Methyl-directed repair of mismatched small heterologous sequences in cell extracts from Escherichia coli

Abstract

The methyl-directed DNA repair efficiency of a set of M13mp18 heteroduplexes containing 1-8 or 22 unpaired bases was determined by using an in vitro DNA mismatch repair assay. The unpaired bases of each heteroduplex residing at overlapping recognition sites of two restriction endonucleases allow independent assay of repair on either DATA strand. Our results showed that the repair of small nucleotide heterologies in Escherichia coli extracts was very similar to base-base mismatch repair, being strand-specific and highly biased to the unmethylated strand. The in vitro activity was also dependent on products of mutH, mutL, mutS, and uvrD loci and was equally efficient on nucleotide insertions and deletions. The repair levels of small heterologies were affected by base composition of the heterologies. However, the extent of repair of heteroduplexes containing small heterologous sequences was found to decrease with an increase in the number of unpaired bases. Heteroduplexes containing an extra nucleotide of 22 bases provoked very low level of methyl-directed repair.