McIntyre, J.I.; (2007) Analysis of cohesin architecture and function in Saccharomyces cerevisiae. Doctoral thesis , University of London.

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Abstract

In this thesis I have undertaken a detailed molecular analysis of a conserved protein complex instrumental in genome stability. I have used the budding yeast Saccharomyces cerevisiae in conjunction with FRET (Fluorescence resonance energy transfer) to come up with a refined molecular architecture of the cohesin complex. This analysis involved the construction of an extensive panel of strains combining pair-wise FRET donor and acceptor fluorophores on subunits of the cohesin complex. This study has revealed many new and interesting insights into the functioning of the complex, which would not have been possible by many conventional biochemical techniques. For example we have shown that cohesin complexes exist as monomers using this analysis. The Sccl subunit could be mapped to the heads in a conformation somewhat different to current models. Current thinking depicts Sccl as a bridge between the otherwise distal Smcl and Smc3 heads. Here we show instead that Sccl likely adopts a conformation with its C-terminus sitting in a groove between the Smcl and Smc3 heads. It was also revealed that the Smcl and Smc3 heads are constitutively together. We also provide new information on the mode of interaction of Pds5 with the cohesin complex. Additionally I have generated mutant alleles of the Smcl and Smc3 subunits of the cohesin complex. These mutant proteins are impaired in ATP hydrolysis. Such mutants have afforded us the opportunity to assess when during the cohesin cycle ATP hydrolysis is required. I have shown that ATP hydrolysis is crucial for cohesin loading onto chromatin, with the mutant alleles showing substantially reduced kinetics of chromatin recruitment. Additionally, ATPase activity seemed not to be required for either cohesion establishment or cohesin relocation along chromosomes. This study provides us with crucial new molecular details on the functioning of the cohesin complex - a master regulator of genome stability.

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