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J. Biol. Chem., Vol. 279, Issue 7, 5934-5946, February 13, 2004

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CENP-B Interacts with CENP-C Domains Containing Mif2 Regions Responsible for Centromere Localization^*

Nobutaka Suzuki, Megumi Nakano, Naohito Nozaki¶, Shin-ichiro Egashira||, Tuneko Okazaki, and Hiroshi Masumoto**

From the Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602 and ^¶Kanagawa Dental College, Yokosuka, Kanagawa 238-8580, Japan

Recently, human artificial chromosomes featuring functional centromeres have been generated efficiently from naked synthetic alphoid DNA containing CENP-B boxes as a de novo mechanism in a human cultured cell line, but not from the synthetic alphoid DNA only containing mutations within CENP-B boxes, indicating that CENP-B has some functions in assembling centromere/kinetochore components on alphoid DNA. To investigate whether any interactions exist between CENP-B and the other centromere proteins, we screened a cDNA library by yeast two-hybrid analysis. An interaction between CENP-B and CENP-C was detected, and the CENP-C domains required were determined to overlap with three Mif2 homologous regions, which were also revealed to be involved in the CENP-C assembly of centromeres by expression of truncated polypeptides in cultured cells. Overproduction of truncated CENP-B containing no CENP-C interaction domains caused abnormal duplication of CENP-C domains at G₂ and cell cycle delay at metaphase. These results suggest that the interaction between CENP-B and CENP-C may be involved in the correct assembly of CENP-C on alphoid DNA. In other words, a possible molecular linkage may exist between one of the kinetochore components and human centromere DNA through CENP-B/CENP-B box interaction.

Received for publication, June 18, 2003 , and in revised form, November 10, 2003.

^* This work was supported by a grant-in-aid for Scientific Research on Priority Areas (B), Core Research for Evolutional Science and Technology, and Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Science, Sports and Culture of Japan, as well as a grant-in-aid (Basic Research 21) for Breakthroughs in Info-Communications, and a grant-in-aid from the Cell Science Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Fujita Health University Inst. for Comprehensive Medical Science, Toyoake, Aichi 470-1192, Japan.

^|| Present address: Tsukuba Research Institute, Banyu Pharmaceutical Co. Ltd., Okubo, Tsukuba 300-2611, Japan.

^** To whom correspondence should be addressed. Tel: 81-52-789-2985; Fax: 81-52-789-5732; E-mail: g44478a{at}nucc.cc.nagoya-u.ac.jp.

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