Biomaterial Database

Steady-state and pre-steady-state kinetic analysis of 8-oxo-7,8-dihydroguanosine triphosphate incorporation and extension by replicative and repair DNA polymerases. 1998

H J Einolf, and N Schnetz-Boutaud, and F P Guengerich

Department of Biochemistry, Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.

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.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D003848	Deoxyguanine Nucleotides	Guanine nucleotides which contain deoxyribose as the sugar moiety.	Deoxyguanosine Phosphates,Nucleotides, Deoxyguanine,Phosphates, Deoxyguanosine
D004256	DNA Polymerase I	A DNA-dependent DNA polymerase characterized in prokaryotes and may be present in higher organisms. It has both 3'-5' and 5'-3' exonuclease activity, but cannot use native double-stranded DNA as template-primer. It is not inhibited by sulfhydryl reagents and is active in both DNA synthesis and repair.	DNA Polymerase alpha,DNA-Dependent DNA Polymerase I,Klenow Fragment,DNA Pol I,DNA Dependent DNA Polymerase I,Polymerase alpha, DNA
D004257	DNA Polymerase II	A DNA-dependent DNA polymerase characterized in E. coli and other lower organisms. It may be present in higher organisms and has an intrinsic molecular activity only 5% of that of DNA Polymerase I. This polymerase has 3'-5' exonuclease activity, is effective only on duplex DNA with gaps or single-strand ends of less than 100 nucleotides as template, and is inhibited by sulfhydryl reagents.	DNA Polymerase epsilon,DNA-Dependent DNA Polymerase II,DNA Pol II,DNA Dependent DNA Polymerase II
D004259	DNA-Directed DNA Polymerase	DNA-dependent DNA polymerases found in bacteria, animal and plant cells. During the replication process, these enzymes catalyze the addition of deoxyribonucleotide residues to the end of a DNA strand in the presence of DNA as template-primer. They also possess exonuclease activity and therefore function in DNA repair.	DNA Polymerase,DNA Polymerases,DNA-Dependent DNA Polymerases,DNA Polymerase N3,DNA Dependent DNA Polymerases,DNA Directed DNA Polymerase,DNA Polymerase, DNA-Directed,DNA Polymerases, DNA-Dependent,Polymerase N3, DNA,Polymerase, DNA,Polymerase, DNA-Directed DNA,Polymerases, DNA,Polymerases, DNA-Dependent DNA
D004260	DNA Repair	The removal of DNA LESIONS and/or restoration of intact DNA strands without BASE PAIR MISMATCHES, intrastrand or interstrand crosslinks, or discontinuities in the DNA sugar-phosphate backbones.	DNA Damage Response
D004261	DNA Replication	The process by which a DNA molecule is duplicated.	Autonomous Replication,Replication, Autonomous,Autonomous Replications,DNA Replications,Replication, DNA,Replications, Autonomous,Replications, DNA
D001482	Base Composition	The relative amounts of the PURINES and PYRIMIDINES in a nucleic acid.	Base Ratio,G+C Composition,Guanine + Cytosine Composition,G+C Content,GC Composition,GC Content,Guanine + Cytosine Content,Base Compositions,Base Ratios,Composition, Base,Composition, G+C,Composition, GC,Compositions, Base,Compositions, G+C,Compositions, GC,Content, G+C,Content, GC,Contents, G+C,Contents, GC,G+C Compositions,G+C Contents,GC Compositions,GC Contents,Ratio, Base,Ratios, Base
D013698	Templates, Genetic	Macromolecular molds for the synthesis of complementary macromolecules, as in DNA REPLICATION; GENETIC TRANSCRIPTION of DNA to RNA, and GENETIC TRANSLATION of RNA into POLYPEPTIDES.	Genetic Template,Genetic Templates,Template, Genetic
D017123	Bacteriophage T7	Virulent bacteriophage and type species of the genus T7-like phages, in the family PODOVIRIDAE, that infects E. coli. It consists of linear double-stranded DNA, terminally redundant, and non-permuted.	Coliphage T7,Enterobacteria phage T7,Phage T7,T7 Phage,Phage, T7,Phages, T7,T7 Phages