KINETICS OF DEOXYRIBONUCLEOTIDE INSERTION AND EXTENSION AT ABASIC TEMPLATE LESIONS IN DIFFERENT SEQUENCE CONTEXTS USING HIV-1 REVERSE-TRANSCRIPTASE

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 pri mer synthesis beyond the lesion, by addition of the ''next correct'' d eoxynucleotide, were measured as a function of sequence context. Inser tion of purines was favored over pyrimidines, A > G > T is similar to C. Primer extension past the lesion occurred by two distinct mechanism s, either by direct or by misalignment extension. An ''A-rule'' appear ed to hold for the case of direct extension, where the abasic template moiety is intrahelical, aligned opposite the primer 3'-terminus. In m isalignment extension, the primer terminus is realigned from a site di rectly opposite the lesion to a new position opposite a neighboring te mplate base 5' to the lesion. Direct extension efficiencies were measu red in 16 different configurations, by varying 4 bases at the primer 3 '-termini and 4 at the 5'-side (downstream) of the lesion. The predomi nant order of direct extension was A > G > T is similar to C, similar to that observed for insertion. Reduced primer extension rates were no t caused by a reduction in HIV-1 RT-DNA binding. Primers terminating i n C showed inefficient direct extension, but were readily extended via misaligned configurations. The ratios of direct-to-misalignment exten sion 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, mi salignment extension was not observed. A striking result was that whil e primers were extended past an abasic lesion by HIV-1 RT in both dire ct and misalignment modes, avian myeloblastosis virus RT failed to cat alyze significant extension by either mode.