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.
Calmann
Melissa A
MA
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School Worcester, MA 01605, USA.
Evans
James E
JE
Marinus
M G
MG
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