Steady-state and pre-steady-state kinetic analysis of 8-oxo-7,8-dihydroguanosine triphosphate incorporation and extension by replicative and repair DNA polymerases

Abstract

The kinetics of 8-oxo-7,8-dihydroguanosine triphosphate (8-oxo-dGTP) incorporation into DNA by Escherichia coli polymerases I exo- (KF-) and II exo- (Pol II-), HIV-1 RT reverse transcriptase (HIV-1 RT), and bacteriophage T7 exo- (T7(-)) were examined to determine the misincorporation potential for 8-oxo-dGTP and to investigate the role of base pairing symmetry in DNA polymerase fidelity. 8-Oxo-dGTP was found to be a poor substrate for the four polymerases, with insertion efficiencies >10(4)-fold lower than for dGTP incorporation. Insertion efficiencies of 8-oxo-dGTP were also consistently lower than for incorporation of dNTPs opposite template 8-oxo-G, previously studied in this laboratory. In steady-state reactions, T7(-) had a high preference for 8-oxo-dGTP insertion opposite A (97%) and HIV-1 RT, KF-, and Pol II- preferred to insert 8-oxo-dGTP opposite C. Misinsertion frequencies for 8-oxo-dGTP also varied considerably from frequencies of misinsertion at template 8-oxo-G adducts for Pol II-, HIV-1 RT, and T7(-). Pre-steady-state 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. Although extension past template 8-oxo-G adducts had previously been shown to occur preferentially for the mispair, extension past primer 8-oxo-G:template A or C pairs was variable. The low and comparable estimated Kd 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 Kdapp values for 8-oxo-dGTP and dGTP binding to HIV-1 RT.DNA indicates that there are contributions to the kinetically determined Kdapp (such as conformational change and/or phosphodiester bond formation) which may be involved in the selection against 8-oxo-dGTP. The differences in binding (Kdapp), incorporation, and extension kinetics of 8-oxo-dGTP compared to normal dNTP incorporation at template 8-oxo-G adducts indicate that polymerase fidelity does not depend solely upon the overall geometry of Watson-Crick base pairs and reflects the asymmetry of the enzyme active site.