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.
Kitagawa
K
K
Department of Molecular Biology, School of Science, Nagoya University, Japan.
Masumoto
H
H
Ikeda
M
M
Okazaki
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001324:Autoantigens; D001483:Base Sequence; D001665:Binding Sites; D002453:Cell Cycle; D002467:Cell Nucleus / Q000378:metabolism; D002503:Centromere / Q000378:metabolism*; D051936:Centromere Protein B; D002868:Chromosomal Proteins, Non-Histone / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D017124:Conserved Sequence; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D007399:Interphase; D046911:Macromolecular Substances; D051379:Mice; D008938:Mitosis; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D001324:Autoantigens; C497926:CENPB protein, human; C497927:Cenpb protein, mouse; D051936:Centromere Protein B; D002868:Chromosomal Proteins, Non-Histone; D017931:DNA Primers; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D004247:DNA