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J. Biol. Chem., Vol. 276, Issue 6, 3904-3910, February 9, 2001

This Article Full Text Full Text (PDF) All Versions of this Article: 276/6/3904    most recent M008538200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tang, M.-s. Articles by Tang, Y.-Y. Search for Related Content PubMed PubMed Citation Articles by Tang, M.-s. Articles by Tang, Y.-Y. Social Bookmarking                      What's this?

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

Moon-shong Tang, Michael Nazimiec, Xiangcang Ye, Ganesh H. Iyer, Jamie Eveleigh, Yi Zheng, Wenjing Zhou, and Yen-Yee Tang

From the Department of Environmental Medicine, New York University School of Medicine, Tuxedo, New York 10987

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 X-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 (K_a, 9 × 10⁸/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)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.

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