Novel function of HATs and HDACs in homologous recombination through acetylation of human RAD52 at double-strand break sites

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

アフリカツメガエル卵母細胞を用いるタンパク質生合成のための簡易微量注入法(農芸化学部門)

アフリカツメガエル卵母細胞へのRNA, DNA等の細胞成分注入用の簡易な微量注入装置を考案, 試作した。この装置を用い特に植物性mRNAをアフリカツメガエル卵母細胞へ注入しタンパク質生合成を行わせた。その結果, tobacco mosaic virus RNA, brome mosaic virus RNA, イネglutelin前駆体mRNA, エンドウriburose 1,5 bis-phosphate carboxylase mRNAが忠実に翻訳され, 対応するpolypeptideが形成されることをsodium dodecyl sulfate polyacrylamide gel電気泳動(SDS-PAGE)による分子量の測定と免疫学的方法を組合わせることにより確認した。A simplified and convenient microinjection system to inject cell components such as DNA and RNA into Xenopus oocytes was devised and constructed. By use of this system, RNAs of plants were injected into Xenopus oocytes, then their protein syntheses were examined. Tobacco mosaic virus RNA, brome mosaic virus RNA, rice glutelin precusor mRNA, and pea Riburose 1,5bisphosphate carboxylase mRNA were faithfully translated. The nature of the synthesized polypeptide was confirmed by fluorography using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoelctrophoresis

<i>In vitro</i> acetylation of RAD52 is inhibited in the presence of DNA or RPA.

<p><i>In vitro</i> acetylation assays were performed as described in the Supporting Materials and Methods, using HAT buffer A containing sodium butyrate. The full-length (A), N-terminal half (B), or C-terminal half (C, D) of RAD52 (2 μg) was incubated with [¹⁴C] Ac-CoA and CBP-FLAG (500 ng). (A, B, C) RAD52 was premixed with 8,500 pmol (in nucleotides) of linear ssDNA, circular dsDNA, or linear dsDNA before the addition of CBP and Ac-CoA to the reaction mixture. (D) RAD52 was premixed with the indicated amount of RPA, before adding CBP and Ac-CoA to the reaction mixture.</p

Screening of HDACs for RAD52 <i>in vitro</i>.

<p><i>In vitro</i> HDAC assays were performed as described in the Supporting Materials and Methods, using acetylated RAD52 and recombinant HDAC proteins. Reaction mixtures were analyzed by immunoblotting with an anti-acetyl lysine antibody (top) or an anti-RAD52 (bottom) antibody.</p

Effect of RAD52 (10xR) acetylation-deficient mutant protein on cell growth, cell survival and IR-induced sister chromatid exchange.

<p>(A, B) T-Rex-293 (HEK293) cells stably expressing the Wt or 10xR FLAG-RAD52-HA protein were cultured in the absence of the Tet inducer. (A) Endogenous RAD52 was depleted by siRNA treatment with siRAD52 (3'UTR#1). Where indicated, the cells were also subjected to an siRNA treatment with the mixture of siBRCA2 (#1, #2 and #3) at day 0. The cell growth was examined as described in the Supporting Materials and Methods section. The graph shows the mean values and the standard error of the mean from triplicate samples. Asterisks indicate statistically significant differences (*, <i>P</i><0.05 by t-test). (B) Cells were treated with the indicated concentration of cisplatin. Cell survival was assayed as described in the Supporting Materials and Methods section. Means with standard errors of four experiments are shown. Asterisks indicate statistically significant differences (***, <i>p</i><0.001 by t-test). (C) T-Rex-293 (HEK293) cells stably integrated with pT-Rex-DEST30 containing FLAG-RAD52 (Wt)-HA, FLAG-RAD52 (10xR)-HA or its empty vector were cultured in the absence of the Tet inducer. The cells were exposed to X-ray radiation. The sister chromatid exchange assay was performed, as described in the Materials and Methods section. In independent experiments, 50 cells were counted for each condition. The graph shows the mean values and the standard error of the mean from two independent experiments. Asterisks indicate statistically significant differences (*, <i>P</i><0.05 by t-test).</p

Colocalization of RAD51 foci at DSB sites is inhibited in cells expressing RAD52 (10xR) acetylation-deficient mutant protein.

<p>(A, B) MSCs stably expressing the indicated FLAG-RAD52-HA proteins were irradiated with γ-rays (8 Gy), and subjected to immunofluorescent staining 6 h after irradiation. (A) Immunofluorescent images with anti-HA (green), anti-γH2AX (red), and anti-RAD51 (blue) antibodies are shown. (B) The percentages of RAD51 foci colocalized with γH2AX were calculated, as described in the Supporting Materials and Methods. Error bars indicate the standard error of the mean. Asterisks indicate statistically significant differences between the FLAG-RAD52 (Wt)-HA expressing cells and the FLAG-RAD52 (10xR)-HA expressing cells (***, <i>p</i><0.001 by t-test). (C) T-Rex-293 (HEK293) cells stably integrated with pT-Rex-DEST30 containing FLAG-RAD52 (Wt or 10xR)-HA were cultured in the absence of a tetracycline inducer. As a negative control (-), T-Rex-293 cells that did not contain the expression vector were used. Cell extracts were subjected to immunoblotting analyses with the indicated antibodies. (D) T-Rex-293 (HEK293) cells expressing FLAG-RAD52 (Wt or 10xR)-HA were irradiated with γ-rays (8 Gy), and subjected to immunofluorescent staining 4 h after irradiation. Immunofluorescent images with anti-γH2AX (red) and anti-RAD51 (blue or white) antibodies are shown. (E) MSCs stably expressing FLAG-RAD52 (Wt or 10xR)-HA were irradiated with γ-rays (8 Gy), and subjected to immunofluorescent staining 0.5 or 2 h after irradiation. Immunofluorescent images with anti-γH2AX (red) and anti-RAD51 (blue or white) antibodies are shown.</p