Summary Formation of single-strand DNA (ssDNA) tails at a double-strand break (DSB) is a key step in homologous recombination and DNA damage signaling. The enzyme(s) producing ssDNA at DSBs in eukaryotes remains unknown. We monitored 5’-strand resection at inducible DSB ends and identified proteins required for two stages of resection: initiation and long-range 5’-strand resection. The Mre11-Rad50-Xrs2 complex (MRX) initiates 5’ degradation, whereas Sgs1 and Dna2 degrade 5’-strands exposing long 3’-strands at 4.4 kb/h rate. Deletion of SGS1 or DNA2 reduces resection and DSB repair by single strand annealing between distant repeats. Resection in the absence of SGS1 or DNA2 depends on Exo1. In exo1Δ sgs1Δ mutants the MRX complex and Sae2 in a stepwise manner generate only few hundred nucleotides of ssDNA at the break resulting in inefficient gene conversion and G2/M damage checkpoint arrest. We provide the first comprehensive model of the early steps of DSB repair in eukaryotes.

If not properly processed and repaired, DNA double-strand breaks (DSBs) can give rise to deleterious chromosome rearrangements, which could ultimately lead to the tumor phenotype 1, 2. DSB ends are resected in a 5′ to 3′ fashion in cells, to yield single-stranded DNA for the recruitment of factors critical for DNA damage checkpoint activation and repair by homologous recombination2. The resection process involves redundant pathways consisting of nucleases, DNA helicases, and associated proteins3. Being guided by recent genetic studies 4-6 , we have reconstituted the first eukaryotic ATP-dependent DNA end resection machinery comprising the Saccharomyces cerevisiae Mre11-Rad50-Xrs2 (MRX) complex, the Sgs1-Top3-Rmi1 (STR) complex, Dna2 protein and the heterotrimeric single-strand DNA binding protein RPA. We show that DNA strand separation during end resection is mediated by the Sgs1 helicase function, in a manner that is enhanced by Top3-Rmi1 and MRX. In congruence with genetic observations 6 , while the Dna2 nuclease activity is critical for resection, the Mre11 nuclease activity is dispensable. By examining the top3 Y356F allele and its encoded protein, we provide evidence that the topoisomerase activity of Top3, although critical for the suppression of crossover recombination 2,7 , is not needed for resection either in cells or in the reconstituted system. Our results also unveil a multi-faceted role of RPA, in the sequestration of ssDNA generated by DNA unwinding, enhancement of 5′ strand incision, and protection of the 3′ strand. Our reconstituted system should serve as a useful model for delineating the mechanistic intricacy of the DNA break resection process in eukaryotes.The 3′ ssDNA strands derived from DSB resection attract RPA, which promotes the recruitment of checkpoint proteins to effect cell cycle arrest 8 . With the aid of a recombination mediator protein, such as yeast Rad52 or human BRCA2, the Rad51 recombinase displaces RPA from the ssDNA to assemble into a right-handed helical polymer capable of initiating DSB repair by homologous recombination 1,2 . Genetic studies in yeast have shown that DSB resection proceeds in two steps. The MRX complex plays a 3 To whom correspondences and request for materials should be addressed: Gregory Ira: gira@bcm.edu, Patrick Sung: patrick.sung@yale.edu. role in initiation, while the Sgs1 helicase, its associated proteins Top3 and Rmi1, and the helicase/nuclease Dna2, whose nuclease activity is needed for Okazaki fragment processing 9,10 , constitute the DNA motor-driven path of long-range resection. Exo1, a 5′-3′ exonuclease, defines a redundant resection means 4-6 . Here we reconstitute the Sgs1/Dna2-dependent DNA resection machinery and present results germane for understanding its mechanistic underpinnings.The requisite factors, namely, Sgs1, Top3-Rmi1 (TR) complex, MRX complex, Dna2, and RPA were purified and analyzed (see Supplementary Fig. 2 and the Supplementary Information). As shown in Figure 1a, the combination of these factors degraded a 1.9-kb ...

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 0 -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.

The formation of single-stranded DNA (ssDNA) at double-strand break (DSB) ends is essential in repair by homologous recombination and is mediated by DNA helicases and nucleases. Here we estimated the length of ssDNA generated during DSB repair and analyzed the consequences of elimination of processive resection pathways mediated by Sgs1 helicase and Exo1 nuclease on DSB repair fidelity. In wild-type cells during allelic gene conversion, an average of 2–4 kb of ssDNA accumulates at each side of the break. Longer ssDNA is formed during ectopic recombination or break-induced replication (BIR), reflecting much slower repair kinetics. This relatively extensive resection may help determine sequences involved in homology search and prevent recombination within short DNA repeats next to the break. In sgs1Δ exo1Δ mutants that form only very short ssDNA, allelic gene conversion decreases 5-fold and DSBs are repaired by BIR or de novo telomere formation resulting in loss of heterozygosity. The absence of the telomerase inhibitor, PIF1, increases de novo telomere pathway usage to about 50%. Accumulation of Cdc13, a protein recruiting telomerase, at the break site increases in sgs1Δ exo1Δ, and the requirement of the Ku complex for new telomere formation is partially bypassed. In contrast to this decreased and alternative DSB repair, the efficiency and accuracy of gene targeting increases dramatically in sgs1Δ exo1Δ cells, suggesting that transformed DNA is very stable in these mutants. Altogether these data establish a new role for processive resection in the fidelity of DSB repair.

DNA recombination pathways are cell cycle regulated to coordinate with replication. Cyclin-dependent kinase (Cdk1) promotes efficient 5'-strand resection at DNA double strand breaks (DSBs), the initial step of homologous recombination and damage checkpoint activation. The Mre11–Rad50–Xrs2 complex with Sae2 initiates resection, whereas two nucleases, Exo1 and Dna2, and the DNA helicase/topoisomerase complex Sgs1–Top3–Rmi1 generate longer ssDNA at DSBs. Using Saccharomyces cerevisiae we provide evidence for Cdk1-dependent phosphorylation of the resection nuclease Dna2 at Thr4, Ser17 and Ser237 that stimulates its recruitment to DSBs, resection and subsequent Mec1-dependent phosphorylation. Poorly recruited dna2T4A S17A S237A and dna2ΔN248 mutant proteins promote resection only in the presence of Exo1, suggesting crosstalk between Dna2- and Exo1-dependent resection pathways.