Yeast MPH1 Gene Functions in an Error-Free DNA Damage Bypass Pathway That Requires Genes From Homologous Recombination, but Not From Postreplicative Repair

Abstract

The MPH1 gene from Saccharomyces cerevisiae, encoding a member of the DEAH family of proteins, had been identified by virtue of the spontaneous mutator phenotype of respective deletion mutants. Genetic analysis suggested that MPH1 functions in a previously uncharacterized DNA repair pathway that protects the cells from damage-induced mutations. We have now analyzed genetic interactions of mph1 with a variety of mutants from different repair systems with respect to spontaneous mutation rates and sensitivities to different DNA-damaging agents. The dependence of the mph1 mutator phenotype on REV3 and REV1 and the synergy with mutations in base and nucleotide excision repair suggest an involvement of MPH1 in error-free bypass of lesions. However, although we observed an unexpected partial suppression of the mph1 mutator phenotype by rad5, genetic interactions with other mutations in postreplicative repair imply that MPH1 does not belong to this pathway. Instead, mutations from the homologous recombination pathway were found to be epistatic to mph1 with respect to both spontaneous mutation rates and damage sensitivities. Determination of spontaneous mitotic recombination rates demonstrated that mph1 mutants are not deficient in homologous recombination. On the contrary, in an sgs1 background we found a pronounced hyperrecombination phenotype. Thus, we propose that MPH1 is involved in a branch of homologous recombination that is specifically dedicated to error-free bypass.

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

“…mph1 mutants are not deficient in HR, as determination of spontaneous mitotic recombination rates demonstrated. On the contrary, in an sgs1 background, a hyper-recombination phenotype was found (Schürer et al, 2004). Our data argue for a function of Mph1 at a step after the initiation of recombination and before Sgs1.…”

“…RAD54 codes for a dsDNA-dependent ATPase considered a key factor of HR, probably acting at multiple stages in concert with Rad51 (Heyer et al, 2006). Similarly to the HR mutants we have previously tested (Schürer et al, 2004), rad54 is epistatic to mph1 in terms of DNA damage sensitivity. As shown in Figure 3B, rad54 single mutant and rad54 mph1 double mutant show the same sensitivity to 4-NQO, MMS and camptothecin.…”

“…1; Oxoid, Basingstoke, UK) was also added before autoclaving. YPD, synthetic complete medium, drop-out media, canavanine plates, G418 plates and 5-FOA plates were prepared as previously described (Schürer et al, 2004). Sporulation plates: 0.05% D-glucose (Merck, Darmstadt, or Roth, Karlsruhe, Germany), 0.23% yeast extract (Oxoid), 2% potassium acetate (MERCK, Darmstadt, Germany).…”

“…Three serial dilutions were prepared (1 × 10 6 , 1 × 10 5 and 1 × 10 4 cells/ml), 10 µl of the adjusted cell suspension and of all the serial dilutions were spotted onto YPD plates without added chemicals as control and onto YPD plates containing MMS, 4-NQO and camptothecin. Plates were incubated for 3 days at 30 • C. Mutation rates to canavanine resistance were determined by the method of the median (Lea and Coulson, 1948), as described by Schürer et al (2004). Rates of resistance to 5-fluoroorotic acid (5-FOA; Sigma-Aldrich) were determined accordingly.…”

“…We have previously reported that mph1 mutants show increased sensitivity to genotoxic agents and elevated spontaneous mutation rate, dependent on a functional TLS (Entian et al, 1999;Scheller et al, 2000). 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).…”