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

Abstract

In yeast as in human, DNA helicases play critical roles in assisting replication fork progression. The Saccharomyces cerevisiae MPH1 gene, homologue of human FANCM, has been involved in homologous recombination and DNA repair. We describe a synthetic growth defect of an mph1 deletion if combined with an srs2 deletion that can result -depending on the genetic background -in synthetic lethality. The lethality is suppressed by mutations in homologous recombination (rad51, rad52, rad55, rad57 ) and in the DNA damage checkpoint (rad9, rad24, rad17 ). Importantly, rad54 and mph1, epistatic for damage sensitivity, are subadditive for spontaneous mutator phenotype. Therefore, Mph1 could be placed at the Rad51-mediated strand invasion process, with a function distinct from Rad54. Moreover, siz1 mutation is viable with mph1 and additive for DNA damage sensitivity. mph1 srs2 double mutants, isolated in a background where they are viable, are synergistically sensitive to DNA damage. Moderate overexpression of SGS1 partially suppresses this sensitivity. Finally, we observe an epistatic relationship in terms of sensitivity to camptothecin of mms4 or mus81 to mph1. Overall, our results support a role of Mph1 in assisting replication progression. We propose two models for the resumption of DNA synthesis under replicative stress where Mph1 is placed at the sister chromatid interaction step.

Mte1 interacts with Mph1 and promotes crossover recombination and telomere maintenance

“…3; Supplemental Fig. 2; Scheller et al 2000;Schurer et al 2004;Panico et al 2010). MTE1 deletion did not increase cell sensitivity (Lisby et al 2003).…”

“…Importantly, on its own, Mte1 did not catalyze these reactions. Second, we tested the impact of Mte1 on the ability of Mph1 to displace D loops (Prakash et al 2009;Panico et al 2010;Zheng et al 2011). A D loop was formed by Rad51-catalyzed invasion of a 5 ′ -radiolabeled 90-mer oligo D1 into a homologous negatively supercoiled dsDNA plasmid (pBSC) in the presence of Rad54 (Sebesta et al 2011).…”

“…Mph1 has the ability to disrupt D loops formed by Rad51 and catalyze migration of branched DNA structures (Prakash et al 2005(Prakash et al , 2009Whitby 2010;Zheng et al 2011;Mitchel et al 2013). Independent of its role in D-loop unwinding, the Mph1 helicase promotes fork regression and branch migration during replication-associated recombinational repair (Prakash et al 2005(Prakash et al , 2009Mankouri et al 2009;Panico et al 2010;Zheng et al 2011;Xue et al 2014). The role of Mph1 in replication fork rescue is negatively regulated by the Smc5/6 complex by direct binding to the regulatory domain of Mph1 at its C terminus.…”

Functions and regulation of the multitasking FANCM family of DNA motor proteins

“…Genetic analyses have shown that simultaneously removing any two of these helicases increases crossover level more than ablating just one of them (Prakash et al 2009;Mitchel et al 2013). Nonoverlapping DNA repair roles between Mph1 and either Sgs1 or Srs2 lead to synthetic sickness or lethality of double mutants upon DNA damage occurrence (St Onge et al 2007;Chen et al 2009;Panico et al 2010). Similarly, Mph1 and STR orthologs in fission yeast and A. thaliana also have independent functions in crossover control and/or DNA repair (Sun et al 2008;Knoll et al 2012;Seguela-Arnaud et al 2015).…”

The Fanconi Anemia Ortholog FANCM Ensures Ordered Homologous Recombination in Both Somatic and Meiotic Cells in Arabidopsis

“…Sc-MPH1 is epistatic to a number of budding yeast homologous recombination (HR) genes, and the somatic HR rate is increased in mph1 mutant cells (Schürer et al, 2004). Recently, it was shown that Mph1 participates in D-loop formation in HR, together with the recombinase Rad54 (for radiation sensitive54) (Panico et al, 2010;Ede et al, 2011). In double mutants of MPH1 and SGS1 (for slow growth suppressor1), the sole budding yeast RecQ helicase homolog, the HR rate is higher than the rate of either single mutant, indicating two separate pathways with Mph1 and Sgs1 functioning in either one to suppress somatic HR (Schürer et al, 2004).…”