A simple genetic system has been developed to test the effect of over-expression of wild-type or mutated human MutL homologue 1 (hMLH1) proteins on methyl-directed mismatch repair (MMR) in Escherichia coli. The system relies on detection of Lac+ revertants using MMR-proficient or MMR-deficient E. coli strains carrying a lac +1 frameshift mutation expressing hMLH1 proteins. We report that expression of wild-type hMLH1 protein causes an approx. 19-fold increase in mutation rates. The mutator phenotype was due to the ability of hMLH1 protein to interact with bacterial MutL and MutS proteins, thereby interfering with the formation of complexes between MMR proteins and mismatched DNA. Conversely, expression of proteins encoded by alleles deriving from hereditary-non-polyposis-colon-cancer (HNPCC) families decreases mutation rates, depending on the specific amino acid substitutions. These effects parallel the MutL-and MutS-binding and ATP-binding/hydrolysis activities of the mutated proteins.
Abbreviations used: FEN1, flap structure-specific endonuclease 1; GHL, gyrase b/hsp90/MutL; hMLH1, human MutL homologue 1; hMSH2 and hMSH6, human MutS homologues 2 and 6; HNPCC, hereditary non-polyposis colon cancer; hsp90, heat-shock protein 90; IPTG, isopropyl β-d-thiogalactoside; MMR, mismatch repair; NER, nucleotide excision repair; Ni-NTA, Ni2+-nitrilotriacetate; ORF, open reading frame; PCNA, proliferating-cell nuclear antigen; PMS1, postmeiotic segregation increased 1; RFC, replication factor C; RPA, replication protein A; S93G (etc.), Ser93→Gly mutation (etc.).