Structural basis of the Scc2/cohesin interaction and its implication on cohesin loading

所有生物（从细菌到人类）的生物学特征主要是由遗传自其父母的遗传信息决定的。大量的遗传信息是由一种叫做DNA的大分子携带的，它储存在每个细胞中。当细胞生长和分裂时，DNA被精确地复制成“姐妹细胞”，并平等地传递给两个新生的子细胞。这个过程中的错误会导致灾难性的后果，改变细胞的命运。这会导致细胞死亡或癌症和发育障碍等疾病。为了确保复制的遗传信息的精确共享，在DNA复制后产生的姐妹DNA被固定在一起，直到它们准备好移动到细胞的两极，就在细胞分裂之前。这种现象被称为姐妹染色单体内聚。姐妹染色单体的内聚是由一种叫做内聚蛋白的特殊机器介导的，它由三个蛋白质亚基组成。除姐妹染色单体内聚外，内聚蛋白还在其他过程中发挥重要作用，如调节使用哪些基因，修复受损的DNA。虽然它与DNA一起执行各种任务，但内聚蛋白与DNA的相互作用似乎有一个非常简单的模式。这三个亚基相互连接，形成一个巨大的蛋白质环结构，环可以打开和关闭，允许DNA纤维进入或退出环。精确调节内聚蛋白与DNA的结合和分离是其作用的基础。这种调节的缺陷损害了内聚蛋白的功能，这在人类中会导致癌症和遗传性发育障碍（如Cornelia de Lange CdLS和Roberts综合征）。内聚蛋白在体内不能直接结合DNA，它与DNA的结合需要另一种称为Scc2/4的蛋白质复合物。有趣的是，超过一半的CdLS病例是由于sc2缺陷引起的。虽然黏结蛋白已经被研究了20多年，但我们对其依赖scc2的负载反应的分子细节的了解却进展甚微。我们知道加载反应中的一个关键事件是Scc2/黏结蛋白预加载复合物的组装。因此，揭示该复合体的结构将大大提高我们对Scc2如何向DNA招募内聚蛋白的理解，这是本研究的最终目的。黏结配合物的结构已经建立，最近发表了Scc2/4配合物的伪原子结构。问题是如何将这两个结构连接在一起以获得预加载复合体的整体图像。其中的关键是精确地映射Scc2/内聚接口或交互站点。我们的初步实验发现了黏结蛋白的几个区域，这些区域是scc2相互作用的位点。在这项研究中，我们将使用综合的遗传、生化和生物物理方法来确定这些界面。所有这些结果使我们能够创建Scc2/内聚蛋白预加载复合物的结构模型，这将为内聚蛋白加载的分子机制提供新的视角。对这一基本过程的深入了解将有助于我们理解DNA分离在细胞分裂中有时是如何失败的，比如在癌细胞中，或者我们的发育程序是如何出错的，从而导致了与黏结蛋白相关的疾病。

The biological features of all organisms (from bacteria to humans) are mainly decided by genetic information inherited from their parents. A large amount of genetic information is carried by a macromolecule called DNA, which is stored in each cell. When cells grow and divide, DNA is accurately duplicated into 'sisters' and equally transmitted to the two new-born daughter cells. Mistakes in this process can lead to catastrophic consequences, altering the fate of cells. This can cause cell death or diseases such as cancer and developmental disorders. To ensure the precise sharing out of the duplicated genetic information, sister DNAs produced after DNA replication are held together until they are ready to move to opposites poles of the cell, just before the cell divides. This phenomenon is called sister chromatid cohesion.Sister chromatid cohesion is mediated by a special machine called cohesin, which consists of three protein subunits. Cohesin also plays important roles in processes apart from sister chromatid cohesion, such as regulation of which genes are used, and repair of damaged DNA. Although it performs a variety of tasks with DNA, cohesin appears to have a very simple mode to interact with DNA. The three subunits interconnect each other to form a huge protein ring structure, and the ring can open and close, allowing the DNA fiber to enter or exit the ring. Precise regulation of cohesin's association and dissociation with DNA is fundamental for its actions. Defects in this regulation compromise cohesin's function, which in humans would lead to cancer and inherited developmental disorders (such as Cornelia de Lange CdLS and Roberts syndromes).Cohesin cannot directly bind DNA in vivo and its DNA association requires another protein complex called Scc2/4. Interestingly, more than half of reported CdLS cases are due to defective Scc2. Although cohesin has been studied over twenty years, our knowledge of molecular details in its Scc2-dependent loading reaction has progressed very little. We know a key event during this loading reaction is the assembly of Scc2/cohesin pre-loading complex. Therefore, revealing the structure of this complex will greatly improve our understanding of how Scc2 recruits cohesin to DNA, which is the ultimate goal of this study.The architecture of cohesin complex has been established and a pseudo-atomic structure of the Scc2/4 complex was published recently. The question is how to join these two structures together to get a whole picture of the pre-loading complex. The key to this is to precisely map Scc2/cohesin interfaces or interaction sites. Our preliminary experiments discovered several regions of cohesin which are the Scc2-interacting sites. In this study, we will determine these interfaces using comprehensive genetic, biochemical, and biophysical approaches. All these results allow us to create a structural model of the Scc2/cohesin pre-loading complex, which will cast fresh light on the molecular mechanism of the cohesin loading. Insight into this fundamental process will help us understand how DNA segregation sometimes fails in cell division, as in cancer cells, or how our developmental programme goes wrong, which gives rise to cohesin-related diseases.