p21(waf1/cip1)-null human fibroblasts are deficient in nucleotide excision repair 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 co-factor of DNA polymerases delta and epsilon. 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 efficiency were investigated in p21-null human fibroblasts obtained by targeted homologous recombination. After UV-C irradiation, p21-/- 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 significantly reduced in p21-/- 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-/- fibroblasts, was comparable to that observed in parental p21+/+ cells. However, PCNA remained associated with DNA for a longer period in p21-/- 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.
Stivala
L A
LA
Dipartimento di Medicina Sperimentale, sez. Patologia generale, Università di Pavia, Italy.
Riva
F
F
Cazzalini
O
O
Savio
M
M
Prosperi
E
E
D017209:Apoptosis / Q000502:physiology / Q000528:radiation effects; D002453:Cell Cycle / Q000502:physiology / Q000528:radiation effects; D002460:Cell Line; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000172:deficiency / Q000502:physiology*; D004260:DNA Repair / Q000502:physiology*; D004275:DNA, Ribosomal / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology / Q000528:radiation effects; D006801:Humans; D008168:Lung / Q000166:cytology / Q000528:radiation effects; D005810:Multigene Family; D018809:Proliferating Cell Nuclear Antigen / Q000502:physiology*; D011836:Radiation Tolerance / Q000502:physiology; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism / Q000502:physiology; D014466:Ultraviolet Rays
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004275:DNA, Ribosomal; D018809:Proliferating Cell Nuclear Antigen; D016159:Tumor Suppressor Protein p53