Investigating the role of p300 and CBP in nucleotide excision repair: interaction with, and acetylation of XPG protein.

Nucleotide excision repair (NER) is one of the most important DNA repair mechanisms by which cells removal bulky DNA lesions. Our recent findings have shown that the cell cycle inhibitor p21CDKN1A is directly involved in NER and in base excision repair (BER). In NER, p21 participates in the process thanks to its interaction with PCNA, and with the histone acetyltransferase (HAT) p300. Previous studies have suggested that p300 HAT activity is required in NER to provide chromatin accessibility to DNA repair machinery. In addition, it has been shown that PCNA interacts with p300, thereby inhibiting its HAT activity, while p21 restores this function by disrupting interaction with PCNA. Since p300 may also associate with other NER factors, such as XPA, or DDB2, it has been suggested that p300 may play an additional role by regulating directly some step of the repair process. To better understand the involvement of p300 in DNA repair, and to establish whether its homolog CBP also plays a similar role in the process, we have investigated the interaction of p300 or CBP with another typical NER factor, i.e. XPG. The results have shown that both proteins interact with XPG, and that CBP, similarly to p300, is also able to associate with PCNA. To investigate the influence of p300 and CBP on NER efficiency, expression of these proteins has been knocked-down by RNA interference with specific siRNA to p300 or CBP, in primary human fibroblasts. NER activity has been assessed after UV irradiation by analyzing unscheduled DNA synthesis (UDS) through autoradiography of 3H-thimidine incorporation. Immunohistochemical staining for p300 or CBP has been performed in the same samples to verify knock-down of protein levels. The results have indicated that silencing of either p300 or CBP, significantly reduces the number of autoradiographic grains, as compared to cells treated with control siRNA. To clarify whether p300 is required in a particular step of NER, we have analyzed the recruitment of XPG to sites of local UV-irradiation (3-?m pores), after siRNA silencing of both p300 or CBP. No significant difference in the initial recruitment, but persistence of XPG in foci at late times after irradiation, has been found in knocked-down cells. Finally, the possibility that XPG is acetylated by p300 was analyzed in vitro with recombinant proteins. The results have shown that XPG is labeled by an anti-acetyllysine antibody after reaction with purified p300. These results suggest that p300/CBP activity is required in NER after the initial recruitment of to DNA damage sites, and suggest that XPG acetylation is required to release XPG protein from the repair site in order to allow the NER process to proceed to the next step of DNA re-synthesis.