Evidence of the role of PARP-1 - p21 complex in DNA repair

The active involvement of PARP-1 in DNA repair is mediated by the ability not only to recognize DNA damage but also to interact with several DNA repair factors, including p21 CDKN1A, a cyclin-dependent kinase inhibitor that plays important roles in DNA damage response by inducing cell cycle arrest, by regulating the apoptotic pathway, and by participating in various DNA repair processes. We have previously shown that p21 binds to the automodification/DNA-binding domain of PARP-1, particularly after DNA damage induced by alkylating agents (1). High levels of poly(ADP-ribosylation) were found in p21-/- human fibroblasts, as compared with parental p21+/+ cells. In addition, this increased activity was accompanied by a persistent binding of PARP-1 to DNA damage sites, thereby promoting a persistent interaction of PARP-1 with XRCC1 and DNA polymerase ?. These results suggested that p21 may regulate PARP-1 activity, although the function of this regulation has been not yet clarified. To study the possible role of p21 as regulator of PARP-1 activity, we have used purified PARP-1, purified BER factors, and synthetic DNA intermediates of BER together with photoaffinity labeling techniques. In this assay, GST-tagged full length p21 protein, but not a C-terminal peptide, was found to stimulate PARP-1 binding to DNA duplex simulating a BER substrate. To investigate whether the interaction between p21 and PARP-1 is mediated by the poly(ADP-ribose) (PAR), we carried on immunoprecipitation experiments with an antibody to PAR in extracts from cells treated with MNNG. The results showed that the association between p21 and PARP-1 may be mediated by PAR because both proteins were detected in the immunoprecipitate, thus further supporting the role of p21 as regulator of PARP-1 activity.