Mutations in motif II of Escherichia coli DNA helicase II render the enzyme nonfunctional in both mismatch repair and excision repair with differential effects on the unwinding reaction.
Site-directed mutagenesis has been employed to address the functional significance of the highly conserved aspartic and glutamic acid residues present in the Walker B (also called motif II) sequence in Escherichia coli DNA helicase II. Two mutant proteins, UvrDE221Q and UvrDD220NE221Q, were expressed and purified to apparent homogeneity. Biochemical characterization of the DNA-dependent ATPase activity of each mutant protein demonstrated a kcat that was < 0.5% of that of the wild-type protein, with no significant change in the apparent Km for ATP. The E221Q mutant protein exhibited no detectable unwinding of either partial duplex or blunt duplex DNA substrates. The D220NE221Q mutant, however, catalyzed unwinding of both partial duplex and blunt duplex substrates, but at a greatly reduced rate compared with that of the wild-type enzyme. Both mutants were able to bind DNA. Thus, the motif II mutants E221Q and D220NE221Q were able to bind ATP and DNA to the same extent as wild-type helicase II but demonstrate a significant reduction in ATP hydrolysis and helicase functions. The mutant uvrD alleles were also characterized by examining their abilities to complement the mutator and UV light-sensitive phenotypes of a uvrD deletion mutant. Neither the uvrDE221Q nor the uvrDD220NE221Q allele, supplied on a plasmid, was able to complement either phenotype. Further genetic characterization of the mutant uvrD alleles demonstrated that uvrDE221Q confers a dominant negative growth phenotype; the uvrDD220NE221Q allele does not exhibit this effect. The observed difference in effect on viability may reflect the gene products' dissimilar kinetics for unwinding duplex DNA substrates in vitro.
Brosh
R M
RM
Department of Biology, University of North Carolina, Chapel Hill 27599-3280, USA.
Matson
S W
SW
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000502:physiology; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair / Q000502:physiology*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development / Q000528:radiation effects; D029968:Escherichia coli Proteins; D005799:Genes, Dominant / Q000235:genetics; D018698:Glutamic Acid / Q000502:physiology; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D017384:Sequence Deletion; D014466:Ultraviolet Rays
D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; C506746:UvrD protein, E coli; D004265:DNA Helicases