Mutation of a highly conserved arginine in motif IV of Escherichia coli DNA helicase II results in an ATP-binding defect.
A site-directed mutation in motif IV of Escherichia coli DNA helicase II (UvrD) was generated to examine the functional significance of this region. The highly conserved arginine at position 284 was replaced with alanine to construct UvrD-R284A. The ability of the mutant allele to function in methyl-directed mismatch repair and UvrABC-mediated nucleotide excision repair was examined by genetic complementation assays. The R284A substitution abolished function in both DNA repair pathways. To identify the biochemical defects responsible for the loss of biological function, UvrD-R284A was purified to apparent homogeneity, and its biochemical properties were compared with wild-type UvrD. UvrD-R284A failed to unwind a 92-base pair duplex region and was severely compromised in unwinding a 20-base pair duplex region. The Km of UvrD-R284A for ATP was significantly greater than 3 mM compared with 80 microM for UvrD. A large decrease in ATP binding was confirmed using a nitrocellulose filter binding assay. These data suggested that the R284A mutation severely reduced the affinity of helicase II for ATP. The reduced unwinding activity and loss of biological function of UvrD-R284A was probably the result of decreased affinity for ATP. These results implicate motif IV of superfamily I helicases in nucleotide binding and represent the first characterization of a helicase mutation outside motifs I and II that severely impacted the Km for ATP.
Hall
M C
MC
Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Matson
S W
SW
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002918:Chymotrypsin / Q000378:metabolism; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D019281:Dimerization; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D014466:Ultraviolet Rays
D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D001120:Arginine; D002918:Chymotrypsin; D000251:Adenosine Triphosphatases; C506746:UvrD protein, E coli; D004265:DNA Helicases