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.
Full text
PDF




Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Baer R., Bankier A. T., Biggin M. D., Deininger P. L., Farrell P. J., Gibson T. J., Hatfull G., Hudson G. S., Satchwell S. C., Séguin C. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature. 1984 Jul 19;310(5974):207–211. doi: 10.1038/310207a0. [DOI] [PubMed] [Google Scholar]
- 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]
- Boulet A., Simon M., Faye G., Bauer G. A., Burgers P. M. Structure and function of the Saccharomyces cerevisiae CDC2 gene encoding the large subunit of DNA polymerase III. EMBO J. 1989 Jun;8(6):1849–1854. doi: 10.1002/j.1460-2075.1989.tb03580.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Clayton L. K., Goodman M. F., Branscomb E. W., Galas D. J. Error induction and correction by mutant and wild type T4 DNA polymerases. Kinetic error discrimination mechanisms. J Biol Chem. 1979 Mar 25;254(6):1902–1912. [PubMed] [Google Scholar]
- 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]
- Drake J. W., Allen E. F., Forsberg S. A., Preparata R. M., Greening E. O. Genetic control of mutation rates in bacteriophageT4. Nature. 1969 Mar 22;221(5186):1128–1132. [PubMed] [Google Scholar]
- Earl P. L., Jones E. V., Moss B. Homology between DNA polymerases of poxviruses, herpesviruses, and adenoviruses: nucleotide sequence of the vaccinia virus DNA polymerase gene. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3659–3663. doi: 10.1073/pnas.83.11.3659. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Engler M. J., Lechner R. L., Richardson C. C. Two forms of the DNA polymerase of bacteriophage T7. J Biol Chem. 1983 Sep 25;258(18):11165–11173. [PubMed] [Google Scholar]
- Fersht A. R., Knill-Jones J. W., Tsui W. C. Kinetic basis of spontaneous mutation. Misinsertion frequencies, proofreading specificities and cost of proofreading by DNA polymerases of Escherichia coli. J Mol Biol. 1982 Mar 25;156(1):37–51. doi: 10.1016/0022-2836(82)90457-0. [DOI] [PubMed] [Google Scholar]
- Freemont P. S., Friedman J. M., Beese L. S., Sanderson M. R., Steitz T. A. Cocrystal structure of an editing complex of Klenow fragment with DNA. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8924–8928. doi: 10.1073/pnas.85.23.8924. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gibbs J. S., Chiou H. C., Hall J. D., Mount D. W., Retondo M. J., Weller S. K., Coen D. M. Sequence and mapping analyses of the herpes simplex virus DNA polymerase gene predict a C-terminal substrate binding domain. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7969–7973. doi: 10.1073/pnas.82.23.7969. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Hibner U., Alberts B. M. Fidelity of DNA replication catalysed in vitro on a natural DNA template by the T4 bacteriophage multi-enzyme complex. Nature. 1980 May 29;285(5763):300–305. doi: 10.1038/285300a0. [DOI] [PubMed] [Google Scholar]
- Kunkel T. A., Loeb L. A., Goodman M. F. On the fidelity of DNA replication. The accuracy of T4 DNA polymerases in copying phi X174 DNA in vitro. J Biol Chem. 1984 Feb 10;259(3):1539–1545. [PubMed] [Google Scholar]
- 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]
- Leavitt M. C., Ito J. T5 DNA polymerase: structural--functional relationships to other DNA polymerases. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4465–4469. doi: 10.1073/pnas.86.12.4465. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Lo K. Y., Bessman M. J. An antimutator deoxyribonucleic acid polymerase. I. Purification and properties of the enzyme. J Biol Chem. 1976 Apr 25;251(8):2475–2479. [PubMed] [Google Scholar]
- McPheeters D. S., Christensen A., Young E. T., Stormo G., Gold L. Translational regulation of expression of the bacteriophage T4 lysozyme gene. Nucleic Acids Res. 1986 Jul 25;14(14):5813–5826. doi: 10.1093/nar/14.14.5813. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Morris C. F., Hama-Inaba H., Mace D., Sinha N. K., Alberts B. Purification of the gene 43, 44, 45, and 62 proteins of the bacteriophage T4 DNA replication apparatus. J Biol Chem. 1979 Jul 25;254(14):6787–6796. [PubMed] [Google Scholar]
- 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]
- 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]
- Pizzagalli A., Valsasnini P., Plevani P., Lucchini G. DNA polymerase I gene of Saccharomyces cerevisiae: nucleotide sequence, mapping of a temperature-sensitive mutation, and protein homology with other DNA polymerases. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3772–3776. doi: 10.1073/pnas.85.11.3772. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Reha-Krantz L. J., Bessman M. J. Studeis on the biochemical basis of mutation. IV. Effect of amino acid substitution on the enzymatic and biological properties of bacteriophage T4 DNA polymerase. J Mol Biol. 1977 Oct 15;116(1):99–113. doi: 10.1016/0022-2836(77)90121-8. [DOI] [PubMed] [Google Scholar]
- 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]
- Reha-Krantz L. J., Liesner E. M., Parmaksizoglu S., Stocki S. Isolation of bacteriophage T4 DNA polymerase mutator mutants. J Mol Biol. 1986 May 20;189(2):261–272. doi: 10.1016/0022-2836(86)90508-5. [DOI] [PubMed] [Google Scholar]
- Reha-Krantz L. J. Locations of amino acid substitutions in bacteriophage T4 tsL56 DNA polymerase predict an N-terminal exonuclease domain. J Virol. 1989 Nov;63(11):4762–4766. doi: 10.1128/jvi.63.11.4762-4766.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sinha N. K., Haimes M. D. Molecular mechanisms of substitution mutagenesis. An experimental test of the Watson-Crick and topal-fresco models of base mispairings. J Biol Chem. 1981 Oct 25;256(20):10671–10683. [PubMed] [Google Scholar]
- Speyer J. F., Karam J. D., Lenny A. B. On the role of DNA polymerase in base selection. Cold Spring Harb Symp Quant Biol. 1966;31:693–697. doi: 10.1101/sqb.1966.031.01.088. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Topal M. D., Fresco J. R. Complementary base pairing and the origin of substitution mutations. Nature. 1976 Sep 23;263(5575):285–289. doi: 10.1038/263285a0. [DOI] [PubMed] [Google Scholar]
- Wang T. S., Wong S. W., Korn D. Human DNA polymerase alpha: predicted functional domains and relationships with viral DNA polymerases. FASEB J. 1989 Jan;3(1):14–21. doi: 10.1096/fasebj.3.1.2642867. [DOI] [PubMed] [Google Scholar]