Journal Article

Use of UvrABC nuclease to quantify benzo[<i>a</i>]pyrene diol epoxide–DNA adduct formation at methylated versus unmethylated CpG sites in the p53 gene

Moon-shong Tang, Jessica B. Zheng, Mikhail F. Denissenko, Gerd P. Pfeifer and Yi Zheng

in Carcinogenesis

Volume 20, issue 6, pages 1085-1089
Published in print June 1999 | ISSN: 0143-3334
Published online June 1999 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/20.6.1085
Use of UvrABC nuclease to quantify benzo[a]pyrene diol epoxide–DNA adduct formation at methylated versus unmethylated CpG sites in the p53 gene

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We have used the UvrABC nuclease incision method in combination with ligation-mediated polymerase chain reaction (LMPCR) techniques to map and quantify (±)anti-7β, 8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene (BPDE) adduct formation in the p53 gene of human cells. We found that BPDE adduct formation, as revealed by UvrABC incision, preferentially occurred at methylated CpG sites that correspond to the mutational hotspots observed in human lung cancers. Our hypothesis is that it is this methylated CpG sequence-dependent preferential adduct formation, rather than selective growth advantage, that is the major determinant of the p53 mutation pattern in human cancers. Given the far reaching ramifications of such conclusions for cancer etiology, a legitimate question is raised regarding the reliability of using the UvrABC incision method for quantifying and determining the sequence-dependency of adduct formation. Is the higher frequency of UvrABC cutting at methylated versus unmethylated CpG sites due to the preference of the nuclease for cutting at those sites or due to the preferential formation of BPDE adducts at those sites? In order to distinguish between these two possibilities, we have analyzed the kinetics of UvrABC incision at BPDE adducts formed at either methylated CpG sites versus other sequences, or unmethylated CpG sites versus other sequences in exon 5 of the p53 gene. We have found that the UvrABC cutting kinetics are identical for both cases. On the basis of these results we conclude that under proper cutting conditions, UvrABC nuclease reacts with and incises with equal efficiency, BPDE adducts formed at methylated or unmethylated CpG sites as well as other sequences, and that the extent of UvrABC incision accurately reflects the extent of BPDE–DNA adduct formation. These conclusions were further supported by results obtained using a DNA synthesis blockage assay.

Keywords: BPDE, (±)anti-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetra-hydrobenzo[a]pyrene; DTT, dithiothreitol; LMPCR, ligation-mediated polymerase chain reaction.

Journal Article.  3136 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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