Recombination at subtelomeres is regulated by physical distance, double-strand break resection and chromatin status

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.

Keywords: heterochromatin; homologous recombination; nuclear organization; subtelomeres; yeast.

Figure 1. An assay to score recombination efficiency reveals prominent BIR repair of subtelomeric DSB

1. 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.

2. The two ura3 alleles are introduced at chosen loci by PCR gene targeting.

3. 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.

4. 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.

5. Schematic of the primers used to test GC, BIR or NRT by PCR.

6. Representative PCR obtained for the TEL6R TEL4R and TEL6R ura3‐1i strains.

7. 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.

8. 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.

9. 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.

Figure 2. Telomeres clustering upon sir3A2Q overexpression specifically favours gene conversion

1. 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.

2. B

   Schematic representation of the experimental design showing telomere organization, chromatin status and DSB localization.

3. C, D

   Survival frequencies observed after induction of a DSB in WT, sir3∆ and sir3A2Q‐overexpressing cells as in Fig 1G and H.

4. 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.

Figure EV1. Telomeres clustering upon sir3A2Q overexpression specifically favours GC at TEL4R

1. Survival frequencies observed after induction of a DSB at TEL4R in WT‐ and sir3A2Q‐overexpressing cells as in Fig 1G and H.

2. 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).

Figure 3. Subtelomeric loci are good recombination donors but poor acceptors for HR

1. Schematic representation of the assay showing DSB localization and telomere at the nuclear periphery in WT cells.

2. 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.

Figure 4. Loss of telomere perinuclear anchoring has no effect on recombination efficiency

1. Schematic representation of Lac^op‐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.

2. Position of ARS609 in WT, sir3∆ cells or cells overexpressing sir3A2Q or SIR3. Asterisks indicate statistical differences compared to WT measured by proportion test (*P < 0.05; ****P < 0.001).

3. Schematic representation of the assay showing DSB localization and telomere at the nuclear centre in cells overexpressing sir3A2Q.

4. Survival frequencies after DSB induction at the intrachromosomal locus LYS2 in WT, sir3∆‐ and sir3A2Q‐overexpressing cells as in Fig 1G and H.

5. Survival frequencies and GC and BIR repair events observed after DSB induction at a subtelomeric position in WT‐, sir3∆‐ and sir3A2Q‐overexpressing cells as in Fig 1G and H.

Data information: Error bars represent the standard error (SEM) of at least three independent experiments. See Table EV1 for statistical analysis of the GC and BIR repair events and Table EV2 for SEM values.

Figure EV2. Loss of telomere perinuclear anchoring has no effect on recombination efficiency at TEL4R

1. Survival frequencies and GC BIR repair events observed after induction of a DSB in WT‐ and sir3A2Q‐overexpressing cells as in Fig 1G and H. Error bars represent the standard error (SEM) for survival of at least three independent experiments.

Figure 5. Loss of DNA from the telomeric fragment limits gene conversion and favours BIR

1. A

   Representative image of Rfa1‐YFP foci in response to an I‐SceI‐induced DSB at TEL6R or at LYS2 in WT donor‐less cells. Scale bars are 2 μm.

2. B

   Quantification of cells with Rfa1‐YFP foci after DSB induction in donor‐less strains. Mean of two independent experiments are shown. Error bars indicate standard deviation (SD).

3. C

   Quantification of Rfa1‐YFP foci intensity using Q‐foci after induction of I‐SceI in donor‐less strains. Data plotted represent the pool of two independent experiments. Asterisks indicate statistical differences using a Mann–Whitney test (****P < 0.001).

4. D

   Schematic representation of TEL6R and LYS2 loci with primer location indicated for DSB cleavage efficiency (blue) and flanking DNA measurement (red and grey). The telomeres are represented as wavy lines.

5. E

   DNA levels measured at 0.9 kb from the I‐SceI cut site over time by qPCR normalized to DNA levels at the OGG1 locus in donor‐less strains. Error bars represent the standard error (SEM) of at least three independent experiments.

6. F

   Schematic representation of the quantitative PCR assay to monitor I‐SceI‐induced DSB end resection. Red arrows show primers used for real‐time PCR. RE: restriction enzyme cut site.

7. G

   Quantification of ssDNA among cut DNA at 0.9 kb from the DSB at each time point relative to t0. The mean values for three independent experiments are plotted and error bars show standard error (SEM).

8. H–K

   Survival frequencies and distribution of the repair events after DSB induction in the indicated strains as in Fig 1G and H. 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.

Figure EV3. Loss of DNA from the telomeric fragment limits gene conversion and favours BIR at TEL4R

1. A

   DNA levels measured at 0.9 kb on both side of the I‐SceI cut site at TEL4R in WT donor‐less cells. qPCR signals were normalized to DNA level at the OGG1 locus and corrected for differences in cleavage efficiency.

2. B, C

   Survival frequencies and GC (dark) or BIR (light grey) repair events observed after induction of a DSB at TEL4R in the indicated strains as in Fig 1G and H.

Data information: 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.

Figure 6. Silent chromatin spreading over DSB site impairs resection

1. A

   Sir3‐binding at TEL6R in WT cells or in cells overexpressing SIR3 (oeSIR3). Binding is probed by ChIP‐chip using antibodies directed against untagged Sir3p. The mean of two independent biological replicates is shown and error bars correspond to the variation between duplicates. The red inserts mark the insertion site of the URA3 recombination cassette.

2. B

   Telomeric silencing assay at TEL6R in WT cells, cells overexpressing SIR3 (oeSIR3) or sir3A2Q (oesir3A2Q). Increased growth on 5‐FOA plates reflects an increase in telomeric silencing.

3. C–E

   Survival frequencies observed after induction of a DSB in the indicated strains as in Fig 1G and H. Survival frequencies for sir3∆, oesir3A2Q and WT from Figs 2C and 4E are plotted for comparison with strains overexpressing SIR3. Error bars represent the standard error (SEM) of at least three independent experiments. Asterisks indicate statistical differences for survival (**P < 0.01; ****P < 0.001). See Table EV1 for statistical analysis of the GC and BIR repair events and Table EV2 for SEM values.

4. F, G

   DNA levels measured at 0.9 kb from the I‐SceI cut site over time by qPCR in donor‐less WT‐, exo1Δ‐ and SIR3‐overexpressing cells after induction of I‐SceI cleavage at TEL6R (F) or LYS2 (G). DNA levels were normalized to DNA levels at the OGG1 locus and corrected for differences in DSB cleavage efficiency (see Materials and Methods for details). Error bars represent the standard error (SEM) of three to five independent experiments.

5. H, I

   Quantification of ssDNA among cut DNA at 0.9 kb from the DSB at each time point relative to t0 at TEL6R (H) and LYS2 (I). The mean values for three independent experiments are plotted, and error bars show standard error (SEM) of at least three independent experiments.

Figure 7. Silent chromatin spreading over TEL4R DSB site impairs resection

1. A

   Sir3‐binding at TEL4R in WT cells or in cells overexpressing SIR3 (oeSIR3). Binding is probed by ChIP‐chip using antibodies directed against untagged Sir3. The mean of two independent biological replicates is shown, and error bars correspond to the variation between duplicates. The red inserts mark the insertion site of the URA3 recombination cassette.

2. B

   Telomeric silencing assay at TEL4R in WT cells, cells overexpressing SIR3 (oeSIR3) or sir3A2Q (oesir3A2Q). Increased growth on 5‐FOA plates reflects an increase in telomeric silencing.

3. C

   Survival frequencies observed after induction of a DSB in the indicated strains as in Fig 1G and H. 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.

4. D

   Schematic of TEL4R locus with primer location indicated for DSB cleavage efficiency (blue) and flanking DNA measurement (red and grey). The telomeres are represented as wavy lines.

5. E, F

   DNA levels measured at 0.9 kb on the telomeric proximal (E) and on the centromeric proximal (F) sides of the I‐SceI cut site at TEL4R in donor‐less WT‐, exo1Δ‐ and SIR3‐overexpressing cells. DNA levels were normalized to DNA level at the OGG1 locus and corrected for differences in DSB cleavage efficiency (see Materials and Methods for details). Error bars represent the standard error (SEM) of three independent experiments.

Figure EV4. Delay in DSB cleavage efficiency at silent chromatin DSB site

1. DSB cleavage efficiency at the two loci in WT‐, exo1∆‐ and SIR3‐overexpressing cells measured as in Fig 1C. Error bars represent the standard deviation (SD) of at least three independent experiments.

Figure EV5. Telomere anchoring is not limiting for recombination

1. Survival frequencies and GC (dark) or BIR (light grey) repair events observed after induction of a DSB at a subtelomeric position in WT and sir4∆ cells as in Fig 1H.

2. Survival frequencies observed after induction of a DSB at an intrachromosomal position in WT and sir4∆ cells as in Fig 1H.

Data information: Error bars represent the standard error (SEM) of at least three independent experiments. See Table EV1 for statistical analysis of the GC and BIR repair events and Table EV2 for SEM values.