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Analysis of p21CDKN1A Recruitment to DNA Excision Repair Foci in the UV-Induced DNA Damage Response

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 281)

Abstract

The cyclin-dependent kinase (CDK) inhibitor p21CDKN1A (also known as p21waf1/cip1) is a well known player of the G1 and G2 phase cell cycle checkpoints, which are activated in response to DNA damage. In addition, p21 interacts directly with proliferating cell nuclear antigen (PCNA), thereby inhibiting DNA replication. More controversial is the role of p21 in DNA repair, since both inhibition of and requirement for nucleotide excision repair have been suggested. Since the DNA repair process occurs at discrete nuclear foci in a chromatin-bound compartment, a suitable extraction procedure is necessary to investigate the association of p21 with PCNA in these structures. This chapter focuses on biochemical and immunofluorescence methods to analyze the recruitment of p21 protein to DNA repair foci. Cellular fractionation and subsequent nuclear extraction procedures are described for Western blot analysis of p21 recruitment, as well as for protein-protein interaction studies. An in situ extraction protocol is also described for immunofluorescence microscopy and flow cytometric analyses of nuclear localization and cell cycle distribution of p21 recruited to DNA repair foci. The combination of these methodologies is extremely powerful to investigate in more detail the role of p21 in the UV-induced DNA damage response.

Key Words

p21CDKN1A p21waf1/cip1 DNA repair PCNA DNA damage UV-C radiation cyclin-dependent kinase inhibitor protein recruitment chromatin-bound proteins DNase I 
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References

  1. 1.
    Gartel, A. L. and Tyner, A. L. (1999) Transcriptional regulation of the p21(waf1/ cip1) gene. Exp. Cell Res. 246, 280–289.PubMedCrossRefGoogle Scholar
  2. 2.
    Dotto, G. P. (2000) p21(WAF1/Cip1): more than a break to the cell cycle? Biochem. Biophys. Acta 1471, M43–M56.PubMedGoogle Scholar
  3. 3.
    Coqueret, O. (2003) New roles for, p21 and p27 cell-cycle inhibitors: a function for each cell compartment? Trends Cell Biol. 13, 65–70.PubMedCrossRefGoogle Scholar
  4. 4.
    Waga, S., Hannon, G. J., Beach, D., and Stillman, B. (1994) The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. Nature 369, 574–578.PubMedCrossRefGoogle Scholar
  5. 5.
    Flores-Rozas, H., Kelman, Z., Dean, F. B., et al. (1994) Cdk-interacting protein 1 directly binds with proliferating cell nuclear antigen and inhibits DNA replication catalyzed by the DNA polymerase δ holoenzyme. Proc. Natl. Acad. Sci. USA 91, 8655–8659.PubMedCrossRefGoogle Scholar
  6. 6.
    Jónsson, Z. O. and Hübscher, U. (1997) Proliferating cell nuclear antigen: more than a clamp for DNA polymerases. BioEssays 19, 967–975.PubMedCrossRefGoogle Scholar
  7. 7.
    Cox, L. S. (1997) Who binds wins: competition for PCNA rings out cell-cycle changes. Trends Cell Biol. 7, 493–498.PubMedCrossRefGoogle Scholar
  8. 8.
    Prosperi, E. (1997) Multiple roles of proliferating cell nuclear antigen: DNA replication, repair and cell cycle control. In Progress in Cell Cycle Research (Meijer, L., Guidet S., and Philippe, M., eds), vol. 3, Plenum, New York, NY, pp. 193–210.Google Scholar
  9. 9.
    Pan, Z. Q., Reardon, J. T., Li, L., Flores-Rozas, H., Legerski, R., Sancar, A., et al. (1995) Inhibition of nucleotide excision repair by the cyclin-dependent kinase inhibitor p21. J. Biol. Chem. 270, 22,008–22,016.PubMedCrossRefGoogle Scholar
  10. 10.
    Podust, V. N, Podust, L. M., Goubin, F., Ducommun, B., and Hübscher, U. (1995) Mechanism of inhibition of proliferating cell nuclear antigen-dependent DNA synthesis by the cyclin-dependent kinase inhibitor p21. Biochemistry 34, 8869–8875.PubMedCrossRefGoogle Scholar
  11. 11.
    Cooper, M. P., Balajee, A. S., and Bohr, V. A. (1999) The C-terminal domain of p21 inhibits nucleotide excision repair in vitro and in vivo. Mol. Biol. Cell 10, 2119–2129.PubMedGoogle Scholar
  12. 12.
    Smith, M. L., Ford, J. M., Hollander, C, et al. (2000) p53-mediated DNA repair responses to UV radiation: studies of mouse cells lacking p53,p21, and/or gadd45 genes. Mol. Cell Biol. 20, 3705–3714.PubMedCrossRefGoogle Scholar
  13. 13.
    Adimoolam, S., Lin, C. X., and Ford, J. M. (2001) The p53-regulated cyclin-dependent kinase inhibitor, p21 (cipl, wafl, sdil), is not required for global genomic and transcription-coupled nucleotide excision repair of UV-induced DNA photoproducts. J. Biol. Chem. 276, 25,813–25,822.PubMedCrossRefGoogle Scholar
  14. 14.
    Li, R., Waga, S., Hannon, G. J., Beach, D., and Stillman, B. (1994) Differential effects by the p21 CDK inhibitor on PCNA-dependent DNA replication and repair. Nature 371, 534–537.PubMedCrossRefGoogle Scholar
  15. 15.
    Shivji, M. K. K., Gray, S. J., Strausfeld, U. P., Wood, R. D., and Blow, J. J. (1994) Cip1 inhibits DNA replication but not PCNA-dependent nucleotide excision repair. Curr. Biol. 4, 1062–1068.PubMedCrossRefGoogle Scholar
  16. 16.
    Shivji, M. K. K., Ferrari, E., Ball, K., Hübscher, U., and Wood, R. D. (1998) Resistance of human nucleotide excision repair synthesis in vitro to p21 (Cdn1). Oncogene 17, 2827–2838.PubMedCrossRefGoogle Scholar
  17. 17.
    McDonald III, E. R., Wu, G. S., Waldman, T., and El-Deiry, W. S. (1996) Repair defect in p21waf1/cip1 −/− human cancer cells. Cancer Res. 56, 2250–2255.PubMedGoogle Scholar
  18. 18.
    Sheikh, M. S., Chen, Y. Q., Smith, M. L., and Fornace, A. J., Jr. (1997) Role of p21waf1/cip1/sdi1 in cell death and dna repair as studied using a tetracycline-inducible system in p53-deficient cells. Oncogene 14, 1875–1882.PubMedCrossRefGoogle Scholar
  19. 19.
    Stivala, L. A., Riva, F., Cazzalini, O., Savio, M., and Prosperi, E. (2001) p21waf1/cip1-null human fibroblasts are deficient in nucleotide excision repair downstream the recruitment of PCNA to DNA repair sites. Oncogene 20, 563–570.PubMedCrossRefGoogle Scholar
  20. 20.
    Li, R., Hannon, G. J., Beach, D., and Stillman, B. (1996) Subcellular distribution of p21 and PCNA in normal and repair-deficient cells following DNA damage. Curr. Biol. 6, 189–199.PubMedCrossRefGoogle Scholar
  21. 21.
    Savio, M., Stivala, L. A., Scovassi, A. I., Bianchi, L., and Prosperi, E. (1996) p21waf1/cip1 protein associates with the detergent-insoluble form of PCNA concomitantly with disassembly of PCNA at nucleotide repair sites. Oncogene 13, 1591–1598.PubMedGoogle Scholar
  22. 22.
    Krishna, T. S. R., Kong, X.-P., Gary, S., Burgers, P. M., and Kuriyan, J. (1994) Crystal structure of the eukaryotic DNA polymerase processivity factor PCNA. Cell 79, 1233–1243.PubMedCrossRefGoogle Scholar
  23. 23.
    Bravo, R. and Macdonald-Bravo, H. (1987) Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites. J. Cell Biol. 105, 1549–1554.PubMedCrossRefGoogle Scholar
  24. 24.
    Toschi, L. and Bravo, R. (1988) Changes in cyclin/proliferating cell nuclear antigen distribution during DNA repair synthesis. J. Cell Biol. 107, 1623–1628.PubMedCrossRefGoogle Scholar
  25. 25.
    Pagano, M., Theodoras, A. M., Tam, S. W., and Draetta, G. (1994) Cyclin D1-mediated inhibition of repair and replicative DNA synthesis in human fibroblasts. Genes Dev. 8, 1627–1639.PubMedCrossRefGoogle Scholar
  26. 26.
    Savio, M., Stivala, L. A., Bianchi, L., Vannini, V., and Prosperi, E. (1998) Involvement of the proliferating cell nuclear antigen (PCNA) in DNA repair induced by alkylating agents and oxidative damage in human fibroblasts. Carcinogenesis 19, 591–596.PubMedCrossRefGoogle Scholar
  27. 27.
    Volker, M., Moné, M. J., Karmakar, P., et al. (2001) Sequential assembly of the nucleotide excision repair factors in vivo. Mol. Cell 8, 213–224.PubMedCrossRefGoogle Scholar
  28. 28.
    Riva, F., Zuco, V., Supino, R., Vink, A. A., and Prosperi E. (2001) UV-induced DNA incision and proliferating cell nuclear antigen recruitment to repair sites occur independently of p53-replication protein A interaction in p53 wild type and mutant ovarian carcinoma cells. Carcinogenesis 22, 1971–1978.PubMedCrossRefGoogle Scholar
  29. 29.
    Sadaie, M. R., and Mathews, M. B. (1986) Immunochemical and biochemical analysis of the proliferating cell nuclear antigen (PCNA) in HeLa cells. Exp. Cell Res. 163, 423–433.PubMedCrossRefGoogle Scholar
  30. 30.
    Prosperi, E., Stivala, L. A., Sala, E., Scovassi, A. I., and Bianchi, L. (1993) Proliferating cell nuclear antigen complex-formation induced by ultraviolet irradiation in human quiescent fibroblasts as detected by immunostaining and flow cytometry. Exp. Cell Res. 205, 320–325.PubMedCrossRefGoogle Scholar
  31. 31.
    Stivala, L. A., Prosperi, E., Rossi, R., and Bianchi, L. (1993) Involvement of proliferating cell nuclear antigen in DNA repair after damage induced by genotoxic agents in human fibroblasts. Carcinogenesis 14, 2569–2573.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  1. 1.Dipartimento di Medicina Sperimentale, sez. Patologia Generale “C. Golgi”Università di PaviaPaviaItaly
  2. 2.Istituto di Genetica Molecolare del CNRsez. Istochimica e CitometriaPaviaItaly

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