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

Biochemistry
L L Furge, F P Guengerich

Abstract

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 che...Continue Reading

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Citations

Apr 13, 2006·Journal of the American Chemical Society·Xiaojun RenShankar Balasubramanian
Jun 20, 2006·Chemical Research in Toxicology·Jeong-Yun ChoiF Peter Guengerich
Oct 17, 2012·Chemical Research in Toxicology·Michelle L HammAmelia M Weaver
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Dec 29, 2005·PLoS Biology·Olga RechkoblitDinshaw J Patel
Oct 18, 2006·Proceedings of the National Academy of Sciences of the United States of America·Karissa D CarlsonM Todd Washington
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Jan 14, 2012·Free Radical Research·Sarah DelaneyCraig J Yennie
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