Characterization of a DNA exit gate in the human cohesin ring

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Analyses of protein complexes are facilitated by methods that enable the generation of recombinant complexes via coexpression of their subunits from multigene DNA constructs. However, low experimental throughput limits the generation of such constructs in parallel. Here we describe a method that allows up to 25 cDNAs to be assembled into a single baculoviral expression vector in only two steps. This method, called biGBac, uses computationally optimized DNA linker sequences that enable the efficient assembly of linear DNA fragments, using reactions developed by Gibson for the generation of synthetic genomes. The biGBac method uses a flexible and modular “mix and match” approach and enables the generation of baculoviruses from DNA constructs at any assembly stage. Importantly, it is simple, efficient, and fast enough to allow the manual generation of many multigene expression constructs in parallel. We have used this method to generate and characterize recombinant forms of the anaphase-promoting complex/cyclosome, cohesin, and kinetochore complexes.

Section: Results Bigbac Enables the Rapid Assembly Of Up To 25 Cdnas mentioning

confidence: 82%

biGBac enables rapid gene assembly for the expression of large multisubunit protein complexes

Weissmann

Petzold

VanderLinden

et al. 2016

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“…5 B and C and Movie S1). Based on a variety of in vitro evidence and mutational analysis, cohesion has been explained by a simple model in which two DNA molecules are entrapped by individual cohesin rings (60,67,68). Other models posit a requirement for interactions among cohesin complexes (reviewed in refs.…”

Section: Discussionmentioning

confidence: 99%

Superresolution microscopy reveals the three-dimensional organization of meiotic chromosome axes in intact Caenorhabditis elegans tissue

Köhler

Wojcik

et al. 2017

When cells enter meiosis, their chromosomes reorganize as linear arrays of chromatin loops anchored to a central axis. Meiotic chromosome axes form a platform for the assembly of the synaptonemal complex (SC) and play central roles in other meiotic processes, including homologous pairing, recombination, and chromosome segregation. However, little is known about the 3D organization of components within the axes, which include cohesin complexes and additional meiosis-specific proteins. Here, we investigate the molecular organization of meiotic chromosome axes in Caenorhabditis elegans through STORM (stochastic optical reconstruction microscopy) and PALM (photo-activated localization microscopy) superresolution imaging of intact germ-line tissue. By tagging one axis protein (HIM-3) with a photoconvertible fluorescent protein, we established a spatial reference for other components, which were localized using antibodies against epitope tags inserted by CRISPR/Cas9 genome editing. Using 3D averaging, we determined the position of all known components within synapsed chromosome axes to high spatial precision in three dimensions. We find that meiosis-specific HORMA domain proteins span a gap between cohesin complexes and the central region of the SC, consistent with their essential roles in SC assembly. Our data further suggest that the two different meiotic cohesin complexes are distinctly arranged within the axes: Although cohesin complexes containing the kleisin REC-8 protrude above and below the plane defined by the SC, complexes containing COH-3 or -4 kleisins form a central core, which may physically separate sister chromatids. This organization may help to explain the role of the chromosome axes in promoting interhomolog repair of meiotic double-strand breaks by inhibiting intersister repair.

“…A likely explanation is related to the increase in cohesin binding stability achieved during S phase (44,45). Coincident with DNA replication, acetylation of cohesin by Eco1 blocks opening of the DNA exit gate between the Smc3 and Mcd1 interface (29,30,46,47). In yeast, acetylated complexes that are locked in a chromatid embrace are released only at anaphase onset by proteolytic cleavage.…”

Section: Discussionmentioning

confidence: 99%

Binding, sliding, and function of cohesin during transcriptional activation

Borrie

Campor

Joshi

et al. 2017

The ring-shaped cohesin complex orchestrates long-range DNA interactions to mediate sister chromatid cohesion and other aspects of chromosome structure and function. In the yeast Saccharomyces cerevisiae, the complex binds discrete sites along chromosomes, including positions within and around genes. Transcriptional activity redistributes the complex to the 3′ ends of convergently oriented gene pairs. Despite the wealth of information about where cohesin binds, little is known about cohesion at individual chromosomal binding sites and how transcription affects cohesion when cohesin complexes redistribute. In this study, we generated extrachromosomal DNA circles to study cohesion in response to transcriptional induction of a model gene, URA3. Functional cohesin complexes loaded onto the locus via a poly(dA:dT) tract in the gene promoter and mediated cohesion before induction. Upon transcription, the fate of these complexes depended on whether the DNA was circular or not. When gene activation occurred before DNA circularization, cohesion was lost. When activation occurred after DNA circularization, cohesion persisted. The presence of a convergently oriented gene also prevented transcription-driven loss of functional cohesin complexes, at least in M phase-arrested cells. The results are consistent with cohesin binding chromatin in a topological embrace and with transcription mobilizing functional complexes by sliding them along DNA.T he protein complex known as cohesin organizes eukaryotic genomes into structures that segregate faithfully between dividing cells. The complex was first discovered for its role in mediating sister chromatid cohesion, but it is now known to participate in numerous aspects of chromosome biology (1, 2). Cohesin is clinically relevant because mutations in subunits of the complex or in factors that load and activate the complex lead to developmental disorders such as Cornelia de Lange syndrome, Roberts syndrome, and Warsaw breakage syndrome (3, 4).Cohesin is a ring-shaped complex composed of four subunits named Smc1, Smc3, Scc3, and Mcd1/Scc1 in budding yeast. The complex is thought to embrace DNA topologically with chromatin fibers passing through a central open channel (5-7). A substantial body of work supports a model in which cohesion arises from single cohesin complexes that embrace both sister chromatids [the double embrace model (6)]. Whether the central channel is large enough to accommodate both chromatids has recently been brought into question (8). In addition, the behavior of cohesin has not always been consistent with a double embrace by single complexes (for examples, see refs. 9-11). These situations usually involve a perturbation that causes loss of cohesion but not a loss of cohesin binding from chromatin. Such results force consideration of alternative models in which cohesin complexes embrace only one chromatid topologically and interact with other cohesin complexes or chromatin features to mediate cohesion (9,11,12).Cohesin binds densely in centromeric regions of chromos...