p21 waf1/cip1 -null human ®broblasts are de®cient in nucleotide excision repair downstream the recruitment of PCNA to DNA repair sites Lucia A Stivala 1,3 , Federica Riva 2,3 , Ornella Cazzalini 1 , Monica Savio 1 and Ennio Prosperi* ,2 1 Dipartimento di Medicina Sperimentale, sez. Patologia generale, Universita Á di Pavia, Italy; 2 Centro di Studio per l'Istochimica del CNR, Piazza Botta 10, 27100 Pavia, Italy The cyclin-dependent kinase inhibitor p21 waf1/cip1 is known to impair DNA synthesis by binding to PCNA, the co- factor of DNA polymerases d and e. However, a positive role for p21 in nucleotide excision repair (NER) has been suggested. In this study, the sensitivity to DNA damage and DNA repair eciency were investigated in p21-null human ®broblasts obtained by targeted homologous recombination. After UV-C irradiation, p21 7/7 cells showed a threefold reduction in clonogenic survival and an increased susceptibility to apoptosis, as compared with parental p21 +/+ cells. Removal of cyclobutane pyrimidine dimers was signi®cantly reduced in p21 7/7 cells both in the whole genome, and at the level of the rDNA gene cluster, as determined by immunoassay and Southern blot, respectively. After DNA damage, the recruitment of PCNA as detergent-insoluble form associated to DNA repair sites in p21 7/7 ®broblasts, was comparable to that observed in parental p21 +/+ cells. However, PCNA remained associated with DNA for a longer period in p21 7/7 than in p21 +/+ cells. These results suggest that in human cells, p21 is required for NER at a step located downstream the recruitment of PCNA to DNA repair sites. Oncogene (2001) 20, 563 ± 570. Keywords: p21 waf1/cip1 ; nucleotide excision repair; PCNA; p21-null ®broblasts Introduction The cyclin-dependent kinase (CDK) inhibitor p21 waf1/cip1 is an important eector of cell cycle arrest in response to DNA damage (Hartwell and Kastan, 1994). This response is thought to be achieved through two distinct mechanisms: (i) CDK inhibition (Sherr and Roberts, 1999); and (ii) binding to the proliferating cell nuclear antigen (PCNA) (Goubin and Ducommun, 1995; Luo et al., 1995; Nakanishi et al., 1995; Fotedar et al., 1996; Chen et al., 1996b), the co-factor of DNA polymerases d and e (JoÂnsson and HuÈbscher, 1997). The latter interaction results in DNA synthesis inhibition (Flores- Rozas et al., 1994; Waga et al., 1994) by impeding the association of PCNA with DNA polymerase (Podust et al., 1995), FEN1 (Chen et al., 1996a; Warbrick et al., 1997), or DNA ligase I (Levin et al., 1997). Since PCNA is required for nucleotide excision repair (NER), and other DNA repair pathways (Shivji et al., 1992; Nichols and Sancar, 1992; Wood, 1996; Wood and Shivji, 1997), it was suggested that p21 may inhibit NER in certain conditions in vitro (Pan et al., 1995), and in electro- porated cells (Cooper et al., 1999). However, other studies reported that NER synthesis is resistant to p21 in vitro (Li et al., 1994; Shivji et al., 1994, 1998) and in vivo co-localization and interaction of p21 with PCNA were detected at later times after UV irradiation (Li et al., 1996; Savio et al., 1996). Recently, the cancer cell lines HCT116 (in which p21 gene was deleted by homologous recombination), and DLD1 (p53-de®cient) were found more sensitive to UV, and showed a reduced NER eciency (McDonald et al., 1996; Sheikh et al., 1997). Expression of wild type p21 protein, or a C- terminal peptide, but not a mutant form lacking the PCNA-binding domain, restored DNA repair in p21 7/7 HCT116 cells (McDonald et al., 1996), thus implying that p21/PCNA interaction was important for NER. Overexpression of p21 also enhanced DNA repair in BCNU and cis-platin damaged glioblastoma cells (Ruan et al., 1998). In contrast, DNA repair was minimally aected in p21 7/7 murine ®broblasts, while loss of both gadd45 and p21 genes resulted in an additive eect in NER de®ciency (Smith et al., 2000). Thus the involvement of p21 in NER is still controversial. In this study, NER eciency was assessed in human ®broblasts in which p21 gene was deleted by targeted homologous recombination (Brown et al., 1997). These cells were chosen because of the intrinsically higher repair eciency of human, as compared with rodent cells (Mitchell and Hartman, 1990). In addition, being derived from normal ®broblasts, they provide a more controlled genetic background than that of p21-de®cient tumor cells (McDonald et al., 1996; Fan et al., 1997). Loss of p21 was found to induce an increase in UV sensitivity, together with a reduction in NER capacity. Results p21 7/7 fibroblasts are more sensitive to UV damage than p21 +/+ parental cells The sensitivity of p21 7/7 ®broblasts to UV-induced DNA damage was determined by a clonogenic assay, Oncogene (2001) 20, 563 ± 570 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc *Correspondence: E Prosperi 3 The ®rst two authors contributed equally to this work Received 15 August 2000; revised 7 November 2000; accepted 23 November 2000