The SWI/SNF ATP-dependent nucleosome remodeler promotes resection initiation at a DNA double-strand break in yeast

Abstract

DNA double-strand breaks (DSBs) are repaired by either the non-homologous end joining (NHEJ) or homologous recombination (HR) pathway. Pathway choice is determined by the generation of 3΄ single-strand DNA overhangs at the break that are initiated by the action of the Mre11-Rad50-Xrs2 (MRX) complex to direct repair toward HR. DSB repair occurs in the context of chromatin, and multiple chromatin regulators have been shown to play important roles in the repair process. We have investigated the role of the SWI/SNF ATP-dependent nucleosome-remodeling complex in the repair of a defined DNA DSB. SWI/SNF was previously shown to regulate presynaptic events in HR, but its function in these events is unknown. We find that in the absence of functional SWI/SNF, the initiation of DNA end resection is significantly delayed. The delay in resection initiation is accompanied by impaired recruitment of MRX to the DSB, and other functions of MRX in HR including the recruitment of long-range resection factors and activation of the DNA damage response are also diminished. These phenotypes are correlated with a delay in the eviction of nucleosomes surrounding the DSB. We propose that SWI/SNF orchestrates the recruitment of a pool of MRX that is specifically dedicated to HR.

Figure 1.

Initiation of DNA end resection at MAT is impaired in snf5Δ cells. OK (A) Asynchronous WT (n = 9) and snf5Δ (n = 10) cells were harvested at 1 h intervals after addition of galactose to induce a MAT DSB. Resection was monitored by qPCR with primers that anneal 0.1 kb to the right of the MAT DSB. (B) Recruitment of RPA 0.1 kb to the right of the MAT DSB was monitored by ChIP (left) and DNA end resection was simultaneously monitored (right) in WT (n = 3) and snf5Δ (n = 3) asynchronous cells after addition of galactose to induce a MAT DSB. (C) Recruitment of Rad51 0.1 kb to the right of the MAT DSB was monitored by ChIP (left) and DNA end resection was simultaneously monitored (right) in WT (n = 3) and snf5Δ (n = 3) asynchronous cell populations as described in B. Error bars denote one standard deviation. Statistical differences between WT and snf5Δ at time points were assessed by two-way ANOVA with Holm–Sidak post-hoc analysis. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2.

SWI/SNF regulates recruitment of MRX to a MAT DSB. (A) Asynchronous WT (n = 7) and snf5Δ (n = 6) cells were harvested at 1 h intervals after addition of galactose to induce a MAT DSB. Recruitment of Mre11 0.1 kb to the right of the MAT DSB was monitored by ChIP (left) and DNA end resection was simultaneously monitored (right). (B) Asynchronous WT (n = 3) and snf5Δ (n = 3) cells were harvested after addition of galactose. Recruitment of Ku70 (left) and DNA end resection (right) were monitored as in A. (C) Asynchronous ku70Δ (n = 5) and snf5Δku70Δ (n = 3) cells were harvested after addition of galactose. Recruitment of Mre11 (left) and DNA end resection (right) were monitored as in A. WT (solid line) and snf5Δ (dashed line) Mre11 ChIP recruitment data from A are overlaid on the graph. Error bars denote one standard deviation. Statistical differences between strains at time points were assessed by two-way ANOVA with Holm–Sidak post-hoc analysis. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3.

Long-range resection is delayed in snf5Δ cells and relies upon Exo1. Asynchronous WT, snf5Δ, exo1Δ, snf5Δexo1Δ, sgs1Δ and snf5Δsgs1Δ cells (n = 3) were harvested at 2 h intervals after addition of galactose to induce a MAT DSB. Resection was monitored by qPCR with primers annealing either 0.1 kb (A) or 12.0 kb (B) to the right of the MAT DSB. (C) The time for 25% resection to occur (0.75 fraction intact) at positions to the right of the MAT DSB. (D) The time for 25% resection to occur immediately adjacent to the MAT was designated as the time to resection initiation, and (E) resection rates were calculated by determining the slopes of the graphs by linear regression analysis. Error bars denote one standard deviation. Statistical comparisons between strains were assessed by Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4.

Recruitment of Exo1 and Dna2 to a MAT DSB is impaired in snf5Δ cells. Asynchronous WT (n = 3) and snf5Δ (n = 3) strains containing Exo1-Myc or Dna2-Myc were harvested at 1 h intervals after addition of galactose to induce a MAT DSB. Recruitment of (A) Exo1-Myc or (B) Dna2-Myc 0.1 kb to the right of the MAT DSB was monitored by ChIP (left) and DNA end resection was simultaneously monitored (right). Error bars denote one standard deviation. Statistical differences between strains at time points were assessed by two-way ANOVA with Holm–Sidak post-hoc analysis. *P < 0.05, **P < 0.01, ***P < 0.001. (C) Recruitment of Exo1-Myc and Dna2-Myc at 5 h in snf5Δ relative to recruitment in WT, which was set as 1. Error bars denote one standard deviation. Statistical differences between recruitment at 5 h in WT and snf5Δ cells were assessed by Student's t-test. *P < 0.05, **P < 0.01.

Figure 5.

Nucleosome eviction at MAT is delayed in snf5Δ cells. Asynchronous WT (n = 4) and snf5Δ (n = 3) cells containing FLAG-H2B were harvested at 1 h intervals after addition of galactose to induce a MAT DSB. H2B eviction was monitored by ChIP (solid lines) using qPCR with primers that anneal (A) 0.1 kb; (B) 3.1 kb and (C) 6.1 kb to the right of the DSB, and resection was simultaneously monitored (dashed lines). Error bars denote one standard deviation. (D) Model for role of SWI/SNF in the initiation of HR repair. Only initial events in the repair of a DSB by NHEJ or HR are shown. After a DSB is formed, KU rapidly associates with broken ends and recruits Dnl4-Lif1, leading to the recruitment of a pool of NHEJ-MRX that tethers broken ends and stimulates end ligation that is essential for repair by NHEJ (left panel). Recruitment of SWI/SNF to a DSB promotes nucleosome eviction in the vicinity of the break, leading to the recruitment or stabilization of a distinct pool of HR-active MRX (right panel). The nuclease activity of MRX promotes the initiation of end resection, which leads to the displacement of KU. Long-range resection factors, Exo1 and Dna2–STR, are then recruited, the ssDNA overhang is coated with RPA, and the DNA damage checkpoint is activated. RPA is replaced with the Rad51 recombinase and the nucleoprotein filament initiates homology search for HR repair.