P21CDKN1A Regulates the Binding of Poly(ADP-Ribose) Polymerase-1 to DNA Repair Intermediates.

Ilaria Dutto,Lucia A Stivala,Micol Tillhon,Maria Sukhanova,Olga Lavrik,Ennio Prosperi,A Ivana Scovassi,Ornella Cazzalini,Malka Cohen-Armon

doi:10.1371/journal.pone.0146031

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.

Highlights

To better understand the mechanism by which p21 can influence the base excision repair (BER) process, we have investigated whether the association between p21 and Poly(ADP-ribose) polymerase-1 (PARP-1) is dependent on PARP-1 interaction with damaged DNA and on the subsequent PARP-1 activation leading to PAR polymer synthesis
In order to investigate the possible modulation of the basic reactions of PARP-1 by p21 during DNA damage, in the present study we have carried out in vitro experiments using model DNA substrates mimicking BER intermediates which have been previously demonstrated to interact with PARP-1 in mouse embryonic fibroblast (MEF) extracts [42,53]
We have found that p21 increases the binding of PARP-1 to these BER intermediates, suggesting that p21 could exert a positive modulation of DNA binding by PARP-1 (Fig 1)

Summary

Introduction

Poly(ADP-ribose) polymerase-1 (PARP-1) belongs to the PARP superfamily [1] and is involved in several pathways, such as DNA replication, DNA repair, cell death, transcription, mitochondrial activity regulation [2,3,4,5,6,7,8,9] and modulation of chromatin structure [10]. The importance of this interaction in the BER process was supported by evidence that p21−/− cells were more sensitive to alkylating agents and showed BER deficiency, possibly due to persistent DNA binding of PARP-1 and an abnormally high PAR production [40] These findings suggested that p21 might be necessary to regulate PARP-1 activity for the correct turnover of BER factors at the DNA damaged sites [41]. We show that in vivo PAR synthesis facilitates the interaction of PARP-1 with p21, as Olaparib significantly reduced this association These results provide novel clues for the regulation of the BER process, as they indicate p21 as a new activator of the DNA damage recognition step of PARP-1, and further highlight the role of p21 as a cooperative factor for DNA repair

Materials and Methods

Findings

Discussion