Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination

Abstract

Eukaryotes possess mechanisms to limit crossing over during mitotic homologous recombination, thus avoiding possible chromosomal rearrangements. We show here that budding yeast Mph1, an ortholog of human FancM helicase, utilizes its helicase activity to suppress spontaneous unequal sister chromatid exchanges and DNA double-strand break-induced chromosome crossovers. Since the efficiency and kinetics of break repair are unaffected, Mph1 appears to channel repair intermediates into a noncrossover pathway. Importantly, Mph1 works independently of two other helicases-Srs2 and Sgs1-that also attenuate crossing over. By chromatin immunoprecipitation, we find targeting of Mph1 to double-strand breaks in cells. Purified Mph1 binds D-loop structures and is particularly adept at unwinding these structures. Importantly, Mph1, but not a helicase-defective variant, dissociates Rad51-made D-loops. Overall, the results from our analyses suggest a new role of Mph1 in promoting the noncrossover repair of DNA double-strand breaks.[Keywords: Genome instability; recombination; DNA helicase; crossing over; Fanconi anemia] Supplemental material is available at http://www.genesdev.org. Received September 8, 2008; revised version accepted November 12, 2008. DNA double-strand breaks (DSBs) that arise during DNA replication or are induced by DNA damaging agents, such as ionizing radiation, are frequently repaired by homologous recombination (HR). In yeast and other eukaryotes, the RAD52 epistasis group of proteins mediate homologous recombination (for reviews, see Paques and Haber 1999;Krogh and Symington 2004). In this process, DSB ends are resected by nucleases to create 39 ssDNA that becomes coated with the recombinase protein Rad51. The Rad51-ssDNA nucleoprotein filament then carries out a search for a homologous donor DNA sequence and promotes strand invasion of the donor molecule to form a D-loop (Sung and Klein 2006). After D-loop formation, there appear to be two alternative pathways that result in DSB repair. One pathway involves the formation of a double Holliday junction (dHJ) that can be resolved by symmetrical strand cleavage into either a crossover or noncrossover gene conversion (Szostak et al. 1983). An alternative mechanism, called synthesis-dependent strand annealing (SDSA), posits formation mostly of noncrossovers, and in most cases does not involve a dHJ intermediate (for review, see Paques and Haber 1999). In support of the SDSA model of gene conversion, we showed recently that both newly synthesized strands are inherited by the broken recipient DNA molecule (Ira et al. 2006). The choice between crossover and noncrossover is tightly regulated in both mitotic and meiotic cells. In meiotic S. cerevisiae cells the correct number of crossovers are required for proper chromosome segregation; the proportion of gene conversion accompanied by crossing over varies between 25% and 50% depending on the locus (Roeder 1995). In mitotic cells, the proportion of crossovers is much lower, ranging between <1% and 7 These authors c...

Genetic evidence for a role of Saccharomyces cerevisiae Mph1 in recombinational DNA repair under replicative stress

“…Therefore, given the incomplete epistatic relationship between mph1 and rad54, it seems reasonable to place Mph1 at the Rad51-mediated strand invasion process, with a function distinct from that of Rad54. In addition, a recent study by Prakash et al (2009) has indicated that Mph1 is involved in dissolving D-loop structures, independently of Srs2 and Sgs1, in order to attenuate cross-over events. …”

“…A wide genetic analysis placed MPH1 in a pathway dedicated to the error-free bypass of DNA lesions via HR (Schürer et al, 2004). A recent work by Prakash et al (2009) described the ability of purified Mph1 to bind D-loop structures and to unwind these DNA structures, although it is probably not its only function since it would not explain the mph1 mutator phenotype (Scheller et al, 2000). S. cerevisiae Mph1 shows sequence similarity to human FANCM and the archaeabacterial Hef (Komori et al, 2002;Meetei et al, 2005).…”

Regulation of Recombination and Genomic Maintenance

“…FANCM protein in plants and its homologs in fission (Fml1) and budding yeast (Mph1) have been implicated in anticrossover control during meiotic HR and in vegetative cells, respectively (Prakash et al 2009;Crismani et al 2012;Lorenz et al 2012). Budding yeast Mph1 has been shown to dissociate nascent D-loops in reconstituted reactions with Rad51 and Rad54 as well as protein-free D-loops in a helicase-dependent manner (Prakash et al 2009).…”

“…Budding yeast Mph1 has been shown to dissociate nascent D-loops in reconstituted reactions with Rad51 and Rad54 as well as protein-free D-loops in a helicase-dependent manner (Prakash et al 2009). Further analysis will be required to determine the exact mechanism used by these enzymes to avoid crossovers.…”

Interplay between the Smc5/6 complex and the Mph1 helicase in recombinational repair

“…An activity shared by Mph1 and its orthologs is the dissociation of DNA D-loop structures, a function important in limiting crossovers during mitotic recombination (21)(22)(23). In addition, Fml1, FANCM, and Hef can catalyze the regression or unwinding of replication forks, which can potentially lead to recombination (22)(23)(24).…”