Analysis of nucleotide insertion and extension at 8-oxo-7,8-dihydroguanine by replicative T7 polymerase exo(-) and human immunodeficiency virus-1 reverse transcriptase using steady-state and pre-steady-state kinetics

Pre-steady-state kinetics of incorporation of dCTP and dATP opposite site-specific 8-oxo-7,8-dihydroguanine (8-oxoGua), in contrast to dCTP insertion opposite G, were examined as well as extension beyond the lesion using the replicative enzymes bacteriophage polymerase T7 exo(-)(T7(-)) and HIV-1 reverse transcriptase (RT). These results were compared to previous findings for Escherichia coli repair polymerases I (KF-) and II (pol II-) exo(-) [Lowe, L. G., & Guengerich, F. P. (1996) Biochemistry 35, 9840-9849]. HIV-1 RT showed a very high preference for insertion of dATP opposite 8-oxoGua, followed by pol II-, T7(-), and KF-. Steady-state assays showed k(cat) consistently lower than pre-steady-state polymerization rates (k(p)) for insertion of dCTP opposite G or 8-oxoGua and insertion of dATP opposite 8-oxoGua. Pre-steady-state kinetic curves for the addition of dCTP opposite 8-oxoGua or G by KF-, pol II-, and T7(-) were all biphasic, with a rapid initial single-turnover burst followed by a slower multiple turnover rate, while addition of dATP opposite 8-oxoGua by these polymerases did not display burst kinetics. With HIV-1 RT, addition of dATP opposite 8-oxoGua displayed burst kinetics while addition of dCTP did not. Analyses of the chemical step by substitution of phosphorothioate analogs for normal dNTPs suggest that the chemistry is rate-limiting during addition of dCTP and dATP opposite 8-oxoGua by KF-, pol II-, and T7(-); HIV-1 RT did not show a chemical rate-limiting step during addition of dATP opposite 8-oxoGua. Kinetic assays performed with various dCTP concentrations indicate that dCTP has a higher K-d and lower k(p) for incorporation opposite 8-oxoGua compared to G with all four enzymes. The K-d,app(dATP) values for KF-, pol II-, and T7(-) incorporation of dATP opposite 8-oxoGua, estimated in competition assays, were found to be 3-10-fold greater than the K-d(dCTP). Likewise, the K-d,app(dCTP) for HIV-1 RT incorporation of dCTP opposite 8-oxoGua was found to be 10-fold greater than the K-d(dATP) The repair enzymes (KF- and pol II-) efficiently extended the 8-oxoGua.A pair; extension of 8-oxoGua.C was severely impaired, whereas the replicative enzymes (T7(-) and HIV-1 RT) extended both pairs, with faster rates for the extension of the 8-oxoGua.A pair. On the basis of these findings, the fidelity of all four enzymes during replication of 8-oxoGua depends on contributions from the apparent K-d, the ease of base pair extension, and either the rate of conformational change before chemistry or the rate of bond formation.