Tang, Minzhe; (2022) The molecular mechanism of condensin in mitotic chromosome formation. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The rearrangement of interphase chromatin into mitotic chromosomes is crucial for faithful chromosome segregation. Defects in this process may cause chromosome entanglement, decompaction, chromosome bridges during anaphase, and eventually genome instability and cell death. The key player for this process was discovered to be condensin, which is a member of the structural maintenance of chromosome (SMC) complex with a conserved pentameric ring structure. However, the exact role of condensin in mitotic chromosome formation is still under debate. Three prevalent models were proposed to explain the role of condensin in mitotic chromosome condensation, namely the diffusion capture model, the torsion-mediated compaction model, and the loop extrusion model. The diffusion capture model proposes that condensin stabilizes stochastic chromatin interactions, via sequential topological entrapment or condensincondensin interaction, thereby forming and maintaining chromatin loops that compact chromatin. The torsion-mediated compaction model proposes that condensin introduces and maintains torsional strain that structures chromatin into a series of plectonemes, thereby compacting the chromatin. The loop extrusion model proposes that condensin and/or other DNA translocators enlarges chromatin loops anchored by condensin, thereby shrinking the chromatin lengthwise. Each model has its own weak points that still require further characterization. Therefore, my PhD project focuses on characterizing the condensin-DNA interaction in vitro with the hope to provide evidence for one or more of these models. Using purified fission yeast condensin, I reconstituted topological condensin loading onto DNA in vitro. I found that topologically loaded condensin can be subsequently unloaded from DNA in an ATP-dependent manner, recapitulating the regulated turnover of condensin on chromatin in vivo. Importantly, I discovered that condensin can sequentially topologically entrap two dsDNA molecules in vitro. Using single-molecule microscopy, I later confirmed that both the topological DNA loading and second dsDNA capture could be mediated by a single condensin complex. These observations provided a solid ground for the diffusion capture model of mitotic chromosome assembly by condensin.

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