Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Centromere Protein B”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed1992

Anti-helix-loop-helix domain antibodies: discovery of autoantibodies that inhibit DNA binding activity of human centromere protein B (CENP-B).

Centromere protein B (CENP-B) is one of the centromere DNA binding proteins constituting centromeric heterochromatin of human chromosomes. This protein 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-centromere 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.

Amino Acid Sequence↗
PubMed1992

Centromere protein B assembles human centromeric alpha-satellite DNA at the 17-bp sequence, CENP-B box.

We purified 15,000-fold from HeLa cell nuclear extract the centromere antigen that reacts specifically with the 17-bp sequence, designated previously as CENP-B box, in human centromeric alpha-satellite (alphoid) DNA by a two-step procedure including an oligonucleotide affinity column. The purified protein was identified as the centromere protein B (CENP-B) by its mobility on SDS-PAGE (80 kD), and reactivities to a monoclonal antibody raised to CENP-B (bacterial fusion protein) and to anticentromere sera from patients with autoimmune diseases. Direct binding by CENP-B of the CENP-B box sequence in the alphoid DNA has been proved using the purified CENP-B by DNA mobility-shift assay, Southwestern blotting, and DNase I protection analysis. The binding constant of the antigen to the CENP-B box sequence is 6 x 10(8) M-1. DNA mobility-shift assays indicated that the major complex formed between the CENP-B and the DNA contains two DNA molecules, suggesting the importance of the CENP-B/CENP-B box interaction in organization of higher ordered chromatin structures in the centromere and/or kinetochore. Location of DNA binding and dimerization domains in CENP-B was discussed based on the DNA mobility-shift assays performed with a protein fraction containing intact and partial cleavage products of CENP-B.

Autoantigens↗
PubMed1994

Functional domain structure of human centromere protein B. Implication of the internal and C-terminal self-association domains in centromeric heterochromatin condensation.

Centromere protein B (CENP-B) is a common centromere DNA-binding protein among mammalian centromeres. CENP-B possesses the specific DNA binding activity to the 17-base pair sequence dispersed in centromeric repetitive DNA sequences. In the previous study, we have shown that its DNA-binding domain exists within the N-terminal 134 amino acid residues. Here, to clarify the whole domain structure, another functional unit required for CENP-B self-association was examined. Recombinant CENP-B was expressed in Escherichia coli. First, a chemical cross-linking reagent, disuccinimidyl suberate, was used to fix the physical association without losing the DNA-binding activity. The complexes with the same molecular weight as homodimer and trimer were identified after a separation by SDS-polyacrylamide gel electrophoresis. With a series of CENP-B deletion constructs, the area responsible for this oligomer formation was located at the internal region. Second, an electrophoretic mobility shift assay was also used to survey the minimum regions required for the CENP-B self-association. Three separate elements were identified by assaying the capacity to form the additional slow-migrating complex, two in the internal region and one in the C terminus. These results suggest that CENP-B molecules interact with each other at the multiple sites to fold the centromeric DNA repeats into a heterochromatin structure.

Autoantigens↗
PubMed1996

Centromere protein B of African green monkey cells: gene structure, cellular expression, and centromeric localization.

Centromere protein B (CENP-B) is a centromeric DNA-binding protein 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 alpha-satellite DNA, but there has been no report of CENP-B boxes or CENP-B in the centromere 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 centromere-specific localization of AGM CENP-B could be demonstrated. We also isolated AGM alpha-satellite DNA with a CENP-B box-like sequence with CENP-B affinity. These results not only prove that CENP-B functionally persists in AGM cells but also suggest 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.

Amino Acid Sequence↗
PubMed1998

Centromeric protein B null mice are viable with no apparent abnormalities.

The centromere protein B (CENP-B) is a centromeric DNA/binding protein. It recognizes a 17-bp sequence motif called the CENP-B box, which is found in the centromeric region of most chromosomes. It binds DNA through its amino terminus and dimerizes through its carboxy terminus. CENP-B protein has been proposed to perform a vital role in organizing chromatin structures at centromeres. However, other evidence does not agree with this view. For example, CENP-B is found at inactive centromeres on stable dicentric chromosomes, and also mitotically stable chromosomes lacking alpha-satellite DNA have been reported. To address the biological function of CENP-B, we generated mouse null mutants of CENP-B by homologous recombination. Mice lacking CENP-B were viable and fertile, indicating that mice without CENP-B undergo normal somatic and germline development. Thus, both mitosis and meiosis are able to proceed normally in the absence of CENP-B.

Animals↗
PubMed1998

Nucleotide specificity at the boundary and size requirement of the target sites recognized by human centromere protein B (CENP-B) in vitro.

Human centromere protein 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-protein 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.

Autoantigens↗
PubMed1994

Functional cloning of centromere protein B (CENP-B) box-enriched alphoid DNA repeats utilizing the sequence-specific DNA binding activity of human CENP-B in vitro.

The centromere is a distinctive portion of the chromosome consisting of 'centromere DNA' and 'centromere proteins'. Recently, a direct molecular interaction was discovered between human centromere protein B (CENP-B) and human centromeric alphoid repeats. This enabled us to isolate the CENP-B-targeted centromeric DNA sequences by positively utilizing the biologic activity of CENP-B in vitro. In the previous model experiment, we found that oligonucleotides covering the CENP-B binding sequences were enriched by the DNA immunoprecipitation procedure. Here we apply the same technique to the direct isolation of a functional part of human centromeric DNA from a genomic DNA library. Restriction digestion of two isolated clones showed the typical repeating pattern of an alphoid family that is known to localize at the centromeric region of all human chromosomes. Sequence analysis showed that these two clones frequently contain the authentic CENP-B binding motif, CTTCGTTGGAAACGGGA, or a new one with one base replaced, CTTCGTTGGAAACGGGT. The frequent distribution of these motifs suggests that the isolated sequences are directly involved in the organization of centromeric heterochromatin at the primary constriction in conjunction with CENP-B.

Autoantigens↗
PubMed2003

Crystal structure of the human centromere protein B (CENP-B) dimerization domain at 1.65-A resolution.

The human centromere protein 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 alpha-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 alpha-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.

Amino Acid Sequence↗
PubMed1992

An antigenic determinant on human centromere protein B (CENP-B) available for production of human-specific anticentromere antibodies in mouse.

Centromere protein B (CENP-B) is one of the centromere DNA binding proteins constituting centromere heterochromatin throughout the cell cycles. Some components of mammalian centromeres 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 anticentromere 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 anticentromere antibodies, mice were immunized with the recombinant human CENP-B product. One serum that exclusively stained human centromere 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.

Amino Acid Sequence↗
PubMed1995

Analysis of protein-DNA and protein-protein interactions of centromere protein B (CENP-B) and properties of the DNA-CENP-B complex in the cell cycle.

We previously reported that centromere protein 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 centromere structures by forming unique DNA-protein complexes at the sites of CENP-B boxes on the centromeric repetitive DNA both in interphase nuclei and on mitotic chromosomes.

Amino Acid Sequence↗
PubMed2005

Human centromere protein B induces translational positioning of nucleosomes on alpha-satellite sequences.

The human centromere proteins A (CENP-A) and B (CENP-B) are the fundamental centromere components of chromosomes. CENP-A is the centromere-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 centromere-specific nucleosome formation in vitro with recombinant proteins, 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 centromere-specific nucleosomes through its binding to the nucleosomal CENP-B box.

Autoantigens↗
PubMed1997

The distribution of binding sites for centromere protein B (CENP-B) is partly conserved among diverged higher order repeating units of human chromosome 6-specific alphoid DNA.

We previously reported the isolation of alphoid satellite clones from a human genomic library using a DNA immunoprecipitation with centromere protein B (CENP-B). Here, we have characterized the distribution of CENP-B-binding sites on the 3-kb BamHI 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 EcoRI 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.

Autoantigens↗
PubMed1998

Centromere protein B null mice are mitotically and meiotically normal but have lower body and testis weights.

CENP-B is a constitutive centromere DNA-binding protein that is conserved in a number of mammalian species and in yeast. Despite this conservation, earlier cytological and indirect experimental studies have provided conflicting evidence concerning the role of this protein in mitosis. The requirement of this protein in meiosis has also not previously been described. To resolve these uncertainties, we used targeted disruption of the Cenpb gene in mouse to study the functional significance of this protein in mitosis and meiosis. Male and female Cenpb null mice have normal body weights at birth and at weaning, but these subsequently lag behind those of the heterozygous and wild-type animals. The weight and sperm content of the testes of Cenpb null mice are also significantly decreased. Otherwise, the animals appear developmentally and reproductively normal. Cytogenetic fluorescence-activated cell sorting and histological analyses of somatic and germline tissues revealed no abnormality. These results indicate that Cenpb is not essential for mitosis or meiosis, although the observed weight reduction raises the possibility that Cenpb deficiency may subtly affect some aspects of centromere assembly and function, and result in reduced rate of cell cycle progression, efficiency of microtubule capture, and/or chromosome movement. A model for a functional redundancy of this protein is presented.

Animals↗
PubMed1998

A helix-turn-helix structure unit in human centromere protein B (CENP-B).

CENP-B has been suggested to organize arrays of centromere satellite DNA into a higher order structure which then directs centromere 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 centromere 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 proteins was not found. The DBD of yeast RAP1, a telomere binding protein, 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.

Amino Acid Sequence↗
PubMed2006

Anti-endothelial cell antibodies from patients with limited cutaneous systemic sclerosis bind to centromeric protein B (CENP-B).

By using a quantitative immunoblotting technique on protein extracts of human macrovascular and microvascular endothelial cells, we have analyzed the self-reactive repertoires of IgG from 20 patients with limited cutaneous SSc, 40 patients with diffuse SSc and 60 age- and sex-matched healthy controls. Serum IgG from 15/20 patients with limited cutaneous SSc and anti-centromere antibodies bound to at least one of the two 75- and 85-kDa protein bands in the different endothelial cell extracts, whereas IgG from healthy controls or patients with diffuse SSc did not. N-terminal sequencing of the 75- and 85-kDa bands identified CENP-B as the sole antigen in both bands. Moreover, IgG from all of the SSc patients who recognized the 75- and/or 85-kDa bands bound to a full-length recombinant CENP-B protein as assessed by ELISA, whereas IgG from other SSc patients did not. The main target of anti-endothelial cell antibodies in patients with limited cutaneous SSc is the nuclear and ubiquitous protein CENP-B.

Antibody Specificity↗