SMC complexes (cohesin, condensin 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.

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

Bürmann F, Löwe J (2023) Structural biology of SMC complexes across the tree of life. Curr Opin Struct Biol. 80:102598

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

Collier JE, Lee BG, Roig MB, Yatskevich S, Petela NJ, Metson J, Voulgaris M, Gonzalez Llamazares A, Löwe J, Nasmyth KA., (2020) Transport of DNA within cohesin involves clamping on top of engaged heads by Scc2 and entrapment within the ring by Scc3. Elife 9:e59560

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., (2020) Cryo-EM structures of holo condensin reveal a subunit flip-flop mechanism. Nat Struct Mol 27:743-751