Evidence that Loading of Cohesin Onto Chromosomes Involves Opening of Its SMC Hinge

Gruber, Stephan; Arumugam, Prakash; Katou, Yuki; Kuglitsch, Daria; Helmhart, Wolfgang; Shirahige, Katsuhiko; Nasmyth, Kim

doi:10.1016/j.cell.2006.08.048

Cited by 269 publications

(302 citation statements)

References 29 publications

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“…Our mutagenesis strategy was based on the premise that the mutations would be expected to have the same effect in SMC2 as they do in bacterial SMC proteins (Hirano et al, 2001;Hirano and Hirano, 2006), cohesin SMCs (Arumugam et al, 2003;Weitzer et al, 2003;Arumugam et al, 2006) ABC transporters and RAD 50 (Hopfner et al, 2000; also see Figure 1B). A Walker A box mutation, K38I, and the D1113A mutation in the Walker B motif are predicted to prevent ATP binding, whereas an E1114Q "transition state" mutation in the Walker B box would slow the rate of ATP hydrolysis that is thought to be required for head disengagement in SMC complexes (Hirano, 2006).…”

Section: Atp Binding and Hydrolysis Are Required For Smc2 Functionmentioning

confidence: 99%

“…The S1086R mutation in the C-terminal signature motif is predicted to allow ATP binding but prevent ATP hydrolysis. These four residues are completely conserved among SMCs and ABC transporters and have been extensively characterized (Hirano et al, 2001;Arumugam et al, 2003Arumugam et al, , 2006Weitzer et al, 2003;Hirano and Hirano, 2006), but little is known of their role in the condensin complex in vivo. Mutation of the conserved Q-loop (Q147L) is predicted to block binding of the attacking nucleophile (water) and Mg 2ϩ ions involved in ATP hydrolysis (Hopfner et al, 2000).…”

Section: Atp Binding and Hydrolysis Are Required For Smc2 Functionmentioning

confidence: 99%

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Molecular and Genetic Analysis of Condensin Function in Vertebrate Cells

Hudson

Ohta

Freisinger

et al. 2008

MBoC

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We engineered mutants into residues of SMC2 to dissect the role of ATPase function in the condensin complex. These residues are predicted to be involved in ATP binding or hydrolysis and in the Q-loop, which is thought to act as a mediator of conformational changes induced by substrate binding. All the engineered ATPase mutations resulted in lethality when introduced into SMC2 null cells. We found that ATP binding, but not hydrolysis, is essential to allow stable condensin association with chromosomes. How SMC proteins bind and interact with DNA is still a major question. Cohesin may form a ring structure that topologically encircles DNA. We examined whether condensin behaves in an analogous way to its cohesin counterpart, and we have generated a cleavable form of biologically active condensin with PreScission protease sites engineered into the SMC2 protein. This has allowed us to demonstrate that topological integrity of the SMC2-SMC4 heterodimer is not necessary for the stability of the condensin complex in vitro or for its stable association with mitotic chromosomes. Thus, despite their similar molecular organization, condensin and cohesin exhibit fundamental differences in their structure and function.

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Section: Atp Binding and Hydrolysis Are Required For Smc2 Functionmentioning

confidence: 99%

Section: Atp Binding and Hydrolysis Are Required For Smc2 Functionmentioning

confidence: 99%

Molecular and Genetic Analysis of Condensin Function in Vertebrate Cells

Hudson

Ohta

Freisinger

et al. 2008

MBoC

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“…In considering this work, it is perhaps most important to bear in mind that our yeastbased mislocalization system represents a variation on a simple and expandable theme: protein heterodimerization is linked to a chemical stimulus, and a single well-characterized compound, such as rapamycin, may be used to regulate the activity of a wide variety of gene products. Related systems have been employed previously in metazoans and cell lines to regulate protein stability (Stankunas et al, 2003;Liu et al, 2007;Stankunas et al, 2007;Janse et al, 2004;Banaszynski et al, 2006a;Maynard-Smith et al, 2007) and in both metazoans and in yeast to regulate protein-protein interactions (Gruber et al, 2006;Karpova et al, 2005;Mallet et al, 2002;Welm et al, 2002;Pownall et al, 2003;Gallagher et al, 2007;Schwartz et al, 2007). We expect future modifications of this dimerization approach to advance not only this system (e.g.…”

Section: Discussionmentioning

confidence: 97%

A small molecule‐directed approach to control protein localization and function

Geda

Patury

et al. 2008

Yeast

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Protein localization is tightly linked with function, such that the subcellular distribution of a protein serves as an important control point regulating activity. Exploiting this regulatory mechanism, we present here a general approach by which protein location, and hence function, may be controlled on demand in the budding yeast. In this system a small molecule, rapamycin, is used to temporarily recruit a strong cellular address signal to the target protein, placing subcellular localization under control of the selective chemical stimulus. The kinetics of this system are rapid: rapamycin-directed nucleo-cytoplasmic transport is evident 10-12 min posttreatment and the process is reversible upon removal of rapamycin. Accordingly, we envision this platform as a promising approach for the systematic construction of conditional loss-of-function mutants. As proof of principle, we used this system to direct nuclear export of the essential heat shock transcription factor Hsf1p, thereby mimicking the cell-cycle arrest phenotype of an hsf1 temperature-sensitive mutant. Our drug-induced localization platform also provides a method by which protein localization can be uncoupled from endogenous cell signalling events, addressing the necessity or sufficiency of a given localization shift for a particular cell process. To illustrate, we directed the nuclear import of the calcineurin-dependent transcription factor Crz1p in the absence of native stimuli; this analysis directly substantiates that nuclear translocation of this protein is insufficient for its transcriptional activity. In total, this technology represents a powerful method for the generation of conditional alleles and directed mislocalization studies in yeast, with potential applicability on a genome-wide scale.

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“…Protein fusion and chemical crosslinking studies in budding yeast have proposed an alternative possibility for cohesin’s DNA entry [42]. Covalent fusions of Smc3-Scc1 or Smc1-Scc1 have only a minor impact on chromatin loading of cohesin or sister chromatid cohesion establishment.…”

Section: Topological Dna Binding By Cohesinmentioning

confidence: 99%

“…Covalent fusions of Smc3-Scc1 or Smc1-Scc1 have only a minor impact on chromatin loading of cohesin or sister chromatid cohesion establishment. In contrast, hinge closing by insertion of rapamycin induced dimerization proteins, or replacing the hinge domains with unrelated dimerizing proteins, ablates the chromosomal functions of cohesin [42,43]. These findings suggest that the Smc1-Smc3 hinge interface operates as the DNA entry site.…”

Section: Topological Dna Binding By Cohesinmentioning

confidence: 99%

DNA entry, exit and second DNA capture by cohesin: insights from biochemical experiments

Murayama

2018

Nucleus

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Cohesin is a ring-shaped, multi-subunit ATPase assembly that is fundamental to the spatiotemporal organization of chromosomes. The ring establishes a variety of chromosomal structures including sister chromatid cohesion and chromatin loops. At the core of the ring is a pair of highly conserved SMC (Structural Maintenance of Chromosomes) proteins, which are closed by the flexible kleisin subunit. In common with other essential SMC complexes including condensin and the SMC5-6 complex, cohesin encircles DNA inside its cavity, with the aid of HEAT (Huntingtin, elongation factor 3, protein phosphatase 2A and TOR) repeat auxiliary proteins. Through this topological embrace, cohesin is thought to establish a series of intra- and interchromosomal interactions by tethering more than one DNA molecule. Recent progress in biochemical reconstitution of cohesin provides molecular insights into how this ring complex topologically binds and mediates DNA-DNA interactions. Here, I review these studies and discuss how cohesin mediates such chromosome interactions.

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Evidence that Loading of Cohesin Onto Chromosomes Involves Opening of Its SMC Hinge

Cited by 269 publications

References 29 publications

Molecular and Genetic Analysis of Condensin Function in Vertebrate Cells

Molecular and Genetic Analysis of Condensin Function in Vertebrate Cells

A small molecule‐directed approach to control protein localization and function

DNA entry, exit and second DNA capture by cohesin: insights from biochemical experiments

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