STEADY-STATE AND PRE-STEADY-STATE KINETIC-ANALYSIS OF 8-OXO-7, 8-DIHYDROGUANOSINE TRIPHOSPHATE INCORPORATION AND EXTENSION BY REPLICATIVE AND REPAIR DNA-POLYMERASES

The kinetics of 8-oxo-7,8-dihydroguanosine triphosphate (8-oxo-dGTP) i ncorporation into DNA by Escherichia coli polymerases I exo(-) (KF-) a nd II exo(-) (Pol II-), HIV-1 RT reverse transcriptase (HIV-1 RT), and bacteriophage T7 exo(-) (T7(-)) were examined to determine the misinc orporation potential for 8-oxo-dGTP and to investigate the role of bas e pairing symmetry in DNA polymerase fidelity. 8-Oxo-dGTP was found to be a poor substrate for the four polymerases, with insertion efficien cies >10(4)-fold lower than for dGTP incorporation. Insertion efficien cies of 8-oxo-dGTP were also consistently lower than for incorporation of dNTPs opposite template 8-oxo-G, previously studied in this labora tory. In steady-state reactions, T7- had a high preference for 8-oxo-d GTP insertion opposite A (97%) and HIV-1 RT, KF-, and Pol II- preferre d to insert 8-oxo-dGTP opposite C. Misinsertion frequencies for 8-oxo- dGTP also varied considerably from frequencies of misinsertion at temp late 8-oxo-G adducts for Pol II-, HIV-1 RT, and T7(-). Pre-steady-stat e incorporation of 8-oxo-dGTP opposite C (but not opposite A) by HIV-1 RT, KF-, and Pol II- displayed biphasic curves, with rates of initial incorporation 2- to 11-fold lower than normal dGTP incorporation. Alt hough extension past template 8-oxo-G adducts had previously been show n to occur preferentially for the mispair, extension past primer 8-oxo -G:template A or C pairs was variable. The low and comparable estimate d K-d values for dGTP and 8-oxo-dGTP binding to HIV-1 RT alone or HIV- 1 RT.DNA complexes indicated that the initial binding was nonselective and had high affinity. The large difference (>3 orders of magnitude) in kinetic K-dapp values for 8-oxo-dGTP and dGTP binding to HIV-1 RT.D NA indicates that there are contributions to the kinetically determine d K-dapp (such as conformational change and/or phosphodiester bond for mation) which may be involved in the selection against 8-oxo-dGTP. The differences in binding (K-dapp), incorporation, and extension kinetic s of 8-oxo-dGTP compared to normal cNTP incorporation at template 8-ox o-G adducts indicate that polymerase fidelity does not depend solely u pon the overall geometry of Watson-Crick base pairs and reflects the a symmetry of the enzyme active site.