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

Mark Milutinovich,Douglas Koshland,Chris Ward,Robert V Skibbens,Elçin Ünal

doi:10.1371/journal.pgen.0030012

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.

Highlights

Proper transmission of eukaryotic chromosomes during cell division requires DNA replication and three other DNAdependent processes: recombination-dependent DNA repair, sister chromatid cohesion, and chromosome condensation
To gain insight into the structure and function of structural maintenance of chromosomes (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
The cohesin complex tethers together newly replicated chromosomes. The analyses of these RID mutants suggest that the tethering activity of cohesin is generated by two sequential chromatin-binding events, which are regulated both spatially and temporally

Summary

Introduction

Proper transmission of eukaryotic chromosomes during cell division requires DNA replication and three other DNAdependent processes: recombination-dependent DNA repair, sister chromatid cohesion, and chromosome condensation. Each of these diverse processes requires protein complexes containing two members of the highly conserved structural maintenance of chromosomes (Smc) family of proteins [1,2,3]. Smc monomers fold in half at the Hinge domain, allowing the two a-helices to form a long antiparallel coiled-coil domain [6]. This folding juxtaposes the N- and C-terminal globular domains and the Walker A and B motifs, creating an Smc head domain with ATPase activity. Folded Smc monomers resemble a flexible dumbbell, with the Hinge and head domains separated by ;40 nm of coiled coil [6,7]

Methods

Results

Discussion

Conclusion