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

Estimation of secondary measles transmission from a healthcare worker in a hospital setting.

Measles among healthcare workers (HCWs) is associated with a significant risk of nosocomial transmission to susceptible patients. When a measles case occurs in the healthcare setting, most guidelines recommend exhaustive measures. To evaluate the effects of measures against measles transmission in the healthcare setting precisely, it is essential to determine whether secondary transmission generally occurs. This study describes, for the first time, the actual secondary transmission rate for a measles-infected HCW in a ward with no special air ventilation capacity. The routine treatment of a number of immunocompromised patients occurs in this ward, and thus patients as well as HCWs have a thorough understanding and practice of standard and extended precautions. Our paired serum sample study revealed that none of the people in the ward exposed to the HCW at the catarrhal stage over a period of 4 days exhibited elevated levels of antibodies against measles. We suggest that strict adherence to standard and expanded precautions among patients and HCWs may be effective for preventing the transmission of a highly airborne disease, such as measles

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

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