The Experts below are selected from a list of 7368 Experts worldwide ranked by ideXlab platform
Alfred Wittinghofer - One of the best experts on this subject based on the ideXlab platform.
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structural basis for guanine nucleotide exchange on ran by the regulator of Chromosome Condensation rcc1
Cell, 2001Co-Authors: Louis Renault, Jurgen Kuhlmann, Andreas W Henkel, Alfred WittinghoferAbstract:Abstract RCC1 (regulator of Chromosome Condensation), a β propeller chromatin-bound protein, is the guanine nucleotide exchange factor (GEF) for the nuclear GTP binding protein Ran. We report here the 1.8 A crystal structure of a Ran•RCC1 complex in the absence of nucleotide, an intermediate in the multistep GEF reaction. In contrast to previous structures, the phosphate binding region of the nucleotide binding site is perturbed only marginally, possibly due to the presence of a polyvalent anion in the P loop. Biochemical experiments show that a sulfate ion stabilizes the Ran•RCC1 complex and inhibits dissociation by guanine nucleotides. Based on the available structural and biochemical evidence, we present a unified scenario for the GEF mechanism where interaction of the P loop lysine with an acidic residue is a crucial element for the overall reaction.
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structural basis for guanine nucleotide exchange on ran by the regulator of Chromosome Condensation rcc1
Cell, 2001Co-Authors: Louis Renault, Jurgen Kuhlmann, Andreas W Henkel, Alfred WittinghoferAbstract:RCC1 (regulator of Chromosome Condensation), a beta propeller chromatin-bound protein, is the guanine nucleotide exchange factor (GEF) for the nuclear GTP binding protein Ran. We report here the 1.8 A crystal structure of a Ran*RCC1 complex in the absence of nucleotide, an intermediate in the multistep GEF reaction. In contrast to previous structures, the phosphate binding region of the nucleotide binding site is perturbed only marginally, possibly due to the presence of a polyvalent anion in the P loop. Biochemical experiments show that a sulfate ion stabilizes the Ran*RCC1 complex and inhibits dissociation by guanine nucleotides. Based on the available structural and biochemical evidence, we present a unified scenario for the GEF mechanism where interaction of the P loop lysine with an acidic residue is a crucial element for the overall reaction.
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crystallization and preliminary x ray analysis of human rcc1 the regulator of Chromosome Condensation
Acta Crystallographica Section D-biological Crystallography, 1999Co-Authors: Louis Renault, Nicolas Nassar, Michel Roth, Alfred Wittinghofer, Ingrid R VetterAbstract:RCC1, the regulator of Chromosome Condensation, is the guanine nucleotide-exchange factor (GEF) of the GTP-binding protein Ran. Its GEF activity on Ran makes it a key element in nucleo-cytoplasmic transport and cell-cycle regulation. Crystals of human RCC1 suitable for X-ray analysis have been obtained using the seeding technique in hanging drops with sodium citrate as a precipitant. The crystals diffract to 1.7 A at 100 K and belong to the space group P1, with unit-cell parameters a = 49.5, b = 84.3, c = 84.9 A, α = 113.0, β = 103.9,γ = 103.3°. The Matthews parameter (Vm) and the self-rotation function are consistent with three molecules in the unit cell, which is confirmed by the averaged single isomorphous replacement (SIR) electron-density map.
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crystallization and preliminary x ray analysis of human rcc1 the regulator of Chromosome Condensation
Acta Crystallographica Section D-biological Crystallography, 1999Co-Authors: Louis Renault, Nicolas Nassar, Michel Roth, Alfred Wittinghofer, Ingrid R VetterAbstract:RCC1, the regulator of Chromosome Condensation, is the guanine nucleotide-exchange factor (GEF) of the GTP-binding protein Ran. Its GEF activity on Ran makes it a key element in nucleo-cytoplasmic transport and cell-cycle regulation. Crystals of human RCC1 suitable for X-ray analysis have been obtained using the seeding technique in hanging drops with sodium citrate as a precipitant. The crystals diffract to 1.7 A at 100 K and belong to the space group P1, with unit-cell parameters a = 49.5, b = 84.3, c = 84.9 A, α = 113.0, β = 103.9,γ = 103.3°. The Matthews parameter (Vm) and the self-rotation function are consistent with three molecules in the unit cell, which is confirmed by the averaged single isomorphous replacement (SIR) electron-density map.
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the 1 7 a crystal structure of the regulator of Chromosome Condensation rcc1 reveals a seven bladed propeller
Nature, 1998Co-Authors: Louis Renault, Christian Klebe, Nicolas Nassar, Michel Roth, Ingrid R Vetter, Jörg Becker, Alfred WittinghoferAbstract:The gene encoding the regulator of Chromosome Condensation (RCC1) was cloned by virtue of its ability to complement the temperature-sensitive phenotype of the hamster cell line tsBN2, which undergoes premature Chromosome Condensation or arrest in the G1 phase of the cell cycle at non-permissive temperatures1,2. RCC1 homologues have been identified in many eukaryotes, including budding and fission yeast. Mutations in the gene affect pre-messenger RNA processing and transport3,4, mating5, initiation of mitosis6 and chromatin deCondensation7, suggesting that RCC1 is important in the control of nucleo-cytoplasmic transport and the cell cycle. Biochemically, RCC1 is a guanine-nucleotide-exchange factor for the nuclear Ras homologue Ran8; it increases the dissociation of Ran-bound GDP by 105-fold (ref. 9). It may also bind to DNA via a protein–protein complex2. Here we show that the structure of human RCC1, solved to 1.7-A resolution by X-ray crystallography, consists of a seven-bladed propeller formed from internal repeats of 51–68 residues per blade. The sequence and structure of the repeats differ from those of WD40-domain proteins, which also form seven-bladed propellers and include the β-subunits of G proteins. The nature of the structure explains the consequences of a wide range of known mutations. The region of the protein that is involved in guanine-nucleotide exchange is located opposite the region that is thought to be involved in Chromosome binding.
Louis Renault - One of the best experts on this subject based on the ideXlab platform.
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structural basis for guanine nucleotide exchange on ran by the regulator of Chromosome Condensation rcc1
Cell, 2001Co-Authors: Louis Renault, Jurgen Kuhlmann, Andreas W Henkel, Alfred WittinghoferAbstract:Abstract RCC1 (regulator of Chromosome Condensation), a β propeller chromatin-bound protein, is the guanine nucleotide exchange factor (GEF) for the nuclear GTP binding protein Ran. We report here the 1.8 A crystal structure of a Ran•RCC1 complex in the absence of nucleotide, an intermediate in the multistep GEF reaction. In contrast to previous structures, the phosphate binding region of the nucleotide binding site is perturbed only marginally, possibly due to the presence of a polyvalent anion in the P loop. Biochemical experiments show that a sulfate ion stabilizes the Ran•RCC1 complex and inhibits dissociation by guanine nucleotides. Based on the available structural and biochemical evidence, we present a unified scenario for the GEF mechanism where interaction of the P loop lysine with an acidic residue is a crucial element for the overall reaction.
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structural basis for guanine nucleotide exchange on ran by the regulator of Chromosome Condensation rcc1
Cell, 2001Co-Authors: Louis Renault, Jurgen Kuhlmann, Andreas W Henkel, Alfred WittinghoferAbstract:RCC1 (regulator of Chromosome Condensation), a beta propeller chromatin-bound protein, is the guanine nucleotide exchange factor (GEF) for the nuclear GTP binding protein Ran. We report here the 1.8 A crystal structure of a Ran*RCC1 complex in the absence of nucleotide, an intermediate in the multistep GEF reaction. In contrast to previous structures, the phosphate binding region of the nucleotide binding site is perturbed only marginally, possibly due to the presence of a polyvalent anion in the P loop. Biochemical experiments show that a sulfate ion stabilizes the Ran*RCC1 complex and inhibits dissociation by guanine nucleotides. Based on the available structural and biochemical evidence, we present a unified scenario for the GEF mechanism where interaction of the P loop lysine with an acidic residue is a crucial element for the overall reaction.
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crystallization and preliminary x ray analysis of human rcc1 the regulator of Chromosome Condensation
Acta Crystallographica Section D-biological Crystallography, 1999Co-Authors: Louis Renault, Nicolas Nassar, Michel Roth, Alfred Wittinghofer, Ingrid R VetterAbstract:RCC1, the regulator of Chromosome Condensation, is the guanine nucleotide-exchange factor (GEF) of the GTP-binding protein Ran. Its GEF activity on Ran makes it a key element in nucleo-cytoplasmic transport and cell-cycle regulation. Crystals of human RCC1 suitable for X-ray analysis have been obtained using the seeding technique in hanging drops with sodium citrate as a precipitant. The crystals diffract to 1.7 A at 100 K and belong to the space group P1, with unit-cell parameters a = 49.5, b = 84.3, c = 84.9 A, α = 113.0, β = 103.9,γ = 103.3°. The Matthews parameter (Vm) and the self-rotation function are consistent with three molecules in the unit cell, which is confirmed by the averaged single isomorphous replacement (SIR) electron-density map.
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crystallization and preliminary x ray analysis of human rcc1 the regulator of Chromosome Condensation
Acta Crystallographica Section D-biological Crystallography, 1999Co-Authors: Louis Renault, Nicolas Nassar, Michel Roth, Alfred Wittinghofer, Ingrid R VetterAbstract:RCC1, the regulator of Chromosome Condensation, is the guanine nucleotide-exchange factor (GEF) of the GTP-binding protein Ran. Its GEF activity on Ran makes it a key element in nucleo-cytoplasmic transport and cell-cycle regulation. Crystals of human RCC1 suitable for X-ray analysis have been obtained using the seeding technique in hanging drops with sodium citrate as a precipitant. The crystals diffract to 1.7 A at 100 K and belong to the space group P1, with unit-cell parameters a = 49.5, b = 84.3, c = 84.9 A, α = 113.0, β = 103.9,γ = 103.3°. The Matthews parameter (Vm) and the self-rotation function are consistent with three molecules in the unit cell, which is confirmed by the averaged single isomorphous replacement (SIR) electron-density map.
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the 1 7 a crystal structure of the regulator of Chromosome Condensation rcc1 reveals a seven bladed propeller
Nature, 1998Co-Authors: Louis Renault, Christian Klebe, Nicolas Nassar, Michel Roth, Ingrid R Vetter, Jörg Becker, Alfred WittinghoferAbstract:The gene encoding the regulator of Chromosome Condensation (RCC1) was cloned by virtue of its ability to complement the temperature-sensitive phenotype of the hamster cell line tsBN2, which undergoes premature Chromosome Condensation or arrest in the G1 phase of the cell cycle at non-permissive temperatures1,2. RCC1 homologues have been identified in many eukaryotes, including budding and fission yeast. Mutations in the gene affect pre-messenger RNA processing and transport3,4, mating5, initiation of mitosis6 and chromatin deCondensation7, suggesting that RCC1 is important in the control of nucleo-cytoplasmic transport and the cell cycle. Biochemically, RCC1 is a guanine-nucleotide-exchange factor for the nuclear Ras homologue Ran8; it increases the dissociation of Ran-bound GDP by 105-fold (ref. 9). It may also bind to DNA via a protein–protein complex2. Here we show that the structure of human RCC1, solved to 1.7-A resolution by X-ray crystallography, consists of a seven-bladed propeller formed from internal repeats of 51–68 residues per blade. The sequence and structure of the repeats differ from those of WD40-domain proteins, which also form seven-bladed propellers and include the β-subunits of G proteins. The nature of the structure explains the consequences of a wide range of known mutations. The region of the protein that is involved in guanine-nucleotide exchange is located opposite the region that is thought to be involved in Chromosome binding.
William F Blakely - One of the best experts on this subject based on the ideXlab platform.
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a novel parameter cell cycle progression index for radiation dose absorbed estimation in the premature Chromosome Condensation assay
Radiation Protection Dosimetry, 2014Co-Authors: Tomisato Miura, Akifumi Nakata, Kosuke Kasai, Manabu Nakano, Yu Abe, Eiki Tsushima, Natalia I Ossetrova, Mitsuaki A Yoshida, William F BlakelyAbstract:Abstract The calyculin A-induced premature Chromosome Condensation (PCC) assay is a simple and useful method for assessing the cell-cycle distribution in cells, since calyculin A induces Chromosome Condensation in various phases of the cell cycle. In this study, a novel parameter, the cell-cycle progression index (CPI), in the PCC assay was validated as a novel biomarker for biodosimetry. Peripheral blood was drawn from healthy donors after informed consent was obtained. CPI was investigated using a human peripheral blood lymphocyte (PBL) ex vivo irradiation ((60)Co-gamma rays: ∼0.6 Gy min(-1), or X ray: 1.0 Gy min(-1); 0-10 Gy) model. The calyculin A-induced PCC assay was performed for Chromosome preparation. PCC cells were divided into the following five categories according to cell-cycle stage: non-PCC, G1-PCC, S-PCC, G2/M-PCC and M/A-PCC cells. CPI was calculated as the ratio of G2/M-PCC cells to G1-PCC cells. The PCC-stage distribution varied markedly with irradiation doses. The G1-PCC cell fraction was significantly reduced, and the G2/M-PCC cell fraction increased, in 10-Gy-irradiated PBL after 48 h of culture. CPI levels were fitted to an exponential dose-response curve with gamma-ray irradiation [y = 0.6729 + 0.3934 exp(0.5685D), r = 1.0000, p < 0.0001] and X-ray irradiation [y = -0.3743 + 0.9744 exp(0.3321D), r = 0.9999, p < 0.0001]. There were no significant individual (p = 0.853) or gender effects (p = 0.951) on the CPI in the human peripheral blood ex vivo irradiation model. Furthermore, CPI measurements are rapid (< 15 min per case). These results suggest that the CPI is a useful screening tool for the assessment of radiation doses received ranging from 0 to 10 Gy in radiation exposure early after a radiation event, especially after a mass-casualty radiological incident.
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optimization of calyculin a induced premature Chromosome Condensation assay for Chromosome aberration studies
Cytometry Part A, 2011Co-Authors: Tomisato Miura, William F BlakelyAbstract:Calyculin A-induced premature Chromosome Condensation (PCC) assay is a simple and useful method to assess structural and numerical Chromosome aberrations in cells. Our hypothesis in this study is that suboptimum calyculin A induction of PCC resulting in fuzzy compactness and/or shortened length Chromosomes would decrease the detection sensitivity of numerical and structural Chromosome aberrations such as small PCC rings and small excess fragments. In this study, an optimization of calyculin A exposure on Chromosome morphology and PCC induction frequency was investigated using a human peripheral blood lymphocyte (PBL) ex vivo irradiation (60Co-γ rays; ∼0.6 Gy/min; 0–30 Gy) model. Treatment with calyculin A (50 nM) for 15 and 30 min resulted in 11.3 ± 2.7 and 9.9 ± 1.6-fold increases in the frequency of G2/M-PCC cells with extended length Chromosomes compared with the 60-min treated group over a broad dose range (0 to 20 Gy), respectively. The G2/M-PCC scoring index per PCC in 15- and 30-min treated groups was increased by 1.9 ± 0.2 (P = 0.001) and 1.8 ± 0.2 (P = 0.001) compared with the 60-min treated group over 0–20 Gy, respectively. The G2/M-PCC efficiency of 30-min treated group was highest in the three conditions (i.e., 15-, 30-, and 60-min treatment) of calyculin A exposure. Calyculin A (50 nM) treatment for 30 min before the 48-h harvest of mitogen-stimulated human PBL is optimum for the formation of suitable Chromosome morphology necessary to assess structural Chromosome aberrations induced by exposure to radiation using the chemical induced-PCC assay. Published 2011 Wiley Periodicals, Inc.
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premature Chromosome Condensation in human resting peripheral blood lymphocytes for Chromosome aberration analysis using specific whole Chromosome dna hybridization probes
Methods of Molecular Biology, 2005Co-Authors: Pataje G S Prasanna, William F BlakelyAbstract:: This paper describes a unique, simple, and rapid method for inducing premature Chromosome Condensation (PCC) in "resting" human peripheral blood lymphocytes (HPBLs) and also explains an approach to studying numerical changes and/or structural aberrations involving specific Chromosomes. HPBLs are isolated from whole blood on a density gradient and, to induce PCC, are incubated at 37 degrees C in cell culture medium supplemented with a phosphatase inhibitor (okadaic acid or calyculin A), adenosine triphosphate (ATP), and p34cdc2/cyclin B kinase (an essential component of mitosis-promoting factor [MPF]). PCC spreads are prepared on glass slides after a brief hypotonic treatment of cells and fixing in acetic acid/methanol fixative. Aberrations involving specific Chromosomes are analyzed after in situ hybridization and Chromosome painting by fluorescence microscopy. Normal (undamaged) cells display two fluorescent spots per Chromosome, whereas aneuploid cells, or cells with a structural aberration involving the specific Chromosome corresponding to the painting probe, may show more than two spots. This method may be used in many biological and toxicological fields that require analysis of numerical and structural aberrations involving specific Chromosomes.
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induction of premature Chromosome Condensation by a phosphatase inhibitor and a protein kinase in unstimulated human peripheral blood lymphocytes a simple and rapid technique to study Chromosome aberrations using specific whole Chromosome dna hybridization probes for biological dosimetry
Mutation Research-genetic Toxicology and Environmental Mutagenesis, 2000Co-Authors: Pataje G S Prasanna, Nestor D Escalada, William F BlakelyAbstract:Abstract We developed a simple and rapid method to study Chromosome aberrations involving specific Chromosomes using unstimulated human peripheral blood lymphocytes (HPBL). Premature Chromosome Condensation (PCC) was induced by incubating unstimulated HPBL in the presence of okadaic acid (OA, a phosphatase inhibitor), adenosine triphosphate (ATP), and p34 cdc2 /cyclin B kinase [an essential component of mitosis-promoting factor (MPF)], which eliminated the need for fusion with mitotic cells. OA concentration and duration of incubation for PCC induction was optimized using mitogen-stimulated HPBL; a final concentration of 0.75 μM incubated for 3 h was optimum, resulting in approximately 20% PCC yield. In unstimulated HPBL, PCC was induced by the addition of p34 cdc2 /cyclin B kinase at concentrations as low as 5 units/ml to a cell culture medium containing OA. Increases in the concentration of p34 cdc2 /cyclin B kinase from 5 to 50 units/ml resulted in a concentration-dependent increase in PCC yield (30% to 42%). We demonstrate that this technique of inducing PCC in unstimulated HPBL is suitable for studying radiation-induced aberrations involving a specific Chromosome (Chromosome 1) after 24 h repair using a whole-Chromosome in situ hybridization probe and Chromosome painting. Cells with aberrant Chromosome number 1 are characterized with more than two Chromosome spots. The frequency of cells with aberrant Chromosome 1 increased with 60 Co gamma-radiation doses in the region 0–7.5 Gy. The observed dose–effect relationship for the percentage of cells with aberrant Chromosome 1 ( Y ) was explained by using both a linear [ Y =(2.77±0.230) D +0.90±0.431, r 2 =0.966] and a nonlinear power [ Y =(5.70±0.46) D (0.61±0.05) , r 2 =0.9901) model. This technique can be applied to biological dosimetry of radiation exposures involving uniform whole-body low linear energy transfer (LET) exposures.
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application of the premature Chromosome Condensation assay in simulated partial body radiation exposures evaluation of the use of an automated metaphase finder
Stem Cells, 1995Co-Authors: William F Blakely, Pataje G S Prasanna, Christopher J Kolanko, Mark D Pyle, D M MosbrookAbstract:Abstract : The premature Chromosome Condensation (PCC) assay has been proposed as a useful and rapid end point for biological dosimetry following accidental high-dose radiation overexposures. A major benefit of the PCC assay is that it does not require cells to divide for evaluation of cytogenetic damage. The PCC assay was performed on isolated human peripheral lymphocytes exposed in vitro to doses from 1 to 9 Gy of 250 kVp x-rays. The dose-response relationships of the frequency distribution and the yield of PCC fragments in cells were determined after one day of repair at 37 deg C. A Q(pcc) approach, which involves the analysis of the yield of excess PCC fragments in damaged cells, was used to establish a dose-response calibration curve. This method is identical in concept to the Q(dr) technique introduced by Sasaki for partial-body exposure dose-estimates using asymmetrical Chromosome aberrations (i.e., dicentrics and rings) in metaphase spreads of human lymphocytes. A simulated in vitro test of a partial-body exposure to a 6-Gy dose was performed. The results from this test provided dose estimates of 5.3 + or = 0.6, 4.7 + or = 0.6,5.0 + or = 0.6 and 4.7 + or = 0.8 Gy for the 20,30,50 and 75 percent component of 6-Gy irradiated cells, respectively. An automated metaphase-finding system was evaluated for use with the PCC assay. This system helped to locate PCC spreads among the mitotic inducer Chinese hamster ovary (CHO) metaphase spreads, thereby facilitating rapid scoring of samples. We conclude that the measurement of excess PCC fragments in Giemsa-stained preparations provides useful biological dosimetry information on the size of the irradiated fraction in cases of acute radiation exposures. Use of Q(pcc) analysis is recommended for partial-body exposures to determine dose estimates for the irradiated fraction. Automated metaphasc finding significantly enhances the spced of the PCC assay.
Tatsuya Hirano - One of the best experts on this subject based on the ideXlab platform.
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condensin targets and reduces unwound dna structures associated with transcription in mitotic Chromosome Condensation
Nature Communications, 2015Co-Authors: Takashi Sutani, Tatsuya Hirano, Daisuke Yamashita, Toyonori Sakata, Ryuichiro Nakato, Koji Masuda, Mai Ishibashi, Yutaka Suzuki, Masashige Bando, Katsuhiko ShirahigeAbstract:Chromosome Condensation is a hallmark of mitosis in eukaryotes and is a prerequisite for faithful segregation of genetic material to daughter cells. Here we show that condensin, which is essential for assembling condensed Chromosomes, helps to preclude the detrimental effects of gene transcription on mitotic Condensation. ChIP-seq profiling reveals that the fission yeast condensin preferentially binds to active protein-coding genes in a transcription-dependent manner during mitosis. Pharmacological and genetic attenuation of transcription largely rescue bulk Chromosome segregation defects observed in condensin mutants. We also demonstrate that condensin is associated with and reduces unwound DNA segments generated by transcription, providing a direct link between an in vitro activity of condensin and its in vivo function. The human condensin isoform condensin I also binds to unwound DNA regions at the transcription start sites of active genes, implying that our findings uncover a fundamental feature of condensin complexes.
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mcph1 regulates Chromosome Condensation and shaping as a composite modulator of condensin ii
Journal of Cell Biology, 2011Co-Authors: Daisuke Yamashita, Keishi Shintomi, Takao Ono, Ioannis Gavvovidis, Detlev Schindler, Heidemarie Neitzel, Marc Trimborn, Tatsuya HiranoAbstract:Mutations in human MCPH1 (hMCPH1) cause primary microcephaly, which is characterized by a marked reduction of brain size. Interestingly, hMCPH1 mutant patient cells display unique cellular phenotypes, including premature Chromosome Condensation (PCC), in G2 phase. To test whether hMCPH1 might directly participate in the regulation of Chromosome Condensation and, if so, how, we developed a cell-free assay using Xenopus laevis egg extracts. Our results demonstrate that an N-terminal domain of hMCPH1 specifically inhibits the action of condensin II by competing for its chromosomal binding sites in vitro. This simple and powerful assay allows us to dissect mutations causing primary microcephaly in vivo and evolutionary substitutions among different species. A complementation assay using patient cells revealed that, whereas the N-terminal domain of hMCPH1 is sufficient to rescue the PCC phenotype, its central domain plays an auxiliary role in shaping metaphase Chromosomes by physically interacting with condensin II. Thus, hMCPH1 acts as a composite modulator of condensin II to regulate Chromosome Condensation and shaping.
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Chromosome Condensation by a human condensin complex in Xenopus egg extracts.
The Journal of biological chemistry, 2001Co-Authors: Keiji Kimura, Olivier Cuvier, Tatsuya HiranoAbstract:13S condensin is a five-subunit protein complex that plays a central role in mitotic Chromosome Condensation. The condensin complex was originally identified and purified from Xenopus egg extracts and shown to have an ATP-dependent positive supercoiling activity in vitro. We report here the characterization of a human condensin complex purified from HeLa cell nuclear extracts. The human 13S complex has exactly the same composition as its Xenopus counterpart, being composed of two structural maintenance of Chromosomes (human Chromosome-associated polypeptide (hCAP)-C and hCAP-E) subunits and three non-structural maintenance of Chromosomes (hCAP-D2/CNAP1, hCAP-G, and hCAP-H/BRRN) subunits. Human condensin purified from asynchronous HeLa cell cultures fails to reconfigure DNA structure in vitro. When phosphorylated by purified cdc2-cyclin B, however, it gains the ability to introduce positive supercoils into DNA in the presence of ATP and topoisomerase I. Strikingly, human condensin can induce Chromosome Condensation when added back into a Xenopus egg extract that has been immunodepleted of endogenous condensin. Thus, the structure and function of the condensin complex are highly conserved between Xenopus and humans, underscoring its fundamental importance in mitotic Chromosome dynamics in eukaryotic cells.
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phosphorylation and activation of 13s condensin by cdc2 in vitro
Science, 1998Co-Authors: Keiji Kimura, Ryuji Kobayashi, Michiko Hirano, Tatsuya HiranoAbstract:13S condensin is a multisubunit protein complex essential for mitotic Chromosome Condensation in Xenopus egg extracts. Purified 13S condensin introduces positive supercoils into DNA in the presence of topoisomerase I and adenosine triphosphate in vitro. The supercoiling activity of 13Scondensin was regulated by mitosis-specific phosphorylation. Immunodepletion, in vitro phosphorylation, and peptide-mapping experiments indicated that Cdc2 is likely to be the kinase that phosphorylates and activates 13S condensin. Multiple Cdc2 phosphorylation sites are clustered in the carboxyl-terminal domain of the XCAP-D2 (Xenopus Chromosome-associated polypeptide D2) subunit. These results suggest that phosphorylation of 13Scondensin by Cdc2 may trigger mitotic Chromosome Condensation in vitro.
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atp dependent positive supercoiling of dna by 13s condensin a biochemical implication for Chromosome Condensation
Cell, 1997Co-Authors: Keiji Kimura, Tatsuya HiranoAbstract:13S condensin is a five-subunit protein complex that plays a central role in mitotic Chromosome Condensation in Xenopus egg extracts. Two core subunits of this complex, XCAP-C and XCAP-E, belong to an emerging family of putative ATPases, the SMC family. We report here that 13S condensin has a DNA-stimulated ATPase activity and exhibits a high affinity for structured DNAs such as cruciform DNA. 13S condensin is able to introduce positive supercoils into a closed circular DNA in the presence of bacterial or eukaryotic topoisomerase I. The supercoiling reaction is ATP-dependent. We propose that 13S condensin wraps DNA in a right-handed direction by utilizing the energy of ATP hydrolysis. This reaction may represent a key mechanism underlying the compaction of chromatin fibers during mitosis.
Ingrid R Vetter - One of the best experts on this subject based on the ideXlab platform.
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crystallization and preliminary x ray analysis of human rcc1 the regulator of Chromosome Condensation
Acta Crystallographica Section D-biological Crystallography, 1999Co-Authors: Louis Renault, Nicolas Nassar, Michel Roth, Alfred Wittinghofer, Ingrid R VetterAbstract:RCC1, the regulator of Chromosome Condensation, is the guanine nucleotide-exchange factor (GEF) of the GTP-binding protein Ran. Its GEF activity on Ran makes it a key element in nucleo-cytoplasmic transport and cell-cycle regulation. Crystals of human RCC1 suitable for X-ray analysis have been obtained using the seeding technique in hanging drops with sodium citrate as a precipitant. The crystals diffract to 1.7 A at 100 K and belong to the space group P1, with unit-cell parameters a = 49.5, b = 84.3, c = 84.9 A, α = 113.0, β = 103.9,γ = 103.3°. The Matthews parameter (Vm) and the self-rotation function are consistent with three molecules in the unit cell, which is confirmed by the averaged single isomorphous replacement (SIR) electron-density map.
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crystallization and preliminary x ray analysis of human rcc1 the regulator of Chromosome Condensation
Acta Crystallographica Section D-biological Crystallography, 1999Co-Authors: Louis Renault, Nicolas Nassar, Michel Roth, Alfred Wittinghofer, Ingrid R VetterAbstract:RCC1, the regulator of Chromosome Condensation, is the guanine nucleotide-exchange factor (GEF) of the GTP-binding protein Ran. Its GEF activity on Ran makes it a key element in nucleo-cytoplasmic transport and cell-cycle regulation. Crystals of human RCC1 suitable for X-ray analysis have been obtained using the seeding technique in hanging drops with sodium citrate as a precipitant. The crystals diffract to 1.7 A at 100 K and belong to the space group P1, with unit-cell parameters a = 49.5, b = 84.3, c = 84.9 A, α = 113.0, β = 103.9,γ = 103.3°. The Matthews parameter (Vm) and the self-rotation function are consistent with three molecules in the unit cell, which is confirmed by the averaged single isomorphous replacement (SIR) electron-density map.
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the 1 7 a crystal structure of the regulator of Chromosome Condensation rcc1 reveals a seven bladed propeller
Nature, 1998Co-Authors: Louis Renault, Christian Klebe, Nicolas Nassar, Michel Roth, Ingrid R Vetter, Jörg Becker, Alfred WittinghoferAbstract:The gene encoding the regulator of Chromosome Condensation (RCC1) was cloned by virtue of its ability to complement the temperature-sensitive phenotype of the hamster cell line tsBN2, which undergoes premature Chromosome Condensation or arrest in the G1 phase of the cell cycle at non-permissive temperatures1,2. RCC1 homologues have been identified in many eukaryotes, including budding and fission yeast. Mutations in the gene affect pre-messenger RNA processing and transport3,4, mating5, initiation of mitosis6 and chromatin deCondensation7, suggesting that RCC1 is important in the control of nucleo-cytoplasmic transport and the cell cycle. Biochemically, RCC1 is a guanine-nucleotide-exchange factor for the nuclear Ras homologue Ran8; it increases the dissociation of Ran-bound GDP by 105-fold (ref. 9). It may also bind to DNA via a protein–protein complex2. Here we show that the structure of human RCC1, solved to 1.7-A resolution by X-ray crystallography, consists of a seven-bladed propeller formed from internal repeats of 51–68 residues per blade. The sequence and structure of the repeats differ from those of WD40-domain proteins, which also form seven-bladed propellers and include the β-subunits of G proteins. The nature of the structure explains the consequences of a wide range of known mutations. The region of the protein that is involved in guanine-nucleotide exchange is located opposite the region that is thought to be involved in Chromosome binding.