Dynamics of CENP-N kinetochore binding during the cell cycle
Dynamics of CENP-N kinetochore binding during the cell cycle
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DOI：
10.1242/jcs.088625
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发表时间：
2011-11-15
影响因子：
4
通讯作者：
Diekmann, Stephan
中科院分区：
文献类型：
--
作者：
Hellwig, Daniela;Emmerth, Stephan;Diekmann, Stephan
准确的染色体分离需要着丝点的组装，着丝点是在每个姐妹染色单体的着丝粒上组装的多蛋白复合物。这一过程的关键步骤是将组成着丝粒相关网络（CCAN）与构成着丝粒核小体的组蛋白H3变体CENPA结合。该网络被认为是有丝分裂过程中外着丝点组装的持久结构支架。在这里，我们通过荧光共振能量转移（FRET）发现，在体内，CENP-N的n端非常接近CENP-A的n端，这与体外数据显示的CENP-N与CENP-A直接结合的结果一致。此外，我们证明在活细胞中，CENP-N在S期和G2期间与着丝点结合，但在有丝分裂和G1期间大部分不在着丝点上。通过测量着丝点结合的动力学，我们发现CENP-N在G1期经历快速交换，直到S期中期与着丝点稳定结合。大部分CENP-N在S期被装载，并在G2期再次解离。我们提出了一个模型，其中CENP-N在着丝复制过程中作为保真度因子发挥作用，并揭示了CCAN网络比以前认识到的动态得多。
Accurate chromosome segregation requires the assembly of kinetochores, multiprotein complexes that assemble on the centromere of each sister chromatid. A key step in this process involves binding of the constitutive centromere-associated network (CCAN) to CENPA, the histone H3 variant that constitutes centromeric nucleosomes. This network is proposed to operate as a persistent structural scaffold for assembly of the outer kinetochore during mitosis. Here, we show by fluorescence resonance energy transfer (FRET) that the N-terminus of CENP-N lies in close proximity to the N-terminus of CENP-A in vivo, consistent with in vitro data showing direct binding of CENP-N to CENP-A. Furthermore, we demonstrate in living cells that CENP-N is bound to kinetochores during S phase and G2, but is largely absent from kinetochores during mitosis and G1. By measuring the dynamics of kinetochore binding, we reveal that CENP-N undergoes rapid exchange in G1 until the middle of S phase when it becomes stably associated with kinetochores. The majority of CENP-N is loaded during S phase and dissociates again during G2. We propose a model in which CENP-N functions as a fidelity factor during centromeric replication and reveal that the CCAN network is considerably more dynamic than previously appreciated.