Vafa O, Sullivan K F
Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Curr Biol. 1997 Nov 1;7(11):897-900. doi: 10.1016/s0960-9822(06)00381-2.
The pathway of molecular interactions leading to kinetochore assembly on mammalian chromosomes is unknown. Kinetochores could be specified by structural features of centromeric satellite DNA [1-3] or by specific 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 dependence 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 kinetochore [6,9,10]. Hybridization and DNA sequence analyses of cloned kinetochore DNA fragments revealed alpha-satellite as the predominant sequence associated with CENP-A. A major site of micrococcal nuclease digestion was identified by mapping the termini of alpha-satellite clones, suggesting that the inner kinetochore plate contains phased arrays of CENP-A-alpha-satellite nucleosomes. These experiments demonstrate for the first time that complex satellite DNA is a structural component of the kinetochore. Further, because complex satellite DNA is evolutionarily unconserved, these results suggest that molecular recognition events necessary for kinetochore formation take place at the level of DNA conformation or epigenetic mechanisms rather than DNA sequence per se.
导致哺乳动物染色体上动粒组装的分子相互作用途径尚不清楚。动粒可能由着丝粒卫星DNA的结构特征所确定[1 - 3]，或者由特定的DNA序列所确定，类似于出芽酵母的着丝粒，散布在着丝粒卫星DNA阵列中[4,5]。另外，动粒也可能是不严格依赖DNA序列进行复制的表观遗传结构[6 - 8]。我们通过免疫沉淀CENP - A（一种位于动粒内板的着丝粒特异性组蛋白H3同源物）从人类染色体中纯化了与动粒相关的染色质[6,9,10]。对克隆的动粒DNA片段进行杂交和DNA序列分析，结果显示α - 卫星是与CENP - A相关的主要序列。通过绘制α - 卫星克隆的末端图谱，确定了微球菌核酸酶消化的一个主要位点，这表明动粒内板含有CENP - A - α - 卫星核小体的相位阵列。这些实验首次证明复杂的卫星DNA是动粒的结构组成部分。此外，由于复杂的卫星DNA在进化上不保守，这些结果表明动粒形成所必需的分子识别事件发生在DNA构象或表观遗传机制层面，而非DNA序列本身。
