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

Human RAD52 is acetylated <i>in vivo</i>.

(A, B) An in vitro acetylation assay was performed by incubating RAD52 (0.25 μg) in 10 μl HAT buffer A containing 10 mM sodium butyrate, in the presence of 0.5 μg Ac-CoA and CBP-FLAG (54 ng), at 30°C for 60 min. Unacetylated and acetylated RAD52 were subjected to immunoblotting analyses using the indicated antibodies. (C, D, E) Acetylation of the FLAG-RAD52-HA protein purified from MSCs was detected as described in the Supporting Materials and Methods, using the indicated antibodies. The expression plasmid, pT-Rex-DEST30 containing FLAG-RAD52 (Wt)-HA or FLAG-RAD52 (10xR)-HA, was transfected into the cells. FLAG-RAD52-HA was purified from cell extracts 24 h after transfection. Cells were treated with doxorubicin for 2 h, as indicated. (D) The red arrow indicates the mobility-shifted band. (F) MSCs expressing FLAG-RAD52 (Wt)-HA or FLAG-RAD52 (10xR)-HA were unirradiated or irradiated with γ-rays (8 Gy). At the indicated time after irradiation, the cells were subjected to immunofluorescent staining with anti-HA (green), anti-γH2AX (red), and anti-acetylated RAD52 at K323 (blue or white) antibodies. (G, H) T-Rex-293 cells expressing FLAG-RAD52-HA were transfected with either a negative control siRNA or mixture of p300 and CBP-specific siRNAs. At 24 h after transfection, the cells were treated with doxorubicin for 2 h. Immunoprecipitated FLAG-RAD52-HA proteins from the cell extracts were subjected to immunoblotting analyses with the indicated antibodies. (C, E, G, H) The relative band intensities normalized to those of the HA bands are shown below the immunoblots.</p

Colocalization of SIRT2 and SIIR3 with RAD52 at IR-induced DSB sites.

(A, B, C) T-Rex-293 cells expressing FLAG-RAD52 (Wt)-HA were unirradiated or irradiated with γ-rays (8 Gy), and subjected to immunofluorescent staining at the indicated time after IR, using (A) anti-HA (green), anti-γH2AX (red), and anti-SIRT2 (blue or white) antibodies. (B) Anti-HA (green), anti-γH2AX (red), and anti-SIRT3 (blue or white) antibodies were used. (C) An anti-HA (green) antibody, an anti-HDAC3 (red) antibody, and DAPI (blue) were used. (D, E, F) T-Rex-293 cells expressing HDAC3-HA (D), SIRT2-HA (E) or SIRT3-HA (F) were unirradiated or irradiated with γ-rays (8 Gy). At the indicated time after irradiation, the cells were subjected to immunofluorescent staining with an anti-HA (green) antibody, an anti-γH2AX (red) antibody, and DAPI (blue).</p

SIRT2 and SIRT3 are involved in the deacetylation of RAD52 at DSB sites.

T-Rex-293 cells expressing FLAG-RAD52 (Wt)-HA were treated with either control siRNA (C), SIRT2-specific siRNA (siSIRT2), SIRT3-specific siRNA (siSIRT3), or both siRNAs (siSIRT2 & 3), as described in the Supporting Materials and Methods. (A, B) Cell extracts were prepared 48 h after siRNA transfection, and were subjected to immunoblotting analyses with the indicated antibodies. (C) Cells were irradiated with γ-rays (8 Gy) 48 h after siRNA transfection. At 1 h or 6 h after irradiation, cells were subjected to immunofluorescent staining with anti-HA (green), anti-γH2AX (red), and anti-acetylated RAD52 at K323 (blue or white) antibodies.</p

ATM inhibition impedes the recruitment of p300/CBP at IR-induced DSB sites.

(A, B) MRC5V1 cells were transfected with the expression plasmid for wild-type (Wt) HA-p300 (A) or CBP-HA (B). (E) MRC5V1 cells were transfected with the expression plasmid for either HA-p300 S106A or HA-p300 S106D. (A, B, E) At 24 h after transfection, the KU55933 (ATMi) solution or the same volume of DMSO was added to cells 1 h before irradiation with γ-rays (8 Gy). At 1 h after irradiation, the cells were subjected to immunofluorescent staining. Immunofluorescent images with an anti-HA (green) antibody, an anti-γH2AX (red) antibody, and DAPI (blue). (C, D) The percentages of γH2AX foci colocalization with p300 (C) or CBP (D) in Fig 12A and 12B were calculated, as described in the Supporting Materials and Methods, and are shown in the graphs. The numbers of protein foci per cell are also shown in the graphs. Error bars indicate the standard error of the mean. Asterisks indicate statistically significant differences between the indicated pairs of groups (***, p<0.001 by t-test).</p

Effect of acetylation-deficient mutations on ionizing radiation-induced foci formation by RAD52.

<p>(A, B) MSCs stably expressing FLAG-RAD52-HA proteins were irradiated with γ-rays (8 Gy). At the indicated time after irradiation, the cells were subjected to immunofluorescent staining with an anti-HA (green) antibody, an anti-γH2AX (red) antibody, and DAPI (blue). (A) MSCs expressing NLS-RAD52 (Wt) or NLS-RAD52 (13xR) were used. (B) MSCs expressing RAD52 (Wt) or RAD52 (10xR) were used. (C, D) The percentages of RAD52 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 indicated pairs of groups (***, <i>p</i><0.001 by t-test). (E) The number of γH2AX foci per cell was counted in MSCs expressing RAD52 (Wt) or RAD52 (10xR) at the indicated time after irradiation with γ-rays (8 Gy), as described in the Supporting Materials and Methods (N.S., not significant by t-test).</p

A time course analysis of RAD51 foci colocalization with γH2AX foci in cells expressing RAD52 Wt or 10xR mutant protein.

<p>(A, B) RAD52 (Wt or 10xR) knock-in HEK293 cells, as shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007277#pgen.1007277.s010" target="_blank">S9 Fig</a>, were used. The cells were treated with siRNA (siRAD52 (3'UTR #1)) 2 days before irradiation. (A) At the indicated time after irradiation with γ-rays (8 Gy), the cells were subjected to immunofluorescent staining with an anti-RAD51 (green) antibody, an anti-γH2AX (red) antibody, and DAPI (blue). (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 (N.S., not significant; ***, <i>p</i><0.001 by t-test).</p

ATM inhibition impedes acetylation of human RAD52 and its recruitment at IR-induced DSB sites.

<p>(A) MSCs were transfected with the expression plasmid pT-Rex-DEST30, containing FLAG-RAD52 (Wt)-HA. Cells were treated with doxorubicin for 2 h at 24 h after transfection. Where indicated, KU55933 (ATMi) was added to the cells at 1 h before the addition of doxorubicin. Immunoprecipitated FLAG-RAD52-HA proteins from the cell extracts were subsequently subjected to immunoblotting analyses with anti-Ac-RAD52 (K274), anti-Ac-RAD52 (K323) and anti-HA antibodies. The cell extracts before the immunoprecipitation were also subjected to immunoblotting analyses with anti-phospho-CHK2-T68, anti-CHK2, and anti-GAPDH antibodies. The relative band intensities normalized to those of the HA bands are shown. (B, C, D) MRC5 V1 cells were co-transfected with pT-Rex-DEST30 containing FLAG-RAD52 (Wt)-HA and either control or ATM-specific siRNA, as described in the Supporting Materials and Methods. Cell extracts were prepared 24 h after transfection. (B) Cell extracts were subjected to immunoblotting analyses with the indicated antibodies. (C, D) Where indicated, the cells were treated with doxorubicin for 2 h before preparing the cell extracts. Immunoprecipitated FLAG-RAD52-HA proteins from the cell extracts were subjected to immunoblotting analysis with the indicated antibodies. The relative band intensities normalized to those of the M5 bands (C) or to the HA bands (D) are shown. (E, F) Recruitment of RAD52 to DSBs is dependent on ATM. (E) T-Rex-293 cells expressing FLAG-RAD52 (Wt)-HA were irradiated with γ-rays (8 Gy). The KU55933 solution or the same volume of DMSO was added to the cells 1 h before irradiation. At 1 h after irradiation, the cells were subjected to immunofluorescent staining. Immunofluorescent images with anti-HA (green), anti-γH2AX (red), and anti-acetylated RAD52 at K323 (blue or white) antibodies. (F) The percentages of γH2AX foci colocalized with RAD52 in Fig 11E were calculated, as described in the Supporting Materials and Methods, and are shown in the graph. The numbers of protein foci per cell are also shown in the graph. Error bars indicate the standard error of the mean. Asterisks indicate statistically significant differences between the indicated pairs of groups (***, p<0.001 by t-test).</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