Abstract
Centromere protein A (CENP-A) is a histone H3 variant that marks centromere location on the chromosome. To study the subunit structure and folding of human CENP-A-containing chromatin, we generated a set of nucleosomal arrays with canonical core histones and another set with CENP-A substituted for H3. At the level of quaternary structure and assembly, we find that CENP-A arrays are composed of octameric nucleosomes that assemble in a stepwise mechanism, recapitulating conventional array assembly with canonical histones. At intermediate structural resolution, we find that CENP-A-containing arrays are globally condensed relative to arrays with the canonical histones. At high structural resolution, using hydrogen-deuterium exchange coupled to mass spectrometry (H/DX-MS), we find that the DNA superhelical termini within each nucleosome are loosely connected to CENP-A, and we identify the key amino acid substitution that is largely responsible for this behavior. Also the C terminus of histone H2A undergoes rapid hydrogen exchange relative to canonical arrays and does so in a manner that is independent of nucleosomal array folding. These findings have implications for understanding CENP-A-containing nucleosome structure and higher-order chromatin folding at the centromere.