Physical monitoring of repair process of chromosomal double stand break induced by simultaneous nuclear introduction of I-SceI endonuclease

本研究试图建立一种在特定的染色体位点上同时引入单双链DNA断裂（DSB）的方法。如果这成为可能，那么我们将能够通过基因组Southern印迹、修复和/或检查点因子的募集、修复过程本身的进展等来连续监测末端的物理状态。在酵母细胞中，这种技术是可用的，并且极大地有助于我们理解DSB修复的基本过程。为此，我们计划使用罕见的限制性内切酶I-SceI和在其染色体中具有18个碱基对特异性识别序列的细胞。通过I-SceI将DSB引入整合重组底物SCneo中，并且可以通过将G418敏感细胞转化为抗性细胞来确定修复效率。为了以高效和同步的方式引入I-SceI，将I-SceI编码序列与雌激素受体的两个配体结合结构域融合 关于我们 或来自HIV达特序列和6XHis序列的蛋白转导结构域作为纯化标签。在前一种情况下，融合蛋白I-SceI-ER在细胞系中稳定表达。将重组质粒导入大肠杆菌中，经生化纯化，获得TAT-I-SceI融合蛋白。(1)We成功地在鸡B细胞系DT 40中表达了I-SceI-ER，并检测了用雌激素类似物他莫昔芬处理前后蛋白质的定位。I-SceI-ER蛋白在15 min内从细胞质迅速转移到细胞核。然而，发生相对较少的DSB修复事件，这与ER可能干扰核酸内切酶活性的可能性一致。尽管如此，这种细胞细将是有用的一些研究。我们现在正试图通过制造新的融合蛋白来提高效率。(2)We纯化重组TAT-I-SceI蛋白，但该蛋白似乎自发聚集。我们正在努力寻找最佳条件来纯化和储存蛋白质。少

In this study we tried to develop a method to simultaneously introduce a single double strand DNA break (DSB) into defined chromosomal locus. If this is made possible, then we would be able to continuously monitor physical status of the ends by genomic Southern blotting, recruitment of repair and/or checkpoint factors, progression of the repair process itself and so on. In yeast cells, such technology is available and contributes enormously to our understanding of the basic process of the DSB repair.To this end, we plan to use rare restriction endonuclease I-SceI and cells harbouring the18 base-pair specific recognition sequence in its chromosome. The DSB is introduced in the integrated recombination substrate SCneo by I-SceI, and the repair efficiency can be determined by conversion of the G418-sensitive cells to resistant cells. To introduce I-SceI in high efficiency and in a synchronous manner, the I-SceI coding sequence was fused with two ligand binding domains of estrogen receptor … More or the protein transduction domain derived from HIV TAT sequence and 6XHis sequence for purification tag. In the former case, the fusion protein I-SceI-ER was stably expressed in a cell line. The latter case, the plasmid was introduced into E.coli and the fusion protein TAT-I-SceI was biochemically purifed.(1)We successfully expressed the I-SceI-ER in the chicken B cell line DT40, and examined localization of the protein before and after treatment with estrogen analog tamoxifen. I-SceI-ER protein was rapidly (within 15min) mobilized from cytoplasm to nucleus with high efficiency. However, relatively few DSB repair events occurred, which is consistent with the possibility that ER may interfere with the endonuclease activity. Nonetheless, this cell fine would be useful for some studies. We are now trying to improve the efficiency by making new fusion proteins.(2)We purified the recombinant TAT-I-SceI protein but the protein appeared to aggregate spontaneously. We are trying to find optimal condition to purigy and store the protein. Less