Replication stress as a source of telomere recombination during replicative senescence in Saccharomyces cerevisiae

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during replicative senescence in Saccharomyces cerevisiae

Marie-Noëlle Simon, Dmitri Churikov, Vincent Géli

To cite this version:

Marie-Noëlle Simon, Dmitri Churikov, Vincent Géli. Replication stress as a source of telomere recombination during replicative senescence in Saccharomyces cerevisiae. FEMS Yeast Research, Oxford University Press (OUP), 2016, 16 (7), pp.fow085. �10.1093/femsyr/fow085�. �hal-03085000�

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Replication stress as a source of telomere recombination during replicative senescence in Saccharomyces cerevisiae.

Marie-Noëlle Simon*, Dmitri Churikov and Vincent Géli

Centre de Recherche en Cancérologie de Marseille (CRCM), Inserm, U1068, Marseille, F-13009, France, CNRS, UMR7258, Marseille, F-13009, Institut Paoli-Calmettes, Marseille, F-13009, France, Aix-Marseille

University, UM 105, F-13284, Marseille, France. Equipe labellisée Ligue

*Correspondence to: marie-noelle.simon@inserm.fr

ABSTRACT

Replicative senescence is triggered by short unprotected telomeres that arise in the absence of telomerase. In addition, telomeres are known as difficult regions to replicate due to their repetitive G-rich sequence prone to secondary structures and tightly bound non-histone proteins. Here we review accumulating evidence that telomerase inactivation in yeast immediately unmasks the problems associated with replication stress at telomeres. Early after telomerase inactivation, yeast cells undergo successive rounds of stochastic DNA damages and become dependent on recombination for viability long before the bulk of telomeres are getting critically short. The switch from telomerase to recombination to repair replication stress-induced damage at telomeres creates telomere instability, which may drive further genomic alterations and prepare the ground for telomerase-independent immortalization observed in yeast survivors and in 15% of human cancer.

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INTRODUCTION

In eukaryotic cells the physical ends of linear chromosomes have to be distinguished from accidental DNA double-strand breaks (DSBs). For this purpose, cells have evolved telomeres, the sophisticated structures composed of tandem G-rich DNA repeats bound by a complex of proteins called shelterin that cap chromosome ends. The G-rich strand of the telomeric DNA extends beyond the duplex region making a 3’ end overhang, which serves as substrate for telomerase, a reverse transcriptase that extends the G-rich strand in the 5’ to 3’

direction (Blackburn and Collins 2011). Telomeres prevent DNA damage checkpoint activation (Denchi and de Lange 2007) and undesirable DNA repair activities at the ends of chromosomes. In the budding yeast, Saccharomyces cerevisiae, telomeres are formed by an approximately 300 bp -long array of irregular TG1-3 repeats (McEachern and Blackburn 1994) and a 10-15 bases of 3’ end G-overhang (Wellinger et al. 1993). The capping function of shelterin has been a subject of excellent reviews both in yeast (Dewar and Lydall 2012;

Ribeyre and Shore 2012) and mammals (Giraud-Panis et al. 2010; O'Sullivan and Karlseder 2010). Briefly, in budding yeast, duplex telomeric DNA is bound by Rap1 that serves as a platform to recruit Rif1 and Rif2 (Wotton and Shore 1997). Rap1-Rif1-Rif2 limits the access of exonucleases, binding of replication protein A (RPA) and prevents checkpoint activation (Kupiec, 2014). In addition, Rap1 is essential to block telomere fusions through NHEJ (Pardo and Marcand 2005). The single-stand termini of telomeres are bound by Cdc13, a subunit of the CST (Cdc13/Stn1/Ten1) complex that assumes diverse functions at telomeres including an essential capping function that prevents degradation of the 5’ ends (Churikov et al. 2013;

Greetham et al. 2015). Finally, the yKu70/Ku80 complex contributes to telomere capping by inhibiting resection by Exo1 as it does at DSBs (Bonetti et al. 2010; Vodenicharov et al.

2010). In mammals another level of protection is provided by the t-loop formed by invasion of the double-stranded telomeric region by the G-strand overhang (Griffith et al. 1999;

Doksani et al. 2013). This structure does not appear to occur in wild-type yeast because the limited length of the G-overhangs. Nevertheless, a fold back structure of telomeric heterochromatin has been described in yeast (de Bruin et al. 2000) that reduces the susceptibility to nucleolytic degradation (Poschke et al. 2012).

A unique sequence of telomeric DNA prone to fold into G-quadruplexes (Smith et al.

2011) and stable protein-DNA complexes cause pausing of the replication forks and their slow progression through subtelomeres and telomeres (Ivessa et al. 2002; Makovets et al.

2004). Telomere replication thus requires specific helicases and repair proteins (Ivessa et al.

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2002; Paeschke et al. 2010; Hardy et al. 2014). These proteins are particularly important due to the unidirectional replication of telomeres where no converging forks from a neighboring origin can compensate for defective replication (Gilson and Geli 2007). Nevertheless recombination proteins are not required to maintain telomeres in cells with active telomerase (Shampay and Blackburn 1988). Indeed, intra- or inter-telomere recombination is rare in wild type cell as shown by the low level of recombination foci at telomeres detected in vivo (Khadaroo et al. 2009). This suggests that replication stress-induced damages at telomeres are either limited or compensated by the action of the telomerase in wild type cells. Consistently, it has been shown in S. pombe that stalled replication forks form substrates for telomerase (Dehe et al. 2012).

Evidence is mounting that the first consequence of inactivating telomerase is to unmask the problems that stem from replication stress at telomeres well before the appearance of short uncapped telomeres (Khadaroo et al. 2009; Xie et al. 2015; Xu et al. 2015; Jay et al. 2016). In this review we summarize recent data supporting the idea that in the absence of repair by telomerase, cells become dependent on recombination to repair replication-stress induced damage at telomeres. The switch from telomerase to recombination has a profound impact on telomere (and potentially entire genome) stability, which is manifested by dependency of cell viability on multiple DNA repair pathways. Yeast cells that naturally do not experience the absence of telomerase readily reveal the telomere replication problems, when rendered telomerase-negative, and thus will continue to bring new insight into the mechanisms of telomere replication and recombination at telomeres.

Homologous recombination plays multiple roles at telomeres in the absence of telomerase.

The telomerase enzyme compensates for the loss of terminal DNA which stems from the inability of DNA polymerase to complete the synthesis of the lagging strand at the very end of DNA molecule (Olovnikov 1973) and from 5’ to 3’ exonucleolytic processing of the telomeres generated by the leading strand DNA synthesis (Larrivee et al. 2004; Soudet et al.

2014). Upon inactivation of telomerase in yeast, telomeres shorten on average 3-5 bp per population doubling (PD) (Marcand et al. 1999). Proliferation rate is not immediately affected, but it begins to decline after about 20-30 PDs until it reaches a minimum that is referred to as crisis driven by telomere erosion (Figure 1). When telomeres reach a critical length, cells

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enter an irreversible G2/M arrest due to activation of Mec1, an ortholog of the ataxia telangiectasia- and Rad3-related (ATR) phosphatidylinositol 3 kinases (Enomoto et al. 2002;

IPjma and Greider 2003). It is thought that very short telomeres lose their capping function and are detected as and processed largely as DSBs. Following permanent activation of the checkpoint, few cells can recover growth capacity by regenerating functional telomeres through recombination-based mechanisms. Two types of survivors are described in yeast based on genetic requirements and telomere organization (Le et al. 1999; Chen et al. 2001) (Figure 1). The formation of both types appears to involve break-induced replication (BIR) since both depend on Rad52 and Pol32 (Lydeard et al. 2007). Type I survivors rely on the strand invasion activity of Rad51 and show an amplification of the Y’ subtelomeric elements together with the interstitial telomeric sequences (ITS). Type II survivors carry long heterogeneous in length telomere repeats and depend on Rad59 (Chen et al. 2001) as well as checkpoint activation (Grandin and Charbonneau 2007a) and proteins required for resection of the 5’ end of DSBs (Costelloe et al. 2012; Hardy et al. 2014).

The idea of replicative senescence being triggered by telomere attrition that results exclusively from the “end replication problem” was challenged by unprecedented heterogeneity of the population of senescing cells even when they all descended from a single telomerase minus cell (and thus with the same heritage of telomere length). Recent technical advances in the use of microfluidic devises made it possible to directly observe the dynamics of cell divisions in individual cell lineages upon telomerase loss (Xie et al. 2015; Xu et al.

2015). These studies revealed that heterogeneity of the cell cycle duration is already evident within the first 10 divisions without telomerase (Xie et al. 2015). Stochastically some lineages encounter transient cell cycle arrest followed by resumption of divisions, a pattern totally distinct from the short-telomere driven permanent G2/M arrest observed later during terminal senescence (Xu et al. 2015). Unfortunately, microfluidic devises do not yet allow relating transient arrests to specific telomere defects. Nevertheless, much can be learned from analyses of the mutants. For example, deletion of RAD51 substantially decreases the frequency of early lags in cell cycle progression (Xu et al. 2015) suggesting that Rad51-dependent recombination is responsible for them. This observation is in agreement with relocalization of the essential HR factor Rad52 into telomere foci as early as 20 PDs after inactivation of telomerase (Khadaroo et al. 2009). Premature senescence of the rad52 and rad51 cells in the absence of telomerase (Chen et al. 2001; Lundblad and Blackburn 1993) suggests that these telomere recombination events are essential to sustain proliferation early after telomerase inactivation.

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Telomere repair activities that precede survivor formation rely at least in part on non- reciprocal translocation of terminal telomeric repeats (Figure 2A). Sequencing of telomere DNA revealed unequal inter- and intra-telomeric exchanges that occur before the onset of survivor formation (Teixeira et al. 2004; Lee et al. 2007; Abdallah et al. 2009; Kozak et al.

2010; Chang et al. 2011). In yeast, individual telomeres differ in their precise sequence because telomerase RNA template can be aligned to the G-strand overhang in multiple registers (Forstemann et al. 2000). As a consequence, TG1-3 tracts are well suited for homeologous or microhomology-based recombination. Consistently, inactivation of mismatch repair, which suppresses recombination between sequences that contain mismatches (Spell and Jinks-Robertson 2003), delays senescence (Rizki and Lundblad 2001).

Recently, we analyzed in details another mode of telomere repair that operates in pre- senescent cells (Churikov et al. 2014). We showed that in the absence of telomerase, short terminal TG_1-3repeats engage in pairing with ITSs between subtelomeric X and Y’ elements and initiate Pol32-dependent BIR, which results in non-reciprocal translocation of the entire Y’ element and terminal TG_1-3tract from the donor chromosome (Figure 2B). This repair event is associated with a transient arrest of cell cycle progression but the checkpoint function of Mec1 is not essential for efficient translocation. Y’ acquisition has genetic requirements that are distinct from those described for Y’ amplification in type I post senescence survivors.

First, Y’ translocation is mainly promoted by Rad52 associated with Rad59, while Rad51 has only a modest effect in this pathway (Churikov et al. 2014). Second, the Bloom RecQ helicase homolog Sgs1 favors Y’ translocation while it has an inhibitory role on recombination between Y’ elements in wild type cells (Watt et al. 1996). The dependency on Rad59 and positive effect of Sgs1 are in keeping with recombination between homeologous sequences, but how the G-rich overhang anneals to internal TG1-3 tracts that are mainly double stranded remains to be characterized. Possibly, partial opening of the double helix during replication, especially when the fork pauses at subtelomeric region (Makovets et al. 2004), creates permissive condition for the initiation of recombination.

The involvement of Rad51 in recombination between TG1-3 tracts is uncertain. Yeast Rad51 has a preference for GT-rich sequences (Tracy et al. 1997), but at telomeres, single- stranded TG1-3 repeats are bound by Cdc13 (Mitton-Fry et al. 2002). It remains unclear whether Rad51, even assisted by Rad52 and other mediators, would efficiently strip Cdc13 from the single-stranded TG_1-3 repeats. One possibility is that Rad51 may support telomere replication rather than recombination in pre-senescing cells. Indeed, Rad51 is now recognized as one of the factors that escort replication fork (Carr and Lambert 2013), where it protects

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the nascent DNA at stalled replication forks from Mre11-dependent degradation and facilitates replication fork restart (Hashimoto et al. 2010; Gonzalez-Prieto et al. 2013).

Telomerase inactivation aggravates replication stress at the telomeres.

The experiments conducted in the laboratory of Teresa Teixeira showed that the onset of senescence is similar in two populations of cells descended from one mitosis after inactivation of telomerase but the pattern of transient arrests differs between the two sister populations (Xu et al. 2013; Xu et al. 2015). As the starting populations shared the same set of telomeres, these observations suggest that the terminal arrest is determined by initial telomere length while the transient arrests are not (Xu et al. 2015). Therefore, transient arrests might occur in response to stochastic damage that is independent of gradual telomere shortening. In fact, the frequency of transient cell cycle arrest is increased in a strain with elongated as compared to wild-type telomeres (Xu et al., 2015), indicating that the probability of stochastic damage may positively correlate with telomere length. Alterations of the dynamics of DNA replication have emerged as a major source of genome instability (Magdalou et al. 2014). This raises the possibility that loss of telomerase activity further aggravates replication stress that is already present within telomeric regions in wild-type cells (Geronimo and Zakian 2016). In this speculative model, telomerase might repair damage resulting from replication stress at telomeres either by elongating accidentally broken telomere (Figure 3A) or the G-rich single strand exposed at the regressed replication fork (Figure 3B). In the absence of telomerase, either accidental breakage or enzymatic processing of the stalled fork would generate short recombinogenic telomeres (Figure 3).

The checkpoint response to eroded telomeres share many common partners with the response to DSBs by inducing a cascade of events that leads to the activation of Rad53, Mec1, Mec3, Chk1 and Dun1 that are all required for the terminal cell cycle arrest in G2/M (Enomoto et al. 2002; IPjma and Greider 2003; Grandin and Charbonneau 2007a).

Nevertheless the phosphorylation of Rad53 upon inactivation of telomerase depends on both Rad9 and Mrc1 (Grandin et al. 2005), two adaptor proteins implicated respectively in the response to DSB and replication stress (Vialard et al. 1998; Alcasabas et al. 2001; Osborn and Elledge 2003). Furthermore, inactivation of telomerase immediately creates dependence of the viability on the DNA damage adaptors (Jay et al. 2016). Importantly, this dependence can be alleviated by elevation of dNTPs pools (Jay et al. 2016) that facilitates replication

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indicating that telomerase is indeed essential for resolution of the replication stress at telomeres. Interestingly, MRC1, in contrast to other DDR genes, is not required for the cell cycle arrest elicited by eroded telomeres and instead, its deletion accelerates senescence (Grandin and Charbonneau 2007b). Moderate replication stress linked to a reduction of fork speed lead to recruitment of mediators that are crucial for fork protection but do not block S- phase progression and mitotic onset (Koundrioukoff et al. 2013). It is thus possible that cells are under pressure of moderate replicative stress that requires Mrc1 and recombination to be settled in the absence of telomerase. In line with this possibility, the checkpoint deficient mrc1^AQ allele or deletion of the ATM-homolog TEL1, but not RAD9, exacerbates the precocious heterogeneity of cell cycle duration during pre-senescence (Xie et al. 2015).

The importance of counter-acting replication stress to delay the onset of senescence is further supported by the phenotype of various mutants affecting the repair of replication- generated damages. In budding yeast, fork restart depends on the Smc5/6 and Rtt101-Mms1- Mms22 (Luke et al. 2006; Duro et al. 2008; Irmisch et al. 2009). Notably Smc5/6 is required to slow senescence in telomerase defective cells (Chavez et al. 2010; Noel and Wellinger 2011). smc5-6 mutant cells accumulate recombination intermediates at telomeres in the absence of telomerase suggesting commitment of template switch mechanism to bypass a stall-induced lesion (Chavez et al. 2010). Similarly, MMS1 is required to maintain viability before the onset of senescence (Abdallah et al. 2009; Noel and Wellinger 2011). RAD5 deletion as well accelerates senescence (Fallet et al. 2014) suggesting that the error-free DNA damage tolerance (DDT) and template switch are engaged to prevent loss of viability due to unresolved replication stress at telomeres. Involvement of template switch in bypassing replication stress during senescence is consistent with the observations described above including the accumulation of X-shaped structures at telomeres during senescence and the dissolution of these sister chromatid junctions by Sgs1 (Lee et al. 2007; Branzei et al. 2008;

Karras and Jentsch 2010). It is also consistent with dependence of pre-senescing cells on DNA polymerase Pol (Lydeard et al. 2010; Fallet et al. 2014).

Alternatively, stalled forks might also be stabilized by Rad5-dependent regression (see Figure 3B) although it remains unclear whether regression of the replication forks occurs in checkpoint-proficient S. cerevisiae (Sogo et al. 2002).

Although the pathways responsible for resolution of the replication stress are likely to be shared between the telomeres and other regions of the genome, unique properties of the telomere and subtelomere sequences could impact on both the response to replication stress and the outputs of the repair. For example, it remains unclear to which extent RPA and CST

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would intersect or compete to bind internal and terminal TG_1-3sequences when single stranded (Gao et al. 2007; Lewis et al., 2013). Considering the central role of RPA not only in checkpoint activation but also in the choice of the repair pathway (Marechal and Zou 2015), very little is known whether CST shares or interferes with some of these functions. Another difference that is likely to impact on the resolution of replication stress at telomeres is their terminal position. One could imagine that uncoupling of leading and lagging strand synthesis could generate a long terminal G-tail (Audry et al., 2015) that would favor invasion and copy of either sister chromatid or other telomere in a process involving either Rad51- or Rad59- dependent BIR. This possibility is supported by the synthetic growth defect between rad52 and rad5 deletions in the absence of telomerase (Fallet et al. 2014).

Factors influencing telomere replication in the absence of telomerase

As mentioned above, telomeres and subtelomeres are intrinsic obstacles for replication-forks in both yeast (Ivessa and Zakian 2002; Makovets et al. 2004) and mammals (Sfeir et al. 2009).

The precise hitch to telomere replication is not fully defined. In S. cerevisiae, the telomeric repeats are assembled into a compact nucleoprotein structure maintained by the Rap1 as well as Sir and Rif proteins. Subtelomeric nucleosomes at X and Y’ elements also differ from nucleosomes in most of the others regions of the genome by the binding of Sir proteins that impose transcriptional repression and hetero-chromatization (Kueng et al. 2013). Tight binding of Rap1 to telomere repeats likely creates a barrier to fork progression (Makovets et al. 2004). In addition, due to the uni-directionality of telomere replication, the G-rich strand is replicated by the lagging strand machinery ie by the DNA polymerase  that tends to stall on telomeric G-rich repeats (Lormand et al. 2013; Audry et al. 2015). Concomitantly, chromatin structure might be another actor of either the replication stress or the consecutive recombination at telomeres. Senescence is accompanied by an increase in subtelomeric H4K16 acetylation and inactivation of the H4K16 acetylase Sas2 delays senescence and stimulates HR-mediated telomere elongation (Kozak et al. 2010).

It is possible that modification of the telomeric structure as telomeres shorten in the absence of telomerase further aggravates the replication stress at telomeres. It has been shown that the S. pombe Taz1 protein, which binding is expected to be decreased at short telomeres, facilitates telomere replication (Dehe et al., 2012). Another factor with a potential to impair the passage of the replication fork through subtelomere and telomere sequences is the

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interference between the replication fork and the presence of TERRA transcripts. TERRA are long non-coding heterogeneous in length RNA transcribed in the 5’-3’ direction from the subtelomeric region toward chromosome ends in yeast as well as in mammals (Maicher et al.

2014) and which transcription is increased at short telomeres in both S. cerevisiae and S.

pombe (Iglesias et al. 2011; Pfeiffer and Lingner 2012; Cusanelli et al. 2013; Moravec et al., 2016). The level of TERRA transcripts is very low in wild type cells. TERRA transcription is repressed by the Sir complex and Rif1/2 at X-only telomeres but mainly by Rap1-associated Rif1/2 at telomeres containing Y’ elements (Iglesias et al. 2011). The level of TERRA is further controlled by the 5’-3’ RNA exonuclease Rat1 (Luke et al. 2008) and RNase H suggesting that TERRA forms RNA-DNA hybrids with the C-rich telomere strand (Iglesias et al. 2011). Increased production of TERRA upon telomere shortening might be the consequence of a loss of Rif repression associated with the decreased number of telomere repeats. Accumulation of TERRA RNA/DNA hybrids correlates with an increased frequency of telomere recombination in the absence of telomerase (Balk et al. 2013; Yu et al. 2014).

Conversely, overexpression of RNH1 decreases telomeric recombination (Balk et al. 2013), an effect in line with the ability of Rnh1 to decrease Transcription-Associated recombination (TAR) (Huertas and Aguilera 2003; El Hage et al. 2010). Further work will be needed to understand the mechanistic consequences of the presence of R-loops at telomeres. It would be for example crucial to determine whether the presence of R-loops enhances the replication fork pause at telomeres and subtelomeres and whether it is causatively linked to an accumulation of G-rich ssDNA.

Spatial regulation of telomere recombination during survivor formation

The fact that type I and II survivors appear at an estimated frequency of 10^-4 or less (Lundblad and Blackburn 1993) suggests that they result from rare recombination events rather than dedicated pathways. Type I survivor formation has specific genetic requirements.

Rad59-dependent translocation of Y’ elements onto X-only telomeres constitutes a first step of their production (Churikov et al. 2014), while spreading and amplification of Y’ elements at all telomeres next requires Rad52, Rad51, Rad54 and Rad55 and Pol32 (Le et al. 1999;

Lydeard et al. 2007). This suggests that type I survivors are formed by successive rounds of Rad51-catalyzed strand invasion followed by migration of the D-loop and DNA synthesis up to the end of the chromosome, essentially by multiple rounds of Rad51-dependent BIR. In keeping with BIR mechanism, type I survivor formation also requires Pif1 (Hu et al. 2013),

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which is required for long-range synthesis via bubble migration (Saini et al. 2013; Wilson et al. 2013).

Recombination dependent telomere elongation (RTE) observed in type II survivors has drawn more attention because it shares similarity with the ALT (Alternative Lengthening of Telomere) phenotype that accounts for about 15% of human cancers (Pickett and Reddel 2015). RTE required the strand annealing function of Rad52 reinforced by Rad59 as well as components of the BIR pathway (McEachern and Haber 2006; Lydeard et al. 2010). In contrast to Y’ amplification, which is initiated early after telomerase inactivation, the sudden amplification of TG1-3 repeats takes place only after senescence (Chang et al. 2011; Teng et al.

2000). Most probably, RTE occurs at eroded telomeres that are recognized and processed as DSBs, an assumption in accord with type II recombination dependence on activation of the DNA damage checkpoint (Grandin and Charbonneau 2007a). Homologous recombination at DSBs is initiated by resection of the DNA 5’-ends initiated by MRX (Mre11-Rad50-Xrs2) and Sae2 and extended by the exonuclease Exo1 and/or by the combined action of Dna2 and Sgs1 (Symington, 2014). Inactivation of any of these proteins strongly affects formation of type II survivors (Huang et al. 2001; Bertuch and Lundblad 2004; Hardy et al. 2014; Johnson et al. 2001; Maringele and Lydall 2002).

Another level of regulation of telomere recombination is related to the spatial control of DNA damage repair. In wild type cells, telomeres cluster in 6 to 8 foci at the nuclear periphery in the recombination prohibitive zone enriched in Sir proteins during most of the cell cycle (Gotta et al. 1996). Perinuclear telomere localization depends on two redundant pathways involving Sir4 and the yKu70/80 complex, which interact with the nuclear membrane anchored proteins Esc1 and Mps3, respectively (Hediger et al. 2002; Taddei et al.

2004; Taddei et al. 2010). Although the telomeres remain by and large clustered during senescence, a subset of eroded recombinogenic telomeres revealed by foci of colocalization of Cdc13 and Rad52, relocate from their membrane anchor to the Nuclear Pore Complex (NPC) (Khadaroo et al. 2009). Specialization of the nuclear compartments for specific repair and/or protection has emerged as a major actor of HR regulation (Geli and Lisby 2015). It has long been though that NPCs play a role in tethering hard-to-repair DNA damage, such as DSBs without a donor template for HR, collapsed replication forks (Nagai et al. 2008), and eroded telomeres (Khadaroo et al. 2009). This has been recently extended to tandem CAG repeats prone to secondary structures, which localize to NPCs during their replication in repeat array size-dependent manner (Su et al. 2015). Similarly to other types of damages (Nagai et al.

2008; Su et al. 2015; Horigome et al. 2016), localization of telomeres to NPC, as well as

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formation of type II survivors, depends on the small ubiquitin-like modifier (SUMO)-targeted ubiquitin ligase (STUbL) Slx5-Slx8 (Churikov et al. 2016). Accordingly, SUMOylation of proteins bound to telomeres increases as telomeres shorten in the absence of telomerase and correlates with the recruitment of STUbL to telomeres (Churikov et al. 2016). Since Slx5- Slx8 interacts with the pore protein Nup84 (Nagai et al. 2008), it became apparent that it may tether the telomeres to NPC. The current model for spatial regulation of type II recombination is depicted in the figure 4. A challenging but important task for the future will be to identify the proteins, which SUMOylation regulates NPC localization and the ones that are deSUMOylated or degraded at the pores. Although multiple proteins are likely involved, one prominent candidate is RPA, which (1) binds resected telomeres, (2) is SUMOylated in response to DNA damage and telomere shortening (Cremona et al. 2012; Psakhye and Jentsch 2012; Churikov et al. 2016), and (3) constitutively interacts with Slx5 (Churikov et al. 2016), therefore capable to stabilize transient interactions of the STUbL with polySUMOylated substrates.

Perhaps most enigmatic aspect of type II recombination is the nature of the template for telomere elongation that occurs at a time where the reserve of telomeric sequence is exhausted.

The finding that telomere localization to NPC promotes type II recombination implies that such template might be enriched at the NPCs. The presence of extrachromosomal telomeric circles (t-circles) in type II survivors (Larrivee and Wellinger 2006), K. lactis mutants (Basenko et al. 2009) and ALT cancer cells (Henson et al. 2009) led to a model of roll-and- spread mechanism for fast amplification of telomeric repeats (Natarajan and McEachern, 2002; McEachern and Haber 2006). It is thus tempting to speculate that the t-circles are trapped to the NPC as it has been shown for other circular byproducts of recombination (Denoth-Lippuner et al. 2014).

Concluding remarks.

Unlike most human somatic cells, budding yeast constitutively express telomerase, and therefore, did not evolve specific adaptations to sustain telomere maintenance in the absence of telomerase. As a result, inactivation of telomerase via genetic manipulations in budding yeast not only recapitulates the process of replicative senescence observed in human somatic cells, but might also reveal those problems with telomere maintenance that are either counteracted by specific adaptive mechanisms in human cells or are masked by the action of telomerase in yeast. One such problem appears to be related to replication stress at telomeres,

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which is revealed immediately after inactivation of telomerase and concomitantly with the dependence of yeast growth on checkpoint mediators, recombination factors and post- replication repair. Therefore, telomerase itself is essential for repair of damage resulting from replication stress at telomeres, whereas multiple repair mechanisms are likely required to compensate for the absence of telomerase. In fact, unleashed recombination observed at the telomeres in yeast growing in the absence of telomerase appears to stem from the unresolved replication stress. Similar relationship between replication stress and recombination at telomeres likely exist in human cancers that bypassed senescence barrier and activated recombination-based telomere maintenance mechanism known as ALT and possibly in some genetic diseases linked to accelerated aging. Thus, telomerase-negative yeast serve as a useful model for deciphering the causes and consequences of replication stress at telomeres and its bearing on telomere maintenance by recombination.

FUNDING

VG is supported by the ‘‘Ligue Nationale Contre le Cancer’’ and by ‘‘L’Institut National Du Cancer, « TELOCHROM and PLBIO14-012 ». Dmitri Churikov is supported by the SIRIC (INCa-DGOS-Inserm 6038 grant).

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Figure 1. Replicative senescence in Saccharomyces cerevisiae. (A) In yeast, subtelomeric regions contain repeated sequences called X and Y’ elements. The core X element is present at all chromosome ends with limited sequence and size variations and is proximal to either terminal TG1-3 repeats or Y’ elements that are present in two third of the telomeres as 1 to 4 in tandem repeats (Louis and Haber 1992). Upon inactivation of telomerase, the telomere sequences shorten on average 3-5bp per population doubling (PD). Depending on the telomere length in the initial cell, telomeres reach a critical length (about 70 to 100bp) in about 50 to 60 PDs. Uncapped telomeres activate the DNA damage checkpoint leading to a permanent arrest in G2/M. G2/M arrested cells, often referred to as “monsters”, are characterized by a dumbbell morphology and the localization of the undivided nucleus at the bud neck. Among the population of G2/M arrested cells, few cells succeed in reconstituting functional telomeres by homologous recombination, recover growth capacity and give rise to post-senescence survivors. (B) A schematic curve of the growth capacity of the cells in liquid culture upon inactivation of telomerase is shown. The growth rate of the population starts to decline about 20 PDs after telomerase inactivation until it reaches a minimum after 50-60 PDs where the presence of “monsters” is at its maximum in the cell population (senescence).

Videomicroscopy of telomerase-minus cells shows an enrichment of cells undergoing transient cell cycle arrest during the initial period after telomerase inactivation (Xu et al.

2015). This period, defined here as pre-senescence, lasts until the cell population accumulates the highest proportion of monsters due to permanent G2/M arrest and is immediately followed by the appearance of survivors. C) Telomere organization in the survivors. Type I survivors are characterized by an amplification of Y’ elements separated by ITS and a very short array of terminal TG1-3 repeats. Type II survivors carry long (up to 10 kb) and heterogeneous extensions of telomere repeats.

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Figure 2: Models of the short telomere repair in the absence of telomerase. Short telomeres generated as a result of either breakage or enzymatic cleavage of the replication forks can be repaired by BIR after completion of the replication. Depending on the location of the donor (terminal or interstitial TG_1-3 repeats), BIR at the telomeres may result either in the elongation of the terminal TG1-3 tract (A) or in the non-reciprocal translocation of the Y’

element along with the terminal TG1-3 repeats on the recipient short telomere (B). Since heteroduplexes between TG1-3 tracts are expected to contain multiple mismatches, they are likely to be targeted by mismatch repair and prone to unwinding by Sgs1. Therefore multiple rounds of alignment, synthesis and dissociation could be expected during BIR at telomeres.

The involvement of Rad51 in recombination between short TG1-3 repeats remains uncertain (see text for details).