Methyl-directed repair of DNA base-pair mismatches in vitro

Abstract

An assay has been developed that permits analysis of DNA mismatch repair in cell-free extracts of Escherichia coli. The method relies on repair of heteroduplex molecules of f1 R229 DNA, which contain a base-pair mismatch within the single EcoRI site of the molecule. As observed with mismatch heteroduplexes of lambda DNA [Pukkila, P. J., Peterson, J., Herman, G., Modrich, P. & Meselson, M. (1983) Genetics, in press], in vivo mismatch correction of f1 heteroduplexes is directed by the state of dam methylation of d(G-A-T-C) sequences within the DNA duplex. Thus, the heteroduplex (formula: see book) is repaired in vivo to an EcoRI-sensitive form if the strand bearing the wild-type EcoRI sequence carries the dam modification and the other does not. Such molecules are also subject to mismatch repair by E. coli extracts. The in vitro activity is also dependent on ATP, the state of dam methylation of mismatch heteroduplexes, and products of mutH, mutL, mutS, and uvrE loci. However, crude fractions deficient in these gene products do complement in the cell-free system, thus providing assays for their isolation. The in vitro reaction is accompanied by repair synthesis on the unmethylated DNA strand.