Exploring SMC motor proteins and how to stop them

Abstract

Genetic information encoded in the DNA sequences is maintained, replicated, and transcribed across the tree of life. Organisms evolved multiple hierarchical layers of chromosome organization which ensure that DNA can be contained but also processed within individual cells. This thesis focuses on SMC (Structural Maintenance of Chromosomes) protein complexes, an evolutionarily conserved family of motor proteins that hold sister chromatids together and fold genomes throughout the cell cycle by DNA loop extrusion. These complexes play a key role in a variety of functions in the packaging and regulation of chromosomes, and they have been intensely studied in recent years. Despite their importance, the detailed molecular mechanism for DNA loop extrusion by SMC complexes remains unresolved. In this thesis, we utilized single-molecule fluorescence and magnetic tweezers to study the intricacies of DNA loop extrusion by SMC complexes in a controlled in vitro environment. We observed, described, and analyzed the behaviour of SMCs which yielded detailed yet important contributions to the resolution of the DNA loop extrusion mechanism as well as to the physiological impact on genome structure. In particular, we observed that large DNA-tethered roadblocks cannot stall loop extrusion efficiently and concluded that DNA loop extrusion can occur in a non-topological manner. In contrast, we found that a small DNA-binding protein, CCCTC-binding factor (CTCF) stalls cohesin through direct protein-protein interactions with cohesin’s N-terminal region. Furthermore, we could reconcile previous results that suggested different loop extrusion mechanisms for different members of the SMC protein family. We demonstrated that all eukaryotic SMCs extrude DNA asymmetrically, i.e. only from one side at a time, and that frequent direction switches yield the previously observed ‘symmetric’ loop extrusion. Furthermore, we showed that DNA supercoiling is intimately linked to loop extrusion for all eukaryotic SMC complexes, postulating a common DNA loop extrusion mechanism.