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Molecular and Cellular Biology, April 2002, p. 2229-2241, Vol. 22, No. 7
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.7.2229-2241.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

CENP-A, -B, and -C Chromatin Complex That Contains the I-Type -Satellite Array Constitutes the Prekinetochore in HeLa Cells

Satoshi Ando,^1,2, Hua Yang,³ Naohito Nozaki,⁴ Tuneko Okazaki,^2,3 and Kinya Yoda^1,2*

Bioscience Center, Nagoya University, Chikusa-ku, Nagoya 464-8601,¹ Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012,² Fujita Health University Institute for Comprehensive Medical Science, Toyoake, Aichi 470-1192 ,³ Kanagawa Dental College, Yokosuka, Kanagawa 238-8580, Japan⁴

Received 7 August 2001/ Returned for modification 17 September 2001/ Accepted 10 December 2001

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 -satellite DNA that contains the CENP-B box (I-type array). Mapping of hypersensitive sites for micrococcal nuclease (MNase) digestion indicated that CENP-A nucleosomes were phased on the I-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 -satellite array and constitutes the prekinetochore in HeLa cells.

Present address: Department of Molecular Life Science, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259-1193, Japan.

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