Two forms of UvrC protein with different double-stranded DNA binding affinities

Using phosphocellulose followed by single-stranded DNA-cellulose chromatography for purification of UvrC proteins ii om overproducing cells, we found that UvrC elutes at two peaks: 0.4 m KCl (UvrCI) and 0.6 m KCl (UvrCII). Both forms of UvrC have a major peptide band (>95%) of the same molecular weight and identical N-terminal amino acid sequences, which are consistent with the initiation codon being at the unusual GTG site. Both forms of UvrC are active in incising UV-irradiated, supercoiled phiX-174 replicative form I DNA in the presence of UvrA and UvrB proteins; however, the specific activity of UVrCII is one-fourth that of UvrCI, The molecular weight of UvrCII is four times that of UvrCI on the basis of results of size exclusion chromatography and glutaraldehyde cross-linking reactions, indicating that UvrCH is a tetramer of UvrCI, Functionally, these two forms of UvrC proteins can be distinguished under reaction conditions in which the protein/nucleotide molar ratio is >0.06 by using UV-irradiated, P-32-labeled DNA fragments as substrates; under these conditions UvrCII is inactive in incision, but UvrCI remains active. The activity of UvrCII in incising UV-irradiated, P-32-labeled DNA fragments can be restored by adding unirradiated competitive DNA, and the increased level of incision corresponds to a decreased level of UVrCII binding to the substrate DNA. The sites of incision at the 5' and 3' sides of a W-induced pyrimidine dimer are the same for UvrCI and UvrCII. Nitrocellulose filter binding and gel retardation assays show that UvrCII binds to both UV-irradiated and unirradiated double-stranded DNA with the same affinity (K-a, 9 x 10(8)/m) and in a concentration-dependent manner, whereas UvrCI does not, These two forms of UvrC were also produced by the endogenous uvrC operon, me propose that UvrCII-DNA binding may interfere with Uvr(A)(2)B-DNA damage complex formation. However, because of its low copy number and low binding affinity to DNA, UvrCII may not interfere with UVr(A)(2)B-DNA damage complex formation in vivo, but instead through double-stranded DNA binding UVrCII may become concentrated at genomic: areas and therefore may facilitate nucleotide excision repair.