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

Comparative studies on spontaneous and ionizing radiation induced mutagenesis between somatic and male germ cells

We have carried out mutation assays in germ cells from the transgenic mice after irradiation of various doses of ionizing radiation at the post-meiotic spermatid stage, to learn the difference in sensitivity to ionizing radiation in mutation induction among somatic cells and male germ cells (pre-meiotic spermatogonial stem cell and post-meiotic spermatid stage). The transgenic mice used for the assay are the gpt-delta mouse strain, which carries about 80 copies of the bacterial gpt gene per cell as targets for mutagenesis. To measure the induced mutation frequencies in male germ cells (spermatid stage), sperms were extracted at the 14th day after irradiation of 2.5 or 5 Gy of X rays, corresponding to the spermatid stage at the time of treatment. The spontaneous gpt gene mutation frequency in male germ cells was 0.36 x 10-5. The mutation frequencies in male germ cells irradiated with 2.5 or 5 Gy of X rays at the spermatid stage were 0.60 or 1.03 x 10-5, respectively. The induced mutation frequencies in male germ cells irradiated at the spermatid stage were nearly the same as those irradiated at the spermatogonial stem cell stage (0.53 or 1.05 x 10-5 for 2.5 or 5 Gy of X-ray-treatment, respectively), and about three to four times lower than those in somatic cells (2.43 or 3.46 x 10-5 for 2.5 or 5 Gy of X-ray-treatment, respectively). This difference between somatic and male germ cells in the mutation frequency would be mainly due to the high base excision repair activity in male germ cells.The 13th International Congress of Radiation Researc

Characterization of Xpg mutant mice

1st Japan-US DNA Repair Meetin

Long-range single DNA molecule haplotyping of human clinical radiation sensitivity-associated genes (PTTG1 and CD44)

OBJECTIVES: Haploid chromosomes distinguished by multiple SNP markers in human PTTG1 (Chromosome 5) and CD44 (Chromosome 11) genes have been reported to associate with the severity of adverse effects induced by radiation therapy of breast cancer patients. It is of interest to understand biological consequences of the heterozygotic state by functionally distinct, homologous haploid chromosomes within a nucleus of cells in the patients. To this end, diplotype of these chromosomes must be precisely determined to evaluate their clinical outcomes. \nMETHODS: In this study, a novel methodology of long-range (over 20 kb) haplotype determination involving sensitive amplification of single DNA molecules within agarose gels has been developed. Limited dilution of DNA molecules was performed in heated alkaline agarose solution, avoiding extensive shearing and aggregation during dilution process. Aliquoting the gel solution provided physical separation of DNA molecules derived from homologous chromosomes. The solidified agarose gel pieces were then treated by exogenously supplied Phi29 DNA polymerase and random hexamer oligonucleotides, yielding up to 120,000-fold multiple-displacement amplification of the gel-embedded DNA molecules. The amplified materials were recovered in solution as PCR-ready form by simple heating, making them conveniently available for further multiple locus genotyping. \nRESULTS: and CONCLUSIONS Feasibility of this methodology for isolating long-range single DNA molecules spanning the selected SNP markers (PTTG1: 20 kb, CD44: 80 kb) was further confirmed using DNAs derived from patients with heterozygotic state. Thus, the haploid chromosomes distinguished by the SNP markers of the above clinical radiation sensitivity-associated genes can now be successfully determined in the individual breast cancer patients.48ｔｈ the American Society for Cell Biology Annual Meetin

Generation and characterization of Xpg mutant mice with well characterized point mutations

12th International Congress of Radiation Researc

Vitronection improves cell survival after radiation injury in human umbilical vein endothelial cells.

Vitronectin (VN) is a multi-functional protein involved in extracellular matrix (ECM)-cell binding through integrin receptors on the cell surface, which is an important environmental process for maintaining biological homeostasis. We investigated how VN affects the survival of endothelial cells after radiation damage. VN attenuated radiation-induced expression of p21, an inhibitor of cell cycle progression, and selectively inhibited Erk- and p38 MAPK-dependent p21 induction after radiation exposure through regulation of the activity of GSK-3b. VN also reduced the cleavage of caspase-3, thereby inhibiting radiation-induced apoptotic cell death. These results suggest that VN has important roles in cell survival after radiation damage

<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