Post-translational modifications regulate biological processes by controlling a wide variety of protein functions. Among several post-translational modifications, acetylation occurs on specific lysine residues and is catalyzed by histone acetyltransferases (HATs). Although the well-known target proteins for acetylation are histones, we have newly identified acetylation of human Rad52 protein which is involved in homologous recombination. In an in vitro acetylation assay, Rad52 protein was acetylated by HATs such as CBP and p300. On the other hand, Rad51 protein, the other important protein for homologous recombination, was not acetylated by CBP in vitro. We identified the acetylation sites of Rad52 by mass spectrometry with the in vitro- acetylated purified Rad52 protein. As expected, the substitution of the acetylated lysines with arginines abolished acetylation of Rad52 both in vitro. Rad52 binds to both single- and double-stranded DNA via N-terminal domain. In addition, middle part of Rad52 interacts with RPA. In vitro acetylation of Rad52 was inhibited when single- or double-stranded DNA or RPA was included in the reaction mixture, suggesting that DSB-induced acetylation of human Rad52 takes place before its binding to the damaged DNA. We will discuss the regulation of the acetylation of Rad52. ; The8th 3R symposium