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 specifical ly locates to active centromeres of animal chromosomes and therefore is bel ieved to be a component of the specialized centromeric nucleosomes on which the kinetochores are assembled. Here we report that CENP-A, highly purifie d from HeLa cells, can indeed replace histone H3 in a nucleosome reconstitu tion system mediated by nucleosome assembly protein-1 (NAP-1). The structur e of the nucleosomes reconstituted with recombinant CENP-A, histones H2A, H 2B, and H4, and closed circular DNAs had the following properties. By atomi c force microscopy, "beads on a string" images were obtained that were simi lar to those obtained with nucleosomes reconstituted with four standard his tones, DNA ladders with repeats of approximately 10 bp were produced by DNa se I digestion, indicating that the DNA was wrapped round the protein compl ex. Mononucleosomes isolated by glycerol gradient sedimentation had a relat ive molecular mass of approximate to 200 kDa and were composed of 120-150 b p of DNA and equimolar amounts of CENP-A, and histones H4, H2A, and H2B, Th us, we conclude that CENP-A forms an octameric complex with histones H4, H2 A, and H2B in the presence of DNA.