Molecular mechanisms of cohesin

DNA has entered the modern lexicon and the concept that it instructs much of what our bodies do is now widely accepted. Less appreciated is the fact that each of the individual DNA molecules that are a crucial part of our chromosomes must be folded in space in a manner that enables them to fit within the nucleus, be transcribed under control of distant regulatory elements, be replicated and repaired, and in the case of sister DNAs be segregated to opposite sides of the cell during mitosis. The past decade has witnessed the discovery of a set of related molecular machines concerned exclusively with regulating chromosome topology. At the heart of these machines are a pair of rod-shaped Smc proteins whose association creates V shaped molecules whose vertices are connected by a third subunit called kleisin, forming a gigantic tripartite ring. While topo-isomerases alter the supercoiling or inter-catenation of DNAs, they cannot physically tie them together or organize them in space. This seems to be the function of Smc/kleisin ring complexes. Because they also regulate chromosome morphology in eu- and arche-bacteria, Smc/kleisin complexes are amongst the most conserved molecular machines concerned with controlling the physics and chemistry of DNA. Work on the cohesin complex suggests that its triparatite rings hold DNAs together by entrapping them. If true, it is likely that the condensin complex uses a similar principle. The long term goal of our research is to elucidate the mechanism by which cohesin rings entrap and subsequently release DNAs. This goal cannot be achieved without knowing more about the structure of cohesin's subunits and how they interact, which is the more immediate aim of this proposal. There are few if any aspects of chromosome biology that would not benefit from a better understanding of how Smc/kleisin complexes function at a molecular level. More specifically, cohesin dysfunction during meiosis in oocytes has been implicated in the genesis of aneuploidy, which in addition to causing Down's syndrome (Trisomy 21) is the leading cause of age-related infertility. Meanwhile, defects in the loading of cohesin onto chromosomes is thought to be the cause of Cornelia de Lange syndrome, a genetic disease associated with multi-system developmental defects. Genomic instability due to replication, repair, or segregation defects plays an important part during the genesis of many tumours. Cohesin has roles in all three of these fundamental processes and mutations affecting cohesin are frequently identified in tumours. Our work will therefore have important biomedical implications.

Work on cohesin suggests that its triparatite rings hold DNAs together by entrapping them. The ring is made of a pair of rod-shaped Smc proteins (Smc1 and Smc3) whose association creates V shaped molecules whose vertices are connected by a third subunit called kleisin (Scc1). The long term goal of our research is to elucidate the mechanism by which cohesin rings entrap and subsequently release DNAs. This goal cannot be achieved without knowing more about the structure of cohesin's subunits and how they interact. To understand DNA entry, we need to know more about the structure of a separate Scc2/4 loading complex thought to create an entry gate by opening up the Smc1/Smc3 hinge interface, how it interacts with cohesin, how it cooperates with cohesin's Scc3 subunit during the loading process, and more about the structure of the coiled coil arms of Smc1 and Smc3. Our recent work suggests that DNA escapes from cohesin rings through creation of an exit gate at the interface between Smc3's NBD and the N-terminal domain of Scc1. We have recently solved, at least partially, the structure of this interface. Our next task is to understand how cohesin's Pds5, Scc3, and Wapl subunits cooperate to dissociate it. To do this, we need to elucidate the structure of Pds5, preferably complexed with Wapl, then to visualize, using EM how the Pds5/Wapl subcomplex along with Scc3 interacts with the Smc3/Scc1 interface. Cohesin dysfunction during meiosis in oocytes has been implicated in the genesis of aneuploidy which in addition to causing Down's syndrome (Trisomy 21) is the leading cause of age-related infertility. Defects in the loading of cohesin onto chromosomes is thought to be the cause of Cornelia de Lange syndrome, a genetic disease associated with multi-system developmental defects. Genomic instability due to replication, repair, or segregation defects plays an important part during the genesis of many tumours. Our work will therefore have important biomedical implications.

Our findings on how cohesin functions at the molecular level will help understand the basis of cell division and our work will benefit a large number of academic and non-academic scientists working on different aspects of cell cycle research or on any research area involving cell division. For example, cohesin dysfunction during meiosis in oocytes has been implicated in the genesis of aneuploidy, which in addition to causing Down's syndrome (Trisomy 21) is the leading cause of age-related infertility. Also a defect in the loading of cohesin onto chromosomes is thought to be the cause of Cornelia de Lange syndrome, a genetic disease associated with multi-system developmental defects. In addition mutations affecting cohesin are frequently identified in tumours. Recurrent mutations and deletions involving multiple components of the cohesin complex, including STAG2, RAD21, SMC1A and SMC3, have been reported in different myeloid neoplasms. Our work will therefore have important biomedical implications.
The project will potentially identify new interacting proteins and regulatory pathways. This will be interesting for the scientific community as it will provide new knowledge and understanding of Smc/kleisin complexes like cohesin. This knowledge can be applied to enhance the research of related Smc/kleisin molecular machines concerned exclusively with regulating chromosome topology, like the Condensin and Smc5/6 complexes.
Our findings will also be potentially informative for pharmaceutical companies as dozens of proteins are required to regulate cohesin's activity, some of which are kinases or acetyl transferases that could be useful drug targets. Our research would directly feed into novel drug developments for the treatment of toumorigenic malignancies.
The researchers working on the project will gain extensive experience and advance in highly specialised molecular biology, biochemistry and structural biology techniques like cloning, protein expression and purification, X-ray crystallography, structure determination and analysis. These are highly useful skills that make them attractive assets to other labs and companies. Researchers will be able to continue their own academic careers and obtain desirable positions elsewhere benefitting themselves and the British economy.
We aim to publish the first results of our studies at the end of the first year with other publications following later into the grant. Training of researchers will continue throughout the grant and the impact of this training will live on past the duration of the grant. Novel intellectual property will be created throughout the duration of the grant and may create impact well beyond the duration of the grant.