Differential tolerance to DNA polymerization by HIV-1 reverse transcriptase on N⁶ adenine C₁₀R 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 α-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 immunodeficieney 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 C₁₀R 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-(C₁₀R)-BPDE- adducted template, following replication with either the wild type or mutant enzymes. However, the other two C₁₀R 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 C₁₀R and C₁₀S adducted templates, their binding affinities were similar, yet distinct from the unadducted template.