p21(waf1/cip1)-null human fibroblasts are deficient in nucleotide excisionrepair downstream the recruitment of PCNA to DNA repair sites

The cyclin-dependent kinase inhibitor p21(waf1/cip1) is known to impair DNA synthesis by binding to PCNA, the cofactor of DNA polymerases delta and ep silon. However, a positive role for p21 in nucleotide excision repair (NER) has been suggested. In this study, the sensitivity to DNA damage and DNA r epair efficiency were investigated in p21-null human fibroblasts obtained b y targeted homologous recombination. After UV-C irradiation, p21(sic) cells showed a threefold reduction in clonogenic survival and an increased susce ptibility to apoptosis, as compared with parental p21(+/+) cells. Removal o f cyclobutane pyrimidine dimers was significantly reduced in p21(-/-) cells both in the whole genome, and at the level of the rDNA gene cluster, as de termined by immunoassay and Southern blot, respectively. After DNA damage, the recruitment of PCNA as detergent-insoluble form associated to DNA repai r sites in p21(sic) fibroblasts, was comparable to that observed in parenta l p21(+/+) cells, However, PCNA remained associated with DNA for a longer p eriod in p21(sic) than in p21(+/+) cells, These results suggest that in hum an cells, p21 is required for NER at a step located downstream the recruitm ent of PCNA to DNA repair sites.