Abstract
Cohesin helps mediate sister chromatid cohesion, chromosome condensation, DNA repair, and transcription regulation. We exploited proximity-dependent labeling to define the in vivo interactions of cohesin domains with DNA or with other cohesin domains that lie within the same or in different cohesin complexes. Our results suggest that both cohesin's head and hinge domains are proximal to DNA, and cohesin structure is dynamic with differential folding of its coiled coil regions to generate butterfly confirmations. This method also reveals that cohesins form ordered clusters on and off DNA. The levels of cohesin clusters and their distribution on chromosomes are cell cycle-regulated. Cohesin clustering is likely necessary for cohesion maintenance because clustering and maintenance uniquely require the same subset of cohesin domains and the auxiliary cohesin factor Pds5p. These conclusions provide important new mechanistic and biological insights into the architecture of the cohesin complex, cohesin-cohesin interactions, and cohesin's tethering and loop-extruding activities.
Highlights
Chromosome segregation, DNA damage repair, and the regulation of gene expression require the tethering or folding of chromosomes (Uhlmann, 2016; Onn et al, 2008)
We inserted AviTags at each of the four positions in Smc3p of Saccharomyces cerevisiae. These AviTag reporters were positioned in the head domain, within the coiled coil proximal to the head (CC reporter, in the joint after residue V966), at the elbow-proximal coiled coil, and in the hinge domain (Figure 1B, see Figure 1—figure supplement 1B for tag insertion sites in the elbow folded structure suggested in Burmann et al, 2019)
We present additional data supporting the biotinytlation of doubletagged Smc3p occurs in cis for the fully assembled cohesin complex
Summary
Chromosome segregation, DNA damage repair, and the regulation of gene expression require the tethering or folding of chromosomes (Uhlmann, 2016; Onn et al, 2008) These different types of chromosome organizations are all mediated by a conserved family of protein complexes called structural maintenance of chromosomes (SMC) (Onn et al, 2008; Nolivos and Sherratt, 2014; Hirano, 2016; Hassler et al, 2018). The separation of head and hinge dimers by their intervening long coiled coils allows the Smc dimers to achieve multiple conformations in vitro, including rings, rods, or butterflies (Figure 1A). To begin to answer these many unanswered questions about cohesin clustering and architecture, we interrogated cohesin with the method of proximity-dependent biotinylation (Fernandez-Suarez et al, 2008; Jan et al, 2014)
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