Impact of the stereochemistry of benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide-deoxyadenosine adducts on resistance to digestion by phosphodiesterases I and II and translesion synthesis with HIV-1 reverse transcriptase

Spatial orientations of bulky DNA adducts can influence the extent of resistance to digestion by exonucleases and translesion synthesis by HIV-1 reverse transcriptase (HIV-1 RT). In order to determine how different diastereomers of benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide (BPDE )-adducted DNAs influence the activity of these enzymes, 11-mer and 33-mer oligodeoxyribonucleotides were synthesized bearing site-specific and stereospecific BPDE adducts at adenine N⁶ on position two of the human N-ras codon 61. Phosphodiesterase I, which hydrolyzes DNA in the 3′→5′ direction, exhibited greater resistance opposite the lesion with C₁₀-R BPDE-adducted templates than the corresponding C₁₀-S adducts. However, the opposite stereoselective resistance to digestion was observed with phosphodiesterase II, which hydrolyzes DNA in the 5′→3′ direction. These results are complemented by the in vitro replication pattern exhibited with HIV-1 RT. Primer extension reactions under conditions defining single encounters between polymerase and substrate revealed adduct-dependent termination one base 3′ to each of the lesions. When experimental conditions were altered to permit multiple encounters, HIV-1 RT was able to replicate past the damaged site on four of the six adducted templates, exhibiting little pausing opposite the lesion. Analyses of the replication pattern past these lesions revealed two general categories of replication blockage, which, like the exonucleolytic digestion data, were also based on the C₁₀-R and C₁₀-S configuration of the stereoisomers. Thus, the chirality of BPDE-dA adducts modulates enzymatic functions. Furthermore, the (+)- and (-)-anti-trans-BPDE-dA modified templates exhibited the most facile bypass, while the (+)- and (-)-anti-cis-BPDE adducts were most blocking.