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

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.

Cohesin is amember of Structural maintenance of Chromosome (SMC) family and highly conserved throughout the whole eukaryotic lineage. It regulates various cellular processes, including sister chromatid cohesion, DNA damage repair, DNA condensation and gene transcription. The association and dissociation of cohesin with DNA are highly regulated to determine when and where cohesin functions on DNA. Cohesin cannot directly bind DNA and its DNA association depends on the loading complex Scc2/4. Cohesin interacts with Scc2/4 to form a pre-loading complex, which can be recruited to DNA. Although the architecture of cohesin and the structure of Scc2 have been revealed, how this pre-loading complex is assembled and what is the nature of this interaction still remain enigmatic.
To unveil the configuration of the pre-loading complex, two key questions have to be addressed first. What is the cohesin conformation in the pre-loading complex? Our preliminary experiment revealed that a major of cohesin complex exhibits a conformation with juxtaposed coiled-coils and disengaged heads, which is consistent with other studies from prokaryotic Smc-ScpAB and yeast condensin. However, our previous genetics data implied that the head engagement is essential for the pre-loading process. Given that a key function of Scc2 is to trigger ATP hydrolysis-a process requiring the head engagement, we expected that Scc2 should interact with cohesin in a configuration with apart coiled coils and engaged heads. The first part of this proposal will examine the configuration of coiled-coils and heads of cohesin when interacting with Scc2. In the second part of this study, we will address how Scc2 interacts with this cohesin by mapping their interaction sites in vivo using a site-specific crosslink. All the information obtained from this study allows us to construct a structural model of the pre-loading complex, which will provide novel insights into the molecular mechanism of cohesin loading.

The project aims to unveil the molecular detail of the regulation of cohesin's DNA association dynamics. Because cohesin plays fundamental roles in cell proliferation and development in all eukaryotic organisms and its defects in human cause many diseases including cancer and genetic disorders, this work will benefit not only scientific society but also the general public.
This project will deliver scientific, clinical and educational impact.
This study will reveal a structural basis of Scc2/cohesin interaction, which will greatly bring forward our understanding of how cohesin be recruited to DNA. This information promotes the research involving cohesin and chromosome organisation. In this study, we use yeast as a model organism. Because the function and regulation of cohesin are highly conserved throughout the whole eukaryotic lineage, the knowledge obtained from this study can extend to mammalian system, which will reduce the need for animal experiments. Moreover, the scientific impact will be through stimulation of research in the broader field of study of Smc genes/proteins, which in turn will create employment and training opportunities for the UK scientific community.
The clinical impact will not be immediate, but in the long-term the knowledge gained from this study will feed into clinical studies. Our data will be useful to clinicians in developing precise diagnoses and a better understanding of the molecular cause of disorders such as Cornelia de Lange (CdLS) and Roberts syndromes and cancer. We predict that there will be a future interest within the pharmaceutical industry for the development of drugs that modify cohesin function in a range of different pathways. Thus, the study of cohesin function at molecule level will provide a necessary knowledge to understand their cause and to later develop and to design a new strategy for their prevention and treatment.
Currently, there are more than 300 children and adults suffering from Cornelia de Lange syndrome in Britain and Ireland. The fact of lack of efficient treatment might make the patients and their families feel in despair. This study will elucidate the function of Scc2, a protein accounting for more than 60 % of cases of CdLS. Revealing the cause of their disorders would bring new hope for the new treatment to patients and their families, which could improve the quality of their lives.
This project will provide excellent opportunities for young researchers working on this project to develop his/her research capability. With the support for this project, we will employ a PDRA and a research technician. Every year, at least one 4th year research student from my department will join this research as the final research project. They will receive training in all areas proposed in this project and obtain essential skills for their future career. Clearly, this will make a significant contribution to the continuous success of the UK science and economy.
The general public including school children will also benefit from this study as their knowledge and understanding of science and how their cells work will be increased. The Public Engagement & Impact team at the University of Sheffield creates various platforms and provides many opportunities to support the investigator to communicate with the public about our research projects. This will help the general public to understand what we are doing in the laboratory and why it is important for our society and our future. In return, our research will receive more attention and support from the public. We will work with local secondary schools to run a series of classes to introduce the latest discovery in our research and explain how this contributes to our knowledge of modern biology and how this will influent our life in the future. We will provide A-level students to gain research experience by working with us. This will exploit their potentials in science and inspire them to become the future scientists.