Human centromere protein A (CENP-A) can replace histone H3 in nucleosome reconstitution in vitro

Human centromere protein A (CENP-A) can replace histone H3 in nucleosome reconstitution in vitro

^*Bioscience Center, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan; ^§Japan Bioindustry Association, Hatchobori 2-Chome, Chuo-ku, Tokyo 104-3302, Japan; ^‖Fujita Health University Institute for Comprehensive Medical Science, Kutsukake-cho, Toyoake, Aichi 470-1192, Japan; ^¶Department of Natural Environment Science, Faculty of Integrated Human Studies, Kyoto University, Yoshida Nihonmatsu, Kyoto 606-8501, Japan; and ^†Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST), Moto-machi 4-1-8, Kawaguchi, Saitama 332-0012, Japan

Communicated by Nicholas R. Cozzarelli, University of California, Berkeley, CA (received for review February 10, 2000)

Abstract

Centromere protein A (CENP-A) is a variant of histone H3 with more than 60% sequence identity at the C-terminal histone fold domain. CENP-A specifically locates to active centromeres of animal chromosomes and therefore is believed to be a component of the specialized centromeric nucleosomes on which the kinetochores are assembled. Here we report that CENP-A, highly purified from HeLa cells, can indeed replace histone H3 in a nucleosome reconstitution system mediated by nucleosome assembly protein-1 (NAP-1). The structure of the nucleosomes reconstituted with recombinant CENP-A, histones H2A, H2B, and H4, and closed circular DNAs had the following properties. By atomic force microscopy, “beads on a string” images were obtained that were similar to those obtained with nucleosomes reconstituted with four standard histones. DNA ladders with repeats of approximately 10 bp were produced by DNase I digestion, indicating that the DNA was wrapped round the protein complex. Mononucleosomes isolated by glycerol gradient sedimentation had a relative molecular mass of ≈200 kDa and were composed of 120–150 bp of DNA and equimolar amounts of CENP-A, and histones H4, H2A, and H2B. Thus, we conclude that CENP-A forms an octameric complex with histones H4, H2A, and H2B in the presence of DNA.

Footnotes

↵ ‡ To whom reprint requests should be addressed at: Bioscience Center, Nagoya University Chikusa-ku, Nagoya 464-8601, Japan. E-mail: i45156a{at}nucc.cc.nagoya-u.ac.jp.
Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.130189697.
Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.130189697
Abbreviations:

CENP,

centromere protein;

NAP-1,

nucleosome assembly protein-1;

TCA,

trichloroacetic acid;

EK,

enterokinase;

AFM,

atomic force microscopy;

Topo I,

topoisomerase I