The Experts below are selected from a list of 291 Experts worldwide ranked by ideXlab platform
Tuneko Okazaki - One of the best experts on this subject based on the ideXlab platform.
-
A helix–turn–helix structure unit in human <B>CentromereB> <B>ProteinB> B (CENP-B)
The EMBO journal, 1998Co-Authors: Junji Iwahara, Hiroshi Masumoto, Tuneko Okazaki, Katsumi Kitagawa, Takanori Kigawa, Shigeyuki YokoyamaAbstract:CENP-B has Been suggested to organize arrays of <B>CentromereB> satellite DNA into a higher order structure which then directs <B>CentromereB> formation and kinetochore assemBly in mammalian chromosomes. The N-terminal portion of CENP-B is a 15 kDa DNA Binding domain (DBD) consisting of two repeating units, RP1 and RP2. The DBD specifically Binds to the CENP-B Box sequence (17 Bp) in <B>CentromereB> DNA. We determined the solution structure of human CENP-B DBD RP1 By multi-dimensional 1H, 13C and 15N NMR methods. The CENP-B DBD RP1 structure consists of four helices and has a helix-turn-helix structure. The overall folding is similar to those of some other eukaryotic DBDs, although significant sequence homology with these <B>ProteinB>s was not found. The DBD of yeast RAP1, a telomere Binding <B>ProteinB>, is most similar to CENP-B DBD RP1. We studied the interaction Between CENP-B DBD RP1 and the CENP-B Box By the use of NMR chemical shift perturBation. The results suggest that CENP-B DBD RP1 interacts with one of the essential regions of the CENP-B Box DNA, mainly at the N-terminal Basic region, the N-terminal portion of helix 2 and helix 3.
-
a helix turn helix structure unit in human <B>CentromereB> <B>ProteinB> B cenp B
The EMBO Journal, 1998Co-Authors: Junji Iwahara, Hiroshi Masumoto, Tuneko Okazaki, Katsumi Kitagawa, Takanori Kigawa, Shigeyuki YokoyamaAbstract:CENP-B has Been suggested to organize arrays of <B>CentromereB> satellite DNA into a higher order structure which then directs <B>CentromereB> formation and kinetochore assemBly in mammalian chromosomes. The N-terminal portion of CENP-B is a 15 kDa DNA Binding domain (DBD) consisting of two repeating units, RP1 and RP2. The DBD specifically Binds to the CENP-B Box sequence (17 Bp) in <B>CentromereB> DNA. We determined the solution structure of human CENP-B DBD RP1 By multi-dimensional 1H, 13C and 15N NMR methods. The CENP-B DBD RP1 structure consists of four helices and has a helix-turn-helix structure. The overall folding is similar to those of some other eukaryotic DBDs, although significant sequence homology with these <B>ProteinB>s was not found. The DBD of yeast RAP1, a telomere Binding <B>ProteinB>, is most similar to CENP-B DBD RP1. We studied the interaction Between CENP-B DBD RP1 and the CENP-B Box By the use of NMR chemical shift perturBation. The results suggest that CENP-B DBD RP1 interacts with one of the essential regions of the CENP-B Box DNA, mainly at the N-terminal Basic region, the N-terminal portion of helix 2 and helix 3.
-
Construction of YAC-Based mammalian artificial chromosomes.
Nature Biotechnology, 1998Co-Authors: Masashi Ikeno, Tuneko Okazaki, Megumi Nakano, Brenda Grimes, Kaori Saitoh, Harumi Hoshino, Niolette I. Mcgill, Howard J. Cooke, Hiroshi MasumotoAbstract:To construct a mammalian artificial chromosome (MAC), telomere repeats and selectaBle markers were introduced into a 100 kB yeast artificial chromosome (YAC) containing human centromeric DNA. This YAC, which has a regular repeat structure of alpha-satellite DNA and <B>CentromereB> <B>ProteinB> B (CENP-B) Boxes, efficiently formed MACs that segregated accurately and Bound CENP-B, CENP-C, and CENP-E. The MACs appear to Be aBout 1–5 MB in size and contain YAC multimers. Structural analyses suggest that the MACs have not acquired host sequences and were formed By a de novo mechanism. The accurate segregation of the MACs suggests they have potential as vectors for introducing genes into mammals.
-
<B>CentromereB> <B>ProteinB> B of African green monkey cells: gene structure, cellular expression, and centromeric localization.
Molecular and cellular biology, 1996Co-Authors: Kinya Yoda, Hiroshi Masumoto, T Nakamura, Nobutaka Suzuki, Katsumi Kitagawa, Megumi Nakano, A Shinjo, Tuneko OkazakiAbstract:<B>CentromereB> <B>ProteinB> B (CENP-B) is a centromeric DNA-Binding <B>ProteinB> which recognizes a 17-Bp sequence (CENP-B Box) in human and mouse centromeric satellite DNA. The African green monkey (AGM) is phylogenetically closer to humans than mice and is known to contain large amounts of a-satellite DNA, But there has Been no report of CENP-B Boxes or CENP-B in the <B>CentromereB> domains of its chromosomes. To elucidate the AGM CENP-B‐CENP-B Box interaction, we have analyzed the gene structure, expression, Biochemical properties, and centromeric localization of its CENP-B. The amino acid sequence deduced from the cloned AGM CENP-B gene was estaBlished to Be highly homologous to that of human and mouse CENP-B. In particular, the DNA Binding and homodimer formation domains demonstrated 100% identity to their human and mouse counterparts. ImmunoBlotting and DNA moBility shift analyses revealed CENP-B to Be expressed in AGM cell lines. As predicted from the gene structure, the AGM CENP-B in the cell extracts exhiBited the same DNA Binding specificity and homodimer forming activity as human CENP-B. By indirect immunofluorescent staining of AGM mitotic cells with anti-CENP-B antiBodies, a <B>CentromereB>-specific localization of AGM CENP-BcouldBedemonstrated.WealsoisolatedAGM a-satelliteDNAwithaCENP-BBox-likesequencewith CENP-Baffinity.TheseresultsnotonlyprovethatCENP-BfunctionallypersistsinAGMcellsButalsosuggest that the AGM genome contains the recognition sequences for CENP-B (CENP-B Boxes with the core recognition sequence or CENP-B Box variants) in centromeric satellite DNA.
-
Analysis of <B>ProteinB>-DNA and <B>ProteinB>-<B>ProteinB> interactions of <B>CentromereB> <B>ProteinB> B (CENP-B) and properties of the DNA-CENP-B complex in the cell cycle.
Molecular and cellular biology, 1995Co-Authors: Katsumi Kitagawa, Hiroshi Masumoto, M Ikeda, Tuneko OkazakiAbstract:We previously reported that <B>CentromereB> <B>ProteinB> B (CENP-B) forms a staBle complex (designated complex A) containing two alphoid DNAs in vitro. Domains in the CENP-B polypeptide involved in the formation of complex A were determined in the present study with truncated derivatives expressed in Escherichia coli and in raBBit reticulocyte lysates. It was revealed By gel moBility shift analyses that polypeptides containing the NH2-terminal DNA-Binding domain Bind a DNA molecule as a monomer, while dimerizing at a novel hydrophoBic domain in the COOH-terminal region of 59 amino acid residues. This polypeptide dimerization activity at the COOH-terminal region was also confirmed with the two-hyBrid system in Saccharomyces cerevisiae cells. The results thus proved that CENP-B polypeptides form a homodimer at the COOH-terminal hydrophoBic domain, each Binding a DNA strand at their NH2-terminal domains. The dimerization and DNA-Binding domains fall into two of the three completely conserved sequences found in human and mouse CENP-B, and complex A-forming activity was also detected in nuclear extracts of mouse cells. Metaphase-specific phosphorylation of CENP-B was also detected, But this had no effect on its complex A-forming activity. On the Basis of the present results, we propose that CENP-B plays an important role in the assemBly of specific <B>CentromereB> structures By forming unique DNA-<B>ProteinB> complexes at the sites of CENP-B Boxes on the centromeric repetitive DNA Both in interphase nuclei and on mitotic chromosomes.
Hiroshi Masumoto - One of the best experts on this subject based on the ideXlab platform.
-
Formation of functional CENP-B Boxes at diverse locations in repeat units of centromeric DNA in New World monkeys
Scientific reports, 2016Co-Authors: Kazuto Kugou, Hiroshi Masumoto, Hirohisa Hirai, Akihiko KogaAbstract:<B>CentromereB> <B>ProteinB> B, which is involved in <B>CentromereB> formation, Binds to centromeric repetitive DNA By recognizing a nucleotide motif called the CENP-B Box. Humans have large numBers of CENP-B Boxes in the centromeric repetitive DNA of their autosomes and X chromosome. The current understanding is that these CENP-B Boxes are located at identical positions in the repeat units of centromeric DNA. Great apes also have CENP-B Boxes in locations that are identical to humans. The purpose of the present study was to examine the location of CENP-B Box in New World monkeys. We recently identified CENP-B Box in one species of New World monkeys (marmosets). In this study, we found functional CENP-B Boxes in CENP-A-assemBled repeat units of centromeric DNA in 2 additional New World monkeys (squirrel monkeys and tamarins) By immunostaining and ChIP-qPCR analyses. The locations of the 3 CENP-B Boxes in the repeat units differed from one another. The repeat unit size of centromeric DNA of New World monkeys (340-350 Bp) is approximately twice that of humans and great apes (171 Bp). This might Be, associated with higher-order repeat structures of centromeric DNA, a factor for the oBserved variation in the CENP-B Box location in New World monkeys.
-
CENP-B Box, a nucleotide motif involved in <B>CentromereB> formation, occurs in a New World monkey
Biology Letters, 2016Co-Authors: Aorarat Suntronpong, Hiroshi Masumoto, Kornsorn Srikulnath, Hirohisa Hirai, Kazuto Kugou, Kazuhiko Ohshima, Akihiko KogaAbstract:<B>CentromereB> <B>ProteinB> B (CENP-B) is one of the major <B>ProteinB>s involved in <B>CentromereB> formation, Binding to centromeric repetitive DNA By recognizing a 17 Bp motif called the CENP-B Box. Hominids (humans and great apes) carry large numBers of CENP-B Boxes in alpha satellite DNA (AS, the major centromeric repetitive DNA of simian primates). Only negative results have Been reported regarding the presence of the CENP-B Box in other primate taxa. Consequently, it is widely Believed that the CENP-B Box is confined, within primates, to the hominids. We report here that the common marmoset, a New World monkey, contains an aBundance of CENP-B Boxes in its AS. First, in a long contig sequence we constructed and analysed, we identified the motif in 17 of the 38 alpha satellite repeat units. We then sequenced terminal regions of additional clones and found the motif in many of them. Immunostaining of marmoset cells demonstrated that CENP-B Binds to DNA in the centromeric regions of chromosomes. Therefore, functional CENP-B Boxes are not confined to hominids. Our results indicate that the efficiency of identification of the CENP-B Box may depend largely on the sequencing methods used, and that the CENP-B Box in centromeric repetitive DNA may Be more common than researchers previously thought.
-
Identification of Novel α‑N-Methylation of CENP‑B That Regulates Its Binding to the Centromeric DNA
2016Co-Authors: Xiaoxia Dai, Hiroshi Masumoto, Koichiro Otake, Changjun You, Qian Cai, Zi Wang, Yinsheng WangAbstract:The eukaryotic <B>CentromereB> is an essential chromatin region required for accurate segregation of sister chromatids during cell division. <B>CentromereB> <B>ProteinB> B (CENP-B) is a highly conserved <B>ProteinB> which can Bind to the 17-Bp CENP-B Box on the centromeric DNA. In this study, we found that CENP-B could Be α-N-methylated in human cells. We also showed that the level of the α-N-methylation was stimulated in cells in response to a variety of extracellular stimuli, including increased cell density, heat shock, and arsenite treatment, although the methylation level was not altered upon metaphase arrest. We identified N-terminal RCC1 methyltransferase (NRMT) as a major enzyme required for the CENP-B methylation. Additionally, we found that chromatin-Bound CENP-B was primarily trimethylated and α-N-trimethylation could enhance CENP-B’s Binding to CENP-B Box in cells. Our study also expands the function of <B>ProteinB> α-N-methylation that has Been known for decades and whose function remains largely unexplored
-
Identification of Novel α-N-Methylation of CENP-B That Regulates Its Binding to the Centromeric DNA
Journal of proteome research, 2013Co-Authors: Xiaoxia Dai, Hiroshi Masumoto, Koichiro Otake, Changjun You, Qian Cai, Zi Wang, Yinsheng WangAbstract:The eukaryotic <B>CentromereB> is an essential chromatin region required for accurate segregation of sister chromatids during cell division. <B>CentromereB> <B>ProteinB> B (CENP-B) is a highly conserved <B>ProteinB> which can Bind to the 17-Bp CENP-B Box on the centromeric DNA. In this study, we found that CENP-B could Be α-N-methylated in human cells. We also showed that the level of the α-N-methylation was stimulated in cells in response to a variety of extracellular stimuli, including increased cell density, heat shock, and arsenite treatment, although the methylation level was not altered upon metaphase arrest. We identified N-terminal RCC1 methyltransferase (NRMT) as a major enzyme required for the CENP-B methylation. Additionally, we found that chromatin-Bound CENP-B was primarily trimethylated and α-N-trimethylation could enhance CENP-B’s Binding to CENP-B Box in cells. Our study also expands the function of <B>ProteinB> α-N-methylation that has Been known for decades and whose function remains largely unexplored.
-
Human <B>CentromereB> <B>ProteinB> B Induces Translational Positioning of Nucleosomes on α-Satellite Sequences
The Journal of biological chemistry, 2005Co-Authors: Yoshinori Tanaka, Hitoshi Kurumizaka, Hiroshi Masumoto, Kinya Yoda, Hiroaki Tachiwana, Tsuneko Okazaki, Shigeyuki YokoyamaAbstract:The human <B>CentromereB> <B>ProteinB>s A (CENP-A) and B (CENP-B) are the fundamental <B>CentromereB> components of chromosomes. CENP-A is the <B>CentromereB>-specific histone H3 variant, and CENP-B specifically Binds a 17-Base pair sequence (the CENP-B Box), which appears within every other alpha-satellite DNA repeat. In the present study, we demonstrated <B>CentromereB>-specific nucleosome formation in vitro with recomBinant <B>ProteinB>s, including histones H2A, H2B, H4, CENP-A, and the DNA-Binding domain of CENP-B. The CENP-A nucleosome wraps 147 Base pairs of the alpha-satellite sequence within its nucleosome core particle, like the canonical H3 nucleosome. Surprisingly, CENP-B Binds to nucleosomal DNA when the CENP-B Box is wrapped within the nucleosome core particle and induces translational positioning of the nucleosome without affecting its rotational setting. This CENP-B-induced translational positioning only occurs when the CENP-B Box sequence is settled in the proper rotational setting with respect to the histone octamer surface. Therefore, CENP-B may Be a determinant for translational positioning of the <B>CentromereB>-specific nucleosomes through its Binding to the nucleosomal CENP-B Box.
Shigeyuki Yokoyama - One of the best experts on this subject based on the ideXlab platform.
-
Human <B>CentromereB> <B>ProteinB> B Induces Translational Positioning of Nucleosomes on α-Satellite Sequences
The Journal of biological chemistry, 2005Co-Authors: Yoshinori Tanaka, Hitoshi Kurumizaka, Hiroshi Masumoto, Kinya Yoda, Hiroaki Tachiwana, Tsuneko Okazaki, Shigeyuki YokoyamaAbstract:The human <B>CentromereB> <B>ProteinB>s A (CENP-A) and B (CENP-B) are the fundamental <B>CentromereB> components of chromosomes. CENP-A is the <B>CentromereB>-specific histone H3 variant, and CENP-B specifically Binds a 17-Base pair sequence (the CENP-B Box), which appears within every other alpha-satellite DNA repeat. In the present study, we demonstrated <B>CentromereB>-specific nucleosome formation in vitro with recomBinant <B>ProteinB>s, including histones H2A, H2B, H4, CENP-A, and the DNA-Binding domain of CENP-B. The CENP-A nucleosome wraps 147 Base pairs of the alpha-satellite sequence within its nucleosome core particle, like the canonical H3 nucleosome. Surprisingly, CENP-B Binds to nucleosomal DNA when the CENP-B Box is wrapped within the nucleosome core particle and induces translational positioning of the nucleosome without affecting its rotational setting. This CENP-B-induced translational positioning only occurs when the CENP-B Box sequence is settled in the proper rotational setting with respect to the histone octamer surface. Therefore, CENP-B may Be a determinant for translational positioning of the <B>CentromereB>-specific nucleosomes through its Binding to the nucleosomal CENP-B Box.
-
CpG methylation of the CENP-B Box reduces human CENP-B Binding.
The FEBS journal, 2004Co-Authors: Yoshinori Tanaka, Hitoshi Kurumizaka, Shigeyuki YokoyamaAbstract:In eukaryotes, CpG methylation is an epigenetic DNA modification that is important for heterochromatin formation. <B>CentromereB> <B>ProteinB> B (CENP-B) specifically Binds to the centromeric 17 Base-pair CENP-B Box DNA, which contains two CpG dinucleotides. In this study, we tested complex formation By the DNA-Binding domain of CENP-B with methylated and unmethylated CENP-B Box DNAs, and found that CENP-B preferentially Binds to the unmethylated CENP-B Box DNA. Competition analyses revealed that the affinity of CENP-B for the CENP-B Box DNA is reduced nearly to the level of nonspecific DNA Binding By CpG methylation.
-
Crystal Structure of the Human <B>CentromereB> <B>ProteinB> B (CENP-B) Dimerization Domain at 1.65-Å Resolution
The Journal of biological chemistry, 2003Co-Authors: Maki S. Tawaramoto, Sam-yong Park, Yoshinori Tanaka, Osamu Nureki, Hitoshi Kurumizaka, Shigeyuki YokoyamaAbstract:ABstract The human <B>CentromereB> <B>ProteinB> B (CENP-B), a centromeric heterochromatin component, forms a homodimer that specifically Binds to a distinct DNA sequence (the CENP-B Box), which appears within every other α-satellite repeat. Previously, we determined the structure of the human CENP-B DNA-Binding domain, CENP-B-(1-129), complexed with the CENP-B Box DNA. In the present study, we determined the crystal structure of its dimerization domain (CENP-B-(540-599)), another functional domain of CENP-B, at 1.65-A resolution. CENP-B-(540-599) contains two α-helices, which are folded into an antiparallel configuration. The CENP-B-(540-599) dimer formed a symmetrical, antiparallel, four-helix Bundle structure with a large hydrophoBic patch in which 23 residues of one monomer form van der Waals contacts with the other monomer. In the CENP-B-(540-599) dimer, the N-terminal ends of CENP-B-(540-599) are oriented on opposite sides of the dimer. This CENP-B dimer configuration may Be suitaBle for capturing two distant CENP-B Boxes during centromeric heterochromatin formation.
-
Crystal structure of the CENP‐B <B>ProteinB>–DNA complex: the DNA‐Binding domains of CENP‐B induce kinks in the CENP‐B Box DNA
The EMBO journal, 2001Co-Authors: Yoshinori Tanaka, Osamu Nureki, Hitoshi Kurumizaka, Junji Iwahara, Tsuneko Okazaki, Shuya Fukai, Shinichi Kawaguchi, Mari Ikuta, Shigeyuki YokoyamaAbstract:The human <B>CentromereB> <B>ProteinB> B (CENP-B), one of the <B>CentromereB> components, specifically Binds a 17 Bp sequence (the CENP-B Box), which appears in every other alpha-satellite repeat. In the present study, the crystal structure of the complex of the DNA-Binding region (129 residues) of CENP-B and the CENP-B Box DNA has Been determined at 2.5 A resolution. The DNA-Binding region forms two helix-turn-helix domains, which are Bound to adjacent major grooves of the DNA. The DNA is kinked at the two recognition helix contact sites, and the DNA region Between the kinks is straight. Among the major groove <B>ProteinB>-Bound DNAs, this 'kink-straight-kink' Bend contrasts with ordinary 'round Bends' (gradual Bending Between two <B>ProteinB> contact sites). The larger kink (43 degrees ) is induced By a novel mechanism, 'phosphate Bridging By an arginine-rich helix': the recognition helix with an arginine cluster is inserted perpendicularly into the major groove and Bridges the groove through direct interactions with the phosphate groups. The overall Bending angle is 59 degrees, which may Be important for the <B>CentromereB>-specific chromatin structure.
-
A helix–turn–helix structure unit in human <B>CentromereB> <B>ProteinB> B (CENP-B)
The EMBO journal, 1998Co-Authors: Junji Iwahara, Hiroshi Masumoto, Tuneko Okazaki, Katsumi Kitagawa, Takanori Kigawa, Shigeyuki YokoyamaAbstract:CENP-B has Been suggested to organize arrays of <B>CentromereB> satellite DNA into a higher order structure which then directs <B>CentromereB> formation and kinetochore assemBly in mammalian chromosomes. The N-terminal portion of CENP-B is a 15 kDa DNA Binding domain (DBD) consisting of two repeating units, RP1 and RP2. The DBD specifically Binds to the CENP-B Box sequence (17 Bp) in <B>CentromereB> DNA. We determined the solution structure of human CENP-B DBD RP1 By multi-dimensional 1H, 13C and 15N NMR methods. The CENP-B DBD RP1 structure consists of four helices and has a helix-turn-helix structure. The overall folding is similar to those of some other eukaryotic DBDs, although significant sequence homology with these <B>ProteinB>s was not found. The DBD of yeast RAP1, a telomere Binding <B>ProteinB>, is most similar to CENP-B DBD RP1. We studied the interaction Between CENP-B DBD RP1 and the CENP-B Box By the use of NMR chemical shift perturBation. The results suggest that CENP-B DBD RP1 interacts with one of the essential regions of the CENP-B Box DNA, mainly at the N-terminal Basic region, the N-terminal portion of helix 2 and helix 3.
Katsumi Kitagawa - One of the best experts on this subject based on the ideXlab platform.
-
Sgt1 Dimerization Is Required for Yeast Kinetochore AssemBly
The Journal of biological chemistry, 2008Co-Authors: Parmil K. Bansal, Amanda Nourse, Rashid Abdulle, Katsumi KitagawaAbstract:The kinetochore, which consists of DNA sequence elements and structural <B>ProteinB>s, is essential for high-fidelity chromosome transmission during cell division. In Budding yeast, Sgt1 and Hsp90 help assemBle the core kinetochore complex CBF3 By activating the CBF3 components Skp1 and Ctf13. In this study, we show that Sgt1 forms homodimers By performing in vitro and in vivo immunoprecipitation and analytical ultracentrifugation analyses. Analyses of the dimerization of Sgt1 deletion <B>ProteinB>s showed that the Skp1-Binding domain (amino acids 1–211) contains the Sgt1 homodimerization domain. Also, the Sgt1 mutant <B>ProteinB>s that were unaBle to dimerize also did not Bind Skp1, suggesting that Sgt1 dimerization is important for Sgt1-Skp1 Binding. Restoring dimerization activity of a dimerization-deficient sgt1 mutant (sgt1-L31P) By using the CENP-B (<B>CentromereB> <B>ProteinB>-B) dimerization domain suppressed the temperature sensitivity, the Benomyl sensitivity, and the chromosome missegregation phenotype of sgt1-L31P. These results strongly suggest that Sgt1 dimerization is required for kinetochore assemBly.
-
A helix–turn–helix structure unit in human <B>CentromereB> <B>ProteinB> B (CENP-B)
The EMBO journal, 1998Co-Authors: Junji Iwahara, Hiroshi Masumoto, Tuneko Okazaki, Katsumi Kitagawa, Takanori Kigawa, Shigeyuki YokoyamaAbstract:CENP-B has Been suggested to organize arrays of <B>CentromereB> satellite DNA into a higher order structure which then directs <B>CentromereB> formation and kinetochore assemBly in mammalian chromosomes. The N-terminal portion of CENP-B is a 15 kDa DNA Binding domain (DBD) consisting of two repeating units, RP1 and RP2. The DBD specifically Binds to the CENP-B Box sequence (17 Bp) in <B>CentromereB> DNA. We determined the solution structure of human CENP-B DBD RP1 By multi-dimensional 1H, 13C and 15N NMR methods. The CENP-B DBD RP1 structure consists of four helices and has a helix-turn-helix structure. The overall folding is similar to those of some other eukaryotic DBDs, although significant sequence homology with these <B>ProteinB>s was not found. The DBD of yeast RAP1, a telomere Binding <B>ProteinB>, is most similar to CENP-B DBD RP1. We studied the interaction Between CENP-B DBD RP1 and the CENP-B Box By the use of NMR chemical shift perturBation. The results suggest that CENP-B DBD RP1 interacts with one of the essential regions of the CENP-B Box DNA, mainly at the N-terminal Basic region, the N-terminal portion of helix 2 and helix 3.
-
a helix turn helix structure unit in human <B>CentromereB> <B>ProteinB> B cenp B
The EMBO Journal, 1998Co-Authors: Junji Iwahara, Hiroshi Masumoto, Tuneko Okazaki, Katsumi Kitagawa, Takanori Kigawa, Shigeyuki YokoyamaAbstract:CENP-B has Been suggested to organize arrays of <B>CentromereB> satellite DNA into a higher order structure which then directs <B>CentromereB> formation and kinetochore assemBly in mammalian chromosomes. The N-terminal portion of CENP-B is a 15 kDa DNA Binding domain (DBD) consisting of two repeating units, RP1 and RP2. The DBD specifically Binds to the CENP-B Box sequence (17 Bp) in <B>CentromereB> DNA. We determined the solution structure of human CENP-B DBD RP1 By multi-dimensional 1H, 13C and 15N NMR methods. The CENP-B DBD RP1 structure consists of four helices and has a helix-turn-helix structure. The overall folding is similar to those of some other eukaryotic DBDs, although significant sequence homology with these <B>ProteinB>s was not found. The DBD of yeast RAP1, a telomere Binding <B>ProteinB>, is most similar to CENP-B DBD RP1. We studied the interaction Between CENP-B DBD RP1 and the CENP-B Box By the use of NMR chemical shift perturBation. The results suggest that CENP-B DBD RP1 interacts with one of the essential regions of the CENP-B Box DNA, mainly at the N-terminal Basic region, the N-terminal portion of helix 2 and helix 3.
-
<B>CentromereB> <B>ProteinB> B of African green monkey cells: gene structure, cellular expression, and centromeric localization.
Molecular and cellular biology, 1996Co-Authors: Kinya Yoda, Hiroshi Masumoto, T Nakamura, Nobutaka Suzuki, Katsumi Kitagawa, Megumi Nakano, A Shinjo, Tuneko OkazakiAbstract:<B>CentromereB> <B>ProteinB> B (CENP-B) is a centromeric DNA-Binding <B>ProteinB> which recognizes a 17-Bp sequence (CENP-B Box) in human and mouse centromeric satellite DNA. The African green monkey (AGM) is phylogenetically closer to humans than mice and is known to contain large amounts of a-satellite DNA, But there has Been no report of CENP-B Boxes or CENP-B in the <B>CentromereB> domains of its chromosomes. To elucidate the AGM CENP-B‐CENP-B Box interaction, we have analyzed the gene structure, expression, Biochemical properties, and centromeric localization of its CENP-B. The amino acid sequence deduced from the cloned AGM CENP-B gene was estaBlished to Be highly homologous to that of human and mouse CENP-B. In particular, the DNA Binding and homodimer formation domains demonstrated 100% identity to their human and mouse counterparts. ImmunoBlotting and DNA moBility shift analyses revealed CENP-B to Be expressed in AGM cell lines. As predicted from the gene structure, the AGM CENP-B in the cell extracts exhiBited the same DNA Binding specificity and homodimer forming activity as human CENP-B. By indirect immunofluorescent staining of AGM mitotic cells with anti-CENP-B antiBodies, a <B>CentromereB>-specific localization of AGM CENP-BcouldBedemonstrated.WealsoisolatedAGM a-satelliteDNAwithaCENP-BBox-likesequencewith CENP-Baffinity.TheseresultsnotonlyprovethatCENP-BfunctionallypersistsinAGMcellsButalsosuggest that the AGM genome contains the recognition sequences for CENP-B (CENP-B Boxes with the core recognition sequence or CENP-B Box variants) in centromeric satellite DNA.
-
Analysis of <B>ProteinB>-DNA and <B>ProteinB>-<B>ProteinB> interactions of <B>CentromereB> <B>ProteinB> B (CENP-B) and properties of the DNA-CENP-B complex in the cell cycle.
Molecular and cellular biology, 1995Co-Authors: Katsumi Kitagawa, Hiroshi Masumoto, M Ikeda, Tuneko OkazakiAbstract:We previously reported that <B>CentromereB> <B>ProteinB> B (CENP-B) forms a staBle complex (designated complex A) containing two alphoid DNAs in vitro. Domains in the CENP-B polypeptide involved in the formation of complex A were determined in the present study with truncated derivatives expressed in Escherichia coli and in raBBit reticulocyte lysates. It was revealed By gel moBility shift analyses that polypeptides containing the NH2-terminal DNA-Binding domain Bind a DNA molecule as a monomer, while dimerizing at a novel hydrophoBic domain in the COOH-terminal region of 59 amino acid residues. This polypeptide dimerization activity at the COOH-terminal region was also confirmed with the two-hyBrid system in Saccharomyces cerevisiae cells. The results thus proved that CENP-B polypeptides form a homodimer at the COOH-terminal hydrophoBic domain, each Binding a DNA strand at their NH2-terminal domains. The dimerization and DNA-Binding domains fall into two of the three completely conserved sequences found in human and mouse CENP-B, and complex A-forming activity was also detected in nuclear extracts of mouse cells. Metaphase-specific phosphorylation of CENP-B was also detected, But this had no effect on its complex A-forming activity. On the Basis of the present results, we propose that CENP-B plays an important role in the assemBly of specific <B>CentromereB> structures By forming unique DNA-<B>ProteinB> complexes at the sites of CENP-B Boxes on the centromeric repetitive DNA Both in interphase nuclei and on mitotic chromosomes.
Michio Himeno - One of the best experts on this subject based on the ideXlab platform.
-
Nucleotide specificity at the Boundary and size requirement of the target sites recognized By human <B>CentromereB> <B>ProteinB> B (CENP-B) in vitro
Chromosome Research, 1998Co-Authors: Kenji Sugimoto, Akiko Shibata, Michio HimenoAbstract:Human <B>CentromereB> <B>ProteinB> B (CENP-B) has a sequence-specific DNA Binding activity. We previously reported several CENP-B Binding motifs By analysing synthetic oligonucleotides as well as alphoid DNA isolated from the human genomic liBrary. Here, we examined the size requirement and nucleotide specificity of human CENP-B Binding sequences in vitro. We synthesized three sets of mixed oligonucleotides containing diverged authentic Binding sites (CTTCGTTGGAAACGGGA) in which certain pairs of nucleotides (underlined) were degenerated. Each oligonucleotide with a defined sequence was separately introduced into a plasmid and mixed with GST-fused recomBinant CENP-B. The DNA--<B>ProteinB> complex formed was affinity purified with glutathione Sepharose. Any nucleotide suBstitutions at the positions 1, 2 and 17 did not significantly influence the recovery, while the suBstitutions at positions 3, 4 and 16 did, suggesting that the internal 14-Bp motif (TCGTTGGAAACGGG) constituted the minimum requirement. However, it showed a lower affinity to CENP-B, compared with the authentic motif. The inclusion of T at the 5′ end greatly increased the affinity, and the further addition of A or T at the 3′ end (TTCGTTGGAAACGGGA/T) offered affinity similar to the authentic motif. The first nucleotide of the 17-Bp authentic Binding motif may not Be essential for CENP-B Binding.
-
The distriBution of Binding sites for <B>CentromereB> <B>ProteinB> B (CENP-B) is partly conserved among diverged higher order repeating units of human chromosome 6-specific alphoid DNA
Chromosome Research, 1997Co-Authors: Kenji Sugimoto, Kenji Furukawa, Kayo Kusumi, Michio HimenoAbstract:We previously reported the isolation of alphoid satellite clones from a human genomic liBrary using a DNA immunoprecipitation with <B>CentromereB> <B>ProteinB> B (CENP-B). Here, we have characterized the distriBution of CENP-B-Binding sites on the 3-kB Bam HI repeats of the cos2 clone. Using in situ hyBridization, this alphoid satellite was located primarily at the centromeric region of chromosome 6. The functional Binding sites were mapped By precipitating the restriction fragments with recomBinant CENP-B in vitro . One repeat (2B3-11) consisted of 19 copies of alphoid monomer, eight of which possessed the Binding sites, while another (2B3-9) consisted of 18 copies of the monomer, seven of which possessed the Binding sites. The distriBution of the sites was well conserved Between them, except for the terminus. A similar analysis with the remaining 6-kB region suggested the presence of a continuous 1-kB region with regular spacing of Eco RI sites and the CENP-B-Binding sites. When the nucleotide sequence of 2B3-11 was compared with that of another chromosome 6-specific alphoid repeat (p308) that had Been descriBed previously, this 1-kB region was highly conserved Between them. The distriBution of the CENP-B Binding sites and the order of alphoid monomers might define the folding of alphoid repeats in the centromeric region.
-
Anti-helix-loop-helix domain antiBodies : discovery of autoantiBodies that inhiBit DNA Binding activity of human <B>CentromereB> <B>ProteinB> B (CENP-B)
Journal of biochemistry, 1992Co-Authors: Kenji Sugimoto, Yoshinao Muro, Michio HimenoAbstract:<B>CentromereB> <B>ProteinB> B (CENP-B) is one of the <B>CentromereB> DNA Binding <B>ProteinB>s constituting centromeric heterochromatin of human chromosomes. This <B>ProteinB> was originally identified as the target antigen in autoimmune disease patients (often with scleroderma). In this study, we cloned a human CENP-B cDNA which was longer than the previously isolated one and expressed functional recomBinant CENP-B in Escherichia coli. The DNA Binding domain was finely located within the N-terminal 134-amino-acid residues covering a predicted helix-loop-helix (HLH) structure, By using a set of recomBinant products with stepwise deletions from the C-terminus. From the analysis of their reactivity to anti-<B>CentromereB> sera from autoimmune disease patients, four epitopes were mapped on CENP-B antigen. In addition to two epitopes at the C-terminus, two were found on the HLH region at the N-terminus. In the analysis of the interaction Between the antigen and autoantiBodies, we found that the DNA Binding activity of CENP-B was distorted By the attack of the anti-HLH domain antiBodies in in vitro Binding reactions. Our results suggest that the direct inhiBition of the DNA Binding activity By the autoantiBodies might Be involved in patients' autoimmune reactions in vivo.
-
An Antigenic Determinant on Human <B>CentromereB> <B>ProteinB> B (CENP-B) AvailaBle for Production of Human-Specific Anti<B>CentromereB> AntiBodies in Mouse
Cell structure and function, 1992Co-Authors: Kenji Sugimoto, Hideyuki Migita, Yoshimasa Hagishita, Hiroaki Yata, Michio HimenoAbstract:<B>CentromereB> <B>ProteinB> B (CENP-B) is one of the <B>CentromereB> DNA Binding <B>ProteinB>s constituting <B>CentromereB> heterochromatin throughout the cell cycles. Some components of mammalian <B>CentromereB>s including CENP-B are target antigens for autoimmune disease patients, often those with scleroderma. Recent isolations of CENP-B genes from human and mouse suggested that CENP-B was highly conserved among mammals. From the previous analysis of the reactivity of patient anti<B>CentromereB> sera, two autoepitopes have Been located on the DNA Binding domain at the amino-terminal region. The amino acid sequences for Both the epitopes are perfectly conserved in the two species, human and mouse. In this study, to identify a human-specific antigenic determinant, the remaining two epitopes were further located in separate carBoxyl-terminal regions of human CENP-B. Although the amino acid sequence of one epitope is identical to that of the corresponding region in mouse CENP-B, the other has a less homologous sequence. To confirm that the latter epitope was availaBle for production of human-specific anti<B>CentromereB> antiBodies, mice were immunized with the recomBinant human CENP-B product. One serum that exclusively stained human <B>CentromereB> structure, But not that of other mammals, was identified in the immunofluorescence microscopic oBservation. The epitope analysis showed that the less conserved one was recognized By this serum. These results suggested that the corresponding region defines the antigenic determinants for the species specificity.
-
The clinical expression in anti<B>CentromereB> antiBody-positive patients is not specified By the epitope recognition of CENP-B antigen.
The Journal of dermatology, 1992Co-Authors: Yoshinao Muro, Kenji Sugimoto, Michio Himeno, Masaru OhashiAbstract:<B>CentromereB> <B>ProteinB> B (CENP-B), which is an alphoid DNA Binding <B>ProteinB>, is the target antigen in autoimmune disease patients (often with scleroderma). From our previous analysis of the reactivity of anti<B>CentromereB> sera, four independent epitopes were identified on recomBinant CENP-B. The anti<B>CentromereB> sera displayed heterogeneity in their patterns of reactivity to the four epitopes. We have investigated to what extent this heterogeneity of the target autoepitope on CENP-B accounts for the clinical diversity of anti<B>CentromereB> antiBody (ACA)-positive patients. A major autoepitope, epitope I, was recognized By all 40 ACA-positive sera; however, the other three epitopes were recognized differently from case to case. We could not find any significant correlation Between the reactivity to CENP-B autoepitopes and the clinical presentation of ACA-positive patients. There was consideraBle clinical diversity, even among the nine patients showing specificity for the single major autoepitope. In conclusion, we found that, although ACA-positive patients were Both clinically and immunologically heterogeneous, in most respects the clinical expression appeared to Be independent of the reactivity to the CENP-B autoepitope, a finding which suggests that identification of the target epitope of CENP-B is unlikely to assist in the clinical classification of the disease in ACA-positive patients. The identification of multiple B cell epitopes on CENP-B is consistent with the concept that the self-antigen drives the antiBody response. However, factors other than CENP-B autoepitope specificity must determine the clinical expression of ACA responses.