The mechanism by which chromosomal DNA molecules are held together: entrapment within cohesin rings?
Co-workers: Mrs Jean Metson, Dr Lana Strmecki, Dr Maurici Brunet Roig,
Dr Thomas Gligoris, Dr Johanna Scheinost, Ms Naomi Petela,
Dr Martin Houlard, Dr Madhusudhan Srinivasan, Mr James Rhodes, Dr Christophe Chapard
One of the most important concepts in biology is that the properties of individual cells are determined by the chromosomes that they inherit. A key observation leading to this notion was that cell division is preceded by the meristemic division of its nucleus, namely the condensation of its chromosomes from interphase chromatin, the splitting of chromosomes into a pair of closely apposed sister chromatids, and their subsequent disjunction to opposite poles of the cell prior to its division, a process known as mitosis. We now know that the hereditary material of chromosomes is DNA and that each chromosome contains a single immensely long molecule that is usually replicated many hours before cells actually enter mitosis. What has remained mysterious until recently is what holds sister DNAs together. It has long been suspected but never proven that this “sister chromatid cohesion” has a crucial role in ensuring that microtubules pull sister DNAs in opposite directions. Equally mysterious has been the trigger for what is arguably the most dramatic and one of the most highly regulated events in the life of a eukaryotic cell, the sudden disjunction of sister chromatids at the metaphase to anaphase transition.
Work in our lab has shown that sister chromatids are held together by a multi-subunit complex called cohesin whose Smc1 and Smc3 subunits are rod shaped proteins with ABC-like ATPases at one end of 50nm long intra-molecular anti-parallel coiled coils. At the other ends are pseudo-symmetrical hinge domains that interact to create V shaped Smc1/Smc3 heterodimers. N- and C-terminal domains within cohesin’s third subunit, known as α kleisin, bind to Smc3 and Smc1 ATPase heads respectively, thereby creating a huge tripartite ring whose integrity is essential for holding sister DNAs together. A thiol protease called separase opens the cohesin ring by cleaving its α kleisin subunit, which causes cohesin’s dissociation from chromosomes and triggers sister chromatid disjunction.
We have proposed that cohesin holds sister DNAs together by trapping them within its ring structure. As predicted by this hypothesis, linearization of a small circular chromosome permits chromatin fibres to slide through the cohesin ring and severs the connection between sister DNAs, while introduction of site-specific chemical cross links at the three interfaces between cohesin’s Smc1, Smc3, and a kleisin subunits traps circular sister DNAs inside a single, albeit huge, circular cohesin molecule even after protein denaturation.
The ring hypothesis raises many questions: does transient dissociation of cohesin’s hinge domains permit DNA entry, do its ABC-like ATPases facilitate this process and if so what is the role of the Scc2/Scc4 complex, how are sister DNAs trapped within a single ring, how are DNAs prevented from exiting the ring, how is the ring opened in prophase during mitosis when much cohesin (at least in animal cells) dissociates from chromosome arms without cleavage by separase, how is the latter prevented at centromeres by Sgo1, how stable are the connections between sister DNAs mediated by cohesin and can they be regenerated in the decade long period between pre-meiotic DNA replication and ovulation in humans, and finally does cohesin have an important role in regulating gene expression during interphase as suggested by the developmental defects associated with Cornelia de Lange syndrome, which is caused by Scc2 haplo-insufficiency? The ring hypothesis proposes a novel topological mechanism for connecting DNAs, namely protein-DNA catenation. It might be appropriate therefore to consider cohesin as a novel type of catenating enzyme or concatenase. A similar principle might lie behind the function of condensin, the Smc5/6 complex, and bacterial Smc-kleisin complexes. If so, protein-DNA catenation might be essential for the organization and segregation of all genomes on this planet. Much of our work is done using the yeast Saccharomyces cerevisiae but we also study the regulation of cohesion in mouse oocytes and the role of cohesin in postmitotic cells in Drosophila.
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Figure 1: Structural model of the tripartite cohesin complex consisting of Smc1 (red), Smc3 (blue) and Scc1 (green) proteins encircling two 10nm sister chromatids, e.g. DNA (gold) wrapped around nucleosomes (grey)
Figure 2: Still image from a live recording of a mouse oocyte at the metaphase II stage. The mitotic spindle is labelled by Tubulin-EGFP in green, chromosomes are labelled by histone H2B-mCherry in red
Figure 3: Polytene chromosome spreads of Drosophila larvae expressing a TEV-cleavable Rad21 cohesin subunit. After induction of TEV-protease, Rad21 (stained in green) is lost from chromosomes while the association of other chromosomal proteins (e.g. the enhancer-blocker protein CTCF, stained in red) remains unaltered.