THE C-TERMINAL REGION OF THE UVRB PROTEIN OF ESCHERICHIA-COLI CONTAINS AN IMPORTANT DETERMINANT FOR UVRC BINDING TO THE PREINCISION COMPLEXBUT NOT THE CATALYTIC SITE FOR 3'-INCISION

The UvrABC endonuclease from Escherichia coli repairs damage in the DN A by dual incision of the damaged strand on both sides of the lesion. The incisions are in an ordered fashion, first on the 3'-side and next on the 5'-side of the damage, and they are the result of binding of U vrC to the UvrB-DNA preincision complex, In this paper, we show that a t least the C-terminal 24 amino acids of UvrB are involved in interact ion with UvrC and that this binding is important for the 3'-incision. The C-terminal region of UvrB, which shows homology with a domain of t he UvrC protein, is part of a region that is predicted to be able to f orm a coiled-coil, We therefore propose that UvrB and UvrC interact th rough the formation of such a structure. The C-terminal region of UvrB only interacts with UvrC when present in the preincision complex, ind icating that the conformational change in UvrB accompanying the format ion of this complex exposes the UvrC binding domain, Binding of UvrC t o the C-terminal region of UvrB is not important for the 5'-incision, suggesting that for this incision a different interaction of UvrC with the UvrB-DNA complex is required, Truncated UvrB mutants that lack up to 99 amino acids from the C terminus still give rise to significant incision (1-2%), indicating that this C-terminal region of UvrB does n ot participate in the formation of the active site for 3'-incision. Th is region, however, contains the residue (Glu-640) that was proposed t o be involved in 3'-catalysis, since a mutation of the residue (E640A) fails to promote 3'-incision (Lin, J. J., Phillips, A. M., Hearst, J. E., and Sancar, A. (1992) J. Biol. Chem. 267, 17695-17700). We have i solated a mutant UvrB with the same E640A substitution, but this prote in shows normal UvrC binding and incision in vitro and also results in normal survival after UV irradiation in vivo. As a consequence of the se results, it is still an open question as to whether the catalytic s ite for 3'-incision is located in UvrB, in UvrC, or is formed by both proteins.