Formation and enzymatic properties of the UvrB.DNA complex.
The UvrA, UvrB, and UvrC proteins collectively catalyze the dual incision of a damaged DNA strand in an ATP-dependent reaction. We previously reported (Orren, D. K., and Sancar, A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5237-5241) that UvrA delivers UvrB to damaged sites in DNA; upon addition of UvrC to these UvrB.DNA complexes, the DNA is incised. In the present study, we have further characterized both the delivery of UvrB to DNA and the subsequent incision process, with emphasis on the role of ATP in these reactions. The UvrA-dependent delivery of UvrB onto damaged DNA is relatively slow (kon approximately 6 x 10(4) M-1 s-1) and requires ATP hydrolysis (Km = 120 microM). Although ATP enhances the stability of UvrB.DNA complexes (koff = 8.5 x 10(-5) s-1), the isolated UvrB.DNA complexes do not contain any covalently attached or stably bound nucleotide. However, ATP binding is required for the UvrC-dependent dual incision of DNA bound by UvrB. Interestingly, adenosine 5'-(3-O-thio)triphosphate can substitute for ATP at this step. The Km for ATP during incision is 2 microM, but ATP is not hydrolyzed at a detectable level during the incision reaction. The incisions made by UvrB-UvrC are on both sides of the adduct and result in the excision of the damaged nucleotide.
Orren
D K
DK
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill 27599.
Sancar
A
A
D000227:Adenine Nucleotides / Q000494:pharmacology; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D004265:DNA Helicases; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D006150:Guanine Nucleotides / Q000494:pharmacology; D007700:Kinetics; D010957:Plasmids
D000227:Adenine Nucleotides; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D006150:Guanine Nucleotides; C058093:UvrB protein, E coli; D004247:DNA; D004265:DNA Helicases