Uhlmann lab Chromosome Segregation Laboratory
: Biochemical reconstitution of topological DNA binding by the cohesin ring
Introduction
The cohesin complex is a central player in chromosome biology. Defects in cohesin and its regulators are responsible for chromosome missegregation in many human malignancies. They are also the cause for Cornelia de Lange syndrome, a severe developmental disorder.
The genomic DNA that makes up the chromosomes is replicated during S-phase of the eukaryotic cell cycle. After replication, the two newly synthesised sister chromatids remain connected with each other by the chromosomal cohesin complex.
Our lab has contributed over the years to understand how this ring-shaped multi-subunit protein complex works to build sister chromatid cohesion. Sister chromatid cohesion forms the basis for the pairwise alignment of DNA replication products on the spindle apparatus in mitosis, to allow their faithful segregation into daughter cells. Cells defective in sister chromatid cohesion make errors in chromosome segregation, giving rise to aneuploid cells that lack or contain extra copies of chromosomes.
Aneuploidy is a hallmark of malignant tumour progression. Human heritable mutations that compromise the fidelity of chromosome segregation are inevitably linked to early onset tumourigenesis. This year, we have made progress towards understanding how cohesin works as a fascinating molecular machine that holds sister chromatids together
The cohesin complex consists of at least four subunits that together form a large proteinaceous ring. It is thought that cohesin holds together sister chromatids by topologically embracing them. While the embrace model provides an important conceptual framework for sister chromatid cohesion, it leaves many fundamental question wide open. If cohesin topologically embraces DNA, how does the DNA get into the ring and where on chromosomes can this reaction happen? Equally, how does DNA come out of the ring again during cohesin's dynamic DNA binding cycle? Finally, cohesin incorporates an ABC-type ATPase that is required for its function, so how does the ATPase fuel cohesin's activities? Definitive answers to these questions require that we can study cohesin's behaviour in vitro, however, the biochemical reconstitution of cohesin activity has remained an unattained goal.
Figure 1: Biochemical reconstitution of topological cohesin loading onto DNA. a) Purification of fission yeast cohesin and the cohesin loader complex after overexpression of their respective subunits. The final gel filtration steps of the purification are shown. Fractions were analysed by SDS polyacrylamide electrophoresis followed by Coomassie blue staining. b) Schematic of the cohesin loading reaction. c) A circular DNA substrate is required for the loading reaction, hinting at the topological nature of cohesin binding. The input and bead-bound fractions, following the loading reaction, are shown and were quantified. The mean and standard deviation of three independent experiments are shown.
We were now successful in expressing and purifying the fission yeast cohesin complex, as well as its Mis4/Ssl3 cohesin loading factor, which is essential for cohesin function in vivo (Figure 1). Incubation of cohesin with DNA led to spontaneous topological loading of cohesin onto DNA, in an ATP hydrolysis-dependent fashion, but this reaction remained inefficient. Addition of the cohesin loader stimulated ATP hydrolysis and cohesin loading onto DNA. We found that the cohesin loader contacts cohesin at multiple sites around the ring circumference. One of these contacts lays on cohesin's Psc3 subunit, an essential yet hitherto enigmatic part of the cohesin complex.
Using mutational analysis and peptide competition experiments, we showed that at least three loader contacts along the cohesin ring coordinately stimulate the cohesin loading reaction. Considering the large dimensions of the cohesin ring, it is likely that a conformational rearrangement must take place to accommodate three simultaneous contacts of the loader with cohesin. The cohesin loader can thus be thought of as a template or mould onto which cohesin holds onto, to facilitate the loading reaction.
Our in vitro reconstitution of cohesin loading onto DNA provides mechanistic insight into the initial steps of the establishment of sister chromatid cohesion and other chromosomal processes mediated by cohesin. The results are important not only to understand cohesin, but also the ubiquitous family of chromosomal structural maintenance of chromosomes (SMC) complexes, of which cohesin is a member, that share essential functions in various chromosomal activities in all organisms from bacteria to humans.
Now that we have gained the ability to investigate cohesin behaviour in vitro, we would like to directly observe cohesin's loading onto DNA. We will use a combination of biochemical, structural, single molecule and imaging approaches to do this. In particular, single molecule FRET-based assays and electron microscopy have the potential to shed unprecedented insight into both the cohesin loading reaction as well as the final product of the reaction, the cohesin ring on DNA. Once cohesin is loaded onto DNA, the probably most exciting time during its residence on chromosomes comes during DNA replication in S-phase. Now two sister chromatids are synthesised that cohesin will hold together. We will extend our biochemical assays to address how cohesin identifies the two replication products and establishes linkages between them.
