Genetic and biochemical studies of bacteriophage T4 DNA polymerase 3'-->5'-exonuclease activity.

Abstract:

DNA polymerase exonucleolytic proofreading is important in attaining high fidelity DNA replication. One of the most well characterized proofreading activities is the 3'-->5'-exonuclease activity of bacteriophage T4 DNA polymerase. We have used genetic analyses and protein sequence comparisons to Escherichia coli DNA polymerase I to identify amino acids in the N-terminal region of T4 DNA polymerase that are required for exonucleolytic proofreading. Mutant DNA polymerases with amino acid substitutions D112A/E114A, D219A, or D324A reduced 3'-->5'-exonuclease activity 10(2)-10(4)-fold in various in vitro assays and decreased DNA replication fidelity in vivo. DNA replication activity was also reduced for the exonuclease-deficient DNA polymerases in vitro and in vivo. Reduction in DNA replication appeared to be due primarily to the interdependence of T4 DNA polymerase replication and proofreading activities; T4 DNA polymerase requires 3'-->5'-exonuclease activity to repair primer termini that are not suitable substrates for extension. Observations reported here provide further evidence in support of the proposal that DNA polymerases have distinct 3'-->5'-exonuclease and polymerase active sites.

Polymerases:

Topics:

Mutational Analysis, Kinetic Parameters, Fidelity, Exonuclease Activity

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
T4 D112AE114A Reha-Krantz LJ1993 Nucleotide Substitution Rate 300 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 D324A Reha-Krantz LJ1993 Nucleotide Substitution Rate 60 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 D219A Reha-Krantz LJ1993 Nucleotide Substitution Rate 500 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay

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