The beta -hairpin motif of UvrB is essential for DNA binding, damage processing, and UvrC-mediated incisions.
UvrB plays a major role in recognition and processing of DNA lesions during nucleotide excision repair. The crystal structure of UvrB revealed a similar fold as found in monomeric DNA helicases. Homology modeling suggested that the beta-hairpin motif of UvrB might be involved in DNA binding (Theis, K., Chen, P. J., Skorvaga, M., Van Houten, B., and Kisker, C. (1999) EMBO J. 18, 6899-6907). To determine a role of the beta-hairpin of Bacillus caldotenax UvrB, we have constructed a deletion mutant, Deltabetah UvrB, which lacks residues Gln-97-Asp-112 of the beta-hairpin. Deltabetah UvrB does not form a stable UvrB-DNA pre-incision complex and is inactive in UvrABC-mediated incision. However, Deltabetah UvrB is able to bind to UvrA and form a complex with UvrA and damaged DNA, competing with wild type UvrB. In addition, Deltabetah UvrB shows wild type-like ATPase activity in complex with UvrA that is stimulated by damaged DNA. In contrast to wild type UvrB, the ATPase activity of mutant UvrB does not lead to a destabilization of the damaged duplex. These results indicate that the conserved beta-hairpin motif is a major factor in DNA binding.
Skorvaga
Milan
M
Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Theis
Karsten
K
Mandavilli
Bhaskar S
BS
Kisker
Caroline
C
Van Houten
Bennett
B
D000251:Adenosine Triphosphatases / Q000378:metabolism; D020816:Amino Acid Motifs; D001407:Bacillus / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015394:Molecular Structure; D017433:Protein Structure, Secondary; D021122:Protein Subunits
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D021122:Protein Subunits; C058093:UvrB protein, E coli; D004247:DNA; D004706:Endodeoxyribonucleases; C066567:UvrC protein, E coli; C068631:UvrA protein, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases