Kinetics of Nucleotide Incorporation Opposite DNA Bulky Guanine N2 Adducts by Processive Bacteriophage T7 DNA Polymerase (Exonuclease–) and HIV-1 Reverse Transcriptase

Abstract

Six oligonucleotides with carcinogen derivatives bound at the N2 atom of deoxyguanosine were prepared, including adducts derived from butadiene, acrolein, crotonaldehyde, and styrene, and examined for effects on the replicative enzymes bacteriophage DNA polymerase T7 ؊ (T7 ؊ ) and HIV-1 reverse transcriptase for comparison with previous work on smaller DNA adducts. All of these adducts strongly blocked dCTP incorporation opposite the adducts. dATP was preferentially incorporated opposite the acrolein and crotonaldehyde adducts, and dTTP incorporation was preferred at the butadiene-and styrene-derived adducts. Steady-state kinetic analysis indicated that the reduced catalytic efficiency with adducted DNA involved both an increased K m and attenuated k cat . Fluorescence estimates of K d and pre-steady-state kinetic measurements of k off showed no significantly decreased affinity of T7 ؊ with the adducted oligonucleotides or the dNTP. Pre-steadystate kinetics showed no burst phase kinetics for dNTP incorporation with any of the modified oligonucleotides. These results indicate that phosphodiester bond formation or a conformational change of the enzyme⅐ DNA complex is rate-limiting instead of the step involving release of the oligonucleotide. Thio elemental effects for dNTP incorporation were generally relatively small but variable, indicating that the presence of adducts may sometimes make phosphodiester bond formation rate-limiting but not always.

Translesion Synthesis Past Acrolein-derived DNA Adducts by Human Mitochondrial DNA Polymerase γ

“…Thus, relative to other eukaryotic DNA polymerases, pol ␥ manifested unprecedented high potential to cause mutations during replication past ␥-HOPdG. Preferential incorporation of dA opposite ␥-HOPdG has been shown for T7 DNA polymerase (44), but the relative efficiency of dA incorporation versus dC incorporation was significantly less than that measured for pol ␥. The efficiency of extension from dA opposite the adduct was not addressed in the T7 polymerase study.…”

Interactions of Carcinogen-Bound DNA with Individual DNA Polymerases

“…[36][37][38]73,81,82,85,86,[94][95][96][97] Although the analysis may seem complicated to those inexperienced with kinetics, the purpose of doing pre-steady-state kinetics is to simplify the analysis. 98 The overall goal in kinetic analysis is to define rates of individual steps in reactions.…”

Interaction of the Ty3 Reverse Transcriptase Thumb Subdomain with Template-Primer

“…5 compare the efficiency of single nucleotide addition of each Ty3 mutant on duplexes containing LNA primer substitutions with the activity of the same mutant on unsubstituted DNA. The method of evaluation is analogous to that recently adopted by Zang et al (49) to study nucleotide addition opposite guanine analogs containing N-2 adducts. The inhibitory effect of LNA substitution at positions Ϫ3 and Ϫ4 was not observed with Ty3 RT mutants F292A, F292W, F292Y, and G294A (Fig.…”