DNA Repair 8 (2009) 778–785 Contents lists available at ScienceDirect DNA Repair journal homepage: www.elsevier.com/locate/dnarepair Degradation of p21 CDKN1A after DNA damage is independent of type of lesion, and is not required for DNA repair Monica Savio a , Tania Coppa a , Ornella Cazzalini a , Paola Perucca a , Daniela Necchi a,b , Tiziana Nardo c , Lucia A. Stivala a , Ennio Prosperi c,b,∗ a Dipartimento di Medicina Sperimentale, sez. Patologia Generale “C. Golgi”, Università di Pavia, Pavia, Italy b Dipartimento di Biologia Animale, Università di Pavia, Pavia, Italy c Istituto di Genetica Molecolare del CNR (IGM-CNR), 27100 Pavia, Italy article info Article history: Received 23 December 2008 Received in revised form 3 February 2009 Accepted 20 February 2009 Available online 24 March 2009 Keywords: p21 Degradation PCNA interaction DNA repair DNA damage abstract The inhibitor of cyclin-dependent kinases p21 CDKN1A plays a fundamental role in several pathways involved in the DNA damage response, like checkpoint-mediated cell cycle arrest, transcription, apoptosis, and DNA repair. Although p21 protein level is regulated by proteasomal degradation, the relationship of this process with DNA repair pathways is not yet clear. In addition, the role of protein/protein interaction in regulating turnover of p21 protein, is controversial. Here, we show that in human fibroblasts treated with agents inducing lesions repaired through nucleotide excision repair (NER), or base excision repair (BER), p21 degradation was triggered more by the extent, than by the type of DNA damage, or consequent DNA repair pathway. In fact, lowering the amount of DNA damage resulted in an increased stability of p21 protein. Overexpression of p21 was found to obscure degradation, both for p21 wt and a p21 mutant unable to bind PCNA (p21 PCNA- ). However, when expressed to lower levels, turnover of p21 protein after DNA damage was greatly influenced by interaction with PCNA, since p21 PCNA- was more efficiently degraded than wild-type protein. Interestingly, a p21 mutant protein unable to localize in the nucleus because of mutations in the NLS region, was not degraded after DNA damage, thus indicating that nuclear localization is necessary for p21 turnover. Removal of p21 was not required for NER activity, since inhibition of p21 degradation by caffeine did not affect the UV-induced recruitment of repair proteins, such as PCNA and DNA polymerase , nor significantly influence DNA repair synthesis, as determined by autoradiography. These results indicate that degradation of p21 is not dependent on a particular DNA repair pathway, and is not required for efficient DNA repair. © 2009 Elsevier B.V. All rights reserved. 1. Introduction The cyclin-dependent kinase (CDK) inhibitor p21 CDKN1A plays a significant role in several aspects of the DNA damage response. In fact, p21 protein is an important regulator of the G1 phase cell cycle checkpoint, both by inducing cell cycle arrest through CDK inhibi- tion, and by impairing DNA replication through interaction with PCNA [1]. However, p21 is also involved in other pathways con- tributing to the cell response to DNA damage, i.e. transcription, and apoptosis [2,3]. In addition, recent findings have suggested a partic- ipation of p21 in DNA repair [4,5], although this aspect has remained for long time controversial, given that different model systems pro- vided contrasting results [6–14]. Intriguingly, p21 was reported to undergo proteasomal degradation after UV-induced DNA damage, ∗ Corresponding author at: IGM-CNR, sez. Istochimica e Citometria, Piazza Botta 10, 27100 Pavia, Italy. Tel.: +39 0382 986267; fax: +39 0382 986430. E-mail address: prosperi@igm.cnr.it (E. Prosperi). suggesting that removal of p21 was a pre-requisite for efficient DNA repair [15]. However, other studies showed that p21 degradation is greatly dependent on the UV dose [16], and protein destruction is not necessarily required for DNA repair [17,18]. Other aspects of p21 regulation after DNA damage remain to be elucidated because it is not clear whether proteasomal degradation is a specific pro- cess triggered exclusively by UV radiation, or whether it occurs independently of the type of DNA lesion. In fact, changes in p21 protein level were induced by agents, like ionizing radiations or reactive oxygen species, but were not observed after other type of DNA damage [19–21]. Furthermore, multiple mechanisms appear to be involved in the regulation of p21 turnover, since both ubiquitin- dependent and independent pathways have been described for p21 proteasomal degradation, both at basal levels [22–26], and after DNA damage [27–29]. Regulation of p21 turnover is a critical aspect that is likely to influence the DNA damage response. The phosphorylation of p21 by glycogen synthase kinase 3 (GSK3), an event directly linked to checkpoint activation by ATR, has been reported to be necessary for 1568-7864/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2009.02.005