Richeldi, Martina; (2024) Mechanical principles of the cohesin-DNA interaction studied at a single-molecule level. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Cohesin forms a proteinaceous ring that is thought to link sister chromatids by entrapping DNA and counteracting the forces generated by the mitotic spindle. Whether individual cohesins encircle both sister DNAs and how cohesin opposes spindle-generated forces remains unknown. Here, to probe the maximum tension that cohesin can withstand, I performed force measurements on individual cohesin complexes either bound to one DNA or holding together two DNAs. By covalently closing the hinge and Smc3Psm3-kleisin interfaces, I find that the mechanical stability of the cohesin ring is determined by the hinge domain, which ruptures at ~20 piconewtons of external force. I also show that individual cohesin molecules can establish both double-stranded to single-stranded, as well as double-stranded to double-stranded DNA interactions. This supports an intuitive topological mechanistic picture in which cohesin physically holds two DNAs by entrapping them inside its ring. Forces of ~20 piconewtons disengage cohesin at the hinge and release DNA, both when bound to one or two DNA molecules, indicating that ~40 cohesin molecules are sufficient to counteract known spindle forces. These findings provide a mechanical framework for how cohesin interacts with sister chromatids and opposes the spindle-generated tension during mitosis, with implications for other force-generating chromosomal processes including transcription and DNA replication.

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