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Titolo:
STEADY-STATE AND PRE-STEADY-STATE KINETIC-ANALYSIS OF 8-OXO-7, 8-DIHYDROGUANOSINE TRIPHOSPHATE INCORPORATION AND EXTENSION BY REPLICATIVE AND REPAIR DNA-POLYMERASES
Autore:
EINOLF HJ; SCHNETZBOUTAUD N; GUENGERICH FP;
Indirizzi:
VANDERBILT UNIV,SCH MED,DEPT BIOCHEM,221 KIRKLAND HALL NASHVILLE TN 37232 VANDERBILT UNIV,SCH MED,DEPT BIOCHEM NASHVILLE TN 37232 VANDERBILT UNIV,SCH MED,CTR MOL TOXICOL NASHVILLE TN 37232
Titolo Testata:
Biochemistry
fascicolo: 38, volume: 37, anno: 1998,
pagine: 13300 - 13312
SICI:
0006-2960(1998)37:38<13300:SAPKO8>2.0.ZU;2-S
Fonte:
ISI
Lingua:
ENG
Soggetto:
VIRUS REVERSE-TRANSCRIPTASE; I KLENOW FRAGMENT; ESCHERICHIA-COLI; 8-HYDROXYGUANINE 7,8-DIHYDRO-8-OXOGUANINE; G.C->T.A TRANSVERSIONS; MUTAGENIC SUBSTRATE; FIDELITY; MECHANISM; INSERTION; NUCLEOTIDE;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
52
Recensione:
Indirizzi per estratti:
Citazione:
H.J. Einolf et al., "STEADY-STATE AND PRE-STEADY-STATE KINETIC-ANALYSIS OF 8-OXO-7, 8-DIHYDROGUANOSINE TRIPHOSPHATE INCORPORATION AND EXTENSION BY REPLICATIVE AND REPAIR DNA-POLYMERASES", Biochemistry, 37(38), 1998, pp. 13300-13312

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 K-d 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 K-dapp values for 8-oxo-dGTP and dGTP binding to HIV-1 RT.DNA indicates that there are contributions to the kinetically determined K-dapp (such as conformational change and/or phosphodiester bond formation) which may be involved in the selection against 8-oxo-dGTP. The differences in binding (K-dapp), incorporation, and extension kinetics of 8-oxo-dGTP compared to normal cNTP 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.

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Documento generato il 11/07/20 alle ore 04:20:56