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

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 chemical step by substitution of phosphorothioate analogs for normal dNTPs suggest that the chemistry is rate-limiting during addition of dCTP and dATP opposite 8-oxoGua by KF-, pol II-, and T7-; HIV- RT did not show a chemical rate-limiting step during addition of dATP opposite 8-oxoGua. Kinetic assays performed with various dCTP concentrations indicate that dCTP has a higher Kd and lower k(p) for incorporation opposite 8-oxoGua compared to G with all four enzymes. The K(d,app)dATP values for KF-, pol II-, and T7- incorporation of dATP opposite 8-oxoGua, estimated in competition assays, were found to be 3-10-fold greater than the K(d)dCTP. Likewise, the K(d,app)dCTP for HIV-1 RT incorporation of dCTP opposite 8-oxoGua was found to be 10-fold greater than the K(d)dATP. The repair enzymes (KF- and pol II-) efficiently extended the 8-oxoGua x A pair; extension of 8-oxoGua x C was severely impaired, whereas the replicative enzymes (T7- and HIV-1 RT) extended both pairs, with faster rates for the extension of the 8-oxoGua x A pair. On the basis of these findings, the fidelity of all four enzymes during replication of 8-oxoGua depends on contributions from the apparent Kd, the ease of base pair extension, and either the rate of conformational change before chemistry or the rate of bond formation.

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

“…Because the DNA⅐enzyme dissociation rate is similar for the various adducts (Table V), the thio elemental effect should primarily reflect the rate-limiting contribution of phosphodiester bond formation. The observed elemental effects were generally moderate in these studies (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16) (Table VII). One exception was the dG-CA-S adduct, in which the elemental effect was ϳ100 (Fig.…”

“…The interpretation of these elemental effects is not without controversy, and the mechanistic interpretation has been questioned (92)(93)(94)(95). On the other hand, we (10,13,96) and others (9,17,19,91,97) have noted values in the range of 50 -100 for elemental effects on abnormal incorporation events and, in the absence of significant effects for normal base incorporation, are of the opinion that these cannot simply be dismissed, even if their interpretation may be more complex than affecting only the rate of phosphodiester bond formation. Even in some of the work that questions the change in electronegativity between oxygen and sulfur as the only basis for kinetic changes, the conclusion is still reached that perturbations due to this substitution are manifested in the phosphodiester bond formation step ("chemistry") (93).…”

“…The overall yield of purified T7 Ϫ protein was about 1 mg liter Ϫ1 . Escherichia coli thioredoxin was expressed and purified as described previously (10).…”

“…However, short oligonucleotides are poor substrates for polymerases, particularly the processive polymerases T7 Ϫ and RT (10,41,48). Therefore, extending the adducted DNA to lengths of 35-40 nucleotides was necessary for performing the polymerization reactions.…”

“…9,10,15,[17][18][19][20] remains ratelimiting in the steady state (as without the adduct); (ii) high K m (dNTP) values are seen in steady-state kinetics, but K d (dNTP) values are not altered; (iii) lack of change in K d (DNA) ; (iv) no change in k off for DNA; (v) partial burst kinetics and also biexponential plots within a single cycle, which can be ratio- 1 The abbreviations used are: RT, human immunodeficiency virus-1 reverse transcriptase; T7 Ϫ , bacteriophage T7 DNA polymerase (T7 Ϫ indicates exonuclease Ϫ ); dCTP␣S, 2Ј-deoxycytidine 5Ј-O-(1-thiotriphosphate); dATP␣S, 2Ј-deoxyadenosine 5Ј-O-(1-thiotriphosphate); dTTP␣S, 2Ј-deoxythymidine 5Ј-O-(1-thiotriphosphate); BSA, bovine serum albumin; DTT, dithiothreitol; FAM, 6-carboxyfluorescein; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; dG-EB-R,R, (R,R)-N 2 -(2,3,4-trihydroxy-1-butyl)-2Ј-deoxyguanosine; dG-EB-S,S, (S,S)-N 2 -(2,3,4-trihydroxy-1-butyl)-2Ј-deoxyguanosine; dG-acrolein, nalized by the addition of a step involving a reversible inactive complex; and (vi) discrimination between a slow conformational change and slow phosphodiester bond formation is difficult, and high elemental "thio" effects in some cases (T7 Ϫ with 8-oxo-7,8-dihydro-G) but not in others (RT with all adducts examined). Even a methyl or ethyl group at the guanine N2 atom strongly disrupted the normal polymerase function of T7 Ϫ and RT (16).…”