CHROMATIN CONTAINING CENP-A AND ALPHA-SATELLITE DNA IS A MAJOR COMPONENT OF THE INNER KINETOCHORE PLATE

The pathway of molecular interactions leading to kinetochore assembly on mammalian chromosomes is unknown. Kinetochores could be specified b y structural features of centromeric satellite DNA [1-3] or by specifi c DNA sequences, analogous to budding yeast centromeres, interspersed in centromeric satellite DNA arrays [4,5]. Alternatively, kinetochores could be epigenetic structures that replicate without strict dependen ce on DNA sequence [6-8]. We purified kinetochore-associated chromatin from human chromosomes by immunoprecipitation of CENP-A, a centromere -specific histone H3 homologue located in the inner plate of the kinet ochore [6,9,10]. Hybridization and DNA sequence analyses of cloned kin etochore DNA fragments revealed alpha-satellite as the predominant seq uence associated with CENP-A. A major site of micrococcal nuclease dig estion was identified by mapping the termini of alpha-satellite clones , suggesting that the inner kinetochore plate contains phased arrays o f CENP-A-alpha-satellite nucleosomes. These experiments demonstrate fo r the first time that complex satellite DNA is a structural component of the kinetochore. Further, because complex satellite DNA is evolutio narily unconserved, these results suggest that molecular recognition e vents necessary for kinetochore formation take place at the level of D NA conformation or epigenetic mechanisms rather than DNA sequence per se.