Abstract
It is proposed that mismatch repair (MMR) mediates the cytotoxic effects of DNA damaging agents by exerting a futile repair pathway which leads to double strand breaks (DSBs). Previous reports indicate that the sensitivity of cells defective in homologous recombination (HR) to DNA alkylation is reduced by defects in MMR genes. We have assessed the contribution of different MMR genes to the processing of alkylation damage in vivo. We have directly visualized recombination complexes formed upon DNA damage using fluorescent protein (FP) fusions. We find that msh6 mutants are more resistant than wild type cells to MNNG, and that an msh6 mutation rescues the sensitivity of rad52 strains more efficiently than an msh3 mutation. Analysis of RAD52-GFP tagged strains indicate that MNNG increases repair foci formation, and that the inactivation of the MHS2 and MSH6 genes but not the MSH3 gene result in a reduction of the number of foci formed. In addition, in the absence of HR, NHEJ could process the MNNG-induced DSBs as indicated by the formation of NHEJ-GFP tagged foci. These data suggest that processing of the alkylation damage by MMR, mainly by MSH2-MSH6, is required for recruitment of recombination proteins to the damage site for repair.
Highlights
Mismatch repair (MMR) plays a crucial role in the maintenance of the genome by repairing nucleotide misincorporations that occur during DNA replication and by preventing recombination between diverged sequences [1] [2], for review see [3]
Msh6 mutants were only slightly affected at this concentration of the alkylating agent (Figure 1(a)). This is more apparent at 60 μM of MNNG where the viability of wild type cells was reduced to 4%, and that of rad52 to 0.5%, while the msh6 mutant was 30% viable (7-fold higher than wild type)
Alkylation damage that results in methylation of O6-G can be processed to double strand breaks (DSBs) in a process that requires misincorporation by the DNA polymerase to form a T-methyl-O6-G that can be recognized by the MMR system
Summary
Mismatch repair (MMR) plays a crucial role in the maintenance of the genome by repairing nucleotide misincorporations that occur during DNA replication and by preventing recombination between diverged sequences [1] [2], for review see [3]. Inactivation of MSH6 or MSH3 results in modest phenotype due to their redundancy, whereas inactivation of MSH2 completely impairs the recognition step Besides their important role in correcting replication errors, MMR proteins have been recently reported to participate in cellular responses to some forms of DNA damage induced by certain anticancer drugs [5]. S. cerevisiae uses NHEJ to repair DSBs. The components of yeast NHEJ pathway are divided into three protein complexes [23] that include the exonuclease complex MRX (Mre11/Rad50/Xrs2) [24], the DNA end recognition protein complex yKu (YKU70/YKU80) which is initially recruited to the DNA damage site [25] and the DNA ligase (Dnl4/ Lif1) [25] [26]. The goal of our study is to determine the role of the different MMR genes in the processing of DNA alkylating damage to DSBs and to visualize the recruitment of the recombination pathways during the repair process. Our data suggest that the MSH2-MSH6 and not the MSH2-MSH3 complex mediates the processing of alkylating DNAdamage to DSBs and that both NHEJ and HR recombination pathways are recruited to the DNA damage site for repair
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