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The ATP-dependent mechanism of cohesin function in chromosome segregation

Weitzer, Stefan; (2004) The ATP-dependent mechanism of cohesin function in chromosome segregation. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Equal chromosome distribution during mitotic cell divisions is necessary for maintaining genomic stability in eukaryotes. An essential prerequisite is the alignment of sister chromatid pairs in metaphase. Pairing or cohesion between sister chromatids is established during DNA replication and is promoted by the chromosomal cohesin complex. The budding yeast Saccharomyces cerevisiae cohesin complex consists of four core subunits, Smcl and Smc3, both members of the Structural Maintenance of Chromosome (SMC) protein family, and the Sccl and Scc3 subunits. At the anaphase onset cohesion is suddenly lost by proteolytic cleavage of cohesin's Sccl subunit, leading to dissociation of cohesin from chromosomes and separation of sister chromatids getting pulled towards opposite cell poles by spindle microtubules. The mechanism by which cohesin binds to DNA initially, how cohesion is established during DNA replication and how cohesin dissociates from chromosomes in anaphase, is unknown. In this study, cohesin bound to chromatin which likely represents the functional pool of the complex, was biochemically characterised. Cohesin was found to associate with chromatin in clusters but size, shape and subunit composition does not change during cohesion establishment. This suggests that the molecular function of cohesin is inherent of the complex and may have to be sought in its characteristic architecture and conserved domains. Cohesin's Smc1 and Smc3 subunits are largely composed of long stretches of antiparallel intramolecular coiled coils which are flanked at one end by putative ATP- Binding Cassette (ABC) ATPase head domain. Heterodimerisation of Smc1 and Smc3 results in the formation of a proteinaceous ring, large enough to embrace two strands of DNA which has lead to the hypothesis that cohesion is mediated by entrapment of both sister chromatids within the ring. This study shows that cohesin has indeed ATP binding activity. The two SMC subunits by themselves form a ring, closed at their interacting ATPase head domains in an ATP-dependent and independent fashion. Disruption of this interaction and opening of the ring is triggered by a cleavage fragment of the Scc1 subunit. To assess the role of ATP in cohesion, point mutations were introduced that were designed to prevent ATP binding or hydrolysis by the Smc1 subunit. ATP binding was found to be essential for cohesin complex assembly whereas a motif implicated in ATP hydrolysis is required for loading of cohesin onto DNA. In addition, an intact SMC ring is indispensable for DNA binding, indicating that ATP hydrolysis may be coupled to DNA transport into the ring. These data suggest that ATP hydrolysis is necessary for loading of cohesin onto chromatin, whereas a prerequisite for DNA unloading of cohesin during anaphase is the disruption of the ring promoted by a Scc1 cleavage fragment. The analysis of ATP function in the context of cohesin's ring structure contributes to a biochemical understanding of the establishment and resolution of sister chromatid cohesion. In addition, since ring structure and functional domains appear to be conserved within related Smc protein complexes, similar mechanisms might apply to their multiple roles in chromosome biology like DNA condensation.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: The ATP-dependent mechanism of cohesin function in chromosome segregation
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Mitotic cell division
URI: https://discovery.ucl.ac.uk/id/eprint/10098557
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