A mutational study of the C-terminal zinc-finger motif of the Escherichia coli UvrA protein.
The cysteine 763 residue in the C-terminal zinc-finger region of Escherichia coli UvrA protein was subjected to random mutagenesis, and the results suggested that the UvrA mutants with a small amino acid (Ser, Ala, or Gly) substituting for the cysteine 763 were almost as active as the wild-type in supporting nucleotide excision repair, but its replacement with a large, bulky amino acid (Tyr, Trp, or Phe) rendered the mutants inactive. The C763F mutant UvrA protein was purified for further characterization, and it was found this mutant UvrA protein lost its DNA binding (single-stranded or double-stranded DNA) activity and those other activities dependent on DNA binding, such as formation of damage-specific UvrA2B complexes and the supercoiling reaction. However, this mutant protein retained vigorous ATPase activity and was capable of negatively complementing the wild-type UvrA in JM109 strain. The purified C763F mutant UvrA protein contains a single zinc ion/molecule, half that of the wild-type. It appears that the C763F mutation destabilizes the zinc-anchored structure in the C-terminal zinc finger region, and as a result, the C763F mutant UvrA protein lost its ability to bind DNA.
Wang
J
J
Department of Biochemistry, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, Maryland 21218.
Mueller
K L
KL
Grossman
L
L
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002852:Chromatography, Ion Exchange; D003545:Cysteine / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000032:analysis; D016335:Zinc Fingers
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; C068631:UvrA protein, E coli; D000251:Adenosine Triphosphatases; D015032:Zinc; D003545:Cysteine