title: 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.
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
authors.forename: L L
authors.initials: LL
authors.lastname: Furge
authors.forename: F P
authors.initials: FP
authors.lastname: Guengerich
mesh_terms: D017123:Bacteriophage T7 / Q000201:enzymology*; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D013379:Substrate Specificity; D017186:Titrimetry
chemical_list: D003838:Deoxyadenine Nucleotides; D003845:Deoxycytosine Nucleotides; C024107:2'-deoxycytidine 5'-triphosphate; C024829:8-hydroxyguanine; D006147:Guanine; D004247:DNA; C034970:bacteriophage T7 induced DNA polymerase; D054303:HIV Reverse Transcriptase; D004259:DNA-Directed DNA Polymerase