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

在这项研究中，我们试图开发一种方法，同时引入单双链DNA断裂（DSB）到确定的染色体位点。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/或检查点因子的招募、修复过程本身的进展等来持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了解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及其染色体中含有18个碱基对特异性识别序列的细胞。I-SceI将DSB引入到集成复合底物SCneo中，并通过g418敏感细胞向抗性细胞的转化来确定修复效率。为了高效、同步地导入I-SceI，我们将I-SceI编码序列与雌激素受体的两个配体结合域进行融合，更多的是将HIV TAT序列和6XHis序列衍生的蛋白转导域作为纯化标签。在前一种情况下，融合蛋白I-SceI-ER在细胞系中稳定表达。后一种情况下，质粒引入大肠杆菌，融合蛋白TAT-I-SceI进行生化纯化。(1)我们在鸡B细胞系DT40中成功表达了I-SceI-ER，并检测了雌激素类似物他莫昔芬治疗前后该蛋白的定位。I-SceI-ER蛋白快速（15min内）从细胞质转移到细胞核，效率高。然而，相对较少的DSB修复事件发生，这与ER可能干扰内切酶活性的可能性是一致的。尽管如此，这种细胞模型对某些研究还是有用的。我们现在正试图通过制造新的融合蛋白来提高效率。(2)我们纯化了重组TAT-I-SceI蛋白，但该蛋白表现为自发聚集。我们正在努力寻找纯化和储存蛋白质的最佳条件。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