Tatsumi Yasutoshi, Ezura Kai, Yoshida Kazumasa, Yugawa Takashi, Narisawa-Saito Mako, Kiyono Tohru, Ohta Satoshi, Obuse Chikashi, Fujita Masatoshi
Virology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuohku, Tokyo 104-0045, Japan.
Genes Cells. 2008 Oct;13(10):1045-59. doi: 10.1111/j.1365-2443.2008.01224.x. Epub 2008 Aug 29.
The origin recognition complex (ORC) binds to replication origins to regulate the cell cycle-dependent assembly of pre-replication complexes (pre-RCs). We have found a novel link between pre-RC assembly regulation and telomere homeostasis in human cells. Biochemical analyses showed that human ORC binds to TRF2, a telomere sequence-binding protein that protects telomeres and functions in telomere length homeostasis, via the ORC1 subunit. Immunostaining further revealed that ORC and TRF2 partially co-localize in nuclei, whereas chromatin immunoprecipitation analyses confirmed that pre-RCs are assembled at telomeres in a cell cycle-dependent manner. Over-expression of TRF2 stimulated ORC and MCM binding to chromatin and RNAi-directed TRF2 silencing resulted in reduced ORC binding and pre-RC assembly at telomeres. As expected from previous reports, TRF2 silencing induced telomere elongation. Interestingly, ORC1 silencing by RNAi weakened the TRF2 binding as well as the pre-RC assembly at telomeres, suggesting that ORC and TRF2 interact with each other to achieve stable binding. Furthermore, ORC1 silencing also resulted in modest telomere elongation. These data suggest that ORC might be involved in telomere homeostasis in human cells.
复制起点识别复合体（ORC）结合到复制起点，以调控复制前复合体（pre-RC）依赖细胞周期的组装。我们在人类细胞中发现了pre-RC组装调控与端粒稳态之间的新联系。生化分析表明，人类ORC通过ORC1亚基与TRF2结合，TRF2是一种端粒序列结合蛋白，可保护端粒并在端粒长度稳态中发挥作用。免疫染色进一步显示，ORC和TRF2在细胞核中部分共定位，而染色质免疫沉淀分析证实pre-RC以细胞周期依赖的方式在端粒处组装。TRF2的过表达刺激了ORC和MCM与染色质的结合，而RNA干扰介导的TRF2沉默导致端粒处ORC结合和pre-RC组装减少。正如先前报道所预期的，TRF2沉默诱导端粒延长。有趣的是，RNA干扰介导的ORC1沉默削弱了TRF2的结合以及端粒处的pre-RC组装，这表明ORC和TRF2相互作用以实现稳定结合。此外，ORC1沉默也导致适度的端粒延长。这些数据表明ORC可能参与人类细胞中的端粒稳态。
