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. 1995 Jun;15(6):3072–3081. doi: 10.1128/mcb.15.6.3072

Mammalian DNA polymerase auxiliary proteins: analysis of replication factor C-catalyzed proliferating cell nuclear antigen loading onto circular double-stranded DNA.

L M Podust 1, V N Podust 1, J M Sogo 1, U Hübscher 1
PMCID: PMC230538  PMID: 7760803

Abstract

To understand the mechanism of action of the two eukaryotic replication auxiliary proteins proliferating cell nuclear antigen (PCNA) and replication factor C (RF-C), we constructed a plasmid for producing PCNA which could be 32P labelled in vitro. This allowed us to analyze the assembly of the auxiliary proteins directly on DNA and to examine this process in the absence of DNA synthesis. By using closed circular double-stranded DNA or gapped circular DNA for protein-DNA complex formation, the following results were obtained, (i) RF-C can load PCNA in an ATP-dependent manner directly on double-stranded DNA, and no 3'-OH ends are required for this reaction; (ii) the RF-C-PCNA complex assembled on closed circular DNA differs from those assembled on gapped or nicked circular DNA; (iii) the stable RF-C-PCNA complex can be assembled on circular but not on linear DNA; and (iv) only gapped DNA can partially retain the assembled RF-C-PCNA complex upon the linearization of the template. We propose that RF-C first binds unspecifically to double-stranded DNA in the presence of ATP and then loads PCNA onto DNA to yield a protein complex able to track along DNA. The RF-C-PCNA complex could slide along the template until it encounters a 3'-OH primer-template junction, where it is likely transformed into a competent clamp. The latter complex, finally, might still be able to slide along double-stranded DNA.

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Selected References

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  1. Bunz F., Kobayashi R., Stillman B. cDNAs encoding the large subunit of human replication factor C. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11014–11018. doi: 10.1073/pnas.90.23.11014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Burbelo P. D., Utani A., Pan Z. Q., Yamada Y. Cloning of the large subunit of activator 1 (replication factor C) reveals homology with bacterial DNA ligases. Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11543–11547. doi: 10.1073/pnas.90.24.11543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Burgers P. M., Yoder B. L. ATP-independent loading of the proliferating cell nuclear antigen requires DNA ends. J Biol Chem. 1993 Sep 25;268(27):19923–19926. [PubMed] [Google Scholar]
  5. 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]
  6. Gogol E. P., Young M. C., Kubasek W. L., Jarvis T. C., von Hippel P. H. Cryoelectron microscopic visualization of functional subassemblies of the bacteriophage T4 DNA replication complex. J Mol Biol. 1992 Mar 20;224(2):395–412. doi: 10.1016/0022-2836(92)91003-8. [DOI] [PubMed] [Google Scholar]
  7. Kaboord B. F., Benkovic S. J. Rapid assembly of the bacteriophage T4 core replication complex on a linear primer/template construct. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10881–10885. doi: 10.1073/pnas.90.22.10881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Krishna T. S., Kong X. P., Gary S., Burgers P. M., Kuriyan J. Crystal structure of the eukaryotic DNA polymerase processivity factor PCNA. Cell. 1994 Dec 30;79(7):1233–1243. doi: 10.1016/0092-8674(94)90014-0. [DOI] [PubMed] [Google Scholar]
  10. Kuriyan J., O'Donnell M. Sliding clamps of DNA polymerases. J Mol Biol. 1993 Dec 20;234(4):915–925. doi: 10.1006/jmbi.1993.1644. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. Li B. L., Langer J. A., Schwartz B., Pestka S. Creation of phosphorylation sites in proteins: construction of a phosphorylatable human interferon alpha. Proc Natl Acad Sci U S A. 1989 Jan;86(2):558–562. doi: 10.1073/pnas.86.2.558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lu Y., Riegel A. T. The human DNA-binding protein, PO-GA, is homologous to the large subunit of mouse replication factor C: regulation by alternate 3' processing of mRNA. Gene. 1994 Aug 5;145(2):261–265. doi: 10.1016/0378-1119(94)90017-5. [DOI] [PubMed] [Google Scholar]
  16. Lu Y., Zeft A. S., Riegel A. T. Cloning and expression of a novel human DNA binding protein, PO-GA. Biochem Biophys Res Commun. 1993 Jun 15;193(2):779–786. doi: 10.1006/bbrc.1993.1693. [DOI] [PubMed] [Google Scholar]
  17. Luckow B., Bunz F., Stillman B., Lichter P., Schütz G. Cloning, expression, and chromosomal localization of the 140-kilodalton subunit of replication factor C from mice and humans. Mol Cell Biol. 1994 Mar;14(3):1626–1634. doi: 10.1128/mcb.14.3.1626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Noskov V., Maki S., Kawasaki Y., Leem S. H., Ono B., Araki H., Pavlov Y., Sugino A. The RFC2 gene encoding a subunit of replication factor C of Saccharomyces cerevisiae. Nucleic Acids Res. 1994 May 11;22(9):1527–1535. doi: 10.1093/nar/22.9.1527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. O'Donnell M., Onrust R., Dean F. B., Chen M., Hurwitz J. Homology in accessory proteins of replicative polymerases--E. coli to humans. Nucleic Acids Res. 1993 Jan 11;21(1):1–3. doi: 10.1093/nar/21.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pan Z. Q., Chen M., Hurwitz J. The subunits of activator 1 (replication factor C) carry out multiple functions essential for proliferating-cell nuclear antigen-dependent DNA synthesis. Proc Natl Acad Sci U S A. 1993 Jan 1;90(1):6–10. doi: 10.1073/pnas.90.1.6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Podust L. M., Podust V. N., Floth C., Hübscher U. Assembly of DNA polymerase delta and epsilon holoenzymes depends on the geometry of the DNA template. Nucleic Acids Res. 1994 Aug 11;22(15):2970–2975. doi: 10.1093/nar/22.15.2970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Podust V. N., Georgaki A., Strack B., Hübscher U. Calf thymus RF-C as an essential component for DNA polymerase delta and epsilon holoenzymes function. Nucleic Acids Res. 1992 Aug 25;20(16):4159–4165. doi: 10.1093/nar/20.16.4159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Podust V. N., Hübscher U. Lagging strand DNA synthesis by calf thymus DNA polymerases alpha, beta, delta and epsilon in the presence of auxiliary proteins. Nucleic Acids Res. 1993 Feb 25;21(4):841–846. doi: 10.1093/nar/21.4.841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Podust V. N., Podust L. M., Müller F., Hübscher U. DNA polymerase delta holoenzyme: action on single-stranded DNA and on double-stranded DNA in the presence of replicative DNA helicases. Biochemistry. 1995 Apr 18;34(15):5003–5010. doi: 10.1021/bi00015a011. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Schmitt B., Buhre U., Vosberg H. P. Characterisation of size variants of type I DNA topoisomerase isolated from calf thymus. Eur J Biochem. 1984 Oct 1;144(1):127–134. doi: 10.1111/j.1432-1033.1984.tb08440.x. [DOI] [PubMed] [Google Scholar]
  27. Stillman B. Smart machines at the DNA replication fork. Cell. 1994 Sep 9;78(5):725–728. doi: 10.1016/s0092-8674(94)90362-x. [DOI] [PubMed] [Google Scholar]
  28. Stukenberg P. T., Studwell-Vaughan P. S., O'Donnell M. Mechanism of the sliding beta-clamp of DNA polymerase III holoenzyme. J Biol Chem. 1991 Jun 15;266(17):11328–11334. [PubMed] [Google Scholar]
  29. Stukenberg P. T., Turner J., O'Donnell M. An explanation for lagging strand replication: polymerase hopping among DNA sliding clamps. Cell. 1994 Sep 9;78(5):877–887. doi: 10.1016/s0092-8674(94)90662-9. [DOI] [PubMed] [Google Scholar]
  30. Tinker R. L., Kassavetis G. A., Geiduschek E. P. Detecting the ability of viral, bacterial and eukaryotic replication proteins to track along DNA. EMBO J. 1994 Nov 15;13(22):5330–5337. doi: 10.1002/j.1460-2075.1994.tb06867.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tinker R. L., Williams K. P., Kassavetis G. A., Geiduschek E. P. Transcriptional activation by a DNA-tracking protein: structural consequences of enhancement at the T4 late promoter. Cell. 1994 Apr 22;77(2):225–237. doi: 10.1016/0092-8674(94)90315-8. [DOI] [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. 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]
  34. 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]
  35. 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]
  36. Tsurimoto T., Stillman B. Replication factors required for SV40 DNA replication in vitro. II. Switching of DNA polymerase alpha and delta during initiation of leading and lagging strand synthesis. J Biol Chem. 1991 Jan 25;266(3):1961–1968. [PubMed] [Google Scholar]
  37. 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]
  38. 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]

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