CENP-A, -B, and -C chromatin complex that contains the I-type alpha-satellite array constitutes the prekinetochore in HeLa cells.
CENP-A is a component of centromeric chromatin and defines active centromere regions by forming centromere-specific nucleosomes. We have isolated centromeric chromatin containing the CENP-A nucleosome, CENP-B, and CENP-C from HeLa cells using anti-CENP-A and/or anti-CENP-C antibodies and shown that the CENP-A/B/C complex is predominantly formed on alpha-satellite DNA that contains the CENP-B box (alphaI-type array). Mapping of hypersensitive sites for micrococcal nuclease (MNase) digestion indicated that CENP-A nucleosomes were phased on the alphaI-type array as a result of interactions between CENP-B and CENP-B boxes, implying a repetitive configuration for the CENP-B/CENP-A nucleosome complex. Molecular mass analysis by glycerol gradient sedimentation showed that MNase digestion released a CENP-A/B/C chromatin complex of three to four nucleosomes into the soluble fraction, suggesting that CENP-C is a component of the repetitive CENP-B/CENP-A nucleosome complex. Quantitative analysis by immunodepletion of CENP-A nucleosomes showed that most of the CENP-C and approximately half the CENP-B took part in formation of the CENP-A/B/C chromatin complex. A kinetic study of the solubilization of CENPs showed that MNase digestion first released the CENP-A/B/C chromatin complex into the soluble fraction, and later removed CENP-B and CENP-C from the complex. This result suggests that CENP-A nucleosomes form a complex with CENP-B and CENP-C through interaction with DNA. On the basis of these results, we propose that the CENP-A/B/C chromatin complex is selectively formed on the I-type alpha-satellite array and constitutes the prekinetochore in HeLa cells.
Ando
Satoshi
S
Bioscience Center, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan.
Yang
Hua
H
Nozaki
Naohito
N
Okazaki
Tuneko
T
Yoda
Kinya
K
D000906:Antibodies / Q000276:immunology; D001324:Autoantigens; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D000076247:Centromere Protein A; D051936:Centromere Protein B; D002843:Chromatin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000276:immunology / Q000378:metabolism*; D002877:Chromosomes, Human / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004276:DNA, Satellite / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000737:chemistry* / Q000378:metabolism*; D046911:Macromolecular Substances; D008836:Micrococcal Nuclease / Q000378:metabolism; D008970:Molecular Weight; D009707:Nucleosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011233:Precipitin Tests; D012995:Solubility
D000906:Antibodies; D001324:Autoantigens; C000619700:CENPA protein, human; C497926:CENPB protein, human; D000076247:Centromere Protein A; D051936:Centromere Protein B; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004276:DNA, Satellite; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009707:Nucleosomes; C060905:centromere protein C; D008836:Micrococcal Nuclease