A dominant negative allele of the Escherichia coli uvrD gene encoding DNA helicase II. A biochemical and genetic characterization.
A site-specific lysine to methionine mutation has been engineered at the invariant Lys35 residue in the ATPase A binding site of the Escherichia coli uvrD gene encoding DNA helicase II. The mutant protein (UvrDK35M) has been purified to apparent homogeneity and characterized. The kcat for DNA-dependent ATP hydrolysis was less than 0.5% that of the wild-type enzyme with no change in the apparent Km for ATP. No unwinding of partial duplex DNA substrates could be detected using the mutant protein. Moreover, the mutant protein inhibited the unwinding reaction catalyzed by the wild-type protein at ratios of mutant enzyme to wild-type enzyme < 1. We conclude that the K35M mutation renders helicase II catalytically inactive as a DNA helicase with little or no effect on the ability of the enzyme to bind ATP, DNA, or other proteins. In vivo complementation assays indicate that the mutant protein cannot substitute for the wild-type protein in methyl-directed mismatch repair, suggesting that the ATPase and/or helicase activity of helicase II is required in this repair pathway. Additional genetic characterization of the uvrDK35M allele, supplied on a plasmid, suggests that expression of the mutant protein, at levels equivalent to that of the wild-type protein, results in a dominant negative phenotype. Expression of lower levels of the mutant protein, both in the presence and absence of wild-type helicase II, results in a constitutive induction of the cellular SOS response and extensive filamentation of cells. This induction of the SOS response is not due to a defect in methyl-directed mismatch repair. Taken together, these data are consistent with the notion that E. coli helicase II may have a role in DNA replication.
George
J W
JW
Department of Biology, University of North Carolina at Chapel Hill 27599-3280.
Brosh
R M
RM
Matson
S W
SW
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000483:Alleles; D001483:Base Sequence; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004261:DNA Replication / Q000502:physiology*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial / Q000235:genetics*; D005799:Genes, Dominant / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype
D029968:Escherichia coli Proteins; D000251:Adenosine Triphosphatases; C506746:UvrD protein, E coli; D004265:DNA Helicases