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. 2008 Oct 9;455(7214):770-4.
doi: 10.1038/nature07312. Epub 2008 Sep 21.

Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing

Eleni P Mimitou  1 , Lorraine S Symington
Affiliations

Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing

Eleni P Mimitou et al. Nature. .

Abstract

DNA ends exposed after introduction of double-strand breaks (DSBs) undergo 5'-3' nucleolytic degradation to generate single-stranded DNA, the substrate for binding by the Rad51 protein to initiate homologous recombination. This process is poorly understood in eukaryotes, but several factors have been implicated, including the Mre11 complex (Mre11-Rad50-Xrs2/NBS1), Sae2/CtIP/Ctp1 and Exo1. Here we demonstrate that yeast Exo1 nuclease and Sgs1 helicase function in alternative pathways for DSB processing. Novel, partially resected intermediates accumulate in a double mutant lacking Exo1 and Sgs1, which are poor substrates for homologous recombination. The early processing step that generates partly resected intermediates is dependent on Sae2. When Sae2 is absent, in addition to Exo1 and Sgs1, unprocessed DSBs accumulate and homology-dependent repair fails. These results suggest a two-step mechanism for DSB processing during homologous recombination. First, the Mre11 complex and Sae2 remove a small oligonucleotide(s) from the DNA ends to form an early intermediate. Second, Exo1 and/or Sgs1 rapidly process this intermediate to generate extensive tracts of single-stranded DNA that serve as substrate for Rad51.

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Figures

Figure 1
Figure 1. Sae2 and Exo1 function at different steps in DSB resection
a. Map of the chromosome XV region containing the ade2 direct repeat. The ade2-I allele is cleaved by I-SceI to yield the 2.3-kb and 1.7-kb cut fragments that disappear over time concomitant with repair to the 5-kb SSA product. b. SSA efficiency as indicated by sectoring on YPD plates and survival on YPRaf/Gal plates. Ade+ (white) colonies arise from ade2 (red) colonies by SSA during growth on YPD due to leaky expression of I-SceI. c. SSA physical assay: Monitoring of resection and repair of the I-SceI induced DSB by southern blot analysis of NheI/EagI digested genomic DNA. d. Quantitation of the ADE2 hybridization signal on native versus denatured DNA samples bound on nylon membrane. The means from multiple experiments from three independent inductions are presented, error bars indicate s.d.. * Denotes statistical significance of P<0.02 (unpaired t-test).
Figure 2
Figure 2. Sgs1 and Exo1 function redundantly in DSB resection
a. SSA plating efficiency as described in Fig. 1b. b. SSA physical assay as described in Fig. 1c. c. Schematic representation of MAT switching: the 0.9-kb MATa StyI fragment is cleaved by HO resulting in a 0.7-kb cut fragment that disappears over time concomitant with repair to an 1.8-kb MATα fragment. d. MAT switching assay: HO cut fragment processing and gene conversion efficiency as monitored by southern blot analysis of StyI digested genomic DNA. e. MAT switching assay on sgs1Δ exo1Δ cultures complemented with mutant or wild-type SGS1.
Figure 3
Figure 3. Single stranded intermediates fail to form in the absence of Exo1 and Sgs1
a. Schematic representation of the method used to detect single stranded intermediates after 5′-3′ resection of the MAT locus, showing the positions of the HO cut site, the probe and the StyI (S) and BstXI (B) sites. Processing of the HO-induced break renders the DNA single-stranded and resistant to digestion with restriction enzymes, giving rise to distinct sets of fragments. b. Alkaline electrophoresis of StyI/BstXI digested genomic DNA: ssDNA intermediates formed after the resection of the HO-induced break were detected by using 3′ or 5′ specific riboprobes. The bracket indicates the smeared 5′-terminated strands observed in the rad51Δ exo1Δ sgs1Δ mutant.
Figure 4
Figure 4. Sae2 is required for creating the minimally resected intermediates
SSA assay (a) and mating type switching assay (b) in cells treated or untreated with doxycycline before I-SceI or HO expression. *Refers to a cross-hybridizing band, which is present at time 0 h and if it were due to recombinants in the population would allow survival of cells on YPRaf/Gal medium.
Figure 5
Figure 5. Two-step mechanism for DSB resection
After a DSB is formed and recognized by the MRX complex, Sae2, in collaboration with MRX, trims the ends to create a minimally resected intermediate that is ‘compatible’ for processive resection by the 5′-3′ exonucleolytic activity of Exo1 or Sgs1 helicase and a single-strand specific nuclease. Cells lacking both the nuclease and the helicase activity accumulate the intermediates from MRX/Sae2 cleavage. Because mre11 and sae2 mutants still show DSB processing, Exo1 and Sgs1 must be able to access unprocessed DNA ends, but with reduced efficiency.
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