Construction and characterization of a bacteriophage T4 DNA polymerase deficient in 3' → 5' exonuclease activity

M. W. Frey, N. G. Nossal, T. L. Capson, S. J. Benkovic

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Abstract

Bacteriophage T4 DNA polymerase has a proofreading 3' → 5' exonuclease that plays an important role in maintaining the accuracy of DNA replication. We have constructed a T4 DNA polymerase deficient in this exonuclease by converting Asp-219 to Ala. The exonuclease activity of the mutant T4 DNA polymerase has been reduced by a factor of at least 107, but it retains a polymerase activity whose kinetic parameters, k(cat), K(d) DNA, and K(d) dATP, are very close to those of the wild-type enzyme. Bacteriophage T4 with the mutant polymerase gene has a markedly increased mutation frequency. Asp- 219 in T4 DNA polymerase is within a sequence similar to those surrounding Asp residues previously shown to be essential for the exonuclease activities of the Klenow fragment of Escherichia coli DNA polymerase I (Asp-424), bacteriophage φ29 DNA polymerase (Asp-66), and Saccharomyces cerevisiae DNA polymerase δ (Asp-405). Thus, these studies support the proposal that there are similar sequences in the active sites for the proofreading exonucleases of these and related DNA polymerases.

Original languageEnglish (US)
Pages (from-to)2579-2583
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume90
Issue number7
DOIs
StatePublished - 1993

All Science Journal Classification (ASJC) codes

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