Differential tolerance to DNA polymerization by HIV-1 reverse transcriptase on N-6 adenine C(10)R and C10S benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide-adducted templates

To determine the effect of various stereoisomers of benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide (BPDE) on translesion bypass by human immunodeficiency virus-1 reverse transcriptase and its alpha-helix H mutants, six 33-mer templates were constructed bearing site- and stereospecific adducts. This in vitro model system was chosen to understand the structure-function relationships between the polymerase and damaged DNA during replication. Comparison of the replication pattern between wild type human immunodeficiency virus-1 reverse transcriptase and its mutants, using primers which were 3' to the lesion, revealed essentially similar patterns, While these primers terminated with all three of the C(10)R and two of the C10S BPDE-adducted templates 1 base 5' and 1 base 3' to the damaged site respectively, (+)-anti-trans-(C10S) BPDE-adducted DNA alone permitted the formation of full-length products. Utilization of a primer with its 3'-hydroxyl 1 base beyond the lesion resulted in full-length products with all the C10S BPDE-adducted templates and the (-)-syn-trans-(C(10)R)-BPDE-adducted template, following replication with either the wild type or mutant enzymes, However, the other two C(10)R BPDE-adducted templates failed to allow any primer extension, even with the wild type enzyme, Although T . P depletion studies further confirmed the differential primer extension abilities using the C(10)R and C10S adducted templates, their binding affinities were similar, yet distinct from the unadducted template.