Chromatin containing CENP-A and alpha-satellite DNA is a major component of the inner kinetochore plate
Chromatin containing CENP-A and alpha-satellite DNA is a major component of the inner kinetochore plate
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DOI：
10.1016/s0960-9822(06)00381-2
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发表时间：
1997-11-01
期刊：
影响因子：
9.2
通讯作者：
Sullivan, KF
中科院分区：
文献类型：
--
作者：
Vafa, O;Sullivan, KF
导致动粒在哺乳动物染色体上组装的分子相互作用的途径尚不清楚。着丝点可以由着丝粒卫星DNA的结构特征来指定[1-3]，或者由特定的DNA序列来指定，类似于发芽的酵母着丝粒，散布在着丝粒卫星DNA阵列中[4，5]。或者，动点可以是表观遗传结构，不严格依赖DNA序列进行复制[6-8]。我们通过CENP-A的免疫沉淀从人类染色体中纯化了动粒相关染色质，CENP-A是着丝粒特异的组蛋白H3同源物，位于着丝粒内板[6，9，10]。克隆的动粒DNA片段的杂交和DNA序列分析表明，与CENP-A相关的优势序列为α-卫星。通过绘制α卫星克隆的末端图谱，确定了微球菌核酸酶的一个主要消化部位，表明内部着丝粒平板含有CENP-A-α卫星核小体的相控阵。这些实验首次证明，复杂的卫星DNA是动粒的一个结构成分。此外，由于复杂的卫星DNA在进化上是不保守的，这些结果表明动粒形成所需的分子识别事件发生在DNA构象或表观遗传机制的水平上，而不是DNA序列本身。
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.