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


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.



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

One line summary:

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


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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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