DNA polymerization in the absence of exonucleolytic proofreading: in vivo and in vitro studies.

Abstract:

Classical genetic selection was combined with site-directed mutagenesis to study bacteriophage T4 DNA polymerase 3'----5' exonuclease activity. A mutant DNA polymerase with very little (less than or equal to 1%) 3'----5' exonuclease activity was generated. In vivo, the 3'----5' exonuclease-deficient DNA polymerase produced the highest level of spontaneous mutation observed in T4, 500- to 1800-fold above that of wild type. The large reduction in 3'----5' exonuclease activity appears to be due to two amino acid substitutions: Glu-191 to Ala and Asp-324 to Gly. Protein sequence similarities have been observed between sequences in the Escherichia coli DNA polymerase I 3'----5' exonuclease domain and conserved sequences in eukaryotic, viral, and phage DNA polymerases. It has been proposed that the conserved sequences contain metal ion binding ligands that are required for 3'----5' exonuclease activity; however, we find that some proposed T4 DNA polymerase metal binding residues are not essential for 3'----5' exonuclease activity. Thus, our T4 DNA polymerase studies do not support the hypothesis by Bernad et al. [Bernad, A., Blanco, L., Lazaro, J.M., Martin, G. & Salas, M. (1989) Cell 59, 219-228] that many DNA polymerases, including T4 DNA polymerase, share an extensively conserved 3'----5' exonuclease motif. Therefore, extrapolation from E. coli DNA polymerase I sequence and structure to other DNA polymerases for which there is no structural information may not be valid.

Polymerases:

Topics:

Mutational Analysis, Structure and Structure/Function, Fidelity, Nucleotide Incorporation, Exonuclease Activity, Alignments

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
T4 D189A Reha-Krantz LJ1991 Nucleotide Substitution Rate 4 Mutation frequency (relative to WT) Technique: ac forward mutational assay
T4 D189A Reha-Krantz LJ1991 Nucleotide Substitution Rate 1 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 D189A Reha-Krantz LJ1991 Nucleotide Substitution Rate 1 Mutation frequency (relative to WT) Technique: T4 rII P7oc reversion assay
T4 E191A Reha-Krantz LJ1991 Nucleotide Substitution Rate 5 Mutation frequency (relative to WT) Technique: ac forward mutational assay
T4 E191A Reha-Krantz LJ1991 Nucleotide Substitution Rate 2 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 E191A Reha-Krantz LJ1991 Nucleotide Substitution Rate 6 Mutation frequency (relative to WT) Technique: T4 rII P7oc reversion assay
T4 D189AE191A Reha-Krantz LJ1991 Nucleotide Substitution Rate 16 Mutation frequency (relative to WT) Technique: ac forward mutational assay
T4 D189AE191A Reha-Krantz LJ1991 Nucleotide Substitution Rate 11 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 D189AE191A Reha-Krantz LJ1991 Nucleotide Substitution Rate 30 Mutation frequency (relative to WT) Technique: T4 rII P7oc reversion assay
T4 A89TD363N Reha-Krantz LJ1991 Nucleotide Substitution Rate 74 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 A89TD363N Reha-Krantz LJ1991 Nucleotide Substitution Rate 36 Mutation frequency (relative to WT) Technique: ac forward mutational assay
T4 D324G Reha-Krantz LJ1991 Nucleotide Substitution Rate 280 Mutation frequency (relative to WT) Technique: T4 rII P7oc reversion assay
T4 D324G Reha-Krantz LJ1991 Nucleotide Substitution Rate 63 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 D324G Reha-Krantz LJ1991 Nucleotide Substitution Rate 56 Mutation frequency (relative to WT) Technique: ac forward mutational assay
T4 E191AD324G Reha-Krantz LJ1991 Nucleotide Substitution Rate 650 Mutation frequency (relative to WT) Technique: ac forward mutational assay
T4 E191AD324G Reha-Krantz LJ1991 Nucleotide Substitution Rate 514 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 E191AD324G Reha-Krantz LJ1991 Nucleotide Substitution Rate 1800 errors/bp Technique: T4 rII P7oc reversion assay

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