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. 1996 Oct 15;93(21):11597–11602. doi: 10.1073/pnas.93.21.11597

p21Cip1/Waf1 disrupts the recruitment of human Fen1 by proliferating-cell nuclear antigen into the DNA replication complex.

U Chen 1, S Chen 1, P Saha 1, A Dutta 1
PMCID: PMC38103  PMID: 8876181

Abstract

Fen1 or maturation factor 1 is a 5'-3' exonuclease essential for the degradation of the RNA primer-DNA junctions at the 5' ends of immature Okazaki fragments prior to their ligation into a continuous DNA strand. The gene is also necessary for repair of damaged DNA in yeast. We report that human proliferating-cell nuclear antigen (PCNA) associates with human Fen1 with a Kd of 60 nM and an apparent stoichiometry of three Fen1 molecules per PCNA trimer. The Fen1-PCNA association is seen in cell extracts without overexpression of either partner and is mediated by a basic region at the C terminus of Fen1. Therefore, the polymerase delta-PCNA-Fen1 complex has all the activities associated with prokaryotic DNA polymerases involved in replication: 5'-3' polymerase, 3'-5' exonuclease, and 5'-3' exonuclease. Although p21, a regulatory protein induced by p53 in response to DNA damage, interacts with PCNA with a comparable Kd (10 nM) and a stoichiometry of three molecules of p21 per PCNA trimer, a p21-PCNA-Fen1 complex is not formed. This mutually exclusive interaction suggests that the conformation of a PCNA trimer switches such that it can either bind p21 or Fen1. Furthermore, overexpression of p21 can disrupt Fen1-PCNA interaction in vivo. Therefore, besides interfering with the processivity of polymerase delta-PCNA, p21 also uncouples Fen1 from the PCNA scaffold.

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Selected References

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

  1. Budd M. E., Campbell J. L. A yeast gene required for DNA replication encodes a protein with homology to DNA helicases. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7642–7646. doi: 10.1073/pnas.92.17.7642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Campbell J. L. Yeast DNA replication. J Biol Chem. 1993 Dec 5;268(34):25261–25264. [PubMed] [Google Scholar]
  3. Chen J., Jackson P. K., Kirschner M. W., Dutta A. Separate domains of p21 involved in the inhibition of Cdk kinase and PCNA. Nature. 1995 Mar 23;374(6520):386–388. doi: 10.1038/374386a0. [DOI] [PubMed] [Google Scholar]
  4. Chen J., Peters R., Saha P., Lee P., Theodoras A., Pagano M., Wagner G., Dutta A. A 39 amino acid fragment of the cell cycle regulator p21 is sufficient to bind PCNA and partially inhibit DNA replication in vivo. Nucleic Acids Res. 1996 May 1;24(9):1727–1733. doi: 10.1093/nar/24.9.1727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dutta A., Ruppert J. M., Aster J. C., Winchester E. Inhibition of DNA replication factor RPA by p53. Nature. 1993 Sep 2;365(6441):79–82. doi: 10.1038/365079a0. [DOI] [PubMed] [Google Scholar]
  6. Dutta A., Stillman B. cdc2 family kinases phosphorylate a human cell DNA replication factor, RPA, and activate DNA replication. EMBO J. 1992 Jun;11(6):2189–2199. doi: 10.1002/j.1460-2075.1992.tb05278.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Flores-Rozas H., Kelman Z., Dean F. B., Pan Z. Q., Harper J. W., Elledge S. J., O'Donnell M., Hurwitz J. Cdk-interacting protein 1 directly binds with proliferating cell nuclear antigen and inhibits DNA replication catalyzed by the DNA polymerase delta holoenzyme. Proc Natl Acad Sci U S A. 1994 Aug 30;91(18):8655–8659. doi: 10.1073/pnas.91.18.8655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Goubin F., Ducommun B. Identification of binding domains on the p21Cip1 cyclin-dependent kinase inhibitor. Oncogene. 1995 Jun 15;10(12):2281–2287. [PubMed] [Google Scholar]
  11. 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]
  12. Harper J. W., Adami G. R., Wei N., Keyomarsi K., Elledge S. J. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 1993 Nov 19;75(4):805–816. doi: 10.1016/0092-8674(93)90499-g. [DOI] [PubMed] [Google Scholar]
  13. Harrington J. J., Lieber M. R. Functional domains within FEN-1 and RAD2 define a family of structure-specific endonucleases: implications for nucleotide excision repair. Genes Dev. 1994 Jun 1;8(11):1344–1355. doi: 10.1101/gad.8.11.1344. [DOI] [PubMed] [Google Scholar]
  14. Hurwitz J., Dean F. B., Kwong A. D., Lee S. H. The in vitro replication of DNA containing the SV40 origin. J Biol Chem. 1990 Oct 25;265(30):18043–18046. [PubMed] [Google Scholar]
  15. 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]
  16. Johnson R. E., Kovvali G. K., Prakash L., Prakash S. Requirement of the yeast RTH1 5' to 3' exonuclease for the stability of simple repetitive DNA. Science. 1995 Jul 14;269(5221):238–240. doi: 10.1126/science.7618086. [DOI] [PubMed] [Google Scholar]
  17. Kelly T. J. SV40 DNA replication. J Biol Chem. 1988 Dec 5;263(34):17889–17892. [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Li R., Waga S., Hannon G. J., Beach D., Stillman B. Differential effects by the p21 CDK inhibitor on PCNA-dependent DNA replication and repair. Nature. 1994 Oct 6;371(6497):534–537. doi: 10.1038/371534a0. [DOI] [PubMed] [Google Scholar]
  21. Li X., Li J., Harrington J., Lieber M. R., Burgers P. M. Lagging strand DNA synthesis at the eukaryotic replication fork involves binding and stimulation of FEN-1 by proliferating cell nuclear antigen. J Biol Chem. 1995 Sep 22;270(38):22109–22112. doi: 10.1074/jbc.270.38.22109. [DOI] [PubMed] [Google Scholar]
  22. Luo Y., Hurwitz J., Massagué J. Cell-cycle inhibition by independent CDK and PCNA binding domains in p21Cip1. Nature. 1995 May 11;375(6527):159–161. doi: 10.1038/375159a0. [DOI] [PubMed] [Google Scholar]
  23. Murante R. S., Huang L., Turchi J. J., Bambara R. A. The calf 5'- to 3'-exonuclease is also an endonuclease with both activities dependent on primers annealed upstream of the point of cleavage. J Biol Chem. 1994 Jan 14;269(2):1191–1196. [PubMed] [Google Scholar]
  24. Murray J. M., Tavassoli M., al-Harithy R., Sheldrick K. S., Lehmann A. R., Carr A. M., Watts F. Z. Structural and functional conservation of the human homolog of the Schizosaccharomyces pombe rad2 gene, which is required for chromosome segregation and recovery from DNA damage. Mol Cell Biol. 1994 Jul;14(7):4878–4888. doi: 10.1128/mcb.14.7.4878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mäkelä T. P., Parvin J. D., Kim J., Huber L. J., Sharp P. A., Weinberg R. A. A kinase-deficient transcription factor TFIIH is functional in basal and activated transcription. Proc Natl Acad Sci U S A. 1995 May 23;92(11):5174–5178. doi: 10.1073/pnas.92.11.5174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nakanishi M., Robetorye R. S., Adami G. R., Pereira-Smith O. M., Smith J. R. Identification of the active region of the DNA synthesis inhibitory gene p21Sdi1/CIP1/WAF1. EMBO J. 1995 Feb 1;14(3):555–563. doi: 10.1002/j.1460-2075.1995.tb07031.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nishitani H., Nurse P. p65cdc18 plays a major role controlling the initiation of DNA replication in fission yeast. Cell. 1995 Nov 3;83(3):397–405. doi: 10.1016/0092-8674(95)90117-5. [DOI] [PubMed] [Google Scholar]
  28. Podust V. N., Podust L. M., Goubin F., Ducommun B., Hübscher U. Mechanism of inhibition of proliferating cell nuclear antigen-dependent DNA synthesis by the cyclin-dependent kinase inhibitor p21. Biochemistry. 1995 Jul 11;34(27):8869–8875. doi: 10.1021/bi00027a039. [DOI] [PubMed] [Google Scholar]
  29. Polyak K., Lee M. H., Erdjument-Bromage H., Koff A., Roberts J. M., Tempst P., Massagué J. Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell. 1994 Jul 15;78(1):59–66. doi: 10.1016/0092-8674(94)90572-x. [DOI] [PubMed] [Google Scholar]
  30. Prelich G., Kostura M., Marshak D. R., Mathews M. B., Stillman B. The cell-cycle regulated proliferating cell nuclear antigen is required for SV40 DNA replication in vitro. Nature. 1987 Apr 2;326(6112):471–475. doi: 10.1038/326471a0. [DOI] [PubMed] [Google Scholar]
  31. Reagan M. S., Pittenger C., Siede W., Friedberg E. C. Characterization of a mutant strain of Saccharomyces cerevisiae with a deletion of the RAD27 gene, a structural homolog of the RAD2 nucleotide excision repair gene. J Bacteriol. 1995 Jan;177(2):364–371. doi: 10.1128/jb.177.2.364-371.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Shivji M. K., Grey S. J., Strausfeld U. P., Wood R. D., Blow J. J. Cip1 inhibits DNA replication but not PCNA-dependent nucleotide excision-repair. Curr Biol. 1994 Dec 1;4(12):1062–1068. doi: 10.1016/s0960-9822(00)00244-x. [DOI] [PubMed] [Google Scholar]
  33. Stillman B. Initiation of eukaryotic DNA replication in vitro. Annu Rev Cell Biol. 1989;5:197–245. doi: 10.1146/annurev.cb.05.110189.001213. [DOI] [PubMed] [Google Scholar]
  34. Tan C. K., Castillo C., So A. G., Downey K. M. An auxiliary protein for DNA polymerase-delta from fetal calf thymus. J Biol Chem. 1986 Sep 15;261(26):12310–12316. [PubMed] [Google Scholar]
  35. Turchi J. J., Bambara R. A. Completion of mammalian lagging strand DNA replication using purified proteins. J Biol Chem. 1993 Jul 15;268(20):15136–15141. [PubMed] [Google Scholar]
  36. Vallen E. A., Cross F. R. Mutations in RAD27 define a potential link between G1 cyclins and DNA replication. Mol Cell Biol. 1995 Aug;15(8):4291–4302. doi: 10.1128/mcb.15.8.4291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Waga S., Hannon G. J., Beach D., Stillman B. The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. Nature. 1994 Jun 16;369(6481):574–578. doi: 10.1038/369574a0. [DOI] [PubMed] [Google Scholar]
  38. Waga S., Stillman B. Anatomy of a DNA replication fork revealed by reconstitution of SV40 DNA replication in vitro. Nature. 1994 May 19;369(6477):207–212. doi: 10.1038/369207a0. [DOI] [PubMed] [Google Scholar]
  39. Warbrick E., Lane D. P., Glover D. M., Cox L. S. A small peptide inhibitor of DNA replication defines the site of interaction between the cyclin-dependent kinase inhibitor p21WAF1 and proliferating cell nuclear antigen. Curr Biol. 1995 Mar 1;5(3):275–282. doi: 10.1016/s0960-9822(95)00058-3. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. el-Deiry W. S., Harper J. W., O'Connor P. M., Velculescu V. E., Canman C. E., Jackman J., Pietenpol J. A., Burrell M., Hill D. E., Wang Y. WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res. 1994 Mar 1;54(5):1169–1174. [PubMed] [Google Scholar]

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