Closing the cohesin ring round sister chromatids
A new study completes the structural and functional picture of the cohesin ring that traps DNA during mitosis until the critical moment of cell division.
Crystal structure of the Smc3 head with coiled coil section (Smc3hdCC) in complex with the N-terminal domain of kleisin (Scc1-N). The Smc1 head domain (Smc1hd) is shown in red in dimer formation with the Smc3 head (Click to enlarge)
The researchers behind the work, from Kim Nasmyth's lab in the department and the lab of Jan Löwe at the MRC LMB in Cambridge, describe their findings in a recent paper in Science (1).
Using the new structural information, the group has confirmed the presence of the tripartite cohesin ring in living cells and can begin to look at the mechanisms behind cohesin's functions.
Cohesin is a highly conserved complex comprised of subunits Smc1 and Smc3 forming a v-shaped conformation. These associate with a kleisin subunit to form what is believed to be a ring that holds sister chromatids together during mitosis until exactly the right time for their separation (2).
The interface between Smc3 and kleisin is a key regulatory one. Smc3 acetylation ensures that the ring is locked tightly shut, but release can be triggered by regulatory subunits that allow exit of the sister DNAs.
As Thomas Gligoris, one of the postdocs in the Nasmyth lab, explains, an understanding of this interface has so far eluded researchers. 'We had the crystal structure of the Smc1/Smc3 interface and also the Smc1/kleisin interface but were missing the structure of the Smc3/kleisin interface.'
He and colleagues in the Nasmyth lab collaborated with Jan Löwe to determine the missing crystal structure. With this in hand, they used biochemical studies to identify the regions of Smc3 and kleisin at the interface. They found that these included a highly conserved coiled coil region of Smc3, emerging from the ATPase head that binds to kleisin's N-terminal domain.
Team effort: Naomi Petela, Thomas Gligoris and Johanna Scheinost from the Nasmyth lab
'We found that this coiled coil region is unique to Smc3 and is critical for Smc3's interaction with kleisin,' says Dr Gligoris. Although both Smc1 and Smc3 have ATPase heads that interact with the kleisin, their interactions turn out to be asymmetrical. 'All biochemical studies so far have been based on the assumption that the two subunits bind in the same way to the kleisin, but this appears not to be the case.'
By following these studies with mutational analysis of the Smc3/kleisin interface in S.cerevisiae, the group confirmed that the interface is functionally important for holding the cohesin ring together. Since the components of the cohesin complex are so well conserved across organisms, the group expects that this will hold true in other eukaryotes - a finding backed up by a paper from a different group also published in Science.
'Another group has been working on human cohesin,' says Dr Gligoris. 'They did mass spectroscopy rather than x-ray crystallography and verified what we found in yeast cohesin. Although they used a different approach, they identified the same important region of Smc3.'
The next step was to use the new structural information to test whether interaction of the three Smc/kleisin interfaces creates rings in living yeast cells. Postdoc Johanna Scheinost, together with DPhil student Naomi Petela, optimised a chemical cross-linking procedure that they had already been using, to get a snapshot of what is going on in living cells.
'We were able to engineer thiol-specific chemical cross-links at the Smc3/kleisin interface as well as at the two other interfaces,' explains Dr Scheinost. 'When we cross-linked all three interfaces, we could generate a stable ring.'
The group believes that most cohesin in the cell adopts a stable single ring structure comprised of the three subunits Smc1, Smc3 and the kleisin.
Using the cross-linking approach but with simple circular chromosomes as templates for cohesin, they went on to look at how the DNA is held within the cohesin ring in living cells. After cross-linking all three interfaces, they pulled down cohesin to identify the active DNA species in the ring. They found both unreplicated and replicated chromosomes trapped within it, as would be expected for a population of cycling cells.
Dr Gligoris comments that the group's findings will enable researchers to start teasing apart the mechanisms underlying cohesin function - for example, how cohesin loads onto DNA. 'The region we've looked at is important - it opens up for DNA and is sealed tightly with acetylation of Smc3. Our work is a necessary step towards understanding the mechanisms involved.'