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Title: Probing the cohesin loading reaction using forward genetics and quantitative ChIP-sequencing
Author: Petela, Naomi
ISNI:       0000 0004 6421 5473
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2016
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Cohesion between sister chromatids that are newly formed in S-phase is crucial to ensure the fidelity of chromosome segregation. The cohesin complex mediates cohesion and is comprised of two SMC proteins, Smc1 and Smc3, with a kleisin subunit, Scc1. The SMCs interact via their hinge domains to form a v-shaped heterodimer, with Scc1 forming a bridge between their head domains to produce a ring structure that topologically entraps sister chromatids. In order to confer cohesion, cohesin needs to be loaded onto DNA, a process that is poorly understood. It is known that cohesin loading depends on a loading complex comprised of Scc2 and Scc4, ATP hydrolysis and presumably opening of at least one of the interfaces of the cohesin ring. Using the recently developed technique, quantitative ChIP-seq, we show that Pds5, a cohesin-associated protein, is displaced at centromeres from the cohesin ring during the loading reaction. This event is accompanied by the engagement of SMC ATPase heads in the presence of ATP. Upon ATP hydrolysis, the loading reaction completes with DNA entrapment, translocation to the pericentromeric sequences and the displacement of Scc2 by Pds5. We also describe mutations in Scc2, Smc1 and histone proteins H2A and H2B that enable loading in the absence of Scc4. The mutations in Scc2 alter the dynamics of the association between the loading complex and cohesin, reducing dissociation during the translocation step. All of the mutations are able to restore loading along chromosome arms but not at centromeres where Scc4 has a particular role. However, the Smc1 mutation hinders loading at the centromere, even in the presence of Scc4, due to a defect in the second step of the loading reaction, namely translocation driven by ATP hydrolysis. We suggest that this discrepancy in functionality at the arms and centromere is due to changes in the hinge accompanying both the first and second steps. The rate limiting reaction is the first step at arms and the second at the centromeres. The histone mutants show that nucleosome occupancy plays an important role, at least on chromosome arms, but we find no evidence that this involves the RSC complex.
Supervisor: Nasmyth, Kim Sponsor: Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available