SMC complexes (cohesin, condensin, MukBEF and others) are essential organisers of genomes in all domains of life. At the centre of their activity is the ability to alter the path of DNA in three dimensions in an energy-dependent fashion. The most conserved function of SMC complexes is their ability to generate DNA loops through a process called loop extrusion, while cohesin also facilitates sister chromatid cohesion during cell division. SMC complexes display a conserved architecture and therefore at least some molecular mechanisms are expected to be shared between SMC complexes such as condensin, cohesin and their bacterial counterparts such as MukBEF.

Several key mechanistic questions have remained enigmatic, such as the role of the SMC-kleisin ring structure for their various activities, including loop extrusion, the precise role of the ATPases and the conformations the complexes adopt during their reactions. The main goal of this project is to combine our recently acquired ability to image SMC complexes at high resolution by cryo-EM with loop extrusion reactions, to resolve the enzyme at near-atomic resolutions while it is generating loops of DNA.

The student will adapt the established cohesin loop extrusion assays and will verify the reactions by TIRF microscopy. They will then transfer the reactions onto EM grids. Imaging loop extrusion directly will use sophisticated modern cryo-EM and will be used to sort apart different states of the enzyme during the reaction cycle. It may be required to shift to electron cryotomography (cryo-ET), if single particle approaches prove difficult, for example because of flexibility. After obtaining relevant EM maps, the student will build atomic models and analyse them. Time permitting, we will also add CTCF protein and include CTCF sites on the DNA, to image site-directed loop formation, which is the basis for the formation of TADs in chromosomes.

References

Mechanism of DNA capture by the MukBEF SMC complex and its inhibition by a viral DNA mimic
Bürmann F, Clifton B, Koekemoer S, Wilkinson OJ, Kimanius D, Dillingham MS, Löwe J
Cell 188(9): 2465-2479.e14 (2025)

Structural biology of SMC complexes across the tree of life
Bürmann F, Löwe J
Current Opinion in Structural Biology 80: 102598 (2023)

Cryo-EM structure of MukBEF reveals DNA loop entrapment at chromosomal unloading sites
Bürmann F, Funke LFH, Chin JW, Löwe J
Molecular Cell 81(23): 4891-4906.e8 (2021)

Cryo-EM structures of holo condensin reveal a subunit flip-flop mechanism
Lee BG, Merkel F, Allegretti M, Hassler M, Cawood C, Lecomte L, O’Reilly FJ, Sinn LR, Gutierrez-Escribano P, Kschonsak M, Bravo S, Nakane T, Rappsilber J, Aragon L, Beck M, Löwe J, Haering CH
Nature Structural & Molecular Biology 27(8): 743-751 (2020)