ABSTRACT
The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break (DSB) sites where they induce histone acetylation, thereby influencing the chromatin structure and DNA repair process. Whether p300/CBP at DSB sites also acetylate non-histone proteins, and how their acetylation affects DSB repair, remain unknown. Here we show that p300/CBP acetylate RAD52, a human homologous recombination (HR) DNA repair protein, at DSB sites. Using in vitro acetylated RAD52, we identified 13 potential acetylation sites in RAD52 by a mass spectrometry analysis. An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation, and that non-acetylated RAD52 initially accumulates at DSB sites, but dissociates prematurely from them. In the absence of RAD52 acetylation, RAD51, which plays a central role in HR, also dissociates prematurely from DSB sites, and hence HR is impaired. Furthermore, inhibition of ataxia telangiectasia mutated (ATM) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity, through the formation of RAD52, p300/CBP, SIRT2, and SIRT3 foci at DSB sites. Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism.
Subject(s)
DNA Breaks, Double-Stranded , Histone Acetyltransferases/physiology , Histone Deacetylases/physiology , Homologous Recombination , Rad52 DNA Repair and Recombination Protein/metabolism , Acetylation , Ataxia Telangiectasia Mutated Proteins/metabolism , Histone Acetyltransferases/genetics , Histone Deacetylases/genetics , Humans , Microscopy, Fluorescence , Two-Hybrid System TechniquesABSTRACT
Vitronectin (VN) is a multi-functional protein involved in extracellular matrix (ECM)-cell binding through integrin receptors on the cell surface, which is an important environmental process for maintaining biological homeostasis. We investigated how VN affects the survival of endothelial cells after radiation damage. VN attenuated radiation-induced expression of p21, an inhibitor of cell cycle progression, and selectively inhibited Erk- and p38 MAPK-dependent p21 induction after radiation exposure through regulation of the activity of GSK-3ß. VN also reduced the cleavage of caspase-3, thereby inhibiting radiation-induced apoptotic cell death. These results suggest that VN has important roles in cell survival after radiation damage.
