Escape of Sgs1 from Rad9 inhibition reduces the requirement for Sae2 and functional MRX in DNA end resection

Abstract

Homologous recombination requires nucleolytic degradation (resection) of DNA double-strand break (DSB) ends. In Saccharomyces cerevisiae, the MRX complex and Sae2 are involved in the onset of DSB resection, whereas extensive resection requires Exo1 and the concerted action of Dna2 and Sgs1. Here, we show that the checkpoint protein Rad9 limits the action of Sgs1/Dna2 in DSB resection by inhibiting Sgs1 binding/persistence at the DSB ends. When inhibition by Rad9 is abolished by the Sgs1-ss mutant variant or by deletion of RAD9, the requirement for Sae2 and functional MRX in DSB resection is reduced. These results provide new insights into how early and long-range resection is coordinated.

Keywords: Rad9; Saccharomyces cerevisiae; Sgs1; double‐strand break; resection.

Figure 1

Suppression of the sensitivity to genotoxic agents of sae2Δ and mre11 nuclease-defective mutants by Sgs1-ss

1. A, B Exponentially growing cells were serially diluted (1:10), and each dilution was spotted out onto YEPD plates with or without camptothecin (CPT), phleomycin or MMS.

2. C, D Meiotic tetrads were dissected on YEPD plates that were incubated at 25°C, followed by spore genotyping.

3. E Exponentially growing cells were serially diluted (1:10), and each dilution was spotted out onto YEPD plates with or without CPT, phleomycin or MMS.

4. F, G Meiotic tetrads were dissected on YEPD plates that were incubated at 25°C, followed by spore genotyping.

Figure 2

Suppression of the adaptation defect of sae2Δ cells by Sgs1-ss

1. A YEPR G1-arrested cell cultures of wild-type JKM139 and otherwise isogenic derivative strains were plated on galactose-containing plates (time zero). At the indicated time points, 200 cells for each strain were analysed to determine the frequency of large budded cells and of cells forming microcolonies of more than two cells. The mean values from three independent experiments are represented (n + 3).

2. B Exponentially growing YEPR cultures of the strains in (A) were transferred to YEPRG (time zero), followed by Western blot analysis with anti-Rad53 antibodies.

3. C ChIP analysis. Exponentially growing YEPR cell cultures of JKM139 derivative strains were transferred to YEPRG, followed by ChIP analysis of the recruitment of Mre11–Myc at the indicated distance from the HO-cut compared to untagged Mre11 (no tag). In all diagrams, the ChIP signals were normalized for each time point to the corresponding input signal. The mean values are represented with error bars denoting s.d. (n + 3). *P < 0.01, t-test.

Figure 3

Sgs1-ss suppresses the resection defect of sae2Δ cells

1. A Method to measure double-strand break (DSB) resection. Gel blots of SspI-digested genomic DNA separated on alkaline agarose gel were hybridized with a single-stranded MAT probe (ss probe) that anneals to the unresected strand. 5′–3′ resection progressively eliminates SspI sites (S), producing larger SspI fragments (r1 through r7) detected by the probe.

2. B DSB resection. YEPR exponentially growing cell cultures of JKM139 derivative strains were transferred to YEPRG at time zero. Genomic DNA was analysed for ssDNA formation at the indicated times after HO induction as described in (A).

3. C Densitometric analyses. The experiment as in (B) has been independently repeated three times, and the mean values are represented with error bars denoting s.d. (n + 3).

4. D DSB repair by single-strand annealing (SSA). In YMV45 strain, the HO-cut site is flanked by homologous leu2 sequences that are 4.6 kb apart. HO-induced DSB formation results in generation of 12- and 2.5-kb DNA fragments (HO-cut) that can be detected by Southern blot analysis with a LEU2 probe of KpnI-digested genomic DNA. DSB repair by SSA generates an 8-kb fragment (product).

5. E Densitometric analysis of the product band signals. The intensity of each band was normalized with respect to a loading control (not shown). The mean values are represented with error bars denoting s.d. (n + 3).

Figure 4

Double-strand break (DSB) resection is accelerated by the same mechanism in SGS1-ss and rad9Δ cells

1. A DSB resection. YEPR exponentially growing cell cultures of JKM139 derivative strains were transferred to YEPRG at time zero. Genomic DNA was analysed for ssDNA formation as described in Fig3A.

2. B Densitometric analyses. The experiment as in (A) has been independently repeated three times, and the mean values are represented with error bars denoting s.d. (n + 3).

3. C Exponentially growing cells were serially diluted (1:10), and each dilution was spotted out onto YEPD plates with or without camptothecin (CPT) or phleomycin.

4. D DSB resection. HO was induced at time zero in α-factor-arrested JKM139 derivative cells that were kept arrested in G1 with α-factor throughout the experiment. Genomic DNA was analysed for ssDNA formation as described in Fig3A.

5. E Densitometric analyses. The experiment as in (D) has been independently repeated three times, and the mean values are represented with error bars denoting s.d. (n + 3).

Figure 5

Rapid resection in rad9Δ cells depends mainly on Sgs1

1. A Double-strand break (DSB) resection. YEPR exponentially growing cell cultures of JKM139 derivative strains were transferred to YEPRG at time zero. Genomic DNA was analysed for ssDNA formation as described in Fig3A.

2. B Densitometric analyses. The experiment as in (A) has been independently repeated three times, and the mean values are represented with error bars denoting s.d. (n + 3).

3. C DSB resection. The experiment was performed as in (A).

4. D Densitometric analyses. The experiment as in (C) has been independently repeated three times, and the mean values are represented with error bars denoting s.d. (n + 3).

Figure 6

Rad9 inhibits Sgs1 association at the double-strand breaks (DSBs)

1. A DSB resection. YEPR exponentially growing cell cultures of JKM139 derivative strains were transferred to YEPRG at time zero. Genomic DNA was analysed for ssDNA formation as described in Fig3A.

2. B Densitometric analyses. The experiment as in (A) has been independently repeated three times, and the mean values are represented with error bars denoting s.d. (n + 3).

3. C ChIP analysis. Exponentially growing YEPR cell cultures of JKM139 derivative strains were transferred to YEPRG, followed by ChIP analysis of the recruitment of Sgs1-HA and Sgs1-ss-HA at the indicated distance from the HO-cut compared to untagged Sgs1 (no tag). In all diagrams, the ChIP signals were normalized for each time point to the corresponding input signal. The mean values are represented with error bars denoting s.d. (n + 3). *P < 0.01, t-test.

4. D ChIP analysis in G1-arrested cells. As in (C), but showing ChIP analysis of the recruitment of Sgs1-HA and Sgs1-ss-HA in cells that were kept arrested in G1 by α-factor. The mean values are represented with error bars denoting s.d. (n + 3). *P < 0.01, t-test.

5. E ChIP analysis in G1-arrested cells. As in (C), but showing ChIP analysis of the recruitment of Exo1–Myc in cells that were kept arrested in G1 by α-factor. The mean values are represented with error bars denoting s.d. (n + 3). *P < 0.01, t-test.