CENP-A, -B, and -C chromatin complex that contains the I-type α-satellite array constitutes the prekinetochore in HeLa cells

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 alpha-satellite DNA that contains the CENP-B box (alphaI-type array). Mapping of hypersensitive sites for micrococcal nuclease (MNase) digestion indicated that CENP-A nucleosomes were phased on the alphaI-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 CENPC from the complex. This result suggests that CENP-A nucleosomes form a complex with CENP-B and CENPC 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 alpha-satellite array and constitutes the prekinetochore in HeLa cells.