A microscopic study of cohesin

Cohesin is a ring-shaped Smc (Structural Maintenance of Chromosomes) complex that mediates DNA-DNA interactions between chromosomes (sister chromatid cohesion) and within chromosomes (looping). The mechanisms behind loop formation and cohesion establishment are not known despite cohesin's discovery 20 years ago. In this thesis findings are presented from two projects: one examining the establishment of sister chromatid cohesion and the other the formation of DNA loops by cohesin.
Cohesin can remain associated with chromosomes during DNA replication – Sister chromatid cohesion is established at the DNA replication fork. It is not known whether cohesin that is loaded ahead of the fork is used to build cohesion or if it is dislodged and cohesion is built from cohesin rings in the soluble pool. To address this the objective was to determine whether cohesin removal from chromatin is a necessary aspect of DNA replication. Cohesin was fluorescently labelled in a cell line in which cohesin’s normal releasing activity was abrogated and cohesin’s association with chromatin was followed across S phase. It was found that cohesin could remain in defined subnuclear areas demonstrating that the complex can remain bound to DNA despite replication fork passage. This finding is consistent with the conversion of cohesin that is loaded ahead of the replication fork into its cohesive state.
The cohesin loading factor Scc2 hops between cohesin rings – The human genome is organised into a series of megabase scale domains that interact with themselves more than sequences outside of the domain. Interactions inside theses Topologically Associating Domains (TADs) depend on the cohesin complex and are postulated to form by progressive loop enlargement through cohesin's lumen (loop extrustion). If this is the mechanism then cohesin must be able to translocate vast distances. Recent evidence suggests that Smc complexes are motor proteins and their translocation depends on ATP hydrolysis. Cohesin’s ATPase is stimulated by Scc2 (Nipbl) however this protein is thought to be involved exclusively during the cohesin loading reaction. Consistently, little colocalisation is detected between cohesin and Scc2 as measured by chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq). The aim of this project was to determine whether cohesin and Scc2 interact on DNA after the loading reaction. Using fluorescence recovery after photobleaching (FRAP) and single particle tracking it has been shown that Scc2 turns over rapidly on chromatin indicative of an association independent of cohesin loading, a process that is infrequent due to cohesin’s long residence time. It was also found that stabilisation of cohesin on DNA leads to increased Scc2 chromosomal association and cohesin depletion leads to reduced association. These findings provide evidence for a post-loading association between cohesin and Scc2. Intriguingly, Scc2 was also found to spread across chromatin and cohesin vermicelli very slowly consistent with hopping between cohesin rings. Fluorescence analysis of Scc2 and Scc1 indicate that Scc2 is substoichiometric to cohesin. It was therefore proposed that due to an abundance of binding sites for Scc2, a low diffusion coefficient and a high on-rate, that Scc2 hops between cohesin rings performing a function distinct from loading.

Advisor: Nasmyth, K