Differences and similarities in the repair of two benzo[a]pyrenediol epoxide isomer-induced DNA adducts by uvrA, uvrB, and uvrC gene products

Tang, Moon Shong; Pierce, James; Doisy, Richard; Nazimiec, Michael; MacLeod, Michael C.

doi:10.1021/bi00151a006

Cited by 73 publications

(65 citation statements)

References 17 publications

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“…Using a viral E. coli transfection system we have found that only uvrC mutant cells show low transfectivity for viral DNA containing N-(guanosin-8-yl)-2-aminofluorene adducts; uvrA and uvrB cells show the same transfectivity as do wild-type cells. In contrast, these three mutant cells have the same low transfectivity for UV-irradiated or N-acetoxy-2-acetylaminofluorene and anti-and syn-benzo(a)pyrene diol epoxide-modified viral DNA (20,35). These results indicate that uvrC gene products may function in a manner more complicated than simply participating in the incision step and may interact with proteins other than UvrA and UvrB in vivo.…”

Section: Discussionmentioning

confidence: 89%

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: 89%

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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“…Racemic BPDE was purchased from the NCI repository (Midwest Research Institute, Kansas City, MO). Modification of DNA with BPDE was done according to published procedures (30,31), which include repeated extractions of adducted DNA with organic solvents (water-saturated diethyl ether and isoamyl alcohol). Control DNA samples were treated with solvent (95% ethanol) only.…”

Section: Methodsmentioning

confidence: 99%

“…Purified genomic DNA was modified with BPDE and then reacted with UvrABC nucleases. UvrABC makes a dual incision seven nucleotides 5Ј and four nucleotides 3Ј to a BPDE adduct (30). The 3Ј incisions can be precisely mapped by amplifying the resulting 5-phosphate-containing DNA using LMPCR with P53-specific primers (6,8,35).…”

Section: Cpg Methylationmentioning

confidence: 99%

Cytosine methylation determines hot spots of DNA damage in the human P 53 gene

Denissenko

Chen

Tang

et al. 1997

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

320

232

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“…Results obtained in a number of animal model systems have strongly suggested that BPDE induced DNA damage initiates carcionogenesis (for review, see Harvey, 1991;Harvey, 1981;Weinstein, 1981;Pelkonen and Nebert, 1982;Beland and Poirier, 1989). The binding BPDE to DNA is greatly a ected by DNA sequence context and nucleosome structure (Kaneko and Cerutti, 1982;Boles and Hogan, 1984;Osborne, 1990;Tang et al, 1992;Smith and MacLeod, 1993;Thrall et al, 1994). Recently, using UvrABC incision and LMPCR methods to map BPDE-DNA adduct distribution in the human p53 gene in cultered HeLa cells, normal human ®broblasts and bronchial epithelial cells, we have found that BPDE-guanine adducts form preferentially at codons 157, 248 and 273 (Denissenko et al, 1996); these preferential binding sites correspond exactly to the major mutational hotspot sites found in smoking-associated human lung cancers (Greenblatt et al, 1994;Hollstein et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

Slow repair of bulky DNA adducts along the nontranscribed strand of the human p53 gene may explain the strand bias of transversion mutations in cancers

et al. 1998

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Using UvrABC incision in combination with ligationmediated PCR (LMPCR) we have previously shown that benzo(a)pyrene diol epoxide (BPDE) adduct formation along the nontranscribed strand of the human p53 gene is highly selective; the preferential binding sites coincide with the major mutation hotspots found in human lung cancers. Both sequence-dependent adduct formation and repair may contribute to these mutation hotspots in tumor tissues. To test this possibility, we have extended our previous studies by mapping the BPDE adduct distribution in the transcribed strand of the p53 gene and quantifying the rates of repair for individual damaged bases in exons 5, 7, and 8 for both DNA strands of this gene in normal human ®broblasts. We found that: (i) on both strands, BPDE adducts preferentially form at CpG sequences, and (ii) repair of BPDE adducts in the transcribed DNA strand is consistently faster than repair of adducts in the nontranscribed strand, while repair at the major damage hotspots (guanines at codons 157, 248 and 273) in the nontranscribed strand is two to four times slower than repair at other damage sites. These results strongly suggest that both preferential adduct formation and slow repair lead to hotspots for mutations at codons 157, 248 and 273, and that the strand bias of bulky adduct repair is primarily responsible for the strand bias of G to T transversion mutations observed in the p53 gene in human cancers.

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Differences and similarities in the repair of two benzo[a]pyrenediol epoxide isomer-induced DNA adducts by uvrA, uvrB, and uvrC gene products

Cited by 73 publications

References 17 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

Cytosine methylation determines hot spots of DNA damage in the human P 53 gene

Slow repair of bulky DNA adducts along the nontranscribed strand of the human p53 gene may explain the strand bias of transversion mutations in cancers

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