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

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 10(7), but it retains a polymerase activity whose kinetic parameters, kcat, Kd DNA, and Kd 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 phi 29 DNA polymerase (Asp-66), and Saccharomyces cerevisiae DNA polymerase delta (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.

Polymerases:

Topics:

Mutational Analysis, Kinetic Parameters, Fidelity, Exonuclease Activity, Methods, Alignments

One line summary:

Exonuclease sites are present on many types of polymerases, not solely limited to T4

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
T4 D219A Frey MW1993 Nucleotide Substitution Rate 760 Mutation frequency (relative to WT) Technique: ac forward mutational assay
T4 D219A Frey MW1993 Kd 40nM Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 D219A Frey MW1993 Kd 10uM Reaction: Nucleotide incorporation; Substrate: dATP
T4 D219A Frey MW1993 kcat 3 /second Reaction: Nucleotide incorporation; Substrate: dATP

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