The 9-1-1 checkpoint clamp coordinates resection at DNA double strand breaks

Abstract

DNA-end resection, the generation of single-stranded DNA at DNA double strand break (DSB) ends, is critical for controlling the many cellular responses to breaks. Here we show that the conserved DNA damage checkpoint sliding clamp (the 9-1-1 complex) plays two opposing roles coordinating DSB resection in budding yeast. We show that the major effect of 9-1-1 is to inhibit resection by promoting the recruitment of Rad9(53BP1) near DSBs. However, 9-1-1 also stimulates resection by Exo1- and Dna2-Sgs1-dependent nuclease/helicase activities, and this can be observed in the absence of Rad9(53BP1). Our new data resolve the controversy in the literature about the effect of the 9-1-1 complex on DSB resection. Interestingly, the inhibitory role of 9-1-1 on resection is not observed near uncapped telomeres because less Rad9(53BP1) is recruited near uncapped telomeres. Thus, 9-1-1 both stimulates and inhibits resection and the effects of 9-1-1 are modulated by different regions of the genome. Our experiments illustrate the central role of the 9-1-1 checkpoint sliding clamp in the DNA damage response network that coordinates the response to broken DNA ends. Our results have implications in all eukaryotic cells.

Figure 1.

The 9-1-1 complex inhibits resection of DSBs. (A) The 9-1-1 complex promotes resection at uncapped telomeres by stimulating Exo1 and Dna2-Sgs1 recruitment to ssDNA (model based on Ngo et al. (24)). (B) The efficiencies of DSB induction at the MAT locus. (C) Yeast strains of the indicated genotypes were subjected to western blot analysis with anti-Rad53 and anti-tubulin antibodies following DSB induction. (D) Map of chromosome III showing an HO endonuclease site and loci examined in this study. (E,F) Analysis of 3′ ssDNA and 5′ ssDNA accumulation in JKM179 strains (see strain list in Supplementary Tables S1 and S2) at the indicated loci. All DSB experiments were performed in nocodazole arrested cells. The data plotted are the means and the range from two strains. P-values were calculated using two-tailed unpaired T test. *P < 0.05, **P < 0.01.

Figure 2.

The 9-1-1 complex inhibits Dna2-Sgs1 but stimulates Exo1 at DSBs. (A-C) Analysis of 3′ ssDNA accumulation in JKM179 strains at the indicated loci. The data plotted and the P-values are as described in Figure 1. (D) The 9-1-1 complex inhibits Dna2-Sgs1 but stimulates Exo1 at DSBs.

Figure 3.

The 9-1-1 complex recruits more Rad9^53BP1 near DSBs than uncapped telomeres. (A, B) ChIP analyses of Rad9-HA recruitment near DSBs (A) and uncapped telomeres (B). The ssDNA data were taken from Figure 1E and Supplementary Figure S1B. The data plotted and the P-values are as described in Figure 1. (C, D) The 9-1-1 complex stimulates the recruitment of Rad9^53BP1 which inhibits Dna2-Sgs1 and Exo1, but Rad9^53BP1 binds more poorly to uncapped telomeres.

Figure 4.

The 9-1-1 complex stimulates resection in rad9Δ cells. (A,C) Analysis of 3′ ssDNA accumulation in JKM179 strains at the indicated loci. The data plotted and the P-values are as described in Figure 1. (B) Yeast strains of the indicated genotypes were subjected to western blot analysis with anti-Rad53 and anti-tubulin antibodies following DSB induction.

Figure 5.

The 9-1-1 complex stimulates both Exo1- and Dna2-Sgs1-dependent resection in rad9Δ cells. (A-C) Analysis of 3′ ssDNA accumulation in rad9Δ background strains at the indicated loci. The data plotted and the P-values are as described in Figure 1. (D) The 9-1-1 complex stimulates both Exo1 and Dna2-Sgs1 in rad9Δ cells.

Figure 6.

The 9-1-1 complex affects the binding of both Exo1 and Dna2-Sgs1 to DNA. (A,B) ChIP analysis of Dna2-Myc and Exo1-Myc recruitment to DSBs in JKM179 background strains. The data plotted and the P-values are as described in Figure 1.

Figure 7.

The 9-1-1 complex coordinates DSB resection at the URA3 locus. (A) Map of chromosome V showing an HO endonuclease site and loci examined in this study. (B,D) Analysis of 3′ ssDNA accumulation in the indicated strains. (C) ChIP analyses of Rad9-HA recruitment near DSBs. All the experiments were performed in nocodazole arrested cells. The data plotted and the P-values are as described in Figure 1.

Figure 8.

Control of resection at DSBs and telomeres. Models for the roles of 9-1-1, Rad9^53BP1, Exo1 and Dna2-Sgs1 on resection near DSBs and at uncapped telomeres. The size of the nucleases in each schematic indicates relative resection activities and is deduced by ssDNA measurements in the different genetic settings. Data supporting these figures are taken from Figures 1–7 and Ngo et al. (24). (A, B) 9-1-1 stimulates recruitment of Exo1 and Dna2-Sgs1 to facilitate resection (pathway 1, p1). 9-1-1 stimulates recruitment of Rad9^53BP1 to inhibit resection (pathway 2, p2). Rad9^53BP1 binds more near DSBs than uncapped telomeres. (C) In mec3Δ cells, there is less Rad9^53BP1 recruitment (lack of p2), but there is no 9-1-1 to stimulate activity of Exo1 and Dna2-Sgs1 (lack of p1). At DSBs Exo1 is less active (lack of p1) but Dna2-Sgs1 is more active (lack of p2) (C compared to A). The overall effect is increased resection in mec3Δ mutants (C compared to A). At telomeres Exo1 is less active (lack of p1) but Dna2-Sgs1 activity remains little changed because little Rad9^53BP1 binds (p2 less active at telomeres), and so the overall effect of mec3Δ is reduced resection (C compared to B). (D) In rad9Δ cells, there is no Rad9^53BP1 recruitment. Therefore, Dna2-Sgs1 and Exo1 are more active than in (A, B). Dna2-Sgs1 is more active than Exo1 in the absence of Rad9^53BP1. (E) In rad9Δ mec3Δ cells, there is no 9-1-1 to stimulate Exo1 or Dna2-Sgs1. Therefore, Dna2-Sgs1 and Exo1 are less active than in D. Exo1 is more dependent on 9-1-1 than Dna2-Sgs1. (F) Mec1^ATR also initiates a checkpoint cascade to inhibit Exo1.