Human centromere protein A (CENP-A) can replace histone H3 in nucleosome reconstitution in vitro.
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 approximately 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.
Yoda
K
K
Bioscience Center, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan. i45156a@nucc.cc.nagoya-u.ac.jp
Ando
S
S
Morishita
S
S
Houmura
K
K
Hashimoto
K
K
Takeyasu
K
K
Okazaki
T
T
D001324:Autoantigens; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006657:Histones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009707:Nucleosomes / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid
D001324:Autoantigens; C000619700:CENPA protein, human; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone; D006657:Histones; D009707:Nucleosomes