Kinetics of deoxyribonucleotide insertion and extension at 8-oxo-7,8-dihydroguanine by T7 polymerase exo- and HIV-1 reverse transcriptase (Meeting abstract).

Important note: Information in this article was accurate in 1997. The state of the art may have changed since the publication date.

Kinetics of deoxyribonucleotide insertion and extension at 8-oxo-7,8-dihydroguanine by T7 polymerase exo- and HIV-1 reverse transcriptase (Meeting abstract).

Proc Annu Meet Am Assoc Cancer Res; 38:A277 1997. Unique Identifier : AIDSLINE MED/97619069
Lowe LG; Einolf HJ; Guengerich FP; Department of Biochemistry and Center in Molecular Toxicology,; Vanderbilt University School of Medicine, Nashville, TN 37232

Abstract: The replicative enzymes bacteriophage T7 pol exo- (T7-) and HIV-1 reverse transcriptase (HIV-1 RT) were used with a 24/36-mer sequence to examine the kinetics of incorporation of C and A at a site-specific 8-oxo-7,8-dihydroguanine (8-oxoGua). HIV-1 RT showed a high preference for insertion of A opposite 8-oxoGua. Steady-state assay kcat values were much less than pre-steady-state kp (polymerization rates). Substitution of phosphorothioate analogs for normal dNTPs suggest chemistry to be rate-limiting during addition of C and A opposite 8-oxoGua by T7-; HIV-1 RT did not show a chemical rate-limiting step during addition of A opposite 8-oxoGua. Both enzymes extended 8-oxoGua:A and 8-oxoGua:C 'mismatches,' with faster rates for the extension of 8-oxoGua:A. The fidelity of these replicative enzymes during replication of 8-oxoGua depends on contributions from the Kd,app the rate of next base addition and either the rate of conformational change before chemistry or chemistry (kp). Fluorescence quenching (stopped-flow) is being used to determine the contributions of the chemistry and conformational change rates to polymerase fidelity and the modified triphosphate 8-oxodGTP has been synthesized and is being used in complementary studies.

Keywords: *Bacteriophage T7/ENZYMOLOGY *DNA-Directed RNA Polymerase/CHEMISTRY *Guanine/ANALOGS & DERIVATIVES *HIV-1 Reverse Transcriptase/METABOLISM *Oligodeoxyribonucleotides/CHEMISTRY
971030
M97A1339

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