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Mutagenic Replication in a Human Cell Extract of DNAs Containing Site-specific and Stereospecific Benzo(a)pyrene-7,8-diol-9,10-epoxide DNA Adducts Placed on the Leading and Lagging Strands¹

Department of Human Biological Chemistry and Genetics, Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1071

The environmental carcinogen benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE) forms DNA adducts with unique stereochemistries that may have divergent biological fates, depending on how they are processed within a cell. To investigate the effect of DNA bulky adduct stereoisomerism on the mutagenic outcome of translesion DNA replication in a human cell extract, oligonucleotides were synthesized that contained (+)- and (-)-anti-cis-BPDE enantiomers on N⁶ adenine at position 2 of the human N-ras 61 codon. Both the nonadducted and BPDE-adducted oligonucleotides were introduced into two double-stranded vectors, replicative forms M13mp2SVoriL and M13mp2SVoriR, which contain SV40 origins of replication in two different orientations relative to the adduct insertion site. Nonadducted and adduct-containing vector DNA constructs were replicated in HeLa cytoplasmic extracts and then screened in bacteria for base substitutions at the adduct site. The mutation frequencies for the adducted DNAs were at least 10 times higher than for the nonadducted DNA and ranged from 5.5 x 10^-4 to 1.5 x 10^-3. The (-)-anti-cis enantiomer was more than twice as mutagenic as the (+)-enantiomer. All three possible base mutations were present, with the AG being the predominant one. No dramatic differences in replication fidelity were found when the adducts were placed on the leading versus the lagging strand of the vector.

¹ This work was supported by Grants P01 ES05355 and P30-ES06676 from NIH, and by the Houston Endowment, Distinguished Chair in Environmental Toxicology, University of Texas.

² To whom requests for reprints should be addressed, at Sealy Center for Molecular Science, University of Texas, 5.142 Medical Research Building, 301 University Boulevard, Galveston, TX 77555-1071. Phone: (409) 772-2179; Fax: (409) 772-1790; E-mail: rslloyd@scms.utmb.edu.

Received 10/30/97. Accepted 1/15/98.

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