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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
. 1992 Oct 15;89(20):9377–9381. doi: 10.1073/pnas.89.20.9377

A 5' to 3' exonuclease functionally interacts with calf DNA polymerase epsilon.

G Siegal 1, J J Turchi 1, T W Myers 1, R A Bambara 1
PMCID: PMC50134  PMID: 1329095

Abstract

Analysis of fractions containing purified DNA polymerase epsilon from calf thymus has revealed the presence of a 5' to 3' exonuclease activity that is specific for a single strand of duplex DNA. This activity is capable of degrading a 3'-labeled oligonucleotide hybridized to M13mp18 DNA. When a second oligonucleotide primer is annealed 3 bases upstream, degradation of the downstream primer is strictly dependent on DNA synthesis from the upstream primer. Replacement of the downstream primer by an oligoribonucleotide of identical sequence results in a similar pattern of exonucleolytic activity. The activity has been highly purified and found to cosediment in glycerol gradients with a peptide of 56 kDa as judged by SDS/PAGE analysis. Effects of calf DNA polymerase alpha and delta on exonuclease activity are also observed but with differences in the pattern of products.

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Selected References

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  1. Bambara R. A., Jessee C. B. Properties of DNA polymerases delta and epsilon, and their roles in eukaryotic DNA replication. Biochim Biophys Acta. 1991 Jan 17;1088(1):11–24. doi: 10.1016/0167-4781(91)90147-e. [DOI] [PubMed] [Google Scholar]
  2. Brutlag D., Atkinson M. R., Setlow P., Kornberg A. An active fragment of DNA polymerase produced by proteolytic cleavage. Biochem Biophys Res Commun. 1969 Dec 4;37(6):982–989. doi: 10.1016/0006-291x(69)90228-9. [DOI] [PubMed] [Google Scholar]
  3. Bullock P. A., Seo Y. S., Hurwitz J. Initiation of simian virus 40 DNA synthesis in vitro. Mol Cell Biol. 1991 May;11(5):2350–2361. doi: 10.1128/mcb.11.5.2350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Eki T., Hurwitz J. Influence of poly(ADP-ribose) polymerase on the enzymatic synthesis of SV40 DNA. J Biol Chem. 1991 Feb 15;266(5):3087–3100. [PubMed] [Google Scholar]
  6. Goulian M., Richards S. H., Heard C. J., Bigsby B. M. Discontinuous DNA synthesis by purified mammalian proteins. J Biol Chem. 1990 Oct 25;265(30):18461–18471. [PubMed] [Google Scholar]
  7. Holland P. M., Abramson R. D., Watson R., Gelfand D. H. Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7276–7280. doi: 10.1073/pnas.88.16.7276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ishimi Y., Claude A., Bullock P., Hurwitz J. Complete enzymatic synthesis of DNA containing the SV40 origin of replication. J Biol Chem. 1988 Dec 25;263(36):19723–19733. [PubMed] [Google Scholar]
  9. Joyce C. M. How DNA travels between the separate polymerase and 3'-5'-exonuclease sites of DNA polymerase I (Klenow fragment). J Biol Chem. 1989 Jun 25;264(18):10858–10866. [PubMed] [Google Scholar]
  10. Klenow H., Henningsen I. Selective elimination of the exonuclease activity of the deoxyribonucleic acid polymerase from Escherichia coli B by limited proteolysis. Proc Natl Acad Sci U S A. 1970 Jan;65(1):168–175. doi: 10.1073/pnas.65.1.168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lee S. H., Eki T., Hurwitz J. Synthesis of DNA containing the simian virus 40 origin of replication by the combined action of DNA polymerases alpha and delta. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7361–7365. doi: 10.1073/pnas.86.19.7361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lee S. H., Pan Z. Q., Kwong A. D., Burgers P. M., Hurwitz J. Synthesis of DNA by DNA polymerase epsilon in vitro. J Biol Chem. 1991 Nov 25;266(33):22707–22717. [PubMed] [Google Scholar]
  13. Longley M. J., Bennett S. E., Mosbaugh D. W. Characterization of the 5' to 3' exonuclease associated with Thermus aquaticus DNA polymerase. Nucleic Acids Res. 1990 Dec 25;18(24):7317–7322. doi: 10.1093/nar/18.24.7317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Morrison A., Araki H., Clark A. B., Hamatake R. K., Sugino A. A third essential DNA polymerase in S. cerevisiae. Cell. 1990 Sep 21;62(6):1143–1151. doi: 10.1016/0092-8674(90)90391-q. [DOI] [PubMed] [Google Scholar]
  15. Myers T. W., Romano L. J. Mechanism of stimulation of T7 DNA polymerase by Escherichia coli single-stranded DNA binding protein (SSB). J Biol Chem. 1988 Nov 15;263(32):17006–17015. [PubMed] [Google Scholar]
  16. Nasheuer H. P., Grosse F. DNA polymerase alpha-primase from calf thymus. Determination of the polypeptide responsible for primase activity. J Biol Chem. 1988 Jun 25;263(18):8981–8988. [PubMed] [Google Scholar]
  17. Nethanel T., Kaufmann G. Two DNA polymerases may be required for synthesis of the lagging DNA strand of simian virus 40. J Virol. 1990 Dec;64(12):5912–5918. doi: 10.1128/jvi.64.12.5912-5918.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Prelich G., Stillman B. Coordinated leading and lagging strand synthesis during SV40 DNA replication in vitro requires PCNA. Cell. 1988 Apr 8;53(1):117–126. doi: 10.1016/0092-8674(88)90493-x. [DOI] [PubMed] [Google Scholar]
  19. Sabatino R. D., Myers T. W., Bambara R. A., Kwon-Shin O., Marraccino R. L., Frickey P. H. Calf thymus DNA polymerases alpha and delta are capable of highly processive DNA synthesis. Biochemistry. 1988 Apr 19;27(8):2998–3004. doi: 10.1021/bi00408a050. [DOI] [PubMed] [Google Scholar]
  20. Sabatino R. D., Myers T. W., Bambara R. A. Substrate specificity of the exonuclease associated with calf DNA polymerase. Cancer Res. 1990 Sep 1;50(17):5340–5344. [PubMed] [Google Scholar]
  21. Sapp M., König H., Riedel H. D., Richter A., Knippers R. A newly detected class of mammalian single strand-specific DNA-binding proteins. Effects on DNA polymerase alpha-catalyzed DNA synthesis. J Biol Chem. 1985 Feb 10;260(3):1550–1556. [PubMed] [Google Scholar]
  22. Siegal G., Turchi J. J., Jessee C. B., Mallaber L. M., Bambara R. A., Myers T. W. Structural relationships between two forms of DNA polymerase epsilon from calf thymus. J Biol Chem. 1992 Feb 25;267(6):3991–3999. [PubMed] [Google Scholar]
  23. Syvaoja J., Linn S. Characterization of a large form of DNA polymerase delta from HeLa cells that is insensitive to proliferating cell nuclear antigen. J Biol Chem. 1989 Feb 15;264(5):2489–2497. [PubMed] [Google Scholar]
  24. Tsurimoto T., Melendy T., Stillman B. Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin. Nature. 1990 Aug 9;346(6284):534–539. doi: 10.1038/346534a0. [DOI] [PubMed] [Google Scholar]
  25. Weinberg D. H., Kelly T. J. Requirement for two DNA polymerases in the replication of simian virus 40 DNA in vitro. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9742–9746. doi: 10.1073/pnas.86.24.9742. [DOI] [PMC free article] [PubMed] [Google Scholar]

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