Human RAD52 is directly acetylated by p300/CBP <i>in vitro</i>.

<p>(A) Physical interaction of human RAD52 with CBP. The RAD52 or GST protein was incubated with or without CBP-FLAG in buffer P, and a pull-down assay was performed as described in the Supporting Materials and Methods. Input or immunoprecipitated (IP) proteins were detected by a mixture of anti-RAD52 and anti-GST antibodies (top) or an anti-FLAG (M2) antibody (bottom). (B) RAD52 (0.2 μg; top) or DNA polymerase β (0.2 μg; bottom) was incubated in 10 μl HAT buffer A containing 10 mM sodium butyrate and 0.4 μg acetyl coenzyme A (Ac-CoA) in the absence (-) or presence of HATs (42.5 ng of FLAG-p300 or 275 ng of CBP-FLAG) at 30°C for 90 min. Reaction mixtures were subjected to immunoblotting analyses. (C, D) <i>In vitro</i> acetylation assays were performed as described in the Supporting Materials and Methods, using HAT buffer A containing sodium butyrate. [¹⁴C]Ac-CoA was added where indicated. The reactions were analyzed by Coomassie Brilliant Blue staining (left) or autoradiography (right). Acetylated proteins can be detected by autoradiography. Bovine serum albumin (BSA), as a negative control of acetylation, was not detected in this assay. (C) RAD52 (3 μg), DNA polymerase β (3 μg), or BSA (3 μg) was incubated with CBP-FLAG (2 μg) where indicated. (D) RAD52 (FL, 2 μg), RAD52 (N, 2 μg), or RAD52 (C, 2 μg) was incubated with CBP-FLAG (1 μg), as indicated.