The purification of the<i>Escherichia coli</i>UvrABC incision system

Yeung, Anthony T.; Mattes, William B.; Oh, Euk Y.; Yoakum, George H.; Grossman, Lawrence I.

doi:10.1093/nar/14.21.8535

Cited by 65 publications

(36 citation statements)

References 15 publications

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“…Purification of UvrC proteins has been achieved by using lysogens with the uvrC gene (36,37) or cells with a plasmid containing the uvrC gene (12). The method of purification is mainly based on the ability of UvrC proteins bind to ss-DNA.…”

Section: Discussionmentioning

confidence: 99%

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

Tang

Nazimiec

et al. 2001

Journal of Biological Chemistry

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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 Nterminal 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, 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 Plabeled 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 ؋ 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.

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Section: Discussionmentioning

confidence: 99%

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

Tang

Nazimiec

et al. 2001

Journal of Biological Chemistry

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“…Nucleotide excision entails the removal of the damaged nucleotide(s) in the form of an oligomer (3,4), "repair synthesis" (5,6) to fill in the single-stranded gap, and eventual ligation to restore the intact duplex. In Escherichia coi, (A)BC excinuclease initiates nucleotide excision by incising the damaged strand in a precise manner on both sides of the adduct: the eighth phosphodiester bond 5' and the fifth phosphodiester bond 3' to the adduct (7,8). Two other mechanisms that involve nicking 5' or 3' to the adduct by an endonuclease followed by exonucleolytic removal of the damaged base can be envisioned.…”

mentioning

confidence: 99%

Human nucleotide excision nuclease removes thymine dimers from DNA by incising the 22nd phosphodiester bond 5' and the 6th phosphodiester bond 3' to the photodimer.

Huang

Svoboda

Reardon

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

416

298

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By using a human cell-free system capable of nucleotide excision repair, a synthetic substrate conI of a plasmid containing four thymine dimers at unique locations, and deoxyribonucleoside 5'-[a-thioltriphosphates for repair synthesis, we obtained DNA fragments containing repair patches with phosphorothioate linkages. Based on the resistance of these linkages to digestion by exonuclease HI and their sensitivity to cleavage by 12, we were able to delineate the borders of the repair patch to single-nucleotide resolution and found an asymmetric patch with sharp boundaries. That the repair patch was produced by filing in a gap generated by an excision nuclease and not by nick-translation was confirmed by the finding that the thymidine dimer was released in a 27-to 29-nucleotide oligomer.

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“…The E. coli UvrABC endonuclease has been highly purified and characterized biochemically (1)(2)(3)(4)(5)(6)(7). In the presence of MgATP, UvrA and UvrB proteins form a high-affinity complex specifically with damaged DNA (2,7).…”

mentioning

confidence: 99%

“…The E. coli UvrA, UvrB, and UvrC proteins used in these studies were purified as described (6) to >95% purity as estimated using SDS/polyacrylamide gel electrophoresis.…”

mentioning

confidence: 99%

The noncovalent complex between DNA and the bifunctional intercalator ditercalinium is a substrate for the UvrABC endonuclease of Escherichia coli.

Lambert

Jones²,

Roques³

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

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We have demonstrated that the noncovalent complex formed between DNA and an antitumor bifunctional intercalator, ditercalinium, is recognized in vitro as bulky covalent DNA lesions by the purified Escherichia coli UvrABC endonuclease. It was established that no covalent drug-DNA adduct was formed during the incubation ofthe drug with DNA or during subsequent incubation with the UvrAB proteins. The nucleoprotein-ditercalinium complexes appear different from those generated by repair of pyrimidine dimers. The UvrA protein is able to form a stable complex with ditercaliniumintercalated DNA in the presence of ATP, whereas both UvrA and UvrB proteins are required to form a stable complex with pyrimidine dimer-containing DNA. The apparent half-life of the UvrA-and UvrAB-ditercalinium-DNA complexes following removal of free ditercalinium is 5 min. However, if the free ditercalinium concentration is maintained to allow the intercalation of one molecule of ditercalinium per 3000 base pairs, the half-life of the UvrA-or UvrAB-ditercalinium-DNA complex is 50 min, comparable to that of the complex of UvrAB proteins formed with pyrimidine dimer-containing DNA.UvrABC endonuclease incises ditercalinium-intercalated DNA as efficiently as pyrimidine dimer-containing DNA. However, unlike repair of pyrimidine diners, the incision reaction is strongly favored by the supercoiling of the DNA substrate. Because UvrA-or UvrAB-ditercalinium-DNA complexes can be formed with relaxed DNA without leading to a subsequent incision reaction, these apparently dead-end nucleoprotein complexes may become lesions in themselves resulting in the cytotoxicity of ditercalinium. Our results show that binding of excision repair proteins to a noncovalent DNA-ligand complex may lead to cell toxicity.

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The purification of theEscherichia coliUvrABC incision system

Cited by 65 publications

References 15 publications

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

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

Human nucleotide excision nuclease removes thymine dimers from DNA by incising the 22nd phosphodiester bond 5' and the 6th phosphodiester bond 3' to the photodimer.

The noncovalent complex between DNA and the bifunctional intercalator ditercalinium is a substrate for the UvrABC endonuclease of Escherichia coli.

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