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

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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.