Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut, United States of America.
PLoS Genet. 2011 May;7(5):e1002068. doi: 10.1371/journal.pgen.1002068. Epub 2011 May 12.
Semi-conservative segregation of nucleosomes to sister chromatids during DNA replication creates gaps that must be filled by new nucleosome assembly. We analyzed the cell-cycle timing of centromeric chromatin assembly in Drosophila, which contains the H3 variant CID (CENP-A in humans), as well as CENP-C and CAL1, which are required for CID localization. Pulse-chase experiments show that CID and CENP-C levels decrease by 50% at each cell division, as predicted for semi-conservative segregation and inheritance, whereas CAL1 displays higher turnover. Quench-chase-pulse experiments demonstrate that there is a significant lag between replication and replenishment of centromeric chromatin. Surprisingly, new CID is recruited to centromeres in metaphase, by a mechanism that does not require an intact mitotic spindle, but does require proteasome activity. Interestingly, new CAL1 is recruited to centromeres before CID in prophase. Furthermore, CAL1, but not CENP-C, is found in complex with pre-nucleosomal CID. Finally, CENP-C displays yet a different pattern of incorporation, during both interphase and mitosis. The unusual timing of CID recruitment and unique dynamics of CAL1 identify a distinct centromere assembly pathway in Drosophila and suggest that CAL1 is a key regulator of centromere propagation.
在 DNA 复制过程中，核小体半保守性地分配到姊妹染色单体上，从而产生必须通过新核小体组装来填补的间隙。我们分析了果蝇着丝粒染色质组装的细胞周期定时，其中包含 H3 变体 CID（人类中的 CENP-A），以及 CENP-C 和 CAL1，它们是 CID 定位所必需的。脉冲追踪实验表明，CID 和 CENP-C 的水平在每次细胞分裂时降低 50%，这与半保守性分离和遗传相一致，而 CAL1 的周转率更高。淬火追踪脉冲实验表明，复制和补充着丝粒染色质之间存在显著的滞后。令人惊讶的是，新的 CID 在有丝分裂中期通过一种不需要完整有丝分裂纺锤体但需要蛋白酶体活性的机制被招募到着丝粒。有趣的是，新的 CAL1 在前期比 CID 更早被招募到着丝粒。此外，CAL1，但不是 CENP-C，与前核小体 CID 形成复合物。最后，CENP-C 在有丝分裂间期和有丝分裂期间表现出不同的掺入模式。CID 招募的异常时间和 CAL1 的独特动力学在果蝇中确定了一种独特的着丝粒组装途径，并表明 CAL1 是着丝粒传播的关键调节剂。
