Mechanism of DNA replication fidelity for three mutants of DNA polymerase I: Klenow fragment KF(exo+), KF(polA5), and KF(exo-)

• Published in: Biochemistry (Easton) Vol. 30; no. 5; pp. 1441 - 1448
• Main Authors: Eger, Bryan T, Kuchta, Robert D, Carroll, Steven S, Benkovic, Patricia A, Dahlberg, Michael E, Joyce, Catherine M, Benkovic, Stephen J
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01-02-1991
• Subjects: Analytical, structural and metabolic biochemistry; Base Sequence; Biological and medical sciences; Deoxyribonucleotides - metabolism; DNA Polymerase I - genetics; DNA Polymerase I - metabolism; DNA Replication; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; In Vitro Techniques; Kinetics; Manganese - pharmacology; Molecular Sequence Data; Mutagenesis; Mutation; Oligodeoxyribonucleotides - chemistry; Structure-Activity Relationship; Templates, Genetic; Thermodynamics; Transferases; Fidelity; Radiolabelling; Computer simulation; Enzyme; Escherichia coli; Base mismatching; Mutagenesis; DNA polymerase 1; Bacteria; Replication; Mutation; Kinetic parameter; Enterobacteriaceae
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00219a039

Inhibition of the pre-steady-state burst of nucleotide incorporation by a single incorrect nucleotide (nucleotide discrimination) was measured with the Klenow fragment of DNA polymerase I [KF(exo+)]. For the eight mispairs studied on three DNA sequences, only low levels of discrimination ranging from none to 23-fold were found. The kinetics of dNTP incorporation into the 9/20-mer at low nucleotide concentrations was also determined. A limit of greater than or equal to 250 s-1 was placed on the nucleotide off-rate from the KF(exo+)-9/20-dTTP complex in accord with nucleotide binding being at equilibrium in the overall kinetic sequence. The influence of the relatively short length of the 9/20-mer on the mechanism of DNA replication fidelity was determined by remeasuring important kinetic parameters on a 30/M13-mer with high homology to the 9/20-mer. Pre-steady-state data on the nucleotide turnover rates, the dATP(alpha S) elemental effect, and the burst of dAMP misincorporation into the 30/M13-mer demonstrated that the kinetics were not affected by the length of the DNA primer/template. The effects on fidelity of two site-specific mutations, KF(polA5) and KF(exo-), were also examined. KF(polA5) showed an increased rate of DNA dissociation and a decreased rate of polymerization resulting in less processive DNA synthesis. Nevertheless, with at least one misincorporation event, that of dAMP into the 9/20-mer, KF(polA5) displays an increased replication fidelity.