Assessing Cell Cycle Independent Function of the CDK Inhibitor p21(CDKN¹A) in DNA Repair.

The cyclin-dependent kinase (CDK) inhibitor p21(CDKN1A) 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.