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 II, 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 II protein.
Washburn
B K
BK
Department of Genetics, University of Georgia, Athens 30602.
Kushner
S R
SR
D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004265:DNA Helicases; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000528:radiation effects; D029968:Escherichia coli Proteins; D017353:Gene Deletion; D005798:Genes, Bacterial; D005822:Genetic Vectors; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014466:Ultraviolet Rays
D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D015636:Magnesium Chloride; D000251:Adenosine Triphosphatases; C506746:UvrD protein, E coli; D004265:DNA Helicases