Abstract
The essential and evolutionarily conserved Smc5-Smc6 complex (Smc5/6) is critical for the maintenance of genome stability. Partial loss of Smc5/6 function yields several defects in DNA repair, which are rescued by inactivation of the homologous recombination (HR) machinery. Thus HR is thought to be toxic to cells with defective Smc5/6. Recent work has highlighted a role for Smc5/6 and the Sgs1 DNA helicase in preventing the accumulation of unresolved HR intermediates. Here we investigate how deletion of MPH1, encoding the orthologue of the human FANCM DNA helicase, rescues the DNA damage sensitivity of smc5/6 but not sgs1Δ mutants. We find that MPH1 deletion diminishes accumulation of HR intermediates within both smc5/6 and sgs1Δ cells, suggesting that MPH1 deletion is sufficient to decrease the use of template switch recombination (TSR) to bypass DNA lesions. We further explain how avoidance of TSR is nonetheless insufficient to rescue defects in sgs1Δ mutants, by demonstrating a requirement for Sgs1, along with the post-replicative repair (PRR) and HR machinery, in a pathway that operates in mph1Δ mutants. In addition, we map the region of Mph1 that binds Smc5, and describe a novel allele of MPH1 encoding a protein unable to bind Smc5 (mph1-Δ60). Remarkably, mph1-Δ60 supports normal growth and responses to DNA damaging agents, indicating that Smc5/6 does not simply restrain the recombinogenic activity of Mph1 via direct binding. These data as a whole highlight a role for Smc5/6 and Sgs1 in the resolution of Mph1-dependent HR intermediates.
Highlights
Their proper mitotic segregation and facilitating HRbased repair of DNA double strand breaks
Given its ability to suppress the temperature sensitivity of smc6-9 mutants, we asked if deletion of MPH1 could rescue the DNA damage sensitivity of cells defective for Smc5/6 function. mms21-sp and smc6-9 mutants, deleted for MPH1, exhibited an increased resistance to the DNA-damaging agents, methyl methanesulfonate (MMS) and HU, as compared with the single mms21-sp and smc6-9 controls (Fig. 1A)
Though this is consistent with our hypothesis that MPH1 deletion can rescue phenotypes caused by loss of Smc5/6 function, it is somewhat surprising given that MPH1 deletion in wild-type cells leads to mild MMS sensitivity (Fig. 1A) [25]
Summary
Their proper mitotic segregation and facilitating HRbased repair of DNA double strand breaks. This report uncovered a direct physical interaction between the Smc5/6 complex and the Mph1 helicase, and documented robust rescue of DNA damage sensitivity upon deletion of Mph1 from smc5/6 but not sgs1⌬ mutant cells.
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