Chromatin immunoprecipitation-like assays in mouse embryonic fibroblasts in the presence or absence of Bax protein.

<p>(<b>A</b>) MEF-WT and MEF-Bax-KO were treated without or with DXR (200 nM) for 3 h. CHIP assays were adapted to detect the association of the specific proteins (Rad51 or Bax) with DNA. The experiment was performed twice in two different days. (<b>B</b>) To exclude the possibility that MEF-WT cells might have less Rad51 protein than MEF-Bax-KO cells, DNA isolated from 10⁷ cells from both (WT and KO) were preincubated with various concentrations of Rad51 blocking peptide before Rad51 immunoprecipitation. No apparent differences between the concentrations of blocking peptide needed to block Rad51 binding in MEF-WT and KO were observed. This indirectly suggests that the amount of Rad51 in these cells is not different from one another. Ab, antibody.</p

DXR induces both single- and double-strand DNA breaks in oocytes; old oocytes are more susceptible to DXR-induced apoptosis; and, some strains of mice are more prone to DXR-induced apoptosis than others.

<p>DNA damage was assessed in oocytes of B6C3F1 mice incubated in the presence of DXR (200 nM) for 24 h. DXR induced both single (<b>A</b>) and double-strand DNA breaks (<b>B</b>). Addition of 1 mM SAM (sodium aurothiomalate; general nuclease inhibitor) completely prevented double-strand breaks (<b>D</b>) but it did not prevent DXR-induced single-strand DNA lesions (<b>C</b>). Quantitative assessment of apoptosis in mature oocytes of young (Y; 6 week old) and old ICR mice (O; 43–44 week old), cultured without (control; CON) or with DXR for 24 h, revealed that old oocytes are more susceptible to DXR-induced apoptosis (<b>E</b>). And, assessment of apoptosis in oocytes of young ICR and B6C3F1 mice cultured without or with DXR show that oocytes collected from B6C3F1 females are more prone to DXR-induced apoptosis than oocytes collected from ICR females (<b>F</b>). After culture, oocytes were fixed and processed for assessment of apoptotic characteristics according to morphological changes (such as cellular condensation, budding, and cellular fragmentation). The percentage of oocytes that showed cellular fragmentation, out of the total number of oocytes cultured in each treatment group, was then determined. The data (mean ± SEM) represent the combined results from at least four independent experiments; the total number of oocytes analyzed per group is indicated over the respective bar.</p

Oocyte resistance to DXR- or aging-induced DNA damage is respectively linked to DNA repair and Rad51.

<p>Approximately 80% of oocytes from young and old ICR mice show considerable DNA damage by the time of isolation (respectively, <b>A & C</b>). However, after 6 h of incubation oocytes from young mice significantly repaired the DNA damage as evidenced by the decrease in the length of the comets during the same incubation time (<b>B</b>; compare A <i>vs</i>. B); oocytes from old mice did not show any repair capacity (<b>D</b>; compare C <i>vs</i>. D). Immunohistochemical localization of Rad51 in ovarian sections from ICR (young and old) mice show that, in young ICR mice (<b>E, F</b>), approximately 90% of primordial and primary follicles stained positive for Rad51. While in old ICR mice (<b>G,H,I</b>) only 50% or less of the follicles showed a positive reaction. Arrows point to follicles showing a negative reaction.</p

Inverse relationship between the presence of Bax and Rad51.

<p>Immunolocalization of Bax in mature (metaphase II) oocytes from young (6 week old) AKR/J (deficient in Rad51-dependent DNA repair) and C57BL/6 mice showed that the levels of Bax protein (red) located nearby the DNA (blue) were lower in oocytes isolated from C57BL/6 (<b>A&B</b>), when compared to levels seen in oocytes isolated from AKR/J mice (<b>C&D</b>). Moreover, immunohistochemical localization of Rad51 in ovaries of bax-null mice show that the majority of primordial oocytes in both young (<b>E,F</b>) and old (<b>G–J</b>) mice stain positive for Rad51 (brown color). (<b>K</b>) primordial follicle showing negative staining. For comparison, in the wild-type mice Rad51 positive staining decreased with age (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009204#pone-0009204-g002" target="_blank">Figure 2<b>G–I</b></a>). Mature oocytes were denuded of cumulus cells and immediately (time 0) processed for Bax immunocytochemistry. Oocytes were serially scanned and optical sections were analyzed using Metamorph software (Universal Imaging Corp.). The average pixel intensity for both red and blue channels was calculated by the software for each region. The ratio of red/blue fluorescence (Bax/DNA content) was recorded for each section. Values for each sample were determined as mean ± SEM per oocyte (AKR/J n = 50; C57BL/6 n = 50), and comparison was made between the two strains using Student's t-test. Oocytes (n = 10) devoid of exposure to primary antibody were used as a negative control to determine background noise. (DNA∶blue; Bax∶red or magenta). Immunohistochemical localization of Rad51 was performed in ovaries of bax-null mice young (6 weeks old) and old (42–44 weeks old).</p

<i>In vitro</i> DNA repair capacity of oocytes from <i>bax</i>-null mice.

<p>Approximately 80% of oocytes from young mutant mice (<b>A</b>) showed considerable DNA damage by the time of isolation. However, after 6 h of incubation in control medium these oocytes from young mice (<b>B</b>) had significantly repaired the DNA damage as evidenced by the decrease in the length of the comets; and even after 6 h in the presence of DXR their DNA appears still intact (<b>F</b>), where as young wild-type oocytes had not after treatment with DXR for 6 h (<b>E</b>). The vast majority of oocytes from old bax-null mice showed no DNA damage by the time of isolation (<b>C</b>) or after 6 h in culture (<b>D</b>); compared with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009204#pone-0009204-g002" target="_blank">Figure 2</a> (panels C and D) where oocytes of aged wild-type mice had sustained DNA damage by the time of isolation and were unable to repair it after 6 h of incubation. Oocytes from old bax-null old also sustained a very low level of DNA damage after a 6 h-incubation with DXR (<b>G</b>), compared with wild-type oocytes (<b>E</b>).</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

<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

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