Transport of DNA within cohesin involves clamping on top of engaged heads by Scc2 and entrapment within the ring by Scc3

In addition to extruding DNA loops, cohesin entraps within its SMC-kleisin ring (S-K) individual DNAs during G1 and sister DNAs during S-phase. All three activities require related hook-shaped proteins called Scc2 and Scc3. Using thiol-specific crosslinking we provide rigorous proof of entrapment activity in vitro. Scc2 alone promotes entrapment of DNAs in the E-S and E-K compartments, between ATP-bound engaged heads and the SMC hinge and associated kleisin, respectively. This does not require ATP hydrolysis nor is it accompanied by entrapment within S-K rings, which is a slower process requiring Scc3. Cryo-EM reveals that DNAs transported into E-S/E-K compartments are ‘clamped’ in a sub-compartment created by Scc2’s association with engaged heads whose coiled coils are folded around their elbow. We suggest that clamping may be a recurrent feature of cohesin complexes active in loop extrusion and that this conformation precedes the S-K entrapment required for sister chromatid cohesion.

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ATP dependent DNA transport within cohesin: Scc2 clamps DNA on top of engaged heads while Scc3 promotes entrapment within the SMC-kleisin ring

10.1101/2020.06.03.132233 ◽

2020 ◽

Author(s):

James E Collier ◽

Byung-Gil Lee ◽

Maurici B Roig ◽

Stanislav Yatskevich ◽

Naomi J Petela ◽

...

Keyword(s):

Sister Chromatid ◽

Atp Hydrolysis ◽

Sister Chromatid Cohesion ◽

S Phase ◽

Coiled Coils ◽

Rigorous Proof ◽

Dna Loops ◽

Dna Transport ◽

Chromatid Cohesion

SUMMARYIn addition to extruding DNA loops, cohesin entraps within its SMC-kleisin ring (S-K) individual DNAs during G1 and sister DNAs during S-phase. All three activities require related hook-shaped proteins called Scc2 and Scc3. Using thiol-specific crosslinking we provide rigorous proof of entrapment activity in vitro. Scc2 alone promotes entrapment of DNAs in the E-S and E-K compartments, between ATP-bound engaged heads and the SMC hinge and associated kleisin, respectively. This does not require ATP hydrolysis nor is it accompanied by entrapment within S-K rings, which is a slower process requiring Scc3. Cryo-EM reveals that DNAs transported into E-S/E-K compartments are “clamped” in a sub-compartment created by Scc2’s association with engaged heads whose coiled coils are folded around their elbow. We suggest that clamping may be a recurrent feature of cohesin complexes active in loop extrusion and that this conformation precedes the S-K entrapment required for sister chromatid cohesion.

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Cohesin Associates with Spindle Poles in a Mitosis-specific Manner and Functions in Spindle Assembly in Vertebrate Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e08-04-0419 ◽

2009 ◽

Vol 20 (5) ◽

pp. 1289-1301 ◽

Cited By ~ 28

Author(s):

Xiangduo Kong ◽

Alexander R. Ball ◽

Eiichiro Sonoda ◽

Jie Feng ◽

Shunichi Takeda ◽

...

Keyword(s):

Chromosome Pairing ◽

Sister Chromatid ◽

Spindle Assembly ◽

Sister Chromatid Cohesion ◽

S Phase ◽

Mitotic Apparatus ◽

Spindle Poles ◽

Chromatid Cohesion ◽

Specific Manner

Cohesin is an essential protein complex required for sister chromatid cohesion. Cohesin associates with chromosomes and establishes sister chromatid cohesion during interphase. During metaphase, a small amount of cohesin remains at the chromosome-pairing domain, mainly at the centromeres, whereas the majority of cohesin resides in the cytoplasm, where its functions remain unclear. We describe the mitosis-specific recruitment of cohesin to the spindle poles through its association with centrosomes and interaction with nuclear mitotic apparatus protein (NuMA). Overexpression of NuMA enhances cohesin accumulation at spindle poles. Although transient cohesin depletion does not lead to visible impairment of normal spindle formation, recovery from nocodazole-induced spindle disruption was significantly impaired. Importantly, selective blocking of cohesin localization to centromeres, which disrupts centromeric sister chromatid cohesion, had no effect on this spindle reassembly process, clearly separating the roles of cohesin at kinetochores and spindle poles. In vitro, chromosome-independent spindle assembly using mitotic extracts was compromised by cohesin depletion, and it was rescued by addition of cohesin that was isolated from mitotic, but not S phase, cells. The combined results identify a novel spindle-associated role for human cohesin during mitosis, in addition to its function at the centromere/kinetochore regions.

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PCNA Controls Establishment of Sister Chromatid Cohesion during S Phase

Molecular Cell ◽

10.1016/j.molcel.2006.07.007 ◽

2006 ◽

Vol 23 (5) ◽

pp. 723-732 ◽

Cited By ~ 186

Author(s):

George-Lucian Moldovan ◽

Boris Pfander ◽

Stefan Jentsch

Keyword(s):

Sister Chromatid ◽

Sister Chromatid Cohesion ◽

S Phase ◽

Chromatid Cohesion

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Hst3 Is Regulated by Mec1-dependent Proteolysis and Controls the S Phase Checkpoint and Sister Chromatid Cohesion by Deacetylating Histone H3 at Lysine 56

Journal of Biological Chemistry ◽

10.1074/jbc.m706384200 ◽

2007 ◽

Vol 282 (52) ◽

pp. 37805-37814 ◽

Cited By ~ 50

Author(s):

Safia Thaminy ◽

Benjamin Newcomb ◽

Jessica Kim ◽

Tonibelle Gatbonton ◽

Eric Foss ◽

...

Keyword(s):

Sister Chromatid ◽

Histone H3 ◽

Sister Chromatid Cohesion ◽

S Phase ◽

Chromatid Cohesion ◽

S Phase Checkpoint

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Two Human Orthologues of Eco1/Ctf7 Acetyltransferases Are Both Required for Proper Sister-Chromatid Cohesion

Molecular Biology of the Cell ◽

10.1091/mbc.e04-12-1063 ◽

2005 ◽

Vol 16 (8) ◽

pp. 3908-3918 ◽

Cited By ~ 131

Author(s):

Fajian Hou ◽

Hui Zou

Keyword(s):

Sister Chromatid ◽

Cell Cycle Regulation ◽

Sister Chromatid Cohesion ◽

Genetic Studies ◽

Conserved Domain ◽

Chromatid Cohesion ◽

The Difference ◽

Cycle Regulation ◽

Acetyltransferase Domain

Genetic studies in yeast and Drosophila have uncovered a conserved acetyltransferase involved in sister-chromatid cohesion. Here, we described the two human orthologues, previously named EFO1/ESCO1 and EFO2/ESCO2. Similar to their yeast (Eco1/Ctf7 and Eso1) and fly (deco) counterparts, both proteins feature a conserved C-terminal domain consisting of a H2C2 zinc finger motif and an acetyltransferase domain that is able to catalyze autoacetylation reaction in vitro. However, no similarity can be detected outside of the conserved domain. RNA interference depletion experiment revealed that EFO1/ESCO1 and EFO2/ESCO2 were not redundant and that both were required for proper sister-chromatid cohesion. The difference between EFO1 and EFO2 also is reflected in their cell cycle regulation. In mitosis, EFO1 is phosphorylated, whereas EFO2 is degraded. Furthermore, both proteins associate with chromosomes, and the chromosome binding depends on the diverse N-terminal domains. We propose that EFO1 and EFO2 are targeted to different chromosome structures to help establish or maintain sister-chromatid cohesion.

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The acetyltransferase activity of San stabilizes the mitotic cohesin at the centromeres in a shugoshin-independent manner

The Journal of Cell Biology ◽

10.1083/jcb.200701043 ◽

2007 ◽

Vol 177 (4) ◽

pp. 587-597 ◽

Cited By ~ 55

Author(s):

Fajian Hou ◽

Chih-Wen Chu ◽

Xiangduo Kong ◽

Kyoko Yokomori ◽

Hui Zou

Keyword(s):

Cell Cycle ◽

Sister Chromatid ◽

Sister Chromatid Cohesion ◽

Independent Manner ◽

Acetyltransferase Activity ◽

Chromatid Cohesion ◽

Interphase Cells ◽

Gain Access

Proper sister chromatid cohesion is critical for maintaining genetic stability. San is a putative acetyltransferase that is important for sister chromatid cohesion in Drosophila melanogaster, but not in budding yeast. We showed that San is critical for sister chromatid cohesion in HeLa cells, suggesting that this mechanism may be conserved in metazoans. Furthermore, although a small fraction of San interacts with the NatA complex, San appears to mediate cohesion independently. San exhibits acetyltransferase activity in vitro, and its activity is required for sister chromatid cohesion in vivo. In the absence of San, Sgo1 localizes correctly throughout the cell cycle. However, cohesin is no longer detected at the mitotic centromeres. Furthermore, San localizes to the cytoplasm in interphase cells; thus, it may not gain access to chromosomes until mitosis. Moreover, in San-depleted cells, further depletion of Plk1 rescues the cohesion along the chromosome arms, but not at the centromeres. Collectively, San may be specifically required for the maintenance of the centromeric cohesion in mitosis.

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The cohesin ring uses its hinge to organize DNA using non-topological as well as topological mechanisms

10.1101/197848 ◽

2017 ◽

Cited By ~ 2

Author(s):

Madhusudhan Srinivasan ◽

Johanna C. Scheinost ◽

Naomi J. Petela ◽

Thomas G. Gligoris ◽

Maria Wissler ◽

...

Keyword(s):

Conformational Changes ◽

Sister Chromatid ◽

Atp Hydrolysis ◽

Sister Chromatid Cohesion ◽

Perfect Correlation ◽

Chromatid Cohesion ◽

Lysine Residues ◽

Positively Charged ◽

Hinge Domain

SummaryAs predicted by the notion that sister chromatid cohesion is mediated by entrapment of sister DNAs inside cohesin rings, there is a perfect correlation between co-entrapment of circular minichromosomes and sister chromatid cohesion in a large variety of mutants. In most cells where cohesin loads onto chromosomes but fails to form cohesion, loading is accompanied by entrapment of individual DNAs. However, cohesin with a hinge domain whose positively charged lumen has been neutralized not only loads onto and translocates along chromatin but also organizes it into chromatid-like threads, despite largely failing to entrap DNAs inside its ring. Thus, cohesin engages chromatin in a non-topological as well as a topological manner. Our finding that hinge mutations, but not fusions between Smc and kleisin subunits, abolish entrapment suggests that DNAs may enter cohesin rings through hinge opening. Lastly, mutation of three highly conserved lysine residues inside the Smc1 moiety of Smc1/3 hinges abolishes all loading without affecting cohesin’s initial recruitment to CEN loading sites or its ability to hydrolyze ATP. We suggest that loading and translocation are mediated by conformational changes in cohesin’s hinge driven by cycles of ATP hydrolysis.

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Acetylation of the SUN protein Mps3 by Eco1 regulates its function in nuclear organization

Molecular Biology of the Cell ◽

10.1091/mbc.e11-07-0600 ◽

2012 ◽

Vol 23 (13) ◽

pp. 2546-2559 ◽

Cited By ~ 15

Author(s):

Suman Ghosh ◽

Jennifer M. Gardner ◽

Christine J. Smoyer ◽

Jennifer M. Friederichs ◽

Jay R. Unruh ◽

...

Keyword(s):

Nuclear Membrane ◽

Sister Chromatid ◽

Transmembrane Domain ◽

Nuclear Organization ◽

Spindle Pole Body ◽

Spindle Pole ◽

Sister Chromatid Cohesion ◽

Chromatid Cohesion

The Saccharomyces cerevisiae SUN-domain protein Mps3 is required for duplication of the yeast centrosome-equivalent organelle, the spindle pole body (SPB), and it is involved in multiple aspects of nuclear organization, including telomere tethering and gene silencing at the nuclear membrane, establishment of sister chromatid cohesion, and repair of certain types of persistent DNA double-stranded breaks. How these diverse SUN protein functions are regulated is unknown. Here we show that the Mps3 N-terminus is a substrate for the acetyltransferase Eco1/Ctf7 in vitro and in vivo and map the sites of acetylation to three lysine residues adjacent to the Mps3 transmembrane domain. Mutation of these residues shows that acetylation is not essential for growth, SPB duplication, or distribution in the nuclear membrane. However, analysis of nonacetylatable mps3 mutants shows that this modification is required for accurate sister chromatid cohesion and for chromosome recruitment to the nuclear membrane. Acetylation of Mps3 by Eco1 is one of the few regulatory mechanisms known to control nuclear organization.

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A Potential Role for Human Cohesin in Mitotic Spindle Aster Assembly

Journal of Biological Chemistry ◽

10.1074/jbc.m103364200 ◽

2001 ◽

Vol 276 (50) ◽

pp. 47575-47582 ◽

Cited By ~ 52

Author(s):

Heather C. Gregson ◽

John A. Schmiesing ◽

Jong-Soo Kim ◽

Toshiki Kobayashi ◽

Sharleen Zhou ◽

...

Keyword(s):

Sister Chromatid ◽

Mitotic Spindle ◽

Potential Role ◽

Metaphase Plate ◽

Spindle Pole ◽

Sister Chromatid Cohesion ◽

Cycle Arrest ◽

Spindle Poles ◽

Chromatid Cohesion

The cohesin multiprotein complex containing SMC1, SMC3, Scc3 (SA), and Scc1 (Rad21) is required for sister chromatid cohesion in eukaryotes. Although metazoan cohesin associates with chromosomes and was shown to function in the establishment of sister chromatid cohesion during interphase, the majority of cohesin was found to be off chromosomes and reside in the cytoplasm in metaphase. Despite its dissociation from chromosomes, however, microinjection of an antibody against human SMC1 led to disorganization of the metaphase plate and cell cycle arrest, indicating that human cohesin still plays an important role in metaphase. To address the mitotic function of human cohesin, the subcellular localization of cohesin components was reexamined in human cells. Interestingly, we found that cohesin localizes to the spindle poles during mitosis and interacts with NuMA, a spindle pole-associated factor required for mitotic spindle organization. The interaction with NuMA persists during interphase. Similar to NuMA, a significant amount of cohesin was found to associate with the nuclear matrix. Furthermore, in the absence of cohesin, mitotic spindle asters failed to formin vitro. Our results raise the intriguing possibility that in addition to its well demonstrated function in sister chromatid cohesion, cohesin may be involved in spindle assembly during mitosis.

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