Identification of a region in the coiled-coil domain of Smc3 that is essential for cohesin activity

Abstract

The cohesin complex plays an important role in sister chromatin cohesion. Cohesin's core is composed of two structural maintenance of chromosome (SMC) proteins, called Smc1 and Smc3. SMC proteins are built from a globular hinge domain, a rod-shaped domain composed of long anti-parallel coiled-coil (CC), and a second globular adenosine triphosphatase domain called the head. The functions of both head and hinge domains have been studied extensively, yet the function of the CC region remains elusive. We identified a mutation in the CC of smc3 (L217P) that disrupts the function of the protein. Cells carrying the smc3-L217P allele have a strong cohesion defect and complexes containing smc3-L217P are not loaded onto the chromosomes. However, the mutation does not affect inter-protein interactions in either the core complex or with the Scc2 loader. We show by molecular dynamics and biochemistry that wild-type Smc3 can adopt distinct conformations, and that adenosine triphosphate (ATP) induces the conformational change. The L217P mutation restricts the ability of the mutated protein to switch between the conformations. We suggest that the function of the CC is to transfer ATP binding/hydrolysis signals between the head and the hinge domains. The results provide a new insight into the mechanism of cohesin activity.

Monomeric cohesin state revealed by live‐cell single‐molecule spectroscopy

“…The strains were constructed using standard molecular and genetic methods. Cells were grown in YPD or SD medium, as indicated [9,38,41]. Auxin-induced degradation [42,43] was done as described in Ref.…”

“…Auxin-induced degradation [42,43] was done as described in Ref. [9,38]. Protein depletion was validated by semi-quantitative growth assays on YPD plates, with and without auxin (IAA) (Sigma-Aldrich), and by Western blot with antibodies against V5 (Invitrogen) and tubulin (Abcam).…”

“…G1 cell synchronization was achieved with alpha-factor mating pheromone (Zymo Research). M phase arrest was performed with nocodazole (Santa Cruz Biotechnology) [9,38,41].…”

The chromatin remodeler Chd1 regulates cohesin in budding yeast and humans

“…Sister chromatid cohesion was measured by using the GFP dot assay as previously reported 43,61,62 . In brief, cells carrying LacI-GFP and LacO array, integrated at the LYS2 locus, about 40 kb from the centromere, were grown to mid-log phase and arrested at G2/M by 5 µM nocodazole.…”

ChickenCCDC152shares an NFYB-regulated bidirectional promoter with agrowth hormone receptorantisense transcript and inhibits cells proliferation and migration

“…The coiled-coil domain-containing (CCDC) proteins exhibit different kinds of functions due to their highly versatile folding motif [ 20 , 21 ]. The coiled-coil motif is found in many proteins, such as skeletal and motor proteins, and is involved in molecular recognition systems and protein refolding [ 20 – 22 ].…”