Mutation detection with MutH, MutL, and MutS mismatch repair proteins.
Escherichia coli methyl-directed mismatch repair is initiated by MutS-, MutL-, and ATP-dependent activation of MutH endonuclease, which cleaves at d(GATC) sites in the vicinity of a mismatch. This reaction provides an efficient method for detection of mismatches in heteroduplexes produced by hybridization of genetically distinct sequences after PCR amplification. Multiple examples of transition and transversion mutations, as well as one, two, and three nucleotide insertion/deletion mutants, have been detected in PCR heteroduplexes ranging in size from 400 bp to 2.5 kb. Background cleavage of homoduplexes is largely due to polymerase errors that occur during amplification, and the MutHLS reaction provides an estimate of the incidence of mutant sequences that arise during PCR.
Smith
J
J
Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
Modrich
P
P
D000251:Adenosine Triphosphatases; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004260:DNA Repair; D045643:DNA Repair Enzymes; D004269:DNA, Bacterial / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D007700:Kinetics; D008745:Methylation; D008957:Models, Genetic; D000070956:MutL Proteins; D051719:MutS DNA Mismatch-Binding Protein; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D013379:Substrate Specificity; D013698:Templates, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D003848:Deoxyguanine Nucleotides; D029968:Escherichia coli Proteins; C074770:MutL protein, E coli; D009692:Nucleic Acid Heteroduplexes; C029603:deoxyguanosine triphosphate; D004706:Endodeoxyribonucleases; C059682:methyl-directed mismatch repair protein, E coli; D000251:Adenosine Triphosphatases; D000070956:MutL Proteins; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli; D045643:DNA Repair Enzymes