Function of the homologous regions of the Escherichia coli DNA excision repair proteins UvrB and UvrC in stabilization of the UvrBC-DNA complex and in 3'-incision.

The nicking of damaged DNA during the nucleotide excision repair reaction in E. coli, is the result of a multi-step process involving three enzymes, UvrA, UvrB and UvrC. The UvrB protein is loaded on the site of the damage by UvrA, forming a stable UvrB-DNA complex. This complex is recognized by UvrC and in the resulting UvrBC-DNA complex dual incision takes place, first on the 3'-side and next on the 5'-side of the damaged nucleotide. A domain in the C-terminal part of UvrB has been identified to be essential for formation of the specific UvrBC-DNA complex that induces the 3'-incision [1]. The N-terminal half of UvrC contains a region that is homologous to this C-terminal domain of UvrB. Using site-directed mutagenesis of a conserved phenylalanine in the homologous regions of UvrB and UvrC two mutants were constructed, UvrB(F652L) and UvrC(F223L). Both proteins were tested in vitro using a DNA substrate with a defined cisplatin lesion. The protein-DNA and protein-protein interactions were studied using bandshift assays and DNAse I footprinting. We show that both domains are important for the binding of UvrC to the UvrB-DNA complex.