An essential role for Orc6 in DNA replication through maintenance of pre-replicative complexes
An essential role for Orc6 in DNA replication through maintenance of pre-replicative complexes
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DOI：
10.1038/sj.emboj.7601391
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发表时间：
2006-11-01
期刊：
影响因子：
11.4
通讯作者：
Duncker, Bernard P.
中科院分区：
文献类型：
--
作者：
Semple, Jeffrey W.;Da-Silva, Lance F.;Duncker, Bernard P.
异六聚体起源识别复合体(ORC)是复制前复合体(Pre-RC)G1期组装的支架。在发芽酵母中，只有ORC1-5亚基似乎是起始结合所必需的，但ORC6是细胞增殖所必需的蛋白质。活细胞中ORC6-YFP的成像显示出与复制起点进入亚核区域的组织一致的点状模式。与后生动物的观察结果不同，在母细胞和子细胞的分裂部位没有检测到ORC6，而且ORC6不是有丝分裂或胞质分裂所必需的。ORC6在DNA复制中的重要作用是通过在特定的细胞周期阶段耗尽它来确定的。有趣的是，在前RC形成后，ORC6是进入S相所必需的，而不是以前的模型，认为ORC在细胞周期的这个时间点是可有可无的。当ORC6在G1末期耗尽时，McM2和Mcm10从染色质中被取代，细胞不能通过S期，溴脱氧尿嘧啶核苷掺入后的DNA梳理分析表明，复制起始激发的效率严重受损。
The heterohexameric origin recognition complex (ORC) acts as a scaffold for the G1 phase assembly of pre-replicative complexes (pre-RC). Only the Orc1-5 subunits appear to be required for origin binding in budding yeast, yet Orc6 is an essential protein for cell proliferation. Imaging of Orc6-YFP in live cells revealed a punctate pattern consistent with the organization of replication origins into subnuclear foci. Orc6 was not detected at the site of division between mother and daughter cells, in contrast to observations for metazoans, and is not required for mitosis or cytokinesis. An essential role for Orc6 in DNA replication was identified by depleting it at specific cell cycle stages. Interestingly, Orc6 was required for entry into S phase after pre-RC formation, in contrast to previous models suggesting ORC is dispensable at this point in the cell cycle. When Orc6 was depleted in late G1, Mcm2 and Mcm10 were displaced from chromatin, cells failed to progress through S phase, and DNA combing analysis following bromodeoxyuridine incorporation revealed that the efficiency of replication origin firing was severely compromised.