Mechanism of DNA Replication Fidelity for Three Mutants of DNA Polymerase I: Klenow Fragment KF(exo+), KF(polA5), and KF(exo)

Bryan T. Eger, Steven S. Carroll, Patricia A. Benkovic, Michael E. Dahlberg, Stephen J. Benkovic, Robert D. Kuchta, Catherine M. Joyce

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Abstract

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 ≥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(αS) 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. By comparison of KF(exo-) to KF(exo+) the exonuclease activity was shown to enhance fidelity by as much as a factor of 30 through contributions to the stages of both internal proofreading (i.e., a stage where the newly elongated enzyme-DNAn+1 complex was unable to dissociate) and external proofreading (i.e., a stage where the DNAn+1 freely dissociated from the enzyme-DNAn+1 complex). The KF(exo-) was also used to demonstrate that Mn2+ decreases fidelity at all three stages by (i) increasing the net rate of insertion of the incorrect nucleotide, (ii) decreasing the level of internal proofreading, and (iii) decreasing the level of external proofreading.

Original languageEnglish (US)
Pages (from-to)1441-1448
Number of pages8
JournalBiochemistry
Volume30
Issue number5
DOIs
StatePublished - Feb 1 1991

All Science Journal Classification (ASJC) codes

  • Biochemistry

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