Recombination at subtelomeres is regulated by physical distance, double‐strand break resection and chromatin status
2017; Springer Nature; Volume: 36; Issue: 17 Linguagem: Inglês
10.15252/embj.201796631
ISSN1460-2075
AutoresAmandine Batté, Clémentine Brocas, Hélène Bordelet, Antoine Hocher, Myriam Ruault, Adouda Adjiri, Angela Taddei, Karine Dubrana,
Tópico(s)Chromosomal and Genetic Variations
ResumoArticle28 July 2017free access Transparent process Recombination at subtelomeres is regulated by physical distance, double-strand break resection and chromatin status Amandine Batté Institute of Molecular and Cellular Radiobiology, CEA/DRF, Fontenay-aux-Roses cedex, France Inserm U967, Fontenay-aux-Roses cedex, France Université Paris-Diderot et Université Paris-Sud, UMR967, Fontenay-aux-Roses cedex, France Search for more papers by this author Clémentine Brocas Institute of Molecular and Cellular Radiobiology, CEA/DRF, Fontenay-aux-Roses cedex, France Inserm U967, Fontenay-aux-Roses cedex, France Université Paris-Diderot et Université Paris-Sud, UMR967, Fontenay-aux-Roses cedex, France Search for more papers by this author Hélène Bordelet Institute of Molecular and Cellular Radiobiology, CEA/DRF, Fontenay-aux-Roses cedex, France Inserm U967, Fontenay-aux-Roses cedex, France Université Paris-Diderot et Université Paris-Sud, UMR967, Fontenay-aux-Roses cedex, France Search for more papers by this author Antoine Hocher Institut Curie, PSL Research University, CNRS, UMR3664, Paris, France Sorbonne Universités, UPMC Univ Paris 06, CNRS, UMR3664, Paris, France Search for more papers by this author Myriam Ruault Institut Curie, PSL Research University, CNRS, UMR3664, Paris, France Sorbonne Universités, UPMC Univ Paris 06, CNRS, UMR3664, Paris, France Search for more papers by this author Adouda Adjiri Institut Curie, PSL Research University, CNRS, UMR3664, Paris, France Sorbonne Universités, UPMC Univ Paris 06, CNRS, UMR3664, Paris, France Search for more papers by this author Angela Taddei Institut Curie, PSL Research University, CNRS, UMR3664, Paris, France Sorbonne Universités, UPMC Univ Paris 06, CNRS, UMR3664, Paris, France Search for more papers by this author Karine Dubrana Corresponding Author [email protected] orcid.org/0000-0002-1269-2564 Institute of Molecular and Cellular Radiobiology, CEA/DRF, Fontenay-aux-Roses cedex, France Inserm U967, Fontenay-aux-Roses cedex, France Université Paris-Diderot et Université Paris-Sud, UMR967, Fontenay-aux-Roses cedex, France Search for more papers by this author Amandine Batté Institute of Molecular and Cellular Radiobiology, CEA/DRF, Fontenay-aux-Roses cedex, France Inserm U967, Fontenay-aux-Roses cedex, France Université Paris-Diderot et Université Paris-Sud, UMR967, Fontenay-aux-Roses cedex, France Search for more papers by this author Clémentine Brocas Institute of Molecular and Cellular Radiobiology, CEA/DRF, Fontenay-aux-Roses cedex, France Inserm U967, Fontenay-aux-Roses cedex, France Université Paris-Diderot et Université Paris-Sud, UMR967, Fontenay-aux-Roses cedex, France Search for more papers by this author Hélène Bordelet Institute of Molecular and Cellular Radiobiology, CEA/DRF, Fontenay-aux-Roses cedex, France Inserm U967, Fontenay-aux-Roses cedex, France Université Paris-Diderot et Université Paris-Sud, UMR967, Fontenay-aux-Roses cedex, France Search for more papers by this author Antoine Hocher Institut Curie, PSL Research University, CNRS, UMR3664, Paris, France Sorbonne Universités, UPMC Univ Paris 06, CNRS, UMR3664, Paris, France Search for more papers by this author Myriam Ruault Institut Curie, PSL Research University, CNRS, UMR3664, Paris, France Sorbonne Universités, UPMC Univ Paris 06, CNRS, UMR3664, Paris, France Search for more papers by this author Adouda Adjiri Institut Curie, PSL Research University, CNRS, UMR3664, Paris, France Sorbonne Universités, UPMC Univ Paris 06, CNRS, UMR3664, Paris, France Search for more papers by this author Angela Taddei Institut Curie, PSL Research University, CNRS, UMR3664, Paris, France Sorbonne Universités, UPMC Univ Paris 06, CNRS, UMR3664, Paris, France Search for more papers by this author Karine Dubrana Corresponding Author [email protected] orcid.org/0000-0002-1269-2564 Institute of Molecular and Cellular Radiobiology, CEA/DRF, Fontenay-aux-Roses cedex, France Inserm U967, Fontenay-aux-Roses cedex, France Université Paris-Diderot et Université Paris-Sud, UMR967, Fontenay-aux-Roses cedex, France Search for more papers by this author Author Information Amandine Batté1,2,3,‡, Clémentine Brocas1,2,3,‡, Hélène Bordelet1,2,3, Antoine Hocher4,5, Myriam Ruault4,5, Adouda Adjiri4,5,†, Angela Taddei4,5,‡ and Karine Dubrana *,1,2,3,‡ 1Institute of Molecular and Cellular Radiobiology, CEA/DRF, Fontenay-aux-Roses cedex, France 2Inserm U967, Fontenay-aux-Roses cedex, France 3Université Paris-Diderot et Université Paris-Sud, UMR967, Fontenay-aux-Roses cedex, France 4Institut Curie, PSL Research University, CNRS, UMR3664, Paris, France 5Sorbonne Universités, UPMC Univ Paris 06, CNRS, UMR3664, Paris, France †Present address: Département de Physique, Faculté des Sciences, Université Ferhat Abbas Sétif 1, Setif, Algérie ‡These authors contributed equally to this work as first authors ‡These authors contributed equally to this work as last authors *Corresponding author. Tel: +33 146549343; E-mail: [email protected] EMBO J (2017)36:2609-2625https://doi.org/10.15252/embj.201796631 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Homologous recombination (HR) is a conserved mechanism that repairs broken chromosomes via intact homologous sequences. How different genomic, chromatin and subnuclear contexts influence HR efficiency and outcome is poorly understood. We developed an assay to assess HR outcome by gene conversion (GC) and break-induced replication (BIR), and discovered that subtelomeric double-stranded breaks (DSBs) are preferentially repaired by BIR despite the presence of flanking homologous sequences. Overexpression of a silencing-deficient SIR3 mutant led to active grouping of telomeres and specifically increased the GC efficiency between subtelomeres. Thus, physical distance limits GC at subtelomeres. However, the repair efficiency between reciprocal intrachromosomal and subtelomeric sequences varies up to 15-fold, depending on the location of the DSB, indicating that spatial proximity is not the only limiting factor for HR. EXO1 deletion limited the resection at subtelomeric DSBs and improved GC efficiency. The presence of repressive chromatin at subtelomeric DSBs also favoured recombination, by counteracting EXO1-mediated resection. Thus, repressive chromatin promotes HR at subtelomeric DSBs by limiting DSB resection and protecting against genetic information loss. Synopsis DNA double-strand break (DSB) repair at subtelomeric regions in yeast can occur via gene conversion (GC) and break-induced recombination (BIR). Subtelomeric DSBs are preferentially repaired by BIR. Telomere clustering mediated by the silencing factor Sir3 favors GC between subtelomeric sequences. Break resection accelerates loss of telomere-proximal sequences, thus limiting GC repair. Repressive chromatin protects subtelomeric DSBs from resection, hence increasing GC repair efficiency. Introduction DNA lesions, arising from either environmental stress or endogenous events, challenge genomic integrity. Double-stranded breaks (DSBs) are a highly genotoxic form of DNA damage, and their improper repair leads to genomic instability or cell death. DSB repair can occur through two different mechanisms: non-homologous end joining (NHEJ) and homologous recombination (HR). HR is engaged at DSBs following resection and the formation of 3′ single strands, required for homology search and strand invasion. MRX/Sae2, Exo1 and Sgs1/Dna2 act together to resect DSBs (Gravel et al, 2008; Mimitou & Symington, 2008; Zhu et al, 2008) and generate 3′ ssDNA overhangs that are rapidly stabilized by RPA (Alani et al, 1992). Subsequent coating by Rad51 engages the broken 3′ ssDNA ends in genome probing, likely through repeated interactions with nearby sequences to find a homologous sequence (Renkawitz et al, 2013). One of the 3′ ssDNA ends invades a homologous template and copies the DNA sequences needed to seal the break. The second 3′ end of the DSB can then anneal with this extended and displaced 3′ end that initiated strand invasion, in repair by the "synthesis-dependent strand annealing" (SDSA) pathway. Alternatively, it can form a double Holliday junction by annealing with the single-stranded DNA in the displacement loop (D-loop) generated by strand invasion. In this case, D-loop resolution yields either non-crossover (NCO) or crossover (CO) products (Heyer et al, 2010). SDSA, NCO and CO repair are usually collectively referred to as gene conversion repair (GC). GC is typically an error-free mechanism that limits loss of heterozygosity (LOH) to a small region surrounding the DSB. However, HR can produce break-induced replication events (BIR) when the formation of the initial displacement loop (D-loop) is followed by conservative replication. In BIR, the D-loop migrates to the end of the chromosome, resulting in kilobase-long tracks of LOH (Llorente et al, 2008; Donnianni & Symington, 2013; Saini et al, 2013; Wilson et al, 2013). BIR is the predominant repair pathway employed when only one DSB end is available for strand invasion. It is also used to restart collapsed replication forks and to elongate telomeres when telomerase is absent or telomeres are uncapped (McEachern & Haber, 2006; Llorente et al, 2008). GC and BIR pathways are well defined at the molecular level, but the factors limiting their usage and relative efficiency in the context of the nucleus are far from being fully understood. The nuclear organization of Saccharomyces cerevisiae is well defined through microscopy and chromosome conformation capture experiments (Taddei & Gasser, 2012). During the exponential growth phase, interphase budding yeast chromosomes assume a Rabl-like conformation, with the 16 centromeres held together by the spindle pole body (SPB) at one nuclear pole. The 32 telomeres are found at the nuclear periphery, forming 3–4 foci where the yeast silent chromatin or heterochromatin factors (SIRs—silent information regulators) concentrate. Although budding yeast lacks the molecular factors associated with heterochromatin found in most other eukaryotes, silent chromatin generated by the SIR complex shares most of the functional features of heterochromatin. These features include late replication timing, heritable repression of transcription, a preferential association with the nuclear envelope and the formation of foci. Foci arise from the trans-association of "heterochromatic" loci (Meister & Taddei, 2013). Although these foci do not contain reproducible subsets of chromosomal ends, telomeres at the extremities of chromosomes with comparable arm lengths interact more frequently (Duan et al, 2010; Therizols et al, 2010; Guidi et al, 2015). Because of this genome organization, some loci are in contact more frequently and may be more prone to recombination. Consistently, HR efficiency generally negatively correlates with the spatial distance between a DSB and its homologous targets (Wilson et al, 1994; Burgess & Kleckner, 1999; Agmon et al, 2013; Lee et al, 2016). However, these studies also revealed outliers, for which spatial distances and HR efficiencies were uncorrelated. These outliers suggest that additional factors, other than spatial distance, limit HR efficiency. Indeed, DSB resection, along with the quality of homology between recipient and donor sequences, was shown to limit HR in S. cerevisiae (Lee et al, 2016). Furthermore, sequence context and chromatin structure have recently emerged as regulatory elements for DNA repair efficiency. Studies in Drosophila and mammals suggest that irradiation-induced DNA damage in heterochromatin is preferentially repaired by HR rather than NHEJ, but whether HR efficiency differs in heterochromatic compared to euchromatic regions remains unclear (Goodarzi et al, 2008; Chiolo et al, 2011; Goodarzi & Jeggo, 2013). In addition, a preference for HR may be specific to IR lesions because, at least in Drosophila tissues, an I-SceI break induced at heterochromatic loci is repaired through both HR and NHEJ (Janssen et al, 2016). In budding yeast, the impact of repressive chromatin structures on HR regulation has not been fully addressed, probably due to the under-representation of loci embedded in heterochromatin-like structures. Finally, how the different limiting factors affect HR efficiency and the balance between GC and BIR remains to be determined. To address these questions, we developed an assay to score DSB-induced recombination events at subtelomeric loci. This assay allows us to measure the competition between GC and BIR. We coupled this assay with genetic modifications that modulate the physical distances between telomeres, and/or spreading of repressive chromatin in subtelomeric regions, to determine how these parameters regulate HR efficiency and outcome (BIR versus GC). We discovered that subtelomeric DSBs are predominantly repaired by BIR, despite the presence of homology with donor sequences on both sides of the break. Further, we found that spatial distance, one of the limiting factors for HR efficiency, mainly impacts GC rate between subtelomeres. However, when recombination is between subtelomeric and intrachromosomal regions, DSB chromosomal location and chromatin structure show a stronger impact than spatial distance on HR efficiency. Our data suggest that DNA loss from the telomere proximal fragment limits GC at subtelomeric DSBs. Moreover, our findings are compatible with a model where silent chromatin favours GC by inhibiting DSB processing and the ensuing loss of sequences surrounding the DSB. Results Subtelomeric DSBs are efficiently repaired through BIR-mediated non-reciprocal translocations We developed a recombination assay that scores DSB-induced repair events between URA3 alleles inserted at different chromosomal positions (Fig 1A and B). This system includes two recombination cassettes of about 1.3-kb: a "recipient cassette" bearing a ura3 allele with a single 30 bp I-SceI cleavage site (Colleaux et al, 1988), and a "donor cassette" with a ura3-1 allele. The 30 bp I-SceI recognition site was efficiently cleaved after induction of I-SceI endonuclease, which is driven by the inducible GAL promoter (Fig 1C). We estimated DSB repair by comparing the colony-forming abilities of cells with I-SceI-induced DSB breaks (grown on galactose-containing medium) to those without I-SceI-induced DSB breaks (grown on glucose-containing medium). Cells that formed colonies on galactose-containing medium lost the I-SceI restriction site on the recipient cassette, as inferred from in vitro digestion of a PCR-amplified cassette. Thus, cleavage was efficient and DSBs were subsequently repaired through inaccurate NHEJ or recombination-based mechanisms (Fig 1D). Plating efficiencies of donor-less strains containing a DSB in the recipient cassette were < 0.5%, indicating that repair through inaccurate NHEJ rarely occurred (Fig 1G). Indeed, the presence of the donor ura3-1 allele at the URA3 locus increased cell survival on galactose plates 17- to 300-fold, indicating that HR is the most frequent repair pathway when a homologous sequence is present (Fig 1G and H). Figure 1. An assay to score recombination efficiency reveals prominent BIR repair of subtelomeric DSB Schematic representation of the two ura3 alleles used to test recombination efficiencies and outcome: i, the recipient ura3-I-SceI has a 30 bp I-SceI sequence inserted out of frame ii, the donor ura3-1 bears a missense mutation. The two ura3 alleles are introduced at chosen loci by PCR gene targeting. DSB cleavage efficiency measured in donor-less strains by qPCR using primers flanking the DSB site. Error bars represent the standard deviation (SD) of at least three independent experiments. Disappearance of the I-SceI cleavage site in survivors on galactose medium assessed by in vitro digestion by I-SceI of PCR products amplified with primers flanking the DSB site. Schematic of the primers used to test GC, BIR or NRT by PCR. Representative PCR obtained for the TEL6R TEL4R and TEL6R ura3-1i strains. Survival frequencies observed after induction of a DSB with or without recombination substrate. Error bars represent the survival standard error (SEM) of at least three independent experiments. Survival frequencies and GC (dark) or Pol32-dependent BIR (light grey) repair events observed after induction of a DSB at a subtelomeric position. Error bars represent the survival standard error (SEM) of at least three independent experiments. Asterisks indicate statistical differences for survival (****P < 0.001). See Table EV1 for statistical analysis of the GC and BIR repair events and Table EV2 for SEM values. BIR and GC events are distinguished on high-resolution pulsed-field gel electrophoresis. Repair by BIR leads to a shift of the chromosome size as indicated. Numbers refer to chromosomes. Download figure Download PowerPoint Subtelomeric DSBs repaired by either GC or BIR will ensure cell viability. In contrast, BIR at intrachromosomal loci would be lethal. To distinguish between the repair pathways used at subtelomeric DSBs, we performed PCR with primers flanking the recombination cassettes (Fig 1E and F). We discovered that up to 75% of subtelomeric DSBs exhibited typical BIR non-reciprocal translocations (NRTs), despite both sides of the break having about 700 bp of homology with the subtelomeric donor (Fig 1A and F). High-resolution pulsed-field gel electrophoresis (PFGE) confirmed NRTs of the expected size for BIR events and revealed neither a change of the donor chromosome size nor additional gross chromosomal rearrangements (Fig 1I). Subtelomeric DSBs also engaged in BIR rather than GC when the donor was inserted at an intrachromosomal site, as long as the orientation of the sequence allowed viable repair events. Indeed, 50% of the survivors exhibited typical BIR rearrangements following induction of a DSB in the TEL6R subtelomeric region, in the presence of a ura3-1 allele in the proper orientation (TEL6R ura3-1i, Fig 1B, H and I). NRTs depended strongly on the DNA Polδ subunit Pol32, a factor required for BIR (Fig 1H; Lydeard et al, 2007; Deem et al, 2008). BIR events between TEL6R and ura3-1i or TEL4R, which required polymerization of 117 kb or 10 kb, respectively, relied entirely on POL32 (Fig 1H). However, in the absence of Pol32, recombination between TEL6R and TEL9R subtelomeres still led to NRTs that account for 5% of survival (Fig 1H). These could correspond to recombination events in which the D-loop extended over 3.2 kb to the end of chromosome 9R. It is noteworthy that although Pol32 has been described as specifically required for BIR, we also observe a twofold to fourfold decrease in GC repair events in the absence of Pol32, suggesting that Pol32 also participates in GC (Fig 1H, Tables EV1 and EV2). In summary, subtelomeric DSBs show limited GC efficiency compared to intrachromosomal DSBs, but are repaired efficiently through BIR, which accounts for 50–75% of repair events. Together, these results show that BIR is favoured at subtelomeric DSBs even in the presence of homologous sequences sufficient to allow significant levels of GC (TEL6R ura3-1i; Fig 1H). Telomere clustering upon sir3A2Q overexpression favours gene conversion but not BIR Strikingly, the survival rate of cells with a subtelomeric DSB varies from 19 to 75%, depending on chromosomal location of the donor sequence (Fig 1H). These differences in HR efficiency may reflect a "barrier" that limits HR between subtelomeric and intrachromosomal loci (Pryde & Louis, 1997), and/or the spatial distance between homologous sequences at different loci (Agmon et al, 2013). In agreement with the second hypothesis, the highest HR efficiency was observed between TEL6R and TEL9R subtelomeric loci. These loci are at similar distances from their centromeres and thus spatially close, as shown both by microscopy and HiC (Duan et al, 2010; Therizols et al, 2010; Guidi et al, 2015). In contrast, the recombination rate between TEL6R and the more distant TEL4R, which lies at the end of a long chromosome arm, was almost two times lower (Fig 1H). These results reinforce the previously observed correlations between HR efficiencies and spatial distances, inferred either from the overlap of positions occupied by loci in the nucleus (Agmon et al, 2013) or from HiC contact maps (Lee et al, 2016). However, in these studies, the relative contribution of BIR versus GC events on survival rate was not monitored. To directly test the impact of the physical distance on recombination rate and choice of repair pathway, we modulated the clustering of telomeres in the nucleus. Specifically, we overexpressed Sir3, which leads to the formation of a "hypercluster" of telomeres at the centre of the nucleus (Ruault et al, 2011; Fig 2A and B). Importantly, this increased clustering can be uncoupled from silencing by overexpressing the sir3A2Q silencing-defective allele (Ruault et al, 2011). To overexpress sir3A2Q, the A2Q mutation was inserted by gene targeting in the endogenous SIR3 gene, along with a strong GDP promoter to drive overexpression. As overexpression of sir3A2Q also led to loss of subtelomeric silencing (Ruault et al, 2011), we used the sir3Δ mutant as a control. As expected, the absence of SIR3 or overexpression of sir3A2Q did not significantly affect HR efficiency between two intrachromosomal cassettes (LYS2 ura3-1; Fig 2C and Table EV1). In addition, deletion of SIR3 did not affect HR levels between TEL6R and TEL4R or TEL9R, indicating that loss of SIR3 and subtelomeric silencing did not have a global impact on recombination efficiency (Fig 2D and Table EV1). However, sir3A2Q overexpression that increased physical proximity between subtelomeric cassettes (TEL6R TEL4R and TEL6R TEL9R) significantly increased HR efficiencies (Fig 2D). Interestingly, our molecular analysis revealed that this effect stemmed from a twofold to threefold increase in GC efficiency, whereas BIR was not affected in these conditions for the two telomere pairs tested (Fig 2E and F, Table EV1). Similarly, GC events increased fourfold in response to a DSB at TEL4R upon sir3A2Q-mediated telomere clustering, showing that this effect is not specific to TEL6R (Fig EV1). These data argue that telomere clustering, which increases spatial proximity, favours recombination between homologous sequences. This posits that homology searching is a limiting factor for GC efficiency. However, BIR efficiency did not increase when the proximity between homologous sequences increased, suggesting that BIR efficiency is limited by another step, such as initiation or progression of new DNA synthesis (Jain et al, 2009; Donnianni & Symington, 2013). Figure 2. Telomeres clustering upon sir3A2Q overexpression specifically favours gene conversion A. Rap1 foci grouping upon sir3A2Q overexpression. The sir3A2Q allele is silencing defective and its overexpression leads to telomere clustering at the centre of the nucleus. Representative fluorescent images of the telomere-associated protein GFP-Rap1 and of the nucleolus visualized through Sik1-mRFP in WT, sir3∆ and sir3A2Q-overexpressing cells. Scale bar is 500 nm. B. Schematic representation of the experimental design showing telomere organization, chromatin status and DSB localization. C, D. Survival frequencies observed after induction of a DSB in WT, sir3∆ and sir3A2Q-overexpressing cells as in Fig 1G and H. E, F. Repair events (GC and BIR) after induction of a DSB at TEL6R with TEL4R (E) orTEL9R (F) as a donor in WT and in cells overexpressing the sir3A2Q mutant protein. Data information: Error bars represent the standard error (SEM) of at least three independent experiments. Asterisks indicate statistical differences (*P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001). See Table EV1 for statistical analysis of the GC and BIR repair events and Table EV2 for SEM values. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Telomeres clustering upon sir3A2Q overexpression specifically favours GC at TEL4R Survival frequencies observed after induction of a DSB at TEL4R in WT- and sir3A2Q-overexpressing cells as in Fig 1G and H. Repair events (GC and BIR) after induction of a DSB at TEL4R with TEL6R as a donor in WT and in cells overexpressing the sir3A2Q mutant protein. Data information: Error bars represent the standard error (SEM) of at least three independent experiments. Asterisks indicate statistical differences (***P < 0.005). Download figure Download PowerPoint DSBs in subtelomeric regions are repaired with low efficiency using intrachromosomal donors As discussed above, the existence of a barrier to recombination has been proposed to explain the low HR rate between subtelomeric and internal loci (Pryde & Louis, 1997). In agreement with this hypothesis and with previous reports (Marvin et al, 2009a,b; Agmon et al, 2013), we observed a lower rate of recombination between a subtelomeric DSB at TEL6R and the URA3 internal locus (10% survival), than between two internal loci (30% survival between LYS2 and URA3) or two subtelomeric loci (survival ranging from 40 to 80% for TEL6R TEL4R and TEL6R TEL9R; Fig 1H). However, these pairs also show different physical distances that could account for the different HR efficiency. To distinguish between the contributions of physical distance and chromosomal location of the DSB, we compared the recombination efficiency between reciprocal pairs of intrachromosomal and subtelomeric loci (Fig 3A). Unexpectedly, we observed a very strong asymmetry in repair efficiency within pairs of intrachromosomal and subtelomeric loci. Indeed, a DSB induced at the intrachromosomal URA3 locus was repaired efficiently with donor sequences inserted at the TEL6R or TEL4R subtelomeres, leading to 54 and 45% survival, respectively (Fig 3B). In contrast, DSBs induced at TEL6R or TEL4R with the URA3 locus as a donor only led to 20 and 3% survival, respectively (Fig 3B). Subtelomeric loci thus appear to be efficient recombination donors but poor acceptors. It is noteworthy that subtelomeric DSBs recombine with intrachromosomal donors with low efficiency, even though BIR repair led to viable progenies and accounted for one-third to half of the survivors for TEL4R and TEL6R, respectively. The low HR efficiency of subtelomeric DSBs with intrachromosomal sequences recapitulated the results of spontaneous recombination analyses (Marvin et al, 2009a,b). However, the efficient HR between intrachromosomal DSBs and subtelomeric donors that we observed is not consistent with a recombination barrier between these two loci. Furthermore, the HR rate varied up to 15-fold for reciprocal pairs of loci—thus at the same spatial distance—depending on which locus is damaged. This demonstrates that the pre-existing physical distance between homologous sequences is not the only limiting factor for recombination efficiency. Figure 3. Subtelomeric loci are good recombination donors but poor acceptors for HR Schematic representation of the assay showing DSB localization and telomere at the nuclear periphery in WT cells. Survival frequencies and GC and BIR repair events after DSB induction as in Fig 1H. Error bars represent the standard error (SEM) of at least three independent experiments. Asterisks indicate statistical differences for survival (****P < 0.001). See Table EV1 for statistical analysis of the GC and BIR repair events and Table EV2 for SEM values. Download figure Download PowerPoint Recombination efficiency between subtelomeric and intrachromosomal loci is independent of telomere perinuclear anchoring The nuclear position of subtelomeric and intrachromosomal loci differs, as telomeres are anchored to the nuclear periphery (Palladino et al, 1993). We previously showed that DSBs relocate to the nuclear periphery, and proposed that this change in position favours repair through a still unknown mechanism (Nagai et al, 2008). We hypothesized that relocalization of an intrachromosomal DSB to the nuclear periphery, close to telomeric foci, would favour its encounter with a subtelomeric donor, thus improving HR efficiency. Conversely, a subtelomeric DSB, restrained at the nuclear periphery, would less frequently encounter and recombine with an intrachromosomal donor. To test this hypothesis, we evaluated recombination efficiencies in cells overexpressing the sir3A2Q variant, which triggers telomere clustering in the nuclear interior, independently of silent chromatin formation (Ruault et al, 2011 and Fig 2A). We first scored the localization of TEL6R in three concentric zones of equal area (Hediger et al, 2002 and Fig 4A). Although more than 60% of WT cells displayed TEL6R in the outermost zone, only 40% of the sir3A2Q-overexpressing cells showed TEL6R at this position. This loss of peripheral localization was not simply a consequence of silencing disruption, since TEL6R is found in the outermost zone in 60% of sir3∆ cells (Fig 4B). Therefore, overexpression of sir3A2Q is indeed sufficient to drive TEL6R from the nuclear periphery to the nuclear interior. Figure 4. Loss of telomere perinuclear anchoring has no effect on recombination efficiency Schematic representation of Lacop-tagged ARS609 on chromosome VI. Position of the GFP-tagged locus was scored relative to the NE (Nup49-mCherry). Ratios of distance from NE and diameter in focal plane are binned into three equal surfaces. Position of ARS609 in WT, sir3∆ cells or cells overexpressing sir3A2Q
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