Vorschau

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Abstract

The multi-subunit condensin protein complex plays essential roles in the structural organization of eukaryotic genomes. Its function is particularly important for the maintenance of mitotic chromosome structure during their segregation into daughter cells. Despite its key roles, the molecular mechanisms of condensin action have remained poorly understood. To shed light into the function of condensin complexes, I have analysed the nature of the interaction of condensin with its chromosomal substrate. The Structural Maintenance of Chromosomes (SMC) and kleisin subunits of condensin form a tripartite ring structure, which has been proposed to encircle chromatin fibres and thereby form intra-chromosomal linkages. To test this ‘ring model’, I generated covalently closed condensin rings by site specific cross-linking the interfaces between the Smc2, Smc4 and Brn1 kleisin subunits of budding yeast condensin complexes and then tested their interaction with small circular yeast minichromosomes. Chemical circularization of condensin complexes produced condensin-DNA species that remained even after protein denaturation. The experiments described in the first part of this thesis hence proof that condensin rings topologically encircle chromatin fibres. The finding that condensin encircles chromosomes implies that entry and exit of chromosomes into and out of condensin rings requires the temporary disengagement of at least one of the three ring subunit interfaces. In the second part of this thesis, I provide evidence that, in yeast, the Smc2-Brn1 interface forms a possible gate for DNA passage. I furthermore describe experiments that aim to test the requirement of this gate to open for either condensin loading or unloading from chromosomes in human cells. How might opening and closing of condensin rings be regulated? In the third part of this thesis, I tested whether the Scc2-Scc4 complex, which is required for the entrapment of sister chromatids in the condensin-related cohesin ring complex, plays a similar role for the topological loading onto chromosomes of condensin rings. My experiments demonstrate that depletion of Scc2-Scc4 from the nucleus, while inhibiting cohesin binding to chromosomes, has no notable effect on condensin’s chromosomal association. This suggests that the first step in loading mechanisms differs between the related complexes.