Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks

Abstract

Single-stranded DNA constitutes an important early intermediate for homologous recombination and damage-induced cell cycle checkpoint activation. In Saccharomyces cerevisiae, efficient double-strand break (DSB) end resection requires several enzymes; Mre11/Rad50/Xrs2 (MRX) and Sae2 are implicated in the onset of 5'-strand resection, whereas Sgs1/Top3/Rmi1 with Dna2 and Exo1 are involved in extensive resection. However, the molecular events leading to a switch from the MRX/Sae2-dependent initiation to the Exo1- and Dna2-dependent resection remain unclear. Here, we show that MRX recruits Dna2 nuclease to DSB ends. MRX also stimulates recruitment of Exo1 and antagonizes excess binding of the Ku complex to DSB ends. Using resection assay with purified enzymes in vitro, we found that Ku and MRX regulate the nuclease activity of Exo1 in an opposite way. Efficient loading of Dna2 and Exo1 requires neither Sae2 nor Mre11 nuclease activities. However, Mre11 nuclease activity is essential for resection in the absence of extensive resection enzymes. The results provide new insights into how MRX catalyses end resection and recombination initiation.

Figure 1

The MRX facilitates binding of Dna2 and Exo1 at a DSB. The enrichment of Dna2–9myc (B), Exo1–9myc (C) or Sgs1–9myc (D) flanking the HO-induced DSB at the MAT locus (A) in wild-type (dotted lines, open symbols), rad50Δ, sae2Δ, mre11Δ or mre11-3 mutants (solid lines, filled symbols) were shown at indicated times post-HO induction (1 h: square, 2 h: triangle and 3 h: circle). Fold immunoprecipitate represents the ratio of the anti-myc IP PCR signal before and after HO induction, normalized by the PCR signal of the PRE1 control and the amount of input DNA. Arrows indicate primers used for ChIP assay. The mean values±s.d. from three independent experiments are shown.

Figure 2

Mre11 and Sae2 nucleases have non-redundant functions in resection of HO-induced DSB. (A) The 5′-strand resection was analysed at the break and 28 kb away from the DSB in indicated mutants. Plots showing average percentage of unprocessed 5′ strand at these sites are shown. (B) The 5′-strand resection was analysed in indicated mutants by Southern blot hybridization. Dna2 and Mre11 nucleases have redundant functions in initial resection. (C) The 5′-strand resection at the break was analysed in mutants lacking both extensive resection enzymes and either Mre11 or Sae2 nuclease by Southern blot hybridization. Products of limited resection occurring in the absence of Exo1 and Sgs1 are indicated by arrows.

Figure 3

MRX suppresses excess Ku protein recruitment at a DSB. Kinetics of Ku recruitment to the HO-induced DSB in wild-type (dotted lines, open symbols), rad50Δ, mre11Δ, sae2Δ and mre11-3 mutants (solid lines, filled symbols) were determined by ChIP assays. The values at 1 h (square), 2 h (triangle) and 3 h (circle) are shown. Fold immunoprecipitate represents the ratio of the Ku IP PCR signal before and after HO induction, normalized by the PCR signal of the PRE1 control and the amount of input DNA. Data represent the mean±s.d. of three or more independent experiments.

Figure 4

Ku inhibits the recruitment of Exo1 at a DSB. The enrichment of Dna2-9myc (A) and Exo1-9myc (B) flanking the HO-induced DSB at the MAT locus in the wild-type, yku70Δ, rad50Δ and yku70Δ rad50Δ mutants were shown at indicated times post-HO induction (1 h: square, 2 h: triangle and 3 h: circle). Fold immunoprecipitate was calculated as described in Figure 1. The mean values±s.d. from three independent experiments were shown. IP values at 1 h post-HO expression was marked with ‘^*' to highlight the level of Exo1-9myc recruitment in yku70Δ and yku70Δ rad50Δ mutants.

Figure 5

Ku complex inhibits Exo1-dependent resection. (A–C) The 5′-strand resection was analysed at the break and 28 kb away from the DSB in indicated mutants. Plots are showing the average percentage of unprocessed 5′ strands at these sites. Resection in wild-type and yku70Δ mutants were compared at 3 and 10 kb away from the break.

Figure 6

Deletion of YKU70 suppresses resection and repair defects of rad50Δ. (A) Initial resection at the break and long-range resection measured 28 kb from DSB were analysed in indicated mutants. Southern blots are shown. (B) Resection in rad50Δ and rad50Δ yku70Δ were compared at 3 and 10 kb away from the break. (C) Analysis of DSB repair through gene conversion in an ectopic recombination assay using indicated mutants.

Figure 7

Ku and MRX complexes oppositely regulate the nuclease activity of Exo1. (A) DNA resection assays were performed with linearized DNA as a substrate (cut with SphI) and analysed by native agarose gel electrophoresis and SYBR green staining for the double-stranded DNA (bottom panel) followed by non-denaturing Southern hybridization with a strand-specific RNA probe for the 3′ strand, as previously described (Hopkins and Paull, 2008) (top panel). Reactions contained 10 ng (0.35 nM) pNO1 DNA, 2 nM yeast wt Exo1, MRX (8.3 or 25 nM), Sae2 (8.6 or 26 nM) and 10 nM Ku heterodimer as indicated. The position of single-stranded DNA in the gel was marked as ‘ss', and that of the un-resected plasmid is marked as double stranded, ‘ds'. Migration of molecular weight markers (kb) are shown in the lane marked ‘M'. (B) A model describing how MRX and Sae2 proteins facilitate end resection at HO breaks.