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p21waf1/cip1-null human fibroblasts are deficient in nucleotide excision repair downstream the recruitment of PCNA to DNA repair sites

Oncogene volume 20, pages 563–570 (2001)Cite this article

Abstract

The cyclin-dependent kinase inhibitor p21waf1/cip1 is known to impair DNA synthesis by binding to PCNA, the co-factor of DNA polymerases δ and ε. 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.

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Acknowledgements

The authors wish to thank Prof J Sedivy (Brown University, Providence, RI, USA) for providing parental and p21-targeted subclones, and helpful suggestions for culture growth. Thanks are also given to Dr AA Vink, TNO, Netherlands for providing H3 monoclonal antibody, and to R Melli for figure preparation.

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  1. Lucia A Stivala and Federica Riva: LA Stivala and F Riva contributed equally to this work

Authors and Affiliations

  1. Dipartimento di Medicina Sperimentale, sez. Patologia generale, Università, di Pavia, Italy

    Lucia A Stivala, Ornella Cazzalini & Monica Savio

  2. Centro di Studio per l'Istochimica del CNR, Piazza Botta 10, Pavia, 27100, Italy

    Federica Riva & Ennio Prosperi

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  1. Lucia A Stivala
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Stivala, L., Riva, F., Cazzalini, O. et al. p21waf1/cip1-null human fibroblasts are deficient in nucleotide excision repair downstream the recruitment of PCNA to DNA repair sites. Oncogene 20, 563–570 (2001). https://doi.org/10.1038/sj.onc.1204132

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