Mismatch repair proteins recruited to ultraviolet light-damaged sites lead to degradation of licensing factor Cdt1 in the G1 phase

<p>Cdt1 is rapidly degraded by CRL4^Cdt2 E3 ubiquitin ligase after UV (UV) irradiation. Previous reports revealed that the nucleotide excision repair (NER) pathway is responsible for the rapid Cdt1-proteolysis. Here, we show that mismatch repair (MMR) proteins are also involved in the degradation of Cdt1 after UV irradiation in the G1 phase. First, compared with the rapid (within ∼15 min) degradation of Cdt1 in normal fibroblasts, Cdt1 remained stable for ∼30 min in NER-deficient XP-A cells, but was degraded within ∼60 min. The delayed degradation was also dependent on PCNA and CRL4^Cdt2. The MMR proteins Msh2 and Msh6 were recruited to the UV-damaged sites of XP-A cells in the G1 phase. Depletion of these factors with small interfering RNAs prevented Cdt1 degradation in XP-A cells. Similar to the findings in XP-A cells, depletion of XPA delayed Cdt1 degradation in normal fibroblasts and U2OS cells, and co-depletion of Msh6 further prevented Cdt1 degradation. Furthermore, depletion of Msh6 alone delayed Cdt1 degradation in both cell types. When Cdt1 degradation was attenuated by high Cdt1 expression, repair synthesis at the damaged sites was inhibited. Our findings demonstrate that UV irradiation induces multiple repair pathways that activate CRL4^Cdt2 to degrade its target proteins in the G1 phase of the cell cycle, leading to efficient repair of DNA damage.</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

ATM inhibition impedes the recruitment of SIRT2/SIRT3, but not 53BP1, at IR-induced DSB sites.

<p>(A, B) T-Rex-293 cells expressing SIRT2-HA (A), or SIRT3-HA (B) were irradiated with γ-rays (8 Gy). The KU55933 (ATMi) 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 an anti-HA (green) antibody, an anti-γH2AX (red) antibody, and DAPI (blue). (C, D) The percentages of γH2AX foci colocalization with SIRT2 (C) or SIRT3 (D) in Fig 13A and 13B 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). (E, F) The KU55933 solution or the same volume of DMSO was added to T-Rex-293 cells, 1 h before irradiation with γ-rays (8 Gy). (E) Cells were subjected to immunofluorescent staining at 1 h after irradiation with an anti-SIRT3 (green) antibody, an anti-γH2AX (red) antibody, and DAPI (blue). (F) Cells were subjected to immunofluorescent staining at 1 h after irradiation with an anti-53BP1 (green) antibody, an anti-γH2AX (red) antibody, and DAPI (blue). As a control, unirradiated cells (0 Gy) were used.</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

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

The RAD52 (10xR) mutant protein does not inhibit the formation of replication protein A (RPA) and BRCA1 foci.

<p>(A) 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-HA (green), anti-γH2AX (red), and anti-RPA1 (blue or white) antibodies are shown. (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. Immunofluorescent images with anti-HA (green), anti-γH2AX (red), and anti-phospho-BRCA1 at Ser1524 (blue or white) antibodies are shown.</p

IR-induced foci formation of RAD52 acetylation-site mutant proteins.

<p>(A, B) MSCs (A) or T-Rex-293 (HEK293) cells (B) expressing FLAG-RAD52 (Wt or the indicated mutants)-HA were irradiated with γ-rays (8 Gy), and subjected to immunofluorescent staining 6 h after irradiation. An anti-HA (green) antibody, an anti-γH2AX (red) antibody, and DAPI (blue) were used for immunofluorescent staining.</p

Effect of 13xR mutation on ionizing radiation-induced foci formation by RAD52.

<p>(A, B) MRC5V1-TR cells were unirradiated or irradiated with γ-rays (8 Gy), and subjected to immunofluorescent staining at the indicated time after irradiation. (A) Cells expressing FLAG-RAD52 (Wt)-HA were used. An anti-HA (green) antibody, an anti-γH2AX (red) antibody, and 4',6-diamidino-2-phenylindole (DAPI, blue) were used for immunofluorescent staining. (B) Cells expressing FLAG-RAD52 (13xR)-HA were used. An anti-HA (green) antibody, an anti-γ-tubulin (red) antibody, and DAPI (blue) were used for immunofluorescent staining. (C) MRC5V1-TR cells expressing FLAG-NLS-RAD52 (Wt)-HA or FLAG-NLS-RAD52 (13xR)-HA were irradiated with γ-rays (8 Gy), and subjected to immunofluorescent staining 6 h after irradiation. An anti-HA (green) antibody, an anti-γH2AX (red) antibody, and DAPI (blue) were used for immunofluorescent staining, and the overlaid images are shown.</p