Kinetics of deoxyribonucleotide insertion and extension at abasic template lesions in different sequence contexts using HIV-1 reverse transcriptase.

Abstract:

Deoxyribonucleotide insertion efficiencies were measured opposite site-directed abasic template lesions using human immunodeficiency virus 1 reverse transcriptase (HIV-1RT), and the efficiencies to continue primer synthesis beyond the lesion, by addition of the "next correct" deoxynucleotide, were measured as a function of sequence context. Insertion of purines was favored over pyrimidines, A > G > T approximately C. Primer extension past the lesion occurred by two distinct mechanisms, either by direct or by misalignment extension. An "A-rule" appeared to hold for the case of direct extension, where the abasic template moiety is intrahelical, aligned opposite the primer 3'-terminus. In misalignment extension, the primer terminus is realigned from a site directly opposite the lesion to a new position opposite a neighboring template base 5' to the lesion. Direct extension efficiencies were measured in 16 different configurations, by varying 4 bases at the primer 3'-termini and 4 at the 5'-side (downstream) of the lesion. The predominant order of direct extension was A > G > T approximately C, similar to that observed for insertion. Reduced primer extension rates were not caused by a reduction in HIV-1 RT-DNA binding. Primers terminating in C showed inefficient direct extension, but were readily extended via misaligned configurations. The ratios of direct-to-misalignment extension efficiencies were 27:1, 2.5:1, and 1:25 for A, G, and C opposite the lesion, respectively. For the case of primers terminating in T, misalignment extension was not observed. A striking result was that while primers were extended past an abasic lesion by HIV-1 RT in both direct and misalignment modes, avian myeloblastosis virus RT failed to catalyze significant extension by either mode.

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