Transported duplex passage DNA gate re-sealing ‘Cleavable complex’ B ATP binding DNA binding and hydrolysis and cleavage 423 802 835 N C KAY Figure 1 Mutant top2 alleles used in this study. (A) Diagram of Top2 catalytic cycle. The Top2 dimer (light blue semi-circles) binds a ‘gate’ DNA duplex, and changes conformation on ATP binding, undergoing ‘closure’ and simultaneously inducing a transient DSB in the gate duplex, to which Top2 remains bound at either end as it transports a second duplex through the DSB. The purple squares represent the covalent bonds formed transiently between the 50 phosphates flanking the DSB and tyr 835 of each Top2 subunit. Clamp re-opening requires ATP hydrolysis and release. (B) Simplified scheme of the Top2 polypeptide chain, pointing out the target sites mutagenized during the course of this study. Ala 802 is mutagenized to valine in the top2-191 allele. The Y835F mutant is designed to kill the cleavage activity of Top2, and the K423Q mutant is designed to inhibit ATPase activity and clamp re-opening. catalytically dead Top2 mutations fail to properly segregate Results chromosomes during mitosis and die. In addition, cells The top2-191 allele acts in a dominant manner harbouring extra-chromosomal circular DNA molecules ac- to suppress taz1D cold sensitivity cumulate catenated circles following replication (Sundin Top2 can either catenate or decatenate DNA circles in vitro et al, 1980; DiNardo et al, 1984). Top2 is also able to relieve the topological constraints induced by RF and transcriptional (Hsieh and Brutlag, 1980; Goto and Wang, 1982). It is therefore progression, but this activity is not essential, as topoisome- conceivable that Top2 could either generate or remove chro- mosome entanglements in vivo. If Top2 generated the entangle- rase I also can perform the same (Brill et al, 1987). þ Studies in higher eukaryotes have led to the generation of ment of taz1D telomeres, a mutation in top2 that suppressed an additional idea that Top2 is the main component of a this defect would be expected to be a loss-of-function, recessive ‘chromosomal scaffold’ (Adolphs et al, 1977; Paulson and allele. Conversely, if Top2 removed or prevented entanglement, Laemmli, 1977). Top2 has been suggested to anchor DNA to a suppressing allele would be expected to be a gain-of-function this scaffold by linking it to specific regions of the genome and dominant allele. We assessed whether top2-191 is recessive (Mirkovitch et al, 1984; Adachi et al, 1989), and to have a by integrating it under control of the inducible nmt81 promoter function in re-setting the spatial organization of replication (an attenuated allele of the strong inducible nmt1 promoter) at origins during mitosis (Lemaitre et al, 2005). However, the the lys1 locus (Figure 2A), in a strain that also expresses wild- þ existence and precise function of this scaffold, and the role of type (wt) endogenous Top2. Surprisingly, deletion of taz1 þ Top2 in chromosomal organization, are yet to be definitively fails to induce cold sensitivity in the top2 lys1:top2-191 back- þ understood. ground (Figure 2B). In contrast, deletion of taz1 induces cold The observation that taz1D cells harbouring the top2-191 sensitivity in a strain bearing an empty nmt81 cassette inte- allele
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