Biochemical Analysis of Sister Chromatid Cohesion

描述（申请人摘要）：姐妹染色单体的凝聚力是一个重要的 确保遗传信息可靠分离的细胞过程 在有丝分裂和减数分裂期间。这个项目的长期目标是了解 该方法的生物化学基础是通过使用来自 非洲爪蟾卵和哺乳动物组织培养细胞。一个 重点将放在结构和功能表征， 粘蛋白是一种高度保守的蛋白复合物，在 建立和维持姐妹染色单体的凝聚力。粘着蛋白复合物是 由两个SMC（染色体结构维持）亚基组成， 至少有两个非S MC亚基，并预测作为分子的一部分， 将两个姐妹染色单体粘合在一起的“胶水”。在本建议中，（1） 生物化学和结构的方法将结合起来，以了解 粘着蛋白复合物作用的分子机制。(2)细胞周期依赖 粘着蛋白和染色质之间的相互作用将在体外重建， 将确定加载和卸载过程的分子基础。（三） BimD和Scc 2的脊椎动物同源物，两种蛋白质在遗传上与 姐妹染色单体凝聚力，将在 无细胞提取物和组织培养细胞。(4)一种新的体外试验将 以确定姐妹染色单体的凝聚力是如何在功能上耦合 到DNA复制。从这项工作中获得的信息将最终 有助于更好地了解人类健康，因为染色体 异常，如非整倍体和易位，与 肿瘤发展和出生缺陷。

DESCRIPTION (APPLICANT'S ABSTRACT): Sister chromatid cohesion is an essential cellular process that ensures the faithful segregation of genetic information during mitosis and meiosis. The long-term goal of this project is to understand the biochemical basis of this process by using cell-free extracts derived from Xenopus laevis (African toad) eggs and mammalian tissue culture cells. An emphasis will be made on the structural and functional characterization of cohesin, a highly conserved protein complex that plays a key role in establishing and maintaining sister chromatid cohesion. The cohesin complex is composed of two SMC (structural maintenance of chromosomes) subunits and at least two non-S MC subunits, and is predicted to act as part of the molecular "glue" that holds two sister chromatids together. In this proposal, (1) Biochemical and structural approaches will be combined to understand the molecular mechanisms of action of the cohesin complex. (2) Cell cycle-dependent interactions between cohesin and chromatin will be reconstituted in vitro and the molecular basis of loading and unloading processes will be determined. (3) Vertebrate homologs of BimD and Scc2, two proteins implicated genetically in sister chromatid cohesion, will be identified and characterized in the cell-free extracts and in tissue culture cells. (4) A novel in vitro assay will be developed to determine how sister chromatid cohesion is functionally coupled to DNA replication. The information obtained from this work will ultimately contribute to a better understanding of human health because chromosome anomalies, such as aneuploidy and translocations, are tightly associated with tumor development and birth defects.