Characterization of DNA Helicase II from a uvrD252 Mutant of Escherichia coli

The loss of DNA helicase II (UvrD) in Escherichia coli results in sensitivity to UV light and increased levels of spontaneous mutagenesis. While the effects of various uvrD alleles have been analyzed in vivo, the proteins produced by these alleles have not been examined in any detail. We have cloned one of these alleles, uvrD252, and determined the site of the mutation conferring the phenotype. In addition, the protein it encodes has been purified to homogeneity and characterized in vitro. The mutation responsible for the phenotype was identified as a glycine-to-aspartic-acid change in the putative ATP-binding domain. In comparison to wild-type DNA helicase H, the UvrD252 enzyme exhibited reduced levels of ATPase activity and a large increase in the Km for ATP. The ability of UvrD252 to unwind DNA containing single-stranded regions, as well as DNA containing only nicks, was reduced in comparison to that of the wild-type enzyme. Possible interpretations of these results in relation to the phenotypes of the uvrD252 mutant are discussed. This represents the first detailed analysis of the biochemical properties of a mutant DNA helicase H protein. The uvrD gene of Escherichia coli encodes an 82-kDa protein with both ATPase and helicase activities (26, 30). This enzyme, DNA helicase II, unwinds DNA duplexes with 3 ' to 5 ' polarity with respect to the bound strand (13, 29) and initiates unwinding most effectively when a single-stranded region is present (1, 29). At higher enzyme concentrations, it can also initiate unwinding from a nick or blunt end (13, 42). The involvement of DNA helicase II in the postincision events of nucleotide excision repair and methyl-directed mismatch repair has been demonstrated through studies o