A human centromere protein, CENP-B, has a DNA binding domain containing four potential alpha helices at the NH2 terminus, which is separable from dimerizing activity.

Abstract

Abstract. The alphoid DNA.CENP-B (centromere protein B) complex is the first sequence-specific DNA/ protein complex detected in the centromeric region of human chromosomes. In the reaction, CENP-B recognizes a 17-bp sequence (CENP-B box) and assembles two alphoid DNA molecules into a complex, which is designated complex A (Muro, Y., H. Masumoto, K. Yoda, N. Nozaki, M. Ohashi, and T. Okazaki. 1992. J. Cell Biol. 116:585-596). Since CENP-B gene is conserved in mammalian species and CENP-B boxes are found also in mouse centromere satellite DNA (minor satellite), this sequence-specific DNAprotein interaction may be important for some kind of common centromere function. In this study we have characterized the structure of CENP-B and CENP-B.alphoid DNA complex. We have shown by chemical cross-linking that CENP-B formed a dimer, and have estimated by molecular weight determination the composition of complex A to be a CENP-B dimer and two molecules of alphoid DNA. The DNA binding domain has been delimited within the NH~-terminal 125-amino acid region containing four potential or-helices using truncated CENP-B made in Escherichia coli cells. We have shown that CENP-B had sites highly sensitive to proteases and that the DNA binding domain was separable from the dimerizing activity by the proteolytic cleavage at 20 kD from the COOH terminus of the molecule. Thus, CENP-B may organize a higher order structure in the centromere by juxtaposing two CENP-B boxes in the alphoid DNA repeat through both the DNA-protein and protein-protein interactions.

Host genome surveillance for retrotransposons by transposon-derived proteins

“…Mammalian CENP-Bs form homodimers through their conserved carboxy-terminal domain 27,28 . We explored whether Abp1 could form dimers that might contribute to 'bundling' Tf2 elements into Tf bodies.…”

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 have found that intact CENP-B forms a stable and unique complex in vitro, which we called complex A, containing two DNA molecules and therefore judged to exist as a dimer in its native state (29,50). Complex A was not formed when the COOHterminal epitope was deleted or altered through protein purification or protease digestion (29,50). Thus, we suggested that the dimerization activity of CENP-B is located in the COOHterminal region and is separable from the NH 2 -terminal DNAbinding domain (50).…”

“…CENP-A (17 kDa) is a histone H3-like protein which seems to form a centromere-specific nucleosome (31). The CENP-B gene has been cloned and shown to be highly conserved between humans and mice (11,44), and the properties of CENP-B (80 kDa) have been studied extensively (26,29,33,43,50). cDNA encoding CENP-C (140 kDa) has also been cloned, and the antigen has been localized to the inner kinetochore plate (38).…”

“…We have previously shown that CENP-B binds specifically to a 17-bp motif (named the CENP-B box) in centromeric alpha-satellite DNA by using CENP-B obtained from HeLa cells and have delimited the DNA-binding domain within the NH 2 -terminal 125 amino acid residues which contained four potential ␣-helices by using truncated CENP-B polypeptides expressed in Escherichia coli (26,29,50). We have found that intact CENP-B forms a stable and unique complex in vitro, which we called complex A, containing two DNA molecules and therefore judged to exist as a dimer in its native state (29,50). Complex A was not formed when the COOHterminal epitope was deleted or altered through protein purification or protease digestion (29,50).…”

Sensitive and Rapid Detection of Centromeric Alphoid DNA in Human Metaphase Chromosomes by PNA Fluorescence <i>In Situ</i> Hybridization and Its Application to Biological Radiation Dosimetry

“…The biological characterization and evolution of alphoid DNA have been extensively investigated in human chromosomes (Earnshaw and Rothfield 1985, Earnshaw et al 1989, Tyler-Smith et al 1993, Yoda et al 1993, Rudd et al 2003, Masumoto et al 2004, Rudd and Willard 2004, Rudd et al 2006. However, because of difficulties in analyzing the highly complicated genomic constitution of centromeric regions, the distribution of alphoid DNA sequences in human chromosomes has not been established.…”