The C-terminal zinc finger of UvrA does not bind DNA directly but regulates damage-specific DNA binding.
In prokaryotic nucleotide excision repair, UvrA recognizes DNA perturbations and recruits UvrB for the recognition and processing steps in the reaction. One of the most remarkable aspects of UvrA is that it can recognize a wide range of DNA lesions that differ in chemistry and structure. However, how UvrA interacts with DNA is unknown. To examine the role that the UvrA C-terminal zinc finger domain plays in DNA binding, an eleven amino acid deletion was constructed (ZnG UvrA). Biochemical characterization of the ZnG UvrA protein was carried out using UvrABC DNA incision, DNA binding and ATPase assays. Although ZnG UvrA was able to bind dsDNA slightly better than wild-type UvrA, the ZnG UvrA mutant only supported 50-75% of wild type incision. Surprisingly, the ZnG UvrA mutant, while retaining its ability to bind dsDNA, did not support damage-specific binding. Furthermore, this mutant protein only provided 10% of wild-type Bca UvrA complementation for UV survival of an uvrA deletion strain. In addition, ZnG UvrA failed to stimulate the UvrB DNA damage-associated ATPase activity. Electrophoretic mobility shift analysis was used to monitor UvrB loading onto damaged DNA with wild-type UvrA or ZnG UvrA. The ZnG UvrA protein showed a 30-60% reduction in UvrB loading as compared with the amount of UvrB loaded by wild-type UvrA. These data demonstrate that the C-terminal zinc finger of UvrA is required for regulation of damage-specific DNA binding.
Croteau
Deborah L
DL
Laboratory of Molecular Genetics, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
DellaVecchia
Matthew J
MJ
Wang
Hong
H
Bienstock
Rachelle J
RJ
Melton
Mark A
MA
Van Houten
Bennett
B
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014466:Ultraviolet Rays; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; C058093:UvrB protein, E coli; D000255:Adenosine Triphosphate; D004247:DNA; C068631:UvrA protein, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D008274:Magnesium