Condensin but not cohesin SMC heterodimer induces DNA reannealing through protein-protein assembly

Sakai, Akiko; Hizume, Kohji; Sutani, Takashi; Takeyasu, Kunio; Yanagida, Mitsuhiro

doi:10.1093/emboj/cdg247

Cited by 97 publications

(94 citation statements)

References 39 publications

(90 reference statements)

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“…Large-scale cultures of mitotic cells (5ϫ10 10 cells) were grown at 20°C for 8 hours, extracts were prepared and anti-FLAG antibodies were used to immunoprecipitate condensin holocomplexes. The precipitates contained the five authentic subunits (Sakai et al, 2003;Sutani et al, 1999) (Fig. 5A), as compared with the silver-staining pattern of condensin isolated with the untagged control (Fig.…”

Section: Condensin Subunits Are Mitotically Phosphorylatedmentioning

confidence: 90%

Condensin phosphorylated by the Aurora-B-like kinase Ark1 is continuously required until telophase in a mode distinct from Top2

Nakazawa

Mehrotra

Ebe

et al. 2011

Journal of Cell Science

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SummaryCondensin is a conserved protein complex that functions in chromosome condensation and segregation. It has not been previously unequivocally determined whether condensin is required throughout mitosis. Here, we examined whether Schizosaccharomyces pombe condensin continuously acts on chromosomes during mitosis and compared its role with that of DNA topoisomerase II (Top2). Using double mutants containing a temperature-sensitive allele of the condensin SMC2 subunit cut14 (cut14-208) or of top2, together with the cold-sensitive nda3-KM311 mutation (in -tubulin), temperature-shift experiments were performed. These experiments allowed inactivation of condensin or Top2 at various stages throughout mitosis, even after late anaphase. The results established that mitotic chromosomes require condensin and Top2 throughout mitosis, even in telophase. We then showed that the Cnd2 subunit of condensin (also known as Barren) is the target subunit of Aurora-B-like kinase Ark1 and that Ark1-mediated phosphorylation of Cnd2 occurred throughout mitosis. The phosphorylation sites in Cnd2 were determined by mass spectrometry, and alanine and glutamate residue replacement mutant constructs for these sites were constructed. Alanine substitution mutants of Cnd2, which mimic the unphosphorylated protein, exhibited broad mitotic defects, including at telophase, and overexpression of these constructs caused a severe dominantnegative effect. By contrast, glutamate substitution mutants, which mimic the phosphorylated protein, alleviated the segregation defect in Ark1-inhibited cells. In telophase, the condensin subunits in cut14-208 mutant accumulated in lumps that contained telomeric DNA and proteins that failed to segregate. Condensin might thus serve to keep the segregated chromosomes apart during telophase.

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Section: Condensin Subunits Are Mitotically Phosphorylatedmentioning

confidence: 90%

Condensin phosphorylated by the Aurora-B-like kinase Ark1 is continuously required until telophase in a mode distinct from Top2

Nakazawa

Mehrotra

Ebe

et al. 2011

Journal of Cell Science

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“…of the mammalian RC-1 complex, its SMC1/3 dimer, a variety of C-terminal SMC domains, Schizosaccharomyces pombe condensin, or the B. subtilis SMC homodimer (15,27,28,41,42). However, in one recent study (44), the S. pombe cohesin or its SMC1/3 core were reportedly not active or were weakly active in a reannealing assay, although condensin or its SMC2/4 dimer was active. It is presently unclear why this differs from the other studies.…”

Section: Fig 7 Network Formation Assaymentioning

confidence: 95%

DNA Interaction and Dimerization of Eukaryotic SMC Hinge Domains

Chiu

Revenkova

Jessberger

2004

Journal of Biological Chemistry

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The eukaryotic SMC1/SMC3 heterodimer is essential for sister chromatid cohesion and acts in DNA repair and recombination. Dimerization depends on the central hinge domain present in all SMC proteins, which is flanked at each side by extended coiled-coil regions that terminate in specific globular domains. Here we report on DNA interactions of the eukaryotic, heterodimeric SMC1/SMC3 hinge regions, using the two known isoforms, SMC1␣/SMC3 and the meiotic SMC1␤/SMC3. Both dimers bind DNA with a preference for double-stranded DNA and DNA rich in potential secondary structures. Both dimers form large protein-DNA networks and promote reannealing of complementary DNA strands. DNA binding but not dimerization depends on approximately 20 amino acids of transitional sequence into the coiledcoil region. Replacement of three highly conserved glycine residues, thought to be required for dimerization, in one of the two hinge domains still allows formation of a stable dimer, but if two hinge domains are mutated dimerization fails. Single-mutant dimers bind DNA, but hinge monomers do not. Together, we show that eukaryotic hinge dimerization does not require conserved glycines in both hinge domains, that only the transition into the coiled-coil region rather than the entire coiledcoil region is necessary for DNA binding, and that dimerization is required but not sufficient for DNA binding of the eukaryotic hinge heterodimer.Eukaryotic SMC (structural maintenance of chromosomes) proteins are essential for sister chromatid cohesion and proper chromosome condensation and also act in response to DNA damage, in DNA recombination and repair, and in gene dosage compensation (for recent reviews see Refs. 1-6).SMC proteins, which are between 120 and 160 kDa in molecular mass, feature a design reminiscent of motor proteins. The C-terminal and N-terminal globular domains are separated by extended coiled-coil regions and a hinge domain at the center. Six members of the eukaryotic SMC protein family are known, named SMC1-SMC6 for most eukaryotic organisms. They form three types of heterodimers: SMC1/SMC3, SMC2/SMC4, and SMC5/SMC6. One heterodimer each constitutes a core component of a larger multiprotein complex that acts in one of the key functions described for SMC proteins. The best described complexes are cohesin (7-9), which is based on SMC1/SMC3, and condensin with its SMC2/SMC4 core (10, 11). The SMC5/SMC6 heterodimer forms a multiprotein complex that acts in postreplicative recombinational repair of DNA damage (12, 13). Also, there are several other complexes such as a mammalian complex called RC-1, which has been implicated in DNA recombination and repair reactions (14, 15), a DNA damage response-dependent complex that forms upon phosphorylation of SMC1 (16, 17), and a gene dosage compensation complex in Caenorhabditis elegans that contains SMC2 and SMC4 homologs and is responsible for maintaining the same level of X chromosome expression in XX and XO animals (18). In meiotic cells, there exist at least two, probably more, meiosis-s...

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“…In this model, conformational changes at the ATPase heads need to be transmitted to the hinge, which is located up to 35 nm away and appears to be connected to the heads merely by a flexible coiled coil. A possible means of achieving this involves a bending back of the hinge towards the heads, as has been observed in atomic force microscopy images of the fission yeast Smc1-Smc3 dimer (Sakai et al, 2003). In this way, the ATPase heads might come into direct contact with the hinge domain; some biochemical evidence has been observed for this (McIntyre et al, 2007).…”

Section: Journal Of Cell Sciencementioning

confidence: 97%

Cohesin loading and sliding

Ocampo-Hafalla

Uhlmann

2011

Journal of Cell Science

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SummaryCohesin is best known as a crucial component of chromosomal stability. Composed of several essential subunits in budding yeast, cohesin forms a ring-like complex that is thought to embrace sister chromatids, thereby physically linking them until their timely segregation during cell division. The ability of cohesin to bind chromosomes depends on the Scc2-Scc4 complex, which is viewed as a loading factor for cohesin onto DNA. Notably, in addition to its canonical function in sister chromatid cohesion, cohesin has also been implicated in gene regulation and development in organisms ranging from yeast to human. Despite its importance, both as a mediator of sister chromatid cohesion and as a modulator of gene expression, the nature of the association of cohesin with chromosomes that enables it to fulfil both of these roles remains incompletely understood. The mechanism by which cohesin is loaded onto chromosomes, and how cohesin and the related condensin and Smc5-Smc6 complexes promote DNA interactions require further elucidation. In this Commentary, we critically review the evidence for cohesin loading and its subsequent apparent sliding along chromosomes, and discuss the implications gained from cohesin localisation studies for its important functions in chromosome biology. Journal of Cell Scienceand lethality. In this case, cohesin is thought to promote transcriptional activity of an ecdysone receptor gene (Pauli et al., 2008;Schuldiner et al., 2008). The requirement for cohesin in differentiated, post-mitotic cells provides clear evidence for a function of cohesin on chromosomes that is distinct from its role in cell division. A recent comprehensive analysis of the consequences of cohesin disruption on gene expression from polytene chromosomes in Drosophila salivary gland cells has revealed a role for cohesin in both the upregulation and downregulation of several genes in the vicinity of its binding sites (Pauli et al., 2010). How cohesin and changes to its expression levels result in distinct transcriptional outcomes at individual gene loci and in different cell types is incompletely understood (Schaaf et al., 2009).In zebrafish, cohesin has been shown to positively regulate the Runx transcription factors, which determine cell fate during early embryonic development. Downregulation of the Scc1 or Smc3 cohesin subunits leads to aberrant expression of Runx proteins in a dose-dependent manner, despite cell division being able to proceed (Horsfield et al., 2007). Finally, in mouse and human cells, cohesin-binding sites on chromosomes significantly overlap with those of the transcriptional insulator CCCTC-binding factor (CTCF) (Parelho et al., 2008;Wendt et al., 2008). This colocalisation is functionally relevant because depletion of cohesin subunits Scc1 or Smc3 recapitulates the loss of insulator function seen after CTCF depletion. Notably, the effect of cohesin on the well-characterised insulator at the H19-Igf2 locus is observed even in the G1 phase of the cell cycle, when sister chromatids are absent. Th...

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Condensin but not cohesin SMC heterodimer induces DNA reannealing through protein-protein assembly

Cited by 97 publications

References 39 publications

Condensin phosphorylated by the Aurora-B-like kinase Ark1 is continuously required until telophase in a mode distinct from Top2

Condensin phosphorylated by the Aurora-B-like kinase Ark1 is continuously required until telophase in a mode distinct from Top2

DNA Interaction and Dimerization of Eukaryotic SMC Hinge Domains

Cohesin loading and sliding

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