Effects of primer-template sequence on ATP-dependent removal of chain-terminating nucleotide analogues by HIV-1 reverse transcriptase

HIV-1 reverse transcriptase can remove chain terminators from blocked DNA ends through a nucleotide-dependent mechanism. We show that the catalytic efficiency of the removal reaction can vary several hundred-fold in different sequence contexts and is most strongly affected by the nature of the base pair at the 3′-primer terminus and the six base pairs upstream of it. Similar effects of the upstream sequence were observed with primer-templates terminated with 2′,3′-dideoxy-AMP, 2′,3′-dideoxy-CMP, or 2′,3′-dideoxy-GMP. However, the removal of 2′,3′- dideoxy-TMP or 3′-azido-2′,3′-dideoxy-TMP was much less influenced by upstream primer-template sequence, and the rate of excision of these thymidylate analogues was greater than or equal to that of the other chain-terminating residues in each sequence context tested. These results strongly indicate that the primer terminus and adjacent upstream base pairs interact with reverse transcriptase in a sequence-dependent manner that affects the removal reaction. We conclude that primer-template sequence context is a major factor to consider when evaluating the removal of different chain terminators by HIV-1 reverse transcriptase.