Construction and characterization of a bacteriophage T4 DNA polymerase deficient in 3'-->5' exonuclease activity.
- 1 April 1993
- journal article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 90 (7) , 2579-2583
- https://doi.org/10.1073/pnas.90.7.2579
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.Keywords
This publication has 32 references indexed in Scilit:
- Kinetic characterization of the polymerase and exonuclease activities of the gene 43 protein of bacteriophage T4Biochemistry, 1992
- Three-dimensional structure of the β subunit of E. coli DNA polymerase III holoenzyme: A sliding DNA clampCell, 1992
- Sequence and expression in Escherichia coli of the 40-kDa subunit of activator 1 (replication factor C) of HeLa cells.Proceedings of the National Academy of Sciences, 1992
- Evidence favouring the hypothesis of a conserved 3′–5′ exonuclease active site in DNA-dependent DNA polymerasesGene, 1992
- Protein‐protein interactions at a DNA replication fork: bacteriophage T4 as a modelThe FASEB Journal, 1992
- Analysis of the ATPase subassembly which initiates processive DNA synthesis by DNA polymerase III holoenzyme.Journal of Biological Chemistry, 1991
- Eukaryotic DNA polymerase amino acid sequence required for 3'----5' exonuclease activity.Proceedings of the National Academy of Sciences, 1991
- Mechanism of DNA replication fidelity for three mutants of DNA polymerase I: Klenow fragment KF(exo+), KF(polA5), and KF(exo-)Biochemistry, 1991
- Compilation and alignment of DNA polymerase sequencesNucleic Acids Research, 1991
- ENZYMATIC SYNTHESIS OF DEOXYRIBONUCLEIC ACID .25. PURIFICATION AND PROPERTIES OF DEOXYRIBONUCLEIC ACID POLYMERASE INDUCED BY INFECTION WITH PHAGE T4+1968