title: Two forms of UvrC protein with different double-stranded DNA binding affinities.
abstract: Using phosphocellulose followed by single-stranded DNA-cellulose chromatography for purification of UvrC proteins from 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 UvrCII 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, (32)P-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, (32)P- 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 UV-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. We 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.
authors.affiliation: Department of Environmental Medicine, New York University School of Medicine, Tuxedo, New York 10987.
authors.lastname: Tang Ms
authors.forename: M
authors.initials: M
authors.lastname: Nazimiec
authors.forename: X
authors.initials: X
authors.lastname: Ye
authors.forename: G H
authors.initials: GH
authors.lastname: Iyer
authors.forename: J
authors.initials: J
authors.lastname: Eveleigh
authors.forename: Y
authors.initials: Y
authors.lastname: Zheng
authors.forename: W
authors.initials: W
authors.lastname: Zhou
authors.forename: Y Y
authors.initials: YY
authors.lastname: Tang
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D010584:Bacteriophage phi X 174 / Q000235:genetics; D002853:Chromatography, Liquid; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases; D029968:Escherichia coli Proteins; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism*; D014466:Ultraviolet Rays
chemical_list: D001426:Bacterial Proteins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D020033:Protein Isoforms; D004706:Endodeoxyribonucleases; C066567:UvrC protein, E coli