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. 1995 Apr 25;23(8):1398–1405. doi: 10.1093/nar/23.8.1398

In vitro replication by prokaryotic and eukaryotic polymerases on DNA templates containing site-specific and stereospecific benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide adducts.

P Chary 1, R S Lloyd 1
PMCID: PMC306868  PMID: 7753632

Abstract

DNA adducts of the environmental carcinogen benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) interact stereospecifically with prokaryotic and eukaryotic polymerases in vitro. Toward understanding the capacity to replicate past different diastereomers of BPDE at specific sites in DNA, six deoxyoligonucleotides, each 33 bases long, were constructed with stereochemically defined BPDE adducts on adenine N6 at position two of the human N-ras codon 61. Four polymerases that were studied under single encounters with the template-primer complex terminated synthesis one base 3' to the lesion with all the adducted templates. When multiple encounters between polymerase and substrate were permitted, each of the polymerases analyzed revealed a unique pattern for a given adducted template. The general replication pattern was encompassed under two categories, reflecting the significance of the R and S configurations of C10 of the pyrenyl ring attached to the single-stranded DNA template. Furthermore, within each of these categories, every polymerase demonstrated distinct quantitative differences in product accumulation at a given site, for the various adducted templates. Among the polymerases utilized in this study, exonuclease-deficient Klenow fragment of polymerase I (exo- KF) exhibited the most efficient translesion synthesis resulting in approximately 16% full-length products with the modified templates bearing adducts with C10-S configuration. In contrast, chain elongation with bacteriophage T4 DNA polymerase bearing an active 3'-->5' exonucleolytic activity was most strongly inhibited by all six BPDE-adducted templates. Misincorporation of A opposite the adduct occurred in all the templates when polymerized with Sequenase, whereas exo- KF preferentially incorporated C opposite the C10-R BPDE adducts and A opposite the C10-S BPDE adducts.

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