Two Forms of UvrC Protein with Different Double-stranded DNA Binding Affinities

• Published in: The Journal of biological chemistry Vol. 276; no. 6; pp. 3904 - 3910
• Main Authors: Tang, Moon-shong, Nazimiec, Michael, Ye, Xiangcang, Iyer, Ganesh H., Eveleigh, Jamie, Zheng, Yi, Zhou, Wenjing, Tang, Yen-Yee
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 09.02.2001; American Society for Biochemistry and Molecular Biology
• Subjects: Bacterial Proteins - metabolism; Bacteriophage phi X 174 - genetics; Chromatography, Liquid; DNA, Viral - metabolism; DNA-Binding Proteins - metabolism; Electrophoresis, Polyacrylamide Gel; Endodeoxyribonucleases; Escherichia coli Proteins; Protein Binding; Protein Isoforms - metabolism; Ultraviolet Rays; UvrA protein; UvrB protein; UvrC protein
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M008538200

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 φX-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,32P-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, 32P- 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 (Ka, 9 × 108/m) and in a concentration-dependent manner, whereas UvrCI does not. These two forms of UvrC were also produced by the endogenousuvrC operon. We propose that UvrCII-DNA binding may interfere with Uvr(A)2B-DNA damage complex formation. However, because of its low copy number and low binding affinity to DNA, UvrCII may not interfere with Uvr(A)2B-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.