Involvement of human ORC and TRF2 in pre-replication complex assembly at telomeres
Involvement of human ORC and TRF2 in pre-replication complex assembly at telomeres
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DOI：
10.1111/j.1365-2443.2008.01224.x
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发表时间：
2008-10-01
期刊：
影响因子：
2.1
通讯作者：
Fujita, Masatoshi
中科院分区：
文献类型：
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作者：
Tatsumi, Yasutoshi;Ezura, Kai;Fujita, Masatoshi
起点识别复合体（ORC）与复制起点结合，调节细胞周期依赖性复制前复合体（pre-RCs）的组装。我们在人类细胞中发现了pre-RC组装调节和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是一种保护端粒并在端粒长度稳态中起作用的端粒序列结合蛋白。免疫染色进一步显示ORC和TRF2部分共定位于细胞核，而染色质免疫沉淀分析证实，pre- rc以细胞周期依赖的方式组装在端粒上。TRF2的过度表达刺激ORC和MCM与染色质结合，rnai定向TRF2沉默导致ORC结合和端粒预rc组装减少。如先前报道所料，TRF2沉默诱导端粒伸长。有趣的是，RNAi沉默ORC1削弱了TRF2的结合以及端粒上的pre-RC组装，这表明ORC和TRF2相互作用以实现稳定的结合。此外，ORC1沉默也导致端粒适度延长。这些数据提示ORC可能参与人类细胞端粒稳态。
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.