Human proliferating cell nuclear antigen, poly(ADP-ribose) polymerase-1, and p21waf1/cip1. A dynamic exchange of partners

Isabelle Frouin, Giovanni Maga, Marco Denegri, Federica Riva, Monica Savio, Silvio Spadari, Ennio Prosperi, A. Ivana Scovassi

Research output: Contribution to journalArticle

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Abstract

We addressed the analysis of the physical and functional association of proliferating cell nuclear antigen (PCNA), a protein involved in many DNA transactions, with poly(ADP-ribose) polymerase (PARP-1), an enzyme that plays a crucial role in DNA repair and interacts with many DNA replication/repair factors. We demonstrated that PARP-1 and PCNA co-immunoprecipitated both from the soluble and the DNA-bound fraction isolated from S-phase-synchronized HeLa cells. Immunoprecipitation experiments with purified proteins further confirmed a physical association between PARP-1 and PCNA. To investigate the effect of this association on PARP-1 activity, an assay based on the incorporation of radioactive NAD was performed. Conversely, the effect of PARP-1 on PCNA-dependent DNA synthesis was assessed by a DNA polymerase δ assay. A marked inhibition of both reactions was found. Unexpectedly, PARP-1 activity also decreased in the presence of p21waf1/cip1. By pull-down experiments, we provided the first evidence for an association between PARP-1 and p21, which involves the C-terminal part of p21 protein. This association was further demonstrated to occur also in vivo in MNNG (N-methyl-N′ -nitro-N-nitrosoguanidine)-treated human fibroblasts. These observations suggest that PARP-1 and p21 could cooperate in regulating the functions of PCNA during DNA replication/repair.

Original languageEnglish
Pages (from-to)39265-39268
Number of pages4
JournalJournal of Biological Chemistry
Volume278
Issue number41
DOIs
Publication statusPublished - Oct 10 2003

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Poly(ADP-ribose) Polymerases
Proliferating Cell Nuclear Antigen
Association reactions
DNA Repair
DNA
Repair
DNA Replication
Assays
Methylnitronitrosoguanidine
Proteins
DNA-Directed DNA Polymerase
S Phase
HeLa Cells
Immunoprecipitation
NAD
Fibroblasts
Poly (ADP-Ribose) Polymerase-1
Experiments
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Human proliferating cell nuclear antigen, poly(ADP-ribose) polymerase-1, and p21waf1/cip1. A dynamic exchange of partners. / Frouin, Isabelle; Maga, Giovanni; Denegri, Marco; Riva, Federica; Savio, Monica; Spadari, Silvio; Prosperi, Ennio; Scovassi, A. Ivana.

In: Journal of Biological Chemistry, Vol. 278, No. 41, 10.10.2003, p. 39265-39268.

Research output: Contribution to journalArticle

Frouin, Isabelle ; Maga, Giovanni ; Denegri, Marco ; Riva, Federica ; Savio, Monica ; Spadari, Silvio ; Prosperi, Ennio ; Scovassi, A. Ivana. / Human proliferating cell nuclear antigen, poly(ADP-ribose) polymerase-1, and p21waf1/cip1. A dynamic exchange of partners. In: Journal of Biological Chemistry. 2003 ; Vol. 278, No. 41. pp. 39265-39268.
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