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 ...

Phosphorylation is a universal mechanism for regulating cell behavior in eukaryotes. Although protein kinases are known to target short linear sequence motifs on their substrates, the rules for kinase substrate recognition are not completely understood. We used a rapid peptide screening approach to determine consensus phosphorylation site motifs targeted by 61 of the 122 kinases in Saccharomyces cerevisae. Correlation of these motifs with kinase primary sequence has uncovered previously unappreciated rules for determining specificity within the kinase family, including a residue determining P−3 Arg specificity among members of the CMGC group of kinases. Furthermore, computational scanning of the yeast proteome enabled the prediction of thousands of new kinase-substrate relationships. We experimentally verified several candidate substrates of the Prk1 family of kinases in vitro and in vivo, and we identified a protein substrate of the kinase Vhs1. Together, these results elucidate how kinase catalytic domains recognize their phosphorylation targets and suggest general avenues for the identification of new kinase substrates across eukaryotes.

During DNA repair by homologous recombination (HR), DNA synthesis copies information from a template DNA molecule. Multiple DNA polymerases have been implicated in repair-specific DNA synthesis1–3, but it has remained unclear whether a DNA helicase is involved in this reaction. A good candidate is Pif1, an evolutionarily conserved helicase in S. cerevisiae important for break-induced replication (BIR)4 as well as HR-dependent telomere maintenance in the absence of telomerase5 found in 10–15% of all cancers6. Pif1 plays a role in DNA synthesis across hard-to-replicate sites7, 8 and in lagging strand synthesis with Polδ9–11. Here we provide evidence that Pif1 stimulates DNA synthesis during BIR and crossover recombination. The initial steps of BIR occur normally in Pif1-deficient cells, but Polδ recruitment and DNA synthesis are decreased, resulting in premature resolution of DNA intermediates into half crossovers. Purified Pif1 protein strongly stimulates Polδ-mediated DNA synthesis from a D-loop made by the Rad51 recombinase. Importantly, Pif1 liberates the newly synthesized strand to prevent the accumulation of topological constraint and to facilitate extensive DNA synthesis via the establishment of a migrating D-loop structure. Our results uncover a novel function of Pif1 and provide insights into the mechanism of HR.

Meiotic recombination differs from mitotic recombination in that DSBs are repaired using homologous chromosomes, rather than sister chromatids. This change in partner choice is due in part to a barrier to sister chromatid repair (BSCR) created by the meiosis-specific kinase, Mek1, in a complex with two other meiosis-specific proteins, Hop1 and Red1. HOP1 contains two functional domains, called the N and C domains. Analysis of a point mutation that specifically inactivates the C domain (hop1-K593A) reveals that the N domain is sufficient for Hop1 localization to chromosomes and for Red1 and Hop1 interactions. The C domain is needed for spore viability, for chromosome synapsis, and for preventing DMC1-independent DSB repair, indicating it plays a role in the BSCR. All of the hop1-K593A phenotypes can be bypassed by fusion of ectopic dimerization domains to Mek1, suggesting that the function of the C domain is to promote Mek1 dimerization. Hop1 is a DSB-dependent phosphoprotein, whose phosphorylation requires the presence of the C domain, but is independent of MEK1. These results suggest a model in which Hop1 phosphorylation in response to DSBs triggers dimerization of Mek1 via the Hop1 C domain, thereby enabling Mek1 to phosphorylate target proteins that prevent repair of DSBs by sister chromatids.