Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1987 Jul;84(14):4865–4869. doi: 10.1073/pnas.84.14.4865

Exonucleolytic proofreading by calf thymus DNA polymerase delta.

T A Kunkel, R D Sabatino, R A Bambara
PMCID: PMC305206  PMID: 3474631

Abstract

The fidelity of DNA synthesis by calf thymus DNA polymerase delta (pol delta) in vitro has been determined using an M13lacZ alpha nonsense codon reversion assay. Pol delta is highly accurate, producing on average less than 1 single-base substitution error for each 10(6) nucleotides polymerized. This accuracy is 10- and 500-fold greater than that of DNA polymerases alpha and beta, respectively, in the same assay. Three observations suggest that this higher fidelity results in part from proofreading of misinserted bases by the 3' to 5' exonuclease associated with pol delta. First, the exonuclease efficiently excises terminally mismatched bases. Second, both terminal mismatch excision and the fidelity of DNA synthesis by pol delta are reduced with increasing concentration of deoxynucleoside triphosphates in the synthesis reaction. These effects result from increasing the rate of polymerization relative to the rate of exonucleolytic excision and are hallmarks of exonuclease proofreading. Third, both terminal mismatch excision and fidelity decrease upon addition to the reaction mixture of adenosine monophosphate, a compound known to selectively inhibit the exonuclease but not the polymerase activity of pol delta. These results suggest that 3' to 5' exonuclease-dependent proofreading enhances the fidelity of DNA synthesis by a mammalian DNA polymerase in vitro.

Full text

PDF
4865

Selected References

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

  1. Abbotts J., Loeb L. A. DNA polymerase alpha and models for proofreading. Nucleic Acids Res. 1985 Jan 11;13(1):261–274. doi: 10.1093/nar/13.1.261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Battula N., Loeb L. A. On the fidelity of DNA replication. Lack of exodeoxyribonuclease activity and error-correcting function in avian myeloblastosis virus DNA polymerase. J Biol Chem. 1976 Feb 25;251(4):982–986. [PubMed] [Google Scholar]
  3. Brosius S., Grosse F., Krauss G. Subspecies of DNA polymerase alpha from calf thymus with different fidelity in copying synthetic template-primers. Nucleic Acids Res. 1983 Jan 11;11(1):193–202. doi: 10.1093/nar/11.1.193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Byrnes J. J., Downey K. M., Black V. L., So A. G. A new mammalian DNA polymerase with 3' to 5' exonuclease activity: DNA polymerase delta. Biochemistry. 1976 Jun 29;15(13):2817–2823. doi: 10.1021/bi00658a018. [DOI] [PubMed] [Google Scholar]
  5. Byrnes J. J., Downey K. M., Que B. G., Lee M. Y., Black V. L., So A. G. Selective inhibition of the 3' to 5' exonuclease activity associated with DNA polymerases: a mechanism of mutagenesis. Biochemistry. 1977 Aug 23;16(17):3740–3746. doi: 10.1021/bi00636a002. [DOI] [PubMed] [Google Scholar]
  6. Chang L. M. DNA polymerases from bakers' yeast. J Biol Chem. 1977 Mar 25;252(6):1873–1880. [PubMed] [Google Scholar]
  7. Chen Y. C., Bohn E. W., Planck S. R., Wilson S. H. Mouse DNA polymerase alpha. Subunit structure and identification of a species with associated exonuclease. J Biol Chem. 1979 Nov 25;254(22):11678–11687. [PubMed] [Google Scholar]
  8. 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]
  9. Crute J. J., Wahl A. F., Bambara R. A. Purification and characterization of two new high molecular weight forms of DNA polymerase delta. Biochemistry. 1986 Jan 14;25(1):26–36. doi: 10.1021/bi00349a005. [DOI] [PubMed] [Google Scholar]
  10. Dresler S. L., Frattini M. G. DNA replication and UV-induced DNA repair synthesis in human fibroblasts are much less sensitive than DNA polymerase alpha to inhibition by butylphenyl-deoxyguanosine triphosphate. Nucleic Acids Res. 1986 Sep 11;14(17):7093–7102. doi: 10.1093/nar/14.17.7093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fersht A. R. Fidelity of replication of phage phi X174 DNA by DNA polymerase III holoenzyme: spontaneous mutation by misincorporation. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4946–4950. doi: 10.1073/pnas.76.10.4946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Grosse F., Krauss G., Knill-Jones J. W., Fersht A. R. Accuracy of DNA polymerase-alpha in copying natural DNA. EMBO J. 1983;2(9):1515–1519. doi: 10.1002/j.1460-2075.1983.tb01616.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kaguni L. S., DiFrancesco R. A., Lehman I. R. The DNA polymerase-primase from drosophila melanogaster embryos. Rate and fidelity of polymerization on single-stranded DNA templates. J Biol Chem. 1984 Jul 25;259(14):9314–9319. [PubMed] [Google Scholar]
  14. Kunkel T. A., Alexander P. S. The base substitution fidelity of eucaryotic DNA polymerases. Mispairing frequencies, site preferences, insertion preferences, and base substitution by dislocation. J Biol Chem. 1986 Jan 5;261(1):160–166. [PubMed] [Google Scholar]
  15. Kunkel T. A., Beckman R. A., Loeb L. A. On the fidelity of DNA synthesis. Pyrophosphate-induced misincorporation allows detection of two proofreading mechanisms. J Biol Chem. 1986 Oct 15;261(29):13610–13616. [PubMed] [Google Scholar]
  16. Kunkel T. A., Loeb L. A. Fidelity of mammalian DNA polymerases. Science. 1981 Aug 14;213(4509):765–767. doi: 10.1126/science.6454965. [DOI] [PubMed] [Google Scholar]
  17. Kunkel T. A. Mutational specificity of depurination. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1494–1498. doi: 10.1073/pnas.81.5.1494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kunkel T. A., Schaaper R. M., Beckman R. A., Loeb L. A. On the fidelity of DNA replication. Effect of the next nucleotide on proofreading. J Biol Chem. 1981 Oct 10;256(19):9883–9889. [PubMed] [Google Scholar]
  19. Kunkel T. A. The mutational specificity of DNA polymerase-beta during in vitro DNA synthesis. Production of frameshift, base substitution, and deletion mutations. J Biol Chem. 1985 May 10;260(9):5787–5796. [PubMed] [Google Scholar]
  20. Kunkel T. A. The mutational specificity of DNA polymerases-alpha and -gamma during in vitro DNA synthesis. J Biol Chem. 1985 Oct 15;260(23):12866–12874. [PubMed] [Google Scholar]
  21. Lee M. Y., Tan C. K., Downey K. M., So A. G. Structural and functional properties of calf thymus DNA polymerase delta. Prog Nucleic Acid Res Mol Biol. 1981;26:83–96. doi: 10.1016/s0079-6603(08)60396-7. [DOI] [PubMed] [Google Scholar]
  22. Lee M. Y., Tan C. K., So A. G., Downey K. M. Purification of deoxyribonucleic acid polymerase delta from calf thymus: partial characterization of physical properties. Biochemistry. 1980 May 13;19(10):2096–2101. doi: 10.1021/bi00551a015. [DOI] [PubMed] [Google Scholar]
  23. Loeb L. A., Kunkel T. A. Fidelity of DNA synthesis. Annu Rev Biochem. 1982;51:429–457. doi: 10.1146/annurev.bi.51.070182.002241. [DOI] [PubMed] [Google Scholar]
  24. Mosbaugh D. W., Linn S. Excision repair and DNA synthesis with a combination of HeLa DNA polymerase beta and DNase V. J Biol Chem. 1983 Jan 10;258(1):108–118. [PubMed] [Google Scholar]
  25. Mosbaugh D. W., Meyer R. R. Interaction of mammalian deoxyribonuclease V, a double strand 3' to 5' and 5' to 3' exonuclease, with deoxyribonucleic acid polymerase-beta from the Novikoff hepatoma. J Biol Chem. 1980 Nov 10;255(21):10239–10247. [PubMed] [Google Scholar]
  26. 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]
  27. Ottiger H. P., Hübscher U. Mammalian DNA polymerase alpha holoenzymes with possible functions at the leading and lagging strand of the replication fork. Proc Natl Acad Sci U S A. 1984 Jul;81(13):3993–3997. doi: 10.1073/pnas.81.13.3993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Que B. G., Downey K. M., So A. G. Mechanisms of selective inhibition of 3' to 5' exonuclease activity of Escherichia coli DNA polymerase I by nucleoside 5'-monophosphates. Biochemistry. 1978 May 2;17(9):1603–1606. doi: 10.1021/bi00602a004. [DOI] [PubMed] [Google Scholar]
  29. Skarnes W., Bonin P., Baril E. Exonuclease activity associated with a multiprotein form of HeLa cell DNA polymerase alpha. Purification and properties of the exonuclease. J Biol Chem. 1986 May 15;261(14):6629–6636. [PubMed] [Google Scholar]
  30. Wahl A. F., Crute J. J., Sabatino R. D., Bodner J. B., Marraccino R. L., Harwell L. W., Lord E. M., Bambara R. A. Properties of two forms of DNA polymerase delta from calf thymus. Biochemistry. 1986 Dec 2;25(24):7821–7827. doi: 10.1021/bi00372a006. [DOI] [PubMed] [Google Scholar]
  31. Wahl A. F., Kowalski S. P., Harwell L. W., Lord E. M., Bambara R. A. Immunoaffinity purification and properties of a high molecular weight calf thymus DNA alpha-polymerase. Biochemistry. 1984 Apr 24;23(9):1895–1899. doi: 10.1021/bi00304a001. [DOI] [PubMed] [Google Scholar]
  32. Yarranton G. T., Banks G. R. A DNA polymerase from Ustilago maydis. Evidence of proof-reading by the associated 3' leads to 5' deoxyribonuclease activity. Eur J Biochem. 1977 Aug 1;77(3):521–527. doi: 10.1111/j.1432-1033.1977.tb11694.x. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES