Assessing Cell Cycle Independent Function of the CDK Inhibitor p21CDKN1A in DNA Repair

  • Ilaria Dutto
  • Micol Tillhon
  • Ennio ProsperiEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1336)


The cyclin-dependent kinase (CDK) inhibitor p21CDKN1A is a small protein that is able to regulate many important cell functions, often independently of its activity of CDK inhibitor. In addition to cell cycle, this protein regulates cell transcription, apoptosis, cell motility, and DNA repair. In particular, p21 may participate in different DNA repair processes, like the nucleotide excision repair (NER), base excision repair (BER), and double-strand breaks (DSB) repair, because of its ability to interact with DNA repair proteins, such as proliferating cell nuclear antigen (PCNA), a master regulator of many DNA transactions. Although this role has been debated for a long time, the influence of p21 in DNA repair has been now established. However, it remain to be clarified how this role is coupled to proteasomal degradation that has been shown to occur after DNA damage. This chapter describes procedures to study p21 protein recruitment to localized DNA damage sites in the cell nucleus. In particular, we describe a technique based on local irrradiation with UV light through a polycarbonate filter with micropores; an in situ lysis procedure to detect chromatin-bound proteins by immunofluorescence; a cell fractionation procedure to study chromatin association of p21 by Western blot analysis, and p21 protein–protein interactions by an immunoprecipitation assay.

Key words

DNA repair Localized DNA damage p21CDKN1A p21 degradation p21 recruitment PCNA 


  1. 1.
    El-Deiry WS, Tokino T, Velculescu VE et al (1993) WAF1, a potential mediator of p53 tumor suppression. Cell 75:817–825PubMedCrossRefGoogle Scholar
  2. 2.
    Harper JW, Adami G, Wei N et al (1993) The p21 cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin dependent kinases. Cell 75:805–816PubMedCrossRefGoogle Scholar
  3. 3.
    Herbig U, Sedivy JM (2006) Regulation of growth arrest in senescence: telomere damage is not the end of the story. Mech Ageing Dev 127:16–24PubMedCrossRefGoogle Scholar
  4. 4.
    Perucca P, Cazzalini O, Madine M et al (2009) Loss of p21 CDKN1A impairs entry to quiescence and activates a DNA damage response in normal fibroblasts induced to quiescence. Cell Cycle 8:105–114PubMedCrossRefGoogle Scholar
  5. 5.
    Stivala A, Cazzalini O, Prosperi E (2012) The cyclin-dependent kinase inhibitor p21CDKN1A as a target of anti-cancer drugs. Curr Cancer Drug Targets 12:85–96PubMedCrossRefGoogle Scholar
  6. 6.
    Dotto GP (2000) p21(WAF1/Cip1): more than a break to cell cycle? Biochim Biophys Acta 1471:43–56Google Scholar
  7. 7.
    Coqueret O (2003) New roles for p21 and p27 cell-cycle inhibitors: a function for each cell compartment? Trends Cell Biol 13:65–70PubMedCrossRefGoogle Scholar
  8. 8.
    Abbas T, Dutta A (2009) p21 in cancer, intricate networks and multiple activities. Nat Rev Cancer 9:400–414PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Cazzalini O, Scovassi AI, Savio M et al (2010) Multiple roles of the cell cycle inhibitor p21CDKN1A in the DNA damage response. Mutat Res Rev Mutat 704:12–20CrossRefGoogle Scholar
  10. 10.
    Warbrick E (2000) The puzzle of PCNA’s many partners. Bioessays 22:997–1006PubMedCrossRefGoogle Scholar
  11. 11.
    Prosperi E (2006) The fellowship of the rings, distinct pools of proliferating cell nuclear antigen (PCNA) trimer at work. FASEB J 20:833–837PubMedCrossRefGoogle Scholar
  12. 12.
    Moldovan GL, Pfander B, Jentsch S (2007) PCNA, the maestro of replication fork. Cell 129:665–679PubMedCrossRefGoogle Scholar
  13. 13.
    Oku T, Ikeda S, Sasaki H et al (1998) Functional sites of human PCNA which interact with p21(Cip1/Waf1), DNA polymerase delta and replication factor C. Genes Cells 3:357–369PubMedCrossRefGoogle Scholar
  14. 14.
    Cazzalini O, Perucca P, Riva F et al (2003) p21CDKN1A does not interfere with loading of PCNA at DNA replication sites, but inhibits subsequent binding of DNA polymerase delta at the G1/S phase transition. Cell Cycle 2:596–603PubMedCrossRefGoogle Scholar
  15. 15.
    Soria G, Gottifredi V (2010) PCNA-coupled p21 degradation after DNA damage: the exception that confirms the rule? DNA Repair 9:358–364PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Perkins ND (2002) Not just a Cdk inhibitor: regulation of transcription of p21WAF1/CIP1/SDI1. Cell Cycle 1:39–41PubMedCrossRefGoogle Scholar
  17. 17.
    Gartel AL (2005) The conflicting roles of the cdk inhibitor p21CIP1/WAF1 in apoptosis. Leuk Res 29:1237–1238PubMedCrossRefGoogle Scholar
  18. 18.
    Tillhon M, Cazzalini O, Stivala A et al (2010) Involvement of the cell cycle inhibitor p21CDKN1A in DNA repair processes. In: Thomas AE (ed) DNA damage repair, repair mechanisms and aging. Nova Science, Hauppauge, NY, pp 123–140Google Scholar
  19. 19.
    Bendjennat M, Boulaire J, Jascur T et al (2003) UV irradiation triggers ubiquitin-dependent degradation of p21WAF1 to promote DNA repair. Cell 114:599–610PubMedCrossRefGoogle Scholar
  20. 20.
    Stivala LA, Riva F, Cazzalini O et al (2001) p21(waf1/cip1)-null human fibroblasts are deficient in nucleotide excision repair downstream the recruitment of PCNA to DNA repair sites. Oncogene 20:563–570PubMedCrossRefGoogle Scholar
  21. 21.
    Perucca P, Cazzalini O, Mortusewicz O et al (2006) Spatiotemporal dynamics of p21CDKN1A protein recruitment to DNA-damage sites and interaction with proliferating cellular nuclear antigen. J Cell Sci 119:1517–1527PubMedCrossRefGoogle Scholar
  22. 22.
    Soria G, Speroni J, Podhajcer O et al (2008) p21 differentially regulates DNA replication and DNA repair associated processes after UV-irradiation. J Cell Sci 121:3271–3282PubMedCrossRefGoogle Scholar
  23. 23.
    Cazzalini O, Perucca P, Savio M et al (2008) Interaction of p21(CDKN1A) with PCNA regulates the histon acetyltransferase activity of p300 in nucleotide excision repair. Nucleic Acids Res 36:1713–1722PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Rubbi CP, Milner J (2003) p53 is a chromatin accessibility factor for nucleotide excision repair of DNA damage. EMBO J 22:975–986PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Hong R, Chakravarti D (2003) The human proliferating cell nuclear antigen regulates transcriptional coactivator p300 activity and promotes transcriptional repression. J Biol Chem 278:44505–44513PubMedCrossRefGoogle Scholar
  26. 26.
    Avkin S, Sevilya Z, Toube L et al (2006) p53 and p21 regulate error-prone DNA repair to yield a lower mutation load. Mol Cell 22:407–413PubMedCrossRefGoogle Scholar
  27. 27.
    Frouin I, Maga G, Denegri M et al (2003) Human proliferating cell nuclear antigen, poly(ADP-ribose) polymerase 1, and p21 Waf1/Cip1. A dynamic exchange of partners. J Biol Chem 278:39265–39268PubMedCrossRefGoogle Scholar
  28. 28.
    Cazzalini O, Donà F, Savio M et al (2010) p21CDKN1A participates in base excision repair by regulating the activity of poly(ADP-ribose) polymerase-1. DNA Repair 9:627–635PubMedCrossRefGoogle Scholar
  29. 29.
    Koike M, Yutoku Y, Koike A (2011) Accumulation of p21 proteins at DNA damage sites independent of p53 and core NHEJ factors following irradiation. Biochem Biophys Res Commun 412:39–43PubMedCrossRefGoogle Scholar
  30. 30.
    Wiese C, Rudolph JK, Jakob B et al (2012) PCNA-dependent accumulation of CDKN1A into nuclear foci after ionizing irradiation. DNA Repair 11:511–521PubMedCrossRefGoogle Scholar
  31. 31.
    Mauro M, Rego MA, Boisvert RA, Esashi F, Cavallo F, Jasin M, Howlett NG (2012) p21 promotes error-free replication-coupled DNA double-strand break repair. Nucleic Acids Res 40:8348–8360Google Scholar
  32. 32.
    Dutto I, Tillhon M, Cazzalini O, Stivala LA, Prosperi E (2015) Biology of the cell cycle inhibitor p21CDKN1A: molecular mechanisms and relevance in chemical toxicology. Arch Toxicol 89:155–178Google Scholar
  33. 33.
    Havens CG, Walter JC (2009) Docking of a specialized PIP Box onto chromatin-bound PCNA creates a degron for the ubiquitin ligase CRL4Cdt2. Mol Cell 35:93–104PubMedCentralPubMedCrossRefGoogle Scholar
  34. 34.
    Abbas T, Sivaprasad U, Terai K et al (2008) PCNA-dependent regulation of p21 ubiquitylation and degradation via CRL4Cdt2 ubiquitin ligase complex. Genes Dev 22:2496–2506PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    Nishitani H, Shiomi Y, Iida H et al (2008) CDK inhibitor p21 is degraded by a PCNA coupled Cul4-DDB1CDT2 pathways during S phase and after UV irradiation. J Biol Chem 283:29045–29052PubMedCentralPubMedCrossRefGoogle Scholar
  36. 36.
    Tillhon M, Cazzalini O, Nardo T, Necchi D, Sommatis S, Stivala LA, Scovassi AI, Prosperi E (2012) p300/CBP acetyltransferases interact with and acetylate the nucleotide excision repair factor XPG. DNA Repair 11:844–852Google Scholar
  37. 37.
    Lukas J, Lukas C, Bartek J (2011) More than just a focus: the chromatin response to DNA damage and its role in genome integrity maintenance. Nat Cell Biol 13:1161–1169PubMedCrossRefGoogle Scholar
  38. 38.
    Katsumi S, Kobayashi N, Imoto K et al (2001) In situ visualization of ultraviolet-light-induced DNA damage repair in locally irradiated human fibroblasts. J Invest Dermatol 117:1156–1161PubMedCrossRefGoogle Scholar
  39. 39.
    Volker M, Monè MJ, Karmakar P et al (2001) Sequential assembly of nucleotide excision repair factors in vivo. Mol Cell 8:213–224PubMedCrossRefGoogle Scholar
  40. 40.
    Vermeulen W (2011) Dynamics of mammalian NER proteins. DNA Repair 10:760–771PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2016

Authors and Affiliations

  1. 1.Genome Stability Group, Department of Biology and Biotechnology, Istituto di Genetica Molecolare del CNRUniversity of PaviaPaviaItaly

Personalised recommendations