Error induction and correction by mutant and wild type T4 DNA polymerases

Journal Article · Sun Mar 25 00:00:00 EST 1979 · J. Biol. Chem.; (United States)

OSTI ID:5437887

Clayton, L K ^[1]; Goodman, M F; Branscomb, E W; Galas, D J

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1. Univ. of Southern California, Los Angeles

The fidelity of DNA synthesis as determined by misincorporation of the base analogue 2-aminopurine in competition with adenine has been measured as a function of deoxynucleoside triphosphate substrate concentrations using purified mutator (L56), antimutator (L141), and wild type (T4D) T4 DNA polymerases. Although the rates of both incorporation and turnover of aminopurine and adenine decrease as substrate concentrations are decreased, the ratio of turnover/polymerase activity is increased. The misincorporation of aminopurine decreases with decreasing substrate concentrations such that all three enzymes approach nearly identical misincorporation frequencies at the lowest substrate concentration. The increased accuracy of DNA synthesis corresponds to conditions producing a high nuclease/polymerase ratio. The misinsertion frequency for aminopurine is independent of substrate concentrations and enzyme phenotype; therefore, the increased accuracy of DNA synthesis is shown to be a result of increased nuclease activity and not increased polymerase or nuclease specificity. The data are analyzed in terms of a kinetic model of DNA polymerase accuracy which proposes that discrimination in nucleotide insertion and removal is based on the free energy difference between matched and mismatched base pairs. A value of 1.1 kcal/mol free energy difference, ..delta..G, between adenine: thymine an aminopurine:thymine base pairs is predicted by model analysis. An independent estimate of this free energy difference also gives a value of 1.1 kcal/mol. It is shown that the aminopurine misinsertion frequency for an enzyme having either extremely low 3'-exonuclease activity, Escherichia coli DNA polymerase I, or no measurable exonuclease activity, calf thymus DNA polymerase ..cap alpha.., is 12 to 15%, which is similar to that for the T4 polymerases and consistent with ..delta..G = 1.1 kcal/mol.

• OSTI ID: 5437887
• Journal Information: J. Biol. Chem.; (United States), Vol. 254:6
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
ADENINES
BIOCHEMICAL REACTION KINETICS
DNA
BIOSYNTHESIS
DNA REPLICATION
ERRORS
POLYMERASES
BACTERIOPHAGES
BIOLOGICAL PATHWAYS
ESCHERICHIA COLI
MUTANTS
NUCLEOSIDES
THYMINE
AMINES
AZINES
BACTERIA
ENZYMES
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
KINETICS
MICROORGANISMS
NUCLEIC ACID REPLICATION
NUCLEIC ACIDS
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PARASITES
PURINES
PYRIMIDINES
REACTION KINETICS
RIBOSIDES
SYNTHESIS
URACILS
VIRUSES
550200* - Biochemistry
550700 - Microbiology