核酸酶LEM-3的生化性质与体内功能研究

Chromatin bridge is a kind of DNA linkage which forms by incompletely segregated chromatin during cell division. The formation of chromatin bridge severely affected the accurate chromosome segregation. If processed improperly, it can cause genome instability. Recently, some progress has been made about the mechanism of chromatin bridge formation and detection. However, little is known about how the chromatin bridge can be resolved. Our previous study has revealed a novel mechanism conferred by LEM-3 nuclease，which is able to process chromatin bridge at the final stages of cell division. In this project, we will use genetics, molecular biology and next-generation sequencing to further characterize the biochemical properties of LEM-3 and its regulation by phosphorylation. We will also explore the genetic interaction between lem-3 and tumor suppressor gene BRCA1/brc-1, and identify the in vivo interacting proteins of LEM-3. This study can help us to better understand how cells preserve the accurate genetic information during divisions, and provide an insight into the association between cell proliferation and tumor occurrence.

染色质桥是指在细胞分裂过程中，染色体没有完全分离开，从而形成的DNA连接。染色质桥的产生严重影响细胞分裂过程中染色体的正常分离，其处理不当可引发基因组的不稳定。近年来，虽然对细胞内染色质桥的形成和探测机理有了初步的了解，但下游的处理机制仍不明确。本项目将在前期工作已鉴定核酸酶LEM-3介导细胞分裂末期染色质桥处理新机制的基础上，综合运用遗传学，分子生物学及下一代测序技术，深入分析核酸酶LEM-3的生化性质，进一步解析LEM-3的磷酸化调控机制，探索lem-3与肿瘤抑制基因BRCA1/brc-1之间的相互作用机理，筛选和鉴定LEM-3体内相互作用蛋白。这对研究细胞如何在分裂的最后关键时刻保证遗传信息的精确传递以及阐明细胞增殖和肿瘤发生发展的病理过程之间的关系都具有重要意义。

细胞增殖中的DNA复制缺陷、染色体间的同源重组异常以及端粒融合都会引发分裂过程中分离的染色体间产生错误的DNA连接，也称之为染色质桥。染色质桥的存在会引发染色体的断裂或异倍体的形成。我们前期的研究发现一种在多细胞动物中非常保守的核酸酶LEM-3/ANKLE1在线虫细胞分裂末期定位于中心体上，并且能够介导染色质桥的切割从而促进染色体的正常分离和细胞分裂。本项目中，我们表达并纯化了LEM-3重组蛋白，发现该蛋白对于多种DNA底物均具有结合活性。同时敲除lem-3和肿瘤抑制基因brc-1会导致线虫对于DNA损伤剂cisplatin和MMS更加敏感。此外我们还证明了LEM-3在中间体的解离受ESCRT复合物以及septins蛋白调控，进一步证实LEM-3是中间体/中间体环的相互作用蛋白或组成因子。本项目的研究为深入阐明细胞分裂末期染色体的精确分离机制奠定了良好的基础。

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