Bloom's helicase (BLM) is thought to prevent crossing-over during DNA double-strand-break repair (DSBR) by disassembling double-Holliday junctions (dHJs) or by preventing their formation. We show that the Saccharomyces cerevisiae BLM ortholog, Sgs1, prevents aberrant crossing-over during meiosis by suppressing formation of joint molecules (JMs) comprising three and four interconnected duplexes. Sgs1 and procrossover factors, Msh5 and Mlh3, are antagonistic since Sgs1 prevents dHJ formation in msh5 cells and sgs1 mutation alleviates crossover defects of both msh5 and mlh3 mutants. We propose that differential activity of Sgs1 and procrossover factors at the two DSB ends effects productive formation of dHJs and crossovers and prevents multichromatid JMs and counterproductive crossing-over. Strand invasion of different templates by both DSB ends may be a common feature of DSBR that increases repair efficiency but also the likelihood of associated crossing-over. Thus, by disrupting aberrant JMs, BLM-related helicases maximize repair efficiency while minimizing the risk of deleterious crossing-over.

Summary The Rad2/XPG family nuclease, Exo1, functions in a variety of DNA repair pathways. During meiosis, Exo1 promotes crossing-over and thereby facilitates chromosome segregation at the first division. Meiotic recombination is initiated by programmed DNA double-strand-breaks (DSBs). Nucleolytic resection of DSBs generates long 3′-single-strand tails that undergo strand-exchange with a homologous chromosome to form Joint Molecule (JM) intermediates. We show that meiotic DSB-resection is dramatically reduced in exo1Δ mutants and test the idea that Exo1-catalyzed resection promotes crossing-over by facilitating formation of crossover-specific JMs called double-Holliday Junctions (dHJs). Contrary to this idea, dHJs form at wild-type levels in exo1Δ mutants implying that Exo1 has a second function that promotes resolution of dHJs into crossovers. Surprisingly, the dHJ resolution function of Exo1 is independent of its nuclease activities, but requires interaction with the putative endonuclease complex, Mlh1-Mlh3. Thus, the DSB-resection and pro-crossover functions of Exo1 during meiosis involve temporally and biochemically distinct activities.

CtIP plays an important role in homologous recombination (HR)–mediated DNA double-stranded break (DSB) repair and interacts with Nbs1 and BRCA1, which are linked to Nijmegen breakage syndrome (NBS) and familial breast cancer, respectively. We identified new CDK phosphorylation sites on CtIP and found that phosphorylation of these newly identified CDK sites induces association of CtIP with the N-terminus FHA and BRCT domains of Nbs1. We further showed that these CDK-dependent phosphorylation events are a prerequisite for ATM to phosphorylate CtIP upon DNA damage, which is important for end resection to activate HR by promoting recruitment of BLM and Exo1 to DSBs. Most notably, this CDK-dependent CtIP and Nbs1 interaction facilitates ATM to phosphorylate CtIP in a substrate-specific manner. These studies reveal one important mechanism to regulate cell-cycle-dependent activation of HR upon DNA damage by coupling CDK- and ATM-mediated phosphorylation of CtIP through modulating the interaction of CtIP with Nbs1, which significantly helps to understand how DSB repair is regulated in mammalian cells to maintain genome stability.

Background: DNA double-stranded break (DSB) repair is critical for the maintenance of genome stability and prevention of cancer. Results: Dimerization of CtIP, a critical DSB repair protein, is important for its localization to chromosomal DSBs in mammalian cells. Conclusion: CtIP dimerization is required for DSB repair. Significance: These studies help to understand the molecular mechanisms of DSB repair in mammalian cells.