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 ...

The SRS2 (Suppressor of RAD Six screen mutant 2) gene encodes an ATP-dependent DNA helicase that regulates homologous recombination in Saccharomyces cerevisiae. Mutations in SRS2 result in a hyper-recombination phenotype, sensitivity to DNA damaging agents and synthetic lethality with mutations that affect DNA metabolism. Several of these phenotypes can be suppressed by inactivating genes of the RAD52 epistasis group that promote homologous recombination, implicating inappropriate recombination as the underlying cause of the mutant phenotype. Consistent with the genetic data, purified Srs2 strongly inhibits Rad51-mediated recombination reactions by disrupting the Rad51-ssDNA presynaptic filament. Srs2 interacts with Rad51 in the yeast two-hybrid assay and also in vitro. To investigate the functional relevance of the Srs2-Rad51 complex, we have generated srs2 truncation mutants that retain full ATPase and helicase activities, but differ in their ability to interact with Rad51. Importantly, the srs2 mutant proteins attenuated for Rad51 interaction are much less capable of Rad51 presynaptic filament disruption. An internal deletion in Srs2 likewise diminishes Rad51 interaction and anti-recombinase activity. We also present evidence that deleting the Srs2 C-terminus engenders a hyper-recombination phenotype. These results highlight the importance of Rad51 interaction in the anti-recombinase function of Srs2, and provide evidence that this Srs2 function can be uncoupled from its helicase activity.