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

Abstract

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 NH 2 -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 NH 2 -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.

In vitro assembly of the CENP‐B/α‐satellite DNA/core histone complex: CENP‐B causes nucleosome positioning

“…Although CENP-B has been well characterized as a sequence specific DNA binding protein for centromeric satellite DNA Kitagawa et al 1995), the physiological role of CENP-B in centromere formation has not been firmly established; CENP-B appears to be absent at the centromere of human or murine Y chromosome, while it is present at the inactive centromere in dicentric chromosomes, and it has a sequence homology to some kinds of transposases (Kipling & Warburton 1997;Pluta et al 1995). All these data, which argue against CENP-B functioning as a centromere protein, by no means specify the alternative functions of CENP-B.…”

“…The DNA binding domain and the dimerization domain of CENP-B are located at the NH 2 -terminal 125 amino acid region and the COOH-terminal 59 amino acid region, respectively, and these domains could be separated by proteolytic cleavage of CENP-B without any loss of the activity in each domain Kitagawa et al 1995). Based on these results, we have proposed that CENP-B may organize a higher order structure in the centromere by juxtaposing two CENP-B boxes in the long ␣-satellite array through both DNAprotein and protein-protein interactions.…”

“…CENP-B is found in the inner chromatin region beneath the kinetochore plates . The properties of CENP-B as a specific DNA binding protein have been extensively characterized (Masumoto et al 1989;Muro et al 1992;Yoda et al 1992;Kitagawa et al 1995). A cDNA encoding CENP-C has been cloned, and the antigen localized to the inner kinetochore plate (Saitoh et al 1992).…”

Cell cycle behavior of human HP1 subtypes: distinct molecular domains of HP1 are required for their centromeric localization during interphase and metaphase

“…The plasmids pGFP-CB1-160c and pCFP-CB1-160c, containing the N-terminal fragment of human CENP-B fused with GFP and CFP, respectively, were generated as follows: The N-terminal fragment of CENP-B (-2 to 481 bp) was excised from pETCBN-160 (Kitagawa et al, 1995) with NcoI (the NcoI digestion product was blunt-ended using Klenow) and BamHI. A BamHI-HindIII adaptor was prepared by annealing oligonucleotides 5′-GATCCATGGCTAGCATGACTG-GTGGACAGCAAATGGGTCGGTAGTCGACA-3′ and 5′-AGC-TTGTCGACTACCGACCCATTTGCTGTCCACCAGTCATGCTAG-CCATG-3′.…”

Crystal Structure of the Human Centromere Protein B (CENP-B) Dimerization Domain at 1.65-Å Resolution

“…It contains DNA-binding domains and a dimerization domain at the N terminus and the C terminus, respectively (32). Electron microscopic observations showed that CENP-B bundles the two distant CENP-B boxes by its dimer formation and DNA binding activities (13).…”