Studio del ruolo dell' interazione p21-PCNA nel processo di riparazione del DNA

The cyclin-dependent kinase inhibitor p21CDK1NA is a protein involved in various cellular processes, such as cell cycle arrest, transcription regulation, apoptosis and cell motility. p21 was discovered to actively participate also to DNA repair process, such as nucleotide excision repair (NER), through the interaction with Proliferative Cell Nuclear Antigen (PCNA). For this study, we used plasmids driving the expression of three different mutated form of p21 with a fluorescent tag (GFP) fused to their C-term: p21DD-GFP (T148D), p21AAA-GFP (KRR154-156AAA) and p212KQ-GFP (K161,163Q). These mutants, reported in literature to be resistant to degradation, have been used to test whether the PCNA-interacting partners dynamics at the DNA damage sites (e.g., the recruitment and subsequent release from the repair site) is influenced by the p21 susceptibility to degradation, while the interaction with PCNA is conserved. We initially performed fluorescence microscopy analysis and immunoprecipitation assays on transfected HeLa cells to characterize the p21-GFP mutants. The results showed that, although both p21DD-GFP and p212KQ-GFP mutants have a clear nuclear localization, unexpectedly p21AAA-GFP did not interact with PCNA inhibiting its recruitment to DNA damage sites. Subsequently, we confirmed the enhanced stability of p21AAA and p212KQ mutants, while p21DD showed a poor resistance to UVC-induced degradation. To test whether p21 could influence the PCNA-interacting partners turnover, we compared the persistence of p212KQ with p21WT at the DNA damage sites performing an immunofluorescence assay on HeLa cells transfected with HA-p21WT and HA-p212KQ. The results showed a longer retention of DNA repair factors (e.g., DNA polymerase δ) at the DNA lesions in the presence of p21 mutant, suggesting a consequent delay in the DNA repair process. Then, we also performed live cell imaging confocal analyses using HeLa cell line stably expressing mCherry-PCNA co-transfected with two plasmids driving the expression of fluorescent-tagged (miRFP) DNA Ligase 1 and either p21WT-GFP or p212KQ-GFP, respectively. PCNA and DNA Lig 1 release kinetics in the absence p21 and in the presence of p212KQ resulted to be remarkably slower as compared to p21WT. Therefore, the data suggest that not only the persistence of p21 at the DNA damage sites, but even the absence of p21 could deregulate the DNA repair process. Along with these results, a significant reduction of NER efficiency was detected in HeLa cells transfected with p212KQ-GFP plasmid and in the absence of p21. Consistently, we observed a similar reduction in DNA re-synthesis after UV-C irradiation in human fibroblasts lacking p21. In conclusion, these results suggest a possible mechanism through which p21 influences the PCNA partner dynamics by coupling to the degradation process, which is fundamental to finely tune p21 cellular levels.