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. 1993 Apr 1;90(7):2579–2583. doi: 10.1073/pnas.90.7.2579

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

M W Frey 1, N G Nossal 1, T L Capson 1, S J Benkovic 1
PMCID: PMC46138  PMID: 8464864

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alberts B. M. Prokaryotic DNA replication mechanisms. Philos Trans R Soc Lond B Biol Sci. 1987 Dec 15;317(1187):395–420. doi: 10.1098/rstb.1987.0068. [DOI] [PubMed] [Google Scholar]
  2. Beese L. S., Steitz T. A. Structural basis for the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism. EMBO J. 1991 Jan;10(1):25–33. doi: 10.1002/j.1460-2075.1991.tb07917.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bernad A., Blanco L., Lázaro J. M., Martín G., Salas M. A conserved 3'----5' exonuclease active site in prokaryotic and eukaryotic DNA polymerases. Cell. 1989 Oct 6;59(1):219–228. doi: 10.1016/0092-8674(89)90883-0. [DOI] [PubMed] [Google Scholar]
  4. Blanco L., Bernad A., Salas M. Evidence favouring the hypothesis of a conserved 3'-5' exonuclease active site in DNA-dependent DNA polymerases. Gene. 1992 Mar 1;112(1):139–144. doi: 10.1016/0378-1119(92)90316-h. [DOI] [PubMed] [Google Scholar]
  5. Capson T. L., Peliska J. A., Kaboord B. F., Frey M. W., Lively C., Dahlberg M., Benkovic S. J. Kinetic characterization of the polymerase and exonuclease activities of the gene 43 protein of bacteriophage T4. Biochemistry. 1992 Nov 17;31(45):10984–10994. doi: 10.1021/bi00160a007. [DOI] [PubMed] [Google Scholar]
  6. Chen M., Pan Z. Q., Hurwitz J. Sequence and expression in Escherichia coli of the 40-kDa subunit of activator 1 (replication factor C) of HeLa cells. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2516–2520. doi: 10.1073/pnas.89.7.2516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Delarue M., Poch O., Tordo N., Moras D., Argos P. An attempt to unify the structure of polymerases. Protein Eng. 1990 May;3(6):461–467. doi: 10.1093/protein/3.6.461. [DOI] [PubMed] [Google Scholar]
  8. Derbyshire V., Freemont P. S., Sanderson M. R., Beese L., Friedman J. M., Joyce C. M., Steitz T. A. Genetic and crystallographic studies of the 3',5'-exonucleolytic site of DNA polymerase I. Science. 1988 Apr 8;240(4849):199–201. doi: 10.1126/science.2832946. [DOI] [PubMed] [Google Scholar]
  9. Derbyshire V., Grindley N. D., Joyce C. M. The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. EMBO J. 1991 Jan;10(1):17–24. doi: 10.1002/j.1460-2075.1991.tb07916.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Eger B. T., Kuchta R. D., Carroll S. S., Benkovic P. A., Dahlberg M. E., Joyce C. M., Benkovic S. J. Mechanism of DNA replication fidelity for three mutants of DNA polymerase I: Klenow fragment KF(exo+), KF(polA5), and KF(exo-). Biochemistry. 1991 Feb 5;30(5):1441–1448. doi: 10.1021/bi00219a039. [DOI] [PubMed] [Google Scholar]
  11. Gillin F. D., Nossal N. G. Control of mutation frequency by bacteriophage T4 DNA polymerase. I. The CB120 antimutator DNA polymerase is defective in strand displacement. J Biol Chem. 1976 Sep 10;251(17):5219–5224. [PubMed] [Google Scholar]
  12. Goulian M., Lucas Z. J., Kornberg A. Enzymatic synthesis of deoxyribonucleic acid. XXV. Purification and properties of deoxyribonucleic acid polymerase induced by infection with phage T4. J Biol Chem. 1968 Feb 10;243(3):627–638. [PubMed] [Google Scholar]
  13. Ito J., Braithwaite D. K. Compilation and alignment of DNA polymerase sequences. Nucleic Acids Res. 1991 Aug 11;19(15):4045–4057. doi: 10.1093/nar/19.15.4045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Johnson K. A. Rapid kinetic analysis of mechanochemical adenosinetriphosphatases. Methods Enzymol. 1986;134:677–705. doi: 10.1016/0076-6879(86)34129-6. [DOI] [PubMed] [Google Scholar]
  15. Kong X. P., Onrust R., O'Donnell M., Kuriyan J. Three-dimensional structure of the beta subunit of E. coli DNA polymerase III holoenzyme: a sliding DNA clamp. Cell. 1992 May 1;69(3):425–437. doi: 10.1016/0092-8674(92)90445-i. [DOI] [PubMed] [Google Scholar]
  16. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  17. Lee S. H., Kwong A. D., Pan Z. Q., Hurwitz J. Studies on the activator 1 protein complex, an accessory factor for proliferating cell nuclear antigen-dependent DNA polymerase delta. J Biol Chem. 1991 Jan 5;266(1):594–602. [PubMed] [Google Scholar]
  18. Lin T. C., Rush J., Spicer E. K., Konigsberg W. H. Cloning and expression of T4 DNA polymerase. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7000–7004. doi: 10.1073/pnas.84.20.7000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Morrison A., Bell J. B., Kunkel T. A., Sugino A. Eukaryotic DNA polymerase amino acid sequence required for 3'----5' exonuclease activity. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9473–9477. doi: 10.1073/pnas.88.21.9473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Muzyczka N., Poland R. L., Bessman M. J. Studies on the biochemical basis of spontaneous mutation. I. A comparison of the deoxyribonucleic acid polymerases of mutator, antimutator, and wild type strains of bacteriophage T4. J Biol Chem. 1972 Nov 25;247(22):7116–7122. [PubMed] [Google Scholar]
  21. Nossal N. G., Hershfield M. S. Nuclease activity in a fragment of bacteriophage T4 deoxyribonucleic acid polymerase induced by the amber mutant am B22. J Biol Chem. 1971 Sep 10;246(17):5414–5426. [PubMed] [Google Scholar]
  22. Nossal N. G. Protein-protein interactions at a DNA replication fork: bacteriophage T4 as a model. FASEB J. 1992 Feb 1;6(3):871–878. doi: 10.1096/fasebj.6.3.1310946. [DOI] [PubMed] [Google Scholar]
  23. Ollis D. L., Brick P., Hamlin R., Xuong N. G., Steitz T. A. Structure of large fragment of Escherichia coli DNA polymerase I complexed with dTMP. 1985 Feb 28-Mar 6Nature. 313(6005):762–766. doi: 10.1038/313762a0. [DOI] [PubMed] [Google Scholar]
  24. Onrust R., Stukenberg P. T., O'Donnell M. Analysis of the ATPase subassembly which initiates processive DNA synthesis by DNA polymerase III holoenzyme. J Biol Chem. 1991 Nov 15;266(32):21681–21686. [PubMed] [Google Scholar]
  25. Patel S. S., Wong I., Johnson K. A. Pre-steady-state kinetic analysis of processive DNA replication including complete characterization of an exonuclease-deficient mutant. Biochemistry. 1991 Jan 15;30(2):511–525. doi: 10.1021/bi00216a029. [DOI] [PubMed] [Google Scholar]
  26. Reha-Krantz L. J. Amino acid changes coded by bacteriophage T4 DNA polymerase mutator mutants. Relating structure to function. J Mol Biol. 1988 Aug 20;202(4):711–724. doi: 10.1016/0022-2836(88)90552-9. [DOI] [PubMed] [Google Scholar]
  27. Reha-Krantz L. J., Lambert J. K. Structure-function studies of the bacteriophage T4 DNA polymerase. Isolation of a novel suppressor mutant. J Mol Biol. 1985 Dec 5;186(3):505–514. doi: 10.1016/0022-2836(85)90125-1. [DOI] [PubMed] [Google Scholar]
  28. Reha-Krantz L. J., Stocki S., Nonay R. L., Dimayuga E., Goodrich L. D., Konigsberg W. H., Spicer E. K. DNA polymerization in the absence of exonucleolytic proofreading: in vivo and in vitro studies. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2417–2421. doi: 10.1073/pnas.88.6.2417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Simon M., Giot L., Faye G. The 3' to 5' exonuclease activity located in the DNA polymerase delta subunit of Saccharomyces cerevisiae is required for accurate replication. EMBO J. 1991 Aug;10(8):2165–2170. doi: 10.1002/j.1460-2075.1991.tb07751.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Spicer E. K., Rush J., Fung C., Reha-Krantz L. J., Karam J. D., Konigsberg W. H. Primary structure of T4 DNA polymerase. Evolutionary relatedness to eucaryotic and other procaryotic DNA polymerases. J Biol Chem. 1988 Jun 5;263(16):7478–7486. [PubMed] [Google Scholar]
  31. Tabor S., Richardson C. C. Selective inactivation of the exonuclease activity of bacteriophage T7 DNA polymerase by in vitro mutagenesis. J Biol Chem. 1989 Apr 15;264(11):6447–6458. [PubMed] [Google Scholar]
  32. Tsurimoto T., Stillman B. Functions of replication factor C and proliferating-cell nuclear antigen: functional similarity of DNA polymerase accessory proteins from human cells and bacteriophage T4. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1023–1027. doi: 10.1073/pnas.87.3.1023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tsurimoto T., Stillman B. Replication factors required for SV40 DNA replication in vitro. I. DNA structure-specific recognition of a primer-template junction by eukaryotic DNA polymerases and their accessory proteins. J Biol Chem. 1991 Jan 25;266(3):1950–1960. [PubMed] [Google Scholar]
  34. Weinberg D. H., Collins K. L., Simancek P., Russo A., Wold M. S., Virshup D. M., Kelly T. J. Reconstitution of simian virus 40 DNA replication with purified proteins. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8692–8696. doi: 10.1073/pnas.87.22.8692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wong S. W., Wahl A. F., Yuan P. M., Arai N., Pearson B. E., Arai K., Korn D., Hunkapiller M. W., Wang T. S. Human DNA polymerase alpha gene expression is cell proliferation dependent and its primary structure is similar to both prokaryotic and eukaryotic replicative DNA polymerases. EMBO J. 1988 Jan;7(1):37–47. doi: 10.1002/j.1460-2075.1988.tb02781.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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