The role of ATP binding and hydrolysis by UvrB during nucleotide excision repair

We have isolated UvrB-DNA complexes by capture of biotinylated damaged DNA substrates on streptavidin-coated magnetic beads. With this method the UvrB -DNA preincision complex remains stable even in the absence of ATP. For the binding of UvrC to the UvrB-DNA complex no cofactor is needed. The subsequ ent induction of 3' incision does require ATP binding by UvrB but not hydro lysis. This ATP binding induces a conformational change in the DNA, resulti ng in the appearance of the DNase I-hypersensitive site at the 5' side of t he damage. In contrast, the 5' incision is not dependent on ATP binding bec ause it occurs with the same efficiency with ADP. We show with competition experiments that both incision reactions are induced by the binding of the same UvrC molecule. A DNA substrate containing damage close to the 5' end o f the damaged strand is specifically bound by UvrB in the absence of UvrA a nd ATP (Moolenaar, G. F., Monaco, V., van der Marel, C:. A, van Boom, J. H. , Visse, R,, and Goosen, N. (2000) J. Biol: Chem. 275, 8038-8043), To initi ate the formation of an active UvrBC-DNA incision complex, however, UvrB fi rst needs to hydrolyze ATP, and subsequently a new ATP molecule must be bou nd. Implications of these findings for the mechanism of the UvrA-mediated f ormation of the UvrB-DNA preincision complex will be discussed.