Human proliferating cell nuclear antigen, poly(ADP-ribose) polymerase-1, and p21waf1/cip1. A dynamic exchange of partners.
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 delta 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.
Frouin
Isabelle
I
Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, Via Abbiategrasso 207, 27100 Pavia, Italy.
Maga
Giovanni
G
Denegri
Marco
M
Riva
Federica
F
Savio
Monica
M
Spadari
Silvio
S
Prosperi
Ennio
E
Scovassi
A Ivana
AI
D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism*; D004258:DNA Polymerase III / Q000037:antagonists & inhibitors; D004260:DNA Repair; D004261:DNA Replication; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000302:isolation & purification / Q000378:metabolism*; D011233:Precipitin Tests; D018809:Proliferating Cell Nuclear Antigen / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D018809:Proliferating Cell Nuclear Antigen; D011994:Recombinant Proteins; D008769:Methylnitronitrosoguanidine; D011065:Poly(ADP-ribose) Polymerases; D004258:DNA Polymerase III