The number of cells <i>N</i>_cell vs. α-tracks (<i>N</i>_α) and α-particles ().

<p>A) The observed number of α-tracks (<i>N</i>_α) emitted from single cells and B) the estimated number of α-particles () corrected with solid angular and critical angular dependencies.</p

Microscope images of cells and emitted α-tracks from those cells on aCR-39 detector.

<p>A) Cells and B) corresponding α-tracks. A dot arrow indicates that no α-tracks are observed in the corresponding cell position. The enlarged images of C) cells and D) α-tracks recognized by ellipse fitting result are inset by the colored circles.</p

LET spectrum of α-particles from single cells.

<p>LET spectrum of α-particles from single cells.</p

Scatter plots and contour maps of cells and α-tracks positions.

<p>Scatter plots of the positions of A) cells and B) α-tracks over the entire surface of the CR-39 detector and C) their overlay, respectively. The macroscopic autoradiography as a contour map by counting the number of D) cells and E) α-tracks in binned positions (Δ<i>x</i>, Δ<i>y</i>) with 200 μm intervals.</p

Schematic of the experimental procedure.

<p>Schematic of the experimental procedure.</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

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

<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

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

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.</p