MutS inhibits RecA-mediated strand transfer with methylated DNA substrates.
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.
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School Worcester, MA 01605, USA.
Melissa A
MA
Calmann
James E
JE
Evans
M G
MG
Marinus
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D010584:Bacteriophage phi X 174 / Q000235:genetics; D020137:Base Pair Mismatch; D004249:DNA Damage; D019175:DNA Methylation; D004260:DNA Repair; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism / Q000502:physiology; D013058:Mass Spectrometry; D008769:Methylnitronitrosoguanidine / Q000633:toxicity; D000070956:MutL Proteins; D051719:MutS DNA Mismatch-Binding Protein; D019853:O(6)-Methylguanine-DNA Methyltransferase; D011938:Rec A Recombinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014157:Transcription Factors
D001426:Bacterial Proteins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C074770:MutL protein, E coli; D014157:Transcription Factors; D008769:Methylnitronitrosoguanidine; C045675:Ada protein, E coli; D019853:O(6)-Methylguanine-DNA Methyltransferase; D011938:Rec A Recombinases; D000251:Adenosine Triphosphatases; D000070956:MutL Proteins; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli