title: p21CDKN1A Regulates the Binding of Poly(ADP-Ribose) Polymerase-1 to DNA Repair Intermediates.
abstract: The cell cycle inhibitor p21CDKN1A was previously found to interact directly with DNA nick-sensor poly(ADP-ribose) polymerase-1 (PARP-1) and to promote base excision repair (BER). However, the molecular mechanism responsible for this BER-related association of p21 with PARP-1 remains to be clarified. In this study we investigate the capability of p21 to influence PARP-1 binding to DNA repair intermediates in a reconstituted BER system in vitro. Using model photoreactive BER substrates containing single-strand breaks, we found that full-length recombinant GST-tagged p21 but not a C-terminal domain truncated form of p21 was able to stimulate the PARP-1 binding to BER intermediates with no significant influence on the catalytic activity of PARP-1. In addition, we investigate whether the activation of PARP-1 through poly(ADP-ribose) (PAR) synthesis, is required for its interaction with p21. We have found that in human fibroblasts and in HeLa cells treated with the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), the interaction of p21 with PARP-1 was greatly dependent on PAR synthesis. In fact, an anti-PAR antibody was able to co-immunoprecipitate p21 and PARP-1 from extracts of MNNG-treated cells, while blocking PAR synthesis with the PARP-1 inhibitor Olaparib, drastically reduced the amount of p21 co-immunoprecipitated by a PARP-1 antibody. Our results provide the first evidence that p21 can stimulate the binding of PARP-1 to DNA repair intermediates, and that this cooperation requires PAR synthesis.
authors.affiliation: Istituto di Genetica Molecolare del CNR, Via Abbiategrasso, 207, Pavia, Italy.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Dutto
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine, Russian Academy of Sciences, Prospekt Lavrentiev 8, Novosibirsk, Russian Federation.
authors.forename: Maria
authors.initials: M
authors.lastname: Sukhanova
authors.affiliation: Istituto di Genetica Molecolare del CNR, Via Abbiategrasso, 207, Pavia, Italy.
authors.forename: Micol
authors.initials: M
authors.lastname: Tillhon
authors.affiliation: Dipartimento di Medicina Molecolare, Immunologia e Patologia, Università di Pavia, Via Ferrata 9, Pavia, Italy.
authors.forename: Ornella
authors.initials: O
authors.lastname: Cazzalini
authors.affiliation: Dipartimento di Medicina Molecolare, Immunologia e Patologia, Università di Pavia, Via Ferrata 9, Pavia, Italy.
authors.forename: Lucia A
authors.initials: LA
authors.lastname: Stivala
authors.affiliation: Istituto di Genetica Molecolare del CNR, Via Abbiategrasso, 207, Pavia, Italy.
authors.forename: A Ivana
authors.initials: AI
authors.lastname: Scovassi
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine, Russian Academy of Sciences, Prospekt Lavrentiev 8, Novosibirsk, Russian Federation.
authors.forename: Olga
authors.initials: O
authors.lastname: Lavrik
authors.affiliation: Istituto di Genetica Molecolare del CNR, Via Abbiategrasso, 207, Pavia, Italy.
authors.forename: Ennio
authors.initials: E
authors.lastname: Prosperi
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism*; D053904:DNA Breaks, Single-Stranded; D004260:DNA Repair; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D009154:Mutation; D010793:Phthalazines / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism; D000067856:Poly(ADP-ribose) Polymerase Inhibitors / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding
chemical_list: C493956:CDKN1A protein, human; D002843:Chromatin; D050759:Cyclin-Dependent Kinase Inhibitor p21; D010793:Phthalazines; D010879:Piperazines; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D008769:Methylnitronitrosoguanidine; D011064:Poly Adenosine Diphosphate Ribose; D011065:Poly(ADP-ribose) Polymerases; C531550:olaparib