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Kim Nasmyth - One of the best experts on this subject based on the ideXlab platform.
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sister dna entrapment between juxtaposed smc heads and kleisin of the cohesin complex
Molecular Cell, 2019Co-Authors: Christophe Chapard, Johanna C Scheinost, Robert Jones, Till Van Oepen, Kim NasmythAbstract:Cohesin entraps sister DNAs within tripartite rings created by pairwise interactions between Smc1, Smc3, and Scc1. Because Smc1/3 ATPase heads can also interact with each other, cohesin rings have the potential to form a variety of sub-compartments. Using in vivo cysteine cross-linking, we show that when Smc1 and Smc3 ATPases are engaged in the presence of ATP (E heads), cohesin rings generate a “SMC (S) compartment” between hinge and E heads and a “kleisin (K) compartment” between E heads and their associated kleisin subunit. Upon ATP hydrolysis, cohesin’s heads associate in a different mode, in which their signature motifs and their coiled coils are closely juxtaposed (J heads), creating alternative S and K compartments. We show that K compartments of either E or J type can entrap single DNAs, that acetylation of Smc3 during S phase is associated with J heads, and that sister DNAs are entrapped in J-K compartments.
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the topology of dna entrapment by cohesin rings
bioRxiv, 2018Co-Authors: Christophe Chapard, Johanna C Scheinost, Robert Jones, Till Van Oepen, Kim NasmythAbstract:Summary Cohesin entraps sister DNAs within tripartite rings created by pairwise interactions between Smc1,Smc3, and Scc1. Because the ATPase heads of Smc1 and Smc3 can interact with each other, cohesin rings in fact have the potential to form a variety of sub-compartments. Using in vivo cysteine crosslinking,we show that when Smc1 and Smc3 ATPases are engaged in the presence of ATP (E heads)cohesin rings generate a “SMC (S) compartment” between hinge and E heads and a “kleisin (K)compartment” between E heads and their associated kleisin subunit. Upon ATP hydrolysis, cohesin’s heads associate with each other in a very different mode, in which their signature motifs and their coiled coils are closely juxtaposed (J heads), creating alternative S and K compartments. We show that all four sub-compartments exist in vivo, that acetylation of Smc3 during S phase is accompanied by an increase in the ratio of J to E heads, and that sister DNAs are entrapped in J-K but not E-K compartments or in either type of S compartment.
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S1Supplemental Data ATP Hydrolysis Is Required for Cohesin’s Association with Chromosomes
2015Co-Authors: Prakash Arumugam, Stephan Gruber, Christian H. Haering, Koichi Tanaka, Karl Mechtler, Kim NasmythAbstract:The Smc1 ATP Hydrolysis Mutant Forms Rings mutation of Scc2 and ATP hydrolysis mutants appear therefore to be similar if not identical; namely, a failure not to form tripartite ringsWe tested the effect of an smc1 mutation predicted to abolish ATP hydrolysis (E1158Q) on the formation of tripartite cohesin rings. To but to load them onto chromosomes. do this, we prepared soluble extracts from cycling cells expressing either wild-type or ATP hydrolysis mutant E1158Q myc-tagged Supplemental Experimental Procedures Smc1 and a functional TEV cleavable Scc1 that has six and three HA epitopes fused at its N and C termini (HA6-Scc1TEV-HA3), respec- Yeast Strains tively, and immunoprecipitated Smc1 by using an antibody against All strains are derivatives of W303. Genotypes used in the individual its myc9 epitope. Aliquots of the immunoprecipitation beads were experiments are given in the figure legends. All genes/markers are incubated in the presence or absence of TEV protease, and proteins as in W303 unless otherwise stated. released into the supernatant (S) were separated from those bound to the beads (B). Copurification of Scc1 with Smc1 was completely Molecular Biology dependent on the tag on Smc1 (Figure S1). N- and C-terminal Scc1 The Smc1 and Smc3 genes were cloned into yeast integrative plas-cleavage fragments remained bound to the beads after TEV cleav- mids YIplac211 and YIplac128, respectively [S3]. Mutations were age in the case of both wild-type Smc1 and its E1158Q hydrolysis introduced by overlap extension PCR. The sequences of the primer
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closing the cohesin ring structure and function of its smc3 kleisin interface
Science, 2014Co-Authors: Thomas G Gligoris, Kim Nasmyth, Johanna C Scheinost, Frank Burmann, Naomi J Petela, Koklung Chan, Pelin Uluocak, Frederic Beckouet, Stephan Gruber, Jan LoweAbstract:Through their association with a kleisin subunit (Scc1), cohesin’s Smc1 and Smc3 subunits are thought to form tripartite rings that mediate sister chromatid cohesion. Unlike the structure of Smc1/Smc3 and Smc1/Scc1 interfaces, that of Smc3/Scc1 is not known. Disconnection of this interface is thought to release cohesin from chromosomes in a process regulated by acetylation. We show here that the N-terminal domain of yeast Scc1 contains two α helices, forming a four-helix bundle with the coiled coil emerging from Smc3’s adenosine triphosphatase head. Mutations affecting this interaction compromise cohesin’s association with chromosomes. The interface is far from Smc3 residues, whose acetylation prevents cohesin’s dissociation from chromosomes. Cohesin complexes holding chromatids together in vivo do indeed have the configuration of hetero-trimeric rings, and sister DNAs are entrapped within these.
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atp hydrolysis is required for relocating cohesin from sites occupied by its scc2 4 loading complex
Current Biology, 2011Co-Authors: Takehiko Itoh, Katsuhiko Shirahige, Koklung Chan, Maurici B Roig, A K Mishra, Yuki Katoh, William Upcher, Camilla Godlee, Kim NasmythAbstract:Summary Background The Cohesin complex that holds sister chromatins together until anaphase is comprised of three core subunits: Smc1 and Smc3, two long-rod-shaped proteins with an ABC-like ATPase head (nucleotide-binding domain [NBD]) and a dimerization domain linked by a 50 nm long intramolecular antiparallel coiled-coil, and Scc1, an α-kleisin subunit interconnecting the NBD domains of Smc1 and Smc3. Cohesin's stable association with chromosomes is thought to involve entrapment of chromatin fibers by its tripartite Smc1-Smc3-Scc1 ring via a poorly understood mechanism dependent on a separate Scc2/4 loading complex. A key issue concerns where entrapment initially takes place: at sites where cohesin is found stably associated or at distinct "loading" sites from which it translocates. Results In this study, we find transition state mutant versions (Smc1E1158Q and SmcE1155Q) defective in disengagement of their nucleotide binding domains (NBDs), unlike functional cohesin, colocalize with Scc2/4 at core centromeres, sites that catalyze wild-type cohesin's recruitment to sequences 20 kb or more away. In addition to Scc2/4, the unstable association of transition state complexes with core centromeres requires Scc1's association with Smc1 and Smc3 NBDs, ATP-driven NBD engagement, cohesin's Scc3 subunit, and its hinge domain. Conclusion We propose that cohesin's association with chromosomes is driven by two key events. NBD engagement driven by ATP binding produces an unstable association with specific loading sites like core centromeres, whereas subsequent ATP hydrolysis triggers DNA entrapment, which permits translocation along chromatin fibers.
Frederic Beckouet - One of the best experts on this subject based on the ideXlab platform.
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releasing activity disengages cohesin s smc3 scc1 interface in a process blocked by acetylation
Molecular Cell, 2016Co-Authors: Thomas G Gligoris, Johanna C Scheinost, Naomi J Petela, Koklung Chan, Frederic Beckouet, Maurici B Roig, Madhusudhan Srinivasan, Paul Batty, Alexandra C SmithAbstract:Sister chromatid cohesion conferred by entrapment of sister DNAs within a tripartite ring formed between cohesin’s Scc1, Smc1, and Smc3 subunits is created during S and destroyed at anaphase through Scc1 cleavage by separase. Cohesin’s association with chromosomes is controlled by opposing activities: loading by Scc2/4 complex and release by a separase-independent releasing activity as well as by cleavage. Coentrapment of sister DNAs at replication is accompanied by acetylation of Smc3 by Eco1, which blocks releasing activity and ensures that sisters remain connected. Because fusion of Smc3 to Scc1 prevents release and bypasses the requirement for Eco1, we suggested that release is mediated by disengagement of the Smc3/Scc1 interface. We show that mutations capable of bypassing Eco1 in Smc1, Smc3, Scc1, Wapl, Pds5, and Scc3 subunits reduce dissociation of N-terminal cleavage fragments of Scc1 (NScc1) from Smc3. This process involves interaction between Smc ATPase heads and is inhibited by Smc3 acetylation.
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closing the cohesin ring structure and function of its smc3 kleisin interface
Science, 2014Co-Authors: Thomas G Gligoris, Kim Nasmyth, Johanna C Scheinost, Frank Burmann, Naomi J Petela, Koklung Chan, Pelin Uluocak, Frederic Beckouet, Stephan Gruber, Jan LoweAbstract:Through their association with a kleisin subunit (Scc1), cohesin’s Smc1 and Smc3 subunits are thought to form tripartite rings that mediate sister chromatid cohesion. Unlike the structure of Smc1/Smc3 and Smc1/Scc1 interfaces, that of Smc3/Scc1 is not known. Disconnection of this interface is thought to release cohesin from chromosomes in a process regulated by acetylation. We show here that the N-terminal domain of yeast Scc1 contains two α helices, forming a four-helix bundle with the coiled coil emerging from Smc3’s adenosine triphosphatase head. Mutations affecting this interaction compromise cohesin’s association with chromosomes. The interface is far from Smc3 residues, whose acetylation prevents cohesin’s dissociation from chromosomes. Cohesin complexes holding chromatids together in vivo do indeed have the configuration of hetero-trimeric rings, and sister DNAs are entrapped within these.
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an smc3 acetylation cycle is essential for establishment of sister chromatid cohesion
Molecular Cell, 2010Co-Authors: Frederic Beckouet, Makiko Komata, Katsuhiko Shirahige, Pelin Uluocak, Bin Hu, Maurici B Roig, Takashi Sutani, Vittorio L Katis, Kim NasmythAbstract:Sister chromatid cohesion is thought to involve entrapment of sister DNAs by a tripartite ring composed of the cohesin subunits Smc1, Smc3, and Scc1. Establishment of cohesion during S phase depends on acetylation of Smc3's nucleotide-binding domain (NBD) by the Eco1 acetyl transferase. It is destroyed at the onset of anaphase due to Scc1 cleavage by separase. In yeast, Smc3 acetylation is reversed at anaphase by the Hos1 deacetylase as a consequence of Scc1 cleavage. Smc3 molecules that remain acetylated after mitosis due to Hos1 inactivation cannot generate cohesion during the subsequent S phase, implying that cohesion establishment depends on de novo acetylation during DNA replication. By inducing Smc3 deacetylation in postreplicative cells due to Hos1 overexpression, we provide evidence that Smc3 acetylation contributes to the maintenance of sister chromatid cohesion. A cycle of Smc3 NBD acetylation is therefore an essential aspect of the chromosome cycle in eukaryotic cells.
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building sister chromatid cohesion smc3 acetylation counteracts an antiestablishment activity
Molecular Cell, 2009Co-Authors: Benjamin D Rowland, Pelin Uluocak, Frederic Beckouet, Maurici B Roig, Alexander Kurze, A K Mishra, Tatsuya Nishino, Philippa Underwood, Jean Metson, Richard ImreAbstract:Summary Cohesin's Smc1, Smc3, and Scc1 subunits form a tripartite ring that entraps sister DNAs. Scc3, Pds5, and Rad61 (Wapl) are regulatory subunits that control this process. We describe here smc3 , scc3 , pds5 , and rad61 mutations that permit yeast cell proliferation and entrapment of sister DNAs by cohesin rings in the absence of Eco1, an acetyl transferase normally essential for establishing sister chromatid cohesion. The smc3 mutations cluster around and include a highly conserved lysine (K113) close to Smc3's ATP-binding pocket, which, together with K112, is acetylated by Eco1. Lethality caused by mutating both residues to arginine is suppressed by the scc3 , pds5 , and rad61 mutants. Scc3, Pds5, and Rad61 form a complex and inhibit entrapment of sister DNAs by a process involving the "K112/K113" surface on Smc3's ATPase. According to this model, Eco1 promotes sister DNA entrapment partly by relieving an antiestablishment activity associated with Scc3, Pds5, and Rad61.
Richard Imre - One of the best experts on this subject based on the ideXlab platform.
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building sister chromatid cohesion smc3 acetylation counteracts an antiestablishment activity
Molecular Cell, 2009Co-Authors: Benjamin D Rowland, Pelin Uluocak, Frederic Beckouet, Maurici B Roig, Alexander Kurze, A K Mishra, Tatsuya Nishino, Philippa Underwood, Jean Metson, Richard ImreAbstract:Summary Cohesin's Smc1, Smc3, and Scc1 subunits form a tripartite ring that entraps sister DNAs. Scc3, Pds5, and Rad61 (Wapl) are regulatory subunits that control this process. We describe here smc3 , scc3 , pds5 , and rad61 mutations that permit yeast cell proliferation and entrapment of sister DNAs by cohesin rings in the absence of Eco1, an acetyl transferase normally essential for establishing sister chromatid cohesion. The smc3 mutations cluster around and include a highly conserved lysine (K113) close to Smc3's ATP-binding pocket, which, together with K112, is acetylated by Eco1. Lethality caused by mutating both residues to arginine is suppressed by the scc3 , pds5 , and rad61 mutants. Scc3, Pds5, and Rad61 form a complex and inhibit entrapment of sister DNAs by a process involving the "K112/K113" surface on Smc3's ATPase. According to this model, Eco1 promotes sister DNA entrapment partly by relieving an antiestablishment activity associated with Scc3, Pds5, and Rad61.
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building sister chromatid cohesion smc3 acetylation counteracts an antiestablishment activity
Molecular Cell, 2009Co-Authors: Enjami D Rowland, Pelin Uluocak, Alexander Kurze, A K Mishra, Tatsuya Nishino, Philippa Underwood, Maurici Roig, Frederic Eckoue, Jea Metso, Richard ImreAbstract:Cohesin's Smc1, Smc3, and Scc1 subunits form a tripartite ring that entraps sister DNAs. Scc3, Pds5, and Rad61 (Wapl) are regulatory subunits that control this process. We describe here smc3, scc3, pds5, and rad61 mutations that permit yeast cell proliferation and entrapment of sister DNAs by cohesin rings in the absence of Eco1, an acetyl transferase normally essential for establishing sister chromatid cohesion. The smc3 mutations cluster around and include a highly conserved lysine (K113) close to Smc3's ATP-binding pocket, which, together with K112, is acetylated by Eco1. Lethality caused by mutating both residues to arginine is suppressed by the scc3, pds5, and rad61 mutants. Scc3, Pds5, and Rad61 form a complex and inhibit entrapment of sister DNAs by a process involving the "K112/K113" surface on Smc3's ATPase. According to this model, Eco1 promotes sister DNA entrapment partly by relieving an antiestablishment activity associated with Scc3, Pds5, and Rad61.
Andreas Friedl - One of the best experts on this subject based on the ideXlab platform.
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Syndecan-1 induction in lung microenvironment supports the establishment of breast tumor metastases
BMC, 2018Co-Authors: Colleen Chute, Ning Yang, Xinhai Yang, Kristy Meyer, Keelin O’neil, Ildiko Kasza, Kevin Eliceiri, Caroline Alexander, Andreas FriedlAbstract:Abstract Background Syndecan-1 (Sdc1), a cell surface heparan sulfate proteoglycan normally expressed primarily by epithelia and plasma cells, is aberrantly induced in stromal fibroblasts of breast carcinomas. Stromal fibroblast-derived Sdc1 participates in paracrine growth stimulation of breast carcinoma cells and orchestrates stromal extracellular matrix fiber alignment, thereby creating a migration and invasion-permissive microenvironment. Here, we specifically tested the role of stromal Sdc1 in metastasis. Methods The metastatic potential of the aggressive mouse mammary carcinoma cell lines, 4T1 and E0776, was tested in wild-type and genetically Sdc1-deficient host animals. Metastatic lesions were characterized by immunohistochemical analysis. Results After orthotopic inoculation, the lung metastatic burden was reduced in Sdc1−/− animals by 97% and more than 99%, in BALB/cJ and C57BL/6 animals, respectively. The difference in metastatic efficiency was maintained when the tumor cells were injected into the tail vein, suggesting that host Sdc1 exerts its effect during later stages of the metastatic cascade. Co-localization studies identified Sdc1 expression in stromal fibroblasts within the metastatic microenvironment and in normal airway epithelial cells but not in other cells (endothelial cells, α-smooth muscle actin positive cells, leucocytes, macrophages). The Ki67 proliferation index and the rate of apoptosis of the metastatic tumor cells were diminished in Sdc1−/− vs. Sdc1+/+ animals, and leucocyte density was indistinguishable. Sdc1-mediated metastatic efficiency was abolished when the animals were housed at a thermoneutral ambient temperature of 31 °C, suggesting that the host Sdc1 effect on metastasis requires mild cold stress. Conclusions In summary, Sdc1 is induced in the lung microenvironment after mammary carcinoma cell dissemination and promotes outgrowth of metastases in a temperature-dependent manner
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syndecan 1 in breast cancer stroma fibroblasts regulates extracellular matrix fiber organization and carcinoma cell motility
American Journal of Pathology, 2011Co-Authors: Ning Yang, Rachel Mosher, Songwon Seo, David J Beebe, Andreas FriedlAbstract:Stromal fibroblasts of breast carcinomas frequently express the cell surface proteoglycan syndecan-1 (Sdc1). In human breast carcinoma samples, stromal Sdc1 expression correlates with an organized, parallel, extracellular matrix (ECM) fiber architecture. To examine a possible link between stromal Sdc1 and the fiber architecture, we generated bioactive cell-free three-dimensional ECMs from cultures of Sdc1-positive and Sdc1-negative murine and human mammary fibroblasts (termed ECM-Sdc1 and ECM-mock, respectively). Indeed, ECM-Sdc1 showed a parallel fiber architecture that contrasted markedly with the random fiber arrangement of ECM-mock. When breast carcinoma cells were seeded into the fibroblast-free ECMs, ECM-Sdc1, but not ECM-mock, promoted their attachment, invasion, and directional movement. We further evaluated the contribution of the structural/compositional modifications in ECM-Sdc1 on carcinoma cell behavior. By microcontact printing of culture surfaces, we forced the Sdc1-negative fibroblasts to produce ECM with parallel fiber organization, mimicking the architecture observed in ECM-Sdc1. We found that the fiber topography governs carcinoma cell migration directionality. Conversely, an elevated fibronectin level in ECM-Sdc1 was responsible for the enhanced attachment of the breast carcinoma cells. These observations suggest that Sdc1 expression in breast carcinoma stromal fibroblasts promotes the assembly of an architecturally abnormal ECM that is permissive to breast carcinoma directional migration and invasion.
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syndecan 1 expression by stromal fibroblasts promotes breast carcinoma growth in vivo and stimulates tumor angiogenesis
Oncogene, 2006Co-Authors: J Desouky, Takashi Maeda, Andreas FriedlAbstract:The induction of the cell surface heparan sulfate proteoglycan syndecan-1 (Sdc1) in stromal fibroblasts is observed in more than 70% of human breast carcinomas. Using a coculture model, we have recently shown that stromal cell-derived Sdc1 stimulates carcinoma cell proliferation in vitro, and that this activity requires Sdc1 glycanation. In the present study, we investigated the effect of stromal cell Sdc1 on breast carcinoma growth in vivo. MDA-MB-231 human breast carcinoma cells were inoculated into the flanks of athymic nude mice either alone, or as mixed suspensions with Sdc1-transfected or mock-transfected 3T3 mouse fibroblasts. The mixed tumors showed an intimate association between carcinoma cells and stromal fibroblasts and histologically closely resembled poorly differentiated human breast carcinomas. The presence of fibroblasts led to significantly accelerated tumor growth, which was further augmented (88% increase) by forced expression of stromal Sdc1. The hyperemic macroscopic appearance of tumors containing Sdc1-positive stromal cells contrasted with pale tumors developing in the presence of mock-transfected fibroblasts, which prompted us to examine tumor microvessels. Stromal Sdc1 expression was associated with a significantly elevated microvessel density (36% increase) and a larger vessel area (153% increase). To evaluate the relevance of this finding in human breast cancer, the relationship between stromal Sdc1 and tumor vascularity was also examined in a tissue array containing 207 human breast carcinoma samples. Similar to the xenografts, stromal Sdc1 expression correlated with both vessel density (P=0.013) and total vessel area (P=0.0026). In conclusion, stromal fibroblast-derived Sdc1 stimulates breast carcinoma growth and angiogenesis in vivo.
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induction of syndecan 1 expression in stromal fibroblasts promotes proliferation of human breast cancer cells
Cancer Research, 2004Co-Authors: Takashi Maeda, Caroline M Alexander, Andreas FriedlAbstract:Infiltrating carcinomas characteristically elicit a reactive stromal response, and accumulating evidence indicates that tumor stroma fibroblasts reciprocally promote tumor development and growth. The cell surface heparan sulfate proteoglycan, syndecan-1 (Sdc1), is thought to function as a coreceptor for growth factor and extracellular matrix interactions, and Sdc1 expression is induced in reactive stromal cells in both mice and man. Mice with a targeted mutation in Sdc1 show reduced tumor development in response to oncogene expression and altered responses to other pathological stimuli that are associated with the induction of stromal Sdc1. Here, we test the hypothesis that Sdc1 is required for the growth-promoting activities of reactive stroma. We found that when highly invasive carcinoma cells (MDA-MB-231) were placed in contact with mouse embryonic fibroblasts (MEFs) in a coculture model, Sdc1 expression was induced. Sdc1 was not induced by less invasive or normal cell lines (T47D and NMuMG). Furthermore, the growth of MDA-MB-231 cells was enhanced by 42% when cocultured with Sdc1+/+ MEFs compared with Sdc1-/- MEFs. When T47D cells were cocultured with fibroblasts that expressed transfected Sdc1, these Sdc1-positive fibroblasts stimulated growth of the breast epithelial cells by 85% compared with untransfected controls. The growth-promoting effect was completely abolished when fibroblasts were transfected with mutant Sdc1 lacking heparan sulfate attachment sites. In conclusion, we have demonstrated that a growth-promoting loop exists between breast cancer cells and their stroma that depends on the activity of glycanated Sdc1.
A K Mishra - One of the best experts on this subject based on the ideXlab platform.
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a positively charged channel within the smc1 smc3 hinge required for sister chromatid cohesion
The EMBO Journal, 2011Co-Authors: Alexander Kurze, Takehiko Itoh, Katsuhiko Shirahige, Christian H. Haering, Katharine A Michie, Sarah E Dixon, A K Mishra, Syma Khalid, Lana Strmecki, Jan LoweAbstract:Cohesin's structural maintenance of chromosome 1 (Smc1) and Smc3 are rod-shaped proteins with 50-nm long intra-molecular coiled-coil arms with a heterodimerization domain at one end and an ABC-like nucleotide-binding domain (NBD) at the other. Heterodimerization creates V-shaped molecules with a hinge at their centre. Inter-connection of NBDs by Scc1 creates a tripartite ring within which, it is proposed, sister DNAs are entrapped. To investigate whether cohesin's hinge functions as a possible DNA entry gate, we solved the crystal structure of the hinge from Mus musculus, which like its bacterial counterpart is characterized by a pseudo symmetric heterodimeric torus containing a small channel that is positively charged. Mutations in yeast Smc1 and Smc3 that together neutralize the channel's charge have little effect on dimerization or association with chromosomes, but are nevertheless lethal. Our finding that neutralization reduces acetylation of Smc3, which normally occurs during replication and is essential for cohesion, suggests that the positively charged channel is involved in a major conformational change during S phase.
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atp hydrolysis is required for relocating cohesin from sites occupied by its scc2 4 loading complex
Current Biology, 2011Co-Authors: Takehiko Itoh, Katsuhiko Shirahige, Koklung Chan, Maurici B Roig, A K Mishra, Yuki Katoh, William Upcher, Camilla Godlee, Kim NasmythAbstract:Summary Background The Cohesin complex that holds sister chromatins together until anaphase is comprised of three core subunits: Smc1 and Smc3, two long-rod-shaped proteins with an ABC-like ATPase head (nucleotide-binding domain [NBD]) and a dimerization domain linked by a 50 nm long intramolecular antiparallel coiled-coil, and Scc1, an α-kleisin subunit interconnecting the NBD domains of Smc1 and Smc3. Cohesin's stable association with chromosomes is thought to involve entrapment of chromatin fibers by its tripartite Smc1-Smc3-Scc1 ring via a poorly understood mechanism dependent on a separate Scc2/4 loading complex. A key issue concerns where entrapment initially takes place: at sites where cohesin is found stably associated or at distinct "loading" sites from which it translocates. Results In this study, we find transition state mutant versions (Smc1E1158Q and SmcE1155Q) defective in disengagement of their nucleotide binding domains (NBDs), unlike functional cohesin, colocalize with Scc2/4 at core centromeres, sites that catalyze wild-type cohesin's recruitment to sequences 20 kb or more away. In addition to Scc2/4, the unstable association of transition state complexes with core centromeres requires Scc1's association with Smc1 and Smc3 NBDs, ATP-driven NBD engagement, cohesin's Scc3 subunit, and its hinge domain. Conclusion We propose that cohesin's association with chromosomes is driven by two key events. NBD engagement driven by ATP binding produces an unstable association with specific loading sites like core centromeres, whereas subsequent ATP hydrolysis triggers DNA entrapment, which permits translocation along chromatin fibers.
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building sister chromatid cohesion smc3 acetylation counteracts an antiestablishment activity
Molecular Cell, 2009Co-Authors: Benjamin D Rowland, Pelin Uluocak, Frederic Beckouet, Maurici B Roig, Alexander Kurze, A K Mishra, Tatsuya Nishino, Philippa Underwood, Jean Metson, Richard ImreAbstract:Summary Cohesin's Smc1, Smc3, and Scc1 subunits form a tripartite ring that entraps sister DNAs. Scc3, Pds5, and Rad61 (Wapl) are regulatory subunits that control this process. We describe here smc3 , scc3 , pds5 , and rad61 mutations that permit yeast cell proliferation and entrapment of sister DNAs by cohesin rings in the absence of Eco1, an acetyl transferase normally essential for establishing sister chromatid cohesion. The smc3 mutations cluster around and include a highly conserved lysine (K113) close to Smc3's ATP-binding pocket, which, together with K112, is acetylated by Eco1. Lethality caused by mutating both residues to arginine is suppressed by the scc3 , pds5 , and rad61 mutants. Scc3, Pds5, and Rad61 form a complex and inhibit entrapment of sister DNAs by a process involving the "K112/K113" surface on Smc3's ATPase. According to this model, Eco1 promotes sister DNA entrapment partly by relieving an antiestablishment activity associated with Scc3, Pds5, and Rad61.
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building sister chromatid cohesion smc3 acetylation counteracts an antiestablishment activity
Molecular Cell, 2009Co-Authors: Enjami D Rowland, Pelin Uluocak, Alexander Kurze, A K Mishra, Tatsuya Nishino, Philippa Underwood, Maurici Roig, Frederic Eckoue, Jea Metso, Richard ImreAbstract:Cohesin's Smc1, Smc3, and Scc1 subunits form a tripartite ring that entraps sister DNAs. Scc3, Pds5, and Rad61 (Wapl) are regulatory subunits that control this process. We describe here smc3, scc3, pds5, and rad61 mutations that permit yeast cell proliferation and entrapment of sister DNAs by cohesin rings in the absence of Eco1, an acetyl transferase normally essential for establishing sister chromatid cohesion. The smc3 mutations cluster around and include a highly conserved lysine (K113) close to Smc3's ATP-binding pocket, which, together with K112, is acetylated by Eco1. Lethality caused by mutating both residues to arginine is suppressed by the scc3, pds5, and rad61 mutants. Scc3, Pds5, and Rad61 form a complex and inhibit entrapment of sister DNAs by a process involving the "K112/K113" surface on Smc3's ATPase. According to this model, Eco1 promotes sister DNA entrapment partly by relieving an antiestablishment activity associated with Scc3, Pds5, and Rad61.