PMID:15972855
| Citation |
Calmann, MA, Evans, JE and Marinus, MG (2005) MutS inhibits RecA-mediated strand transfer with methylated DNA substrates. Nucleic Acids Res. 33:3591-7 |
|---|---|
| Abstract |
DNA mismatch repair (MMR) sensitizes human and Escherichia coli dam cells to the cytotoxic action of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) while abrogation of such repair results in drug resistance. In DNA methylated by MNNG, MMR action is the result of MutS recognition of O6-methylguanine base pairs. MutS and Ada methyltransferase compete for the MNNG-induced O6-methylguanine residues, and MMR-induced cytotoxicity is abrogated when Ada is present at higher concentrations than normal. To test the hypothesis that MMR sensitization is due to decreased recombinational repair, we used a RecA-mediated strand exchange assay between homologous phiX174 substrate molecules, one of which was methylated with MNNG. MutS inhibited strand transfer on such substrates in a concentration-dependent manner and its inhibitory effect was enhanced by MutL. There was no effect of these proteins on RecA activity with unmethylated substrates. We quantified the number of O6-methylguanine residues in methylated DNA by HPLC-MS/MS and 5-10 of these residues in phiX174 DNA (5386 bp) were sufficient to block the RecA reaction in the presence of MutS and MutL. These results are consistent with a model in which methylated DNA is perceived by the cell as homeologous and prevented from recombining with homologous DNA by the MMR system. |
| Links |
PubMed PMC1157099 Online version:10.1093/nar/gki673 |
| Keywords |
Adenosine Triphosphatases/metabolism; Bacterial Proteins/metabolism; Bacteriophage phi X 174/genetics; Base Pair Mismatch; DNA Damage; DNA Methylation; DNA Repair; DNA, Viral/metabolism; DNA-Binding Proteins/metabolism; Escherichia coli/drug effects; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins/metabolism; Escherichia coli Proteins/physiology; Mass Spectrometry; Methylnitronitrosoguanidine/toxicity; MutS DNA Mismatch-Binding Protein; O(6)-Methylguanine-DNA Methyltransferase; Rec A Recombinases/antagonists & inhibitors; Rec A Recombinases/metabolism; Transcription Factors |
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