The cyclin-dependent kinase (CDK) inhibitor p21CDKN1A (also known as p21(waf1/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.
Analysis of p21CDKN1A recruitment to DNA excision repair foci in the UV-induced DNA damage response
STIVALA, LUCIA ANNA;
2004-01-01
Abstract
The cyclin-dependent kinase (CDK) inhibitor p21CDKN1A (also known as p21(waf1/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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
