Investigations into the regulation of sister chromatid cohesion

Thesis

Cohesin, a ring-shaped complex composed of four subunits, is an essential player involved in timely and accurate segregation of genetic material at mitosis and meiosis. Cohesin performs a highly conserved role by topologically embracing sister chromatids until their concerted disjunction at anaphase, when the α-kleisin subunit of the ring is irreversibly cleaved by separase. A central part of the cohesin cycle is its cleavage-independent removal from chromatin through the action of the releasing complex.

In more complex eukaryotes, stabilisation of cohesin on DNA depends on the presence of sororin, a small functionally conserved protein whose association with cohesin is thought to counteract the releasing reaction. The first part of this thesis aimed to address the discrepancy of a sororin orthologue never having been identified in S. cerevisiae. This was undertaken using an imaging-based approach to screen a subset of the yeast genome for proteins with cohesin-like localisation. The screen was streamlined such that candidate genes had periodically cycling mRNA transcripts, similarly to cohesin subunits. No novel cohesin-associated proteins could be identified, although this could be in part due to technical limitations of the screen.

The precise molecular details of cohesin’s function and regulation remain poorly understood. A conclusive way to address many questions about cohesion as a whole would be to set up an in vitro assay capable of reconstructing cohesin loading, cohesion establishment, and the releasing reaction. The second part of this thesis aimed to establish a reliable purification protocol for the S. cerevisiae cohesin complex using a bacterial expression system for use in such biochemical assays. While purification of all components of the cohesin trimer (Smc1, Smc3, Scc1) was achieved successfully, the complex could not be confirmed to assemble functionally, as determined by chemical crosslinking of the Smc3/Scc1 interface.

Authors: Kozyrska, K

• Publication date: 2014
• Type of award: MSc by Research
• Level of award: Masters
• Awarding institution: University of Oxford
• Language: English
• Keywords: protein purification; yeast; cohesin; sister chromatid cohesion; sororin
• Subjects: Biochemistry