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, 286 (7), 5119-25

Homologous Recombination-Dependent Rescue of Deficiency in the Structural Maintenance of Chromosomes (Smc) 5/6 Complex

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Homologous Recombination-Dependent Rescue of Deficiency in the Structural Maintenance of Chromosomes (Smc) 5/6 Complex

Alejandro Chavez et al. J Biol Chem.

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.

Figures

FIGURE 1.
FIGURE 1.
Deletion of MPH1 rescues the DNA damage-sensitivity of smc5/6 mutants. A, spot assay comparing the relative growth rates of various single and double mutants between mph1Δ and mms21-sp or smc6–9 grown on YPAD alone or with the indicated genotoxins. B, Mph1 helicase-dependent functions are necessary for sensitizing smc5/6 mutants to DNA-damaging agents. mms21-sp mph1Δ and smc6–9 mph1Δ cells were transformed with either wild-type MPH1, empty vector, or mph1-DE (helicase dead) containing plasmids and spotted to the indicated media. C, deletion of MPH1 confers no DNA damage resistance to cells with mutant forms of Smc1/3 complex members. Hypomorphic alleles encoding the Smc1/3 complex members Smc1 and Mcd1 were used. The indicated double mutants were transformed with either the vector control or MPH1-containing plasmid, and spotted to the indicated media.
FIGURE 2.
FIGURE 2.
Deletion of MPH1 reduces accumulation of recombination intermediates in mms21-sp mutants. A, representative time course of two-dimensional gel electrophoresis (2DGE) followed by Southern blotting examining replication intermediates near ARS305 in mms21-sp and mms21-sp mph1Δ mutants at each time point after release into 0.016% MMS. B, quantification of the ratio of X-shaped molecules to structures running within the replication arc.
FIGURE 3.
FIGURE 3.
The interaction between Smc5 and Mph1 is dispensable with regard to MMS and HU sensitivity. A, two-hybrid analysis comparing the DBD alone or fused to the N terminus of Smc5 with regard to its ability to interact with either AD alone or AD fused to full-length Mph1 or Mph1-Δ60 (lacking amino acids 751–810). Control selects for the presence of both the DBD and AD containing plasmids, -His media selects for the presence of an interaction between the expressed DBD and AD containing proteins. B, co-IP performed by immunoprecipitating Smc5-Myc and probing for the presence of the C-terminal YFP tag on the various Mph1 proteins. C, spot assays comparing the growth of MPH1, mph1Δ, or mph1-Δ60 cells in SMC5/6 wild-type or smc5/6 mutant backgrounds on various media with or without genotoxic agents. The MPH1 and mph1-Δ60 alleles are integrated at the native MPH1 locus.
FIGURE 4.
FIGURE 4.
Sgs1 is essential for rescue of the DNA damage sensitivity mediated by deletion of MPH1. A, Sgs1 is required for deletion of MPH1 to rescue the DNA damage-sensitivity of smc5/6 mutants. Spot assay comparing the growth rates of the various mutant strains. B, Sgs1 requires its helicase and S-phase checkpoint functions for full rescue of the DNA damage sensitivity within smc5/6 mph1Δ cells. mms21-sp mph1Δ sgs1Δ, and smc6–9 mph1Δ sgs1Δ cells were transformed with either empty vector, wild-type SGS1, sgs1-hd (helicase dead allele of SGS1), or sgs1-ΔC202 (allele of SGS1 lacking the C-terminal 202 amino-acids and defective in intra-S-phase checkpoint activity)-containing plasmids and spotted to the indicated media.
FIGURE 5.
FIGURE 5.
Sgs1 is not required for the decreased levels of recombination intermediates seen upon MPH1 deletion. A, representative time course of 2DGE Southern blots examining replication intermediates near ARS305 in mms21-sp sgs1Δ and mms21-sp sgs1Δ mph1Δ mutants at the indicated times after release into 0.016% MMS. B, quantification of the ratio of X-shaped molecules to structures running within the replication arc.
FIGURE 6.
FIGURE 6.
MPH1 deletion rescues the DNA damage-sensitivity of smc5/6 mutants through a HR- and PRR-dependent pathway. A, spot assays comparing the growth rate of strains mutant for members of the Smc5/6 complex along with deletions in MPH1 and RAD52. B, spot assays comparing the growth of smc6–9 mutants with and without MPH1 and members of the PRR machinery.

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