A Multi-Step Pathway for the Establishment of Sister Chromatid Cohesion

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Abstract
The cohesion of sister chromatids is mediated by cohesin, a protein complex containing members of the structural maintenance of chromosome (Smc) family. How cohesins tether sister chromatids is not yet understood. Here, we mutate SMC1, the gene encoding a cohesin subunit of budding yeast, by random insertion dominant negative mutagenesis to generate alleles that are highly informative for cohesin assembly and function. Cohesins mutated in the Hinge or Loop1 regions of Smc1 bind chromatin by a mechanism similar to wild-type cohesin, but fail to enrich at cohesin-associated regions (CARs) and pericentric regions. Hence, the Hinge and Loop1 regions of Smc1 are essential for the specific chromatin binding of cohesin. This specific binding and a subsequent Ctf7/Eco1-dependent step are both required for the establishment of cohesion. We propose that a cohesin or cohesin oligomer tethers the sister chromatids through two chromatin-binding events that are regulated spatially by CAR binding and temporally by Ctf7 activation, to ensure cohesins crosslink only sister chromatids. Complexes containing members of the structural maintenance of chromosomes (Smc) family regulate higher order chromosome architecture in diverse aspects of DNA metabolism including chromosome condensation, sister chromatid cohesion, DNA repair, and global control of transcription. Smc complexes are thought to regulate higher order chromosome folding by tethering together two strands of chromatin. However, the mechanism of tethering is poorly understood in part because of a poor understanding of the function of the core Smc subunits. To gain insight into the structure and function of Smc subunits, we developed a novel strategy of mutagenesis called random insertion dominant negative (RID), which generates informative alleles with high efficiency and should provide an effective tool to study any multi-subunit complex. Using RID we generated novel alleles of a Smc subunit from the cohesin complex. The cohesin complex tethers together newly replicated chromosomes (sister chromatids). The analyses of these RID mutants suggest that the tethering activity of cohesin (and possibly other Smc complexes) is generated by two sequential chromatin-binding events (first the capture of one piece of chromatin followed by the capture of the second piece of chromatin), which are regulated both spatially and temporally. We speculate that the spatial and temporal regulation of cohesin ensures that it tethers together only sister chromatids rather than randomly crosslinking the entire genome.