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. 1992 Aug 25;20(16):4159–4165. doi: 10.1093/nar/20.16.4159

Calf thymus RF-C as an essential component for DNA polymerase delta and epsilon holoenzymes function.

V N Podust 1, A Georgaki 1, B Strack 1, U Hübscher 1
PMCID: PMC334120  PMID: 1354854

Abstract

By using a complementation assay that enabled DNA polymerase delta and DNA polymerase epsilon to replicate a singly-DNA primed M13 DNA in the presence of proliferating cell nuclear antigen (PCNA) and Escherichia coli single-stranded DNA binding protein (SSB), we have purified from calf thymus in a five step procedure a multipolypeptide complex with molecular masses of polypeptides of 155, 70, 60, 58, 39 (doublet), 38 (doublet) and 36 kDa. The protein is very likely replication factor C (Tsurimoto, T. and Stillman, B. (1989) Mol. Cell. Biol. 9, 609-619). This conclusion is based on biochemical and physicochemical data and the finding that it contains a DNA stimulated ATPase which is under certain conditions stimulated by PCNA. Together RF-C, PCNA and ATP convert DNA polymerases delta and epsilon to holoenzyme forms, which were able to replicate efficiently SSB-covered singly-DNA primed M13 DNA. Calf thymus RF-C could form a primer recognition complex on a 3'-OH primer terminus in the presence of calf thymus PCNA and ATP. Holoenzyme complexes of DNA polymerase delta and epsilon could be isolated suggesting that these enzymes directly interact with the auxiliary proteins in a similar way. Under optimal replication conditions on singly-DNA primed M13 DNA the DNA synthesis rate of DNA polymerase delta was higher than of DNA polymerase epsilon. Based on these functional date possible roles of these two DNA polymerases in eukaryotic DNA replication are discussed.

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Selected References

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

  1. Araki H., Ropp P. A., Johnson A. L., Johnston L. H., Morrison A., Sugino A. DNA polymerase II, the probable homolog of mammalian DNA polymerase epsilon, replicates chromosomal DNA in the yeast Saccharomyces cerevisiae. EMBO J. 1992 Feb;11(2):733–740. doi: 10.1002/j.1460-2075.1992.tb05106.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arezzo F., Rose K. M. Tryptic peptide analysis of nanogram quantities of proteins: radioiodination of proteins detected by silver staining in polyacrylamide gels. Anal Biochem. 1987 Dec;167(2):387–393. doi: 10.1016/0003-2697(87)90181-3. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. Burgers P. M. Saccharomyces cerevisiae replication factor C. II. Formation and activity of complexes with the proliferating cell nuclear antigen and with DNA polymerases delta and epsilon. J Biol Chem. 1991 Nov 25;266(33):22698–22706. [PubMed] [Google Scholar]
  6. Fairman M. P., Stillman B. Cellular factors required for multiple stages of SV40 DNA replication in vitro. EMBO J. 1988 Apr;7(4):1211–1218. doi: 10.1002/j.1460-2075.1988.tb02933.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fien K., Stillman B. Identification of replication factor C from Saccharomyces cerevisiae: a component of the leading-strand DNA replication complex. Mol Cell Biol. 1992 Jan;12(1):155–163. doi: 10.1128/mcb.12.1.155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Focher F., Gassmann M., Hafkemeyer P., Ferrari E., Spadari S., Hübscher U. Calf thymus DNA polymerase delta independent of proliferating cell nuclear antigen (PCNA). Nucleic Acids Res. 1989 Mar 11;17(5):1805–1821. doi: 10.1093/nar/17.5.1805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Focher F., Spadari S., Ginelli B., Hottiger M., Gassmann M., Hübscher U. Calf thymus DNA polymerase delta: purification, biochemical and functional properties of the enzyme after its separation from DNA polymerase alpha, a DNA dependent ATPase and proliferating cell nuclear antigen. Nucleic Acids Res. 1988 Jul 25;16(14A):6279–6295. doi: 10.1093/nar/16.14.6279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Goulian M., Heard C. J. The mechanism of action of an accessory protein for DNA polymerase alpha/primase. J Biol Chem. 1990 Aug 5;265(22):13231–13239. [PubMed] [Google Scholar]
  11. Hübscher U., Thömmes P. DNA polymerase epsilon: in search of a function. Trends Biochem Sci. 1992 Feb;17(2):55–58. doi: 10.1016/0968-0004(92)90499-y. [DOI] [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Lee S. H., Hurwitz J. Mechanism of elongation of primed DNA by DNA polymerase delta, proliferating cell nuclear antigen, and activator 1. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5672–5676. doi: 10.1073/pnas.87.15.5672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Linn S. How many pols does it take to replicate nuclear DNA? Cell. 1991 Jul 26;66(2):185–187. doi: 10.1016/0092-8674(91)90608-2. [DOI] [PubMed] [Google Scholar]
  17. Lohman T. M., Green J. M., Beyer R. S. Large-scale overproduction and rapid purification of the Escherichia coli ssb gene product. Expression of the ssb gene under lambda PL control. Biochemistry. 1986 Jan 14;25(1):21–25. doi: 10.1021/bi00349a004. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Prelich G., Tan C. K., Kostura M., Mathews M. B., So A. G., Downey K. M., Stillman B. Functional identity of proliferating cell nuclear antigen and a DNA polymerase-delta auxiliary protein. Nature. 1987 Apr 2;326(6112):517–520. doi: 10.1038/326517a0. [DOI] [PubMed] [Google Scholar]
  21. Sitney K. C., Budd M. E., Campbell J. L. DNA polymerase III, a second essential DNA polymerase, is encoded by the S. cerevisiae CDC2 gene. Cell. 1989 Feb 24;56(4):599–605. doi: 10.1016/0092-8674(89)90582-5. [DOI] [PubMed] [Google Scholar]
  22. So A. G., Downey K. M. Eukaryotic DNA replication. Crit Rev Biochem Mol Biol. 1992;27(1-2):129–155. doi: 10.3109/10409239209082561. [DOI] [PubMed] [Google Scholar]
  23. Thömmes P., Ferrari E., Jessberger R., Hübscher U. Four different DNA helicases from calf thymus. J Biol Chem. 1992 Mar 25;267(9):6063–6073. [PubMed] [Google Scholar]
  24. Thömmes P., Hübscher U. Eukaryotic DNA replication. Enzymes and proteins acting at the fork. Eur J Biochem. 1990 Dec 27;194(3):699–712. doi: 10.1111/j.1432-1033.1990.tb19460.x. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Tsurimoto T., Stillman B. Multiple replication factors augment DNA synthesis by the two eukaryotic DNA polymerases, alpha and delta. EMBO J. 1989 Dec 1;8(12):3883–3889. doi: 10.1002/j.1460-2075.1989.tb08567.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tsurimoto T., Stillman B. Purification of a cellular replication factor, RF-C, that is required for coordinated synthesis of leading and lagging strands during simian virus 40 DNA replication in vitro. Mol Cell Biol. 1989 Feb;9(2):609–619. doi: 10.1128/mcb.9.2.609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Wang T. S. Eukaryotic DNA polymerases. Annu Rev Biochem. 1991;60:513–552. doi: 10.1146/annurev.bi.60.070191.002501. [DOI] [PubMed] [Google Scholar]
  30. Weiser T., Gassmann M., Thömmes P., Ferrari E., Hafkemeyer P., Hübscher U. Biochemical and functional comparison of DNA polymerases alpha, delta, and epsilon from calf thymus. J Biol Chem. 1991 Jun 5;266(16):10420–10428. [PubMed] [Google Scholar]
  31. Wobbe C. R., Weissbach L., Borowiec J. A., Dean F. B., Murakami Y., Bullock P., Hurwitz J. Replication of simian virus 40 origin-containing DNA in vitro with purified proteins. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1834–1838. doi: 10.1073/pnas.84.7.1834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wold M. S., Kelly T. Purification and characterization of replication protein A, a cellular protein required for in vitro replication of simian virus 40 DNA. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2523–2527. doi: 10.1073/pnas.85.8.2523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yoder B. L., Burgers P. M. Saccharomyces cerevisiae replication factor C. I. Purification and characterization of its ATPase activity. J Biol Chem. 1991 Nov 25;266(33):22689–22697. [PubMed] [Google Scholar]

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