The Cdc45.Mcm2-7.GINS Protein Complex in Trypanosomes Regulates DNA Replication and Interacts with Two Orc1-like Proteins in the Origin Recognition Complex
The Cdc45.Mcm2-7.GINS Protein Complex in Trypanosomes Regulates DNA Replication and Interacts with Two Orc1-like Proteins in the Origin Recognition Complex
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DOI：
10.1074/jbc.m111.240143
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发表时间：
2011-09-16
影响因子：
4.8
通讯作者：
Li, Ziyin
中科院分区：
文献类型：
--
作者：
Dang, Hung Quang;Li, Ziyin
准确的DNA复制需要在复制起始处许多调节蛋白的复杂相互作用。CMG （Cdc45.Mcm2-7.GINS）复合体由Cdc45、Mcm2-7和由Sld5和Psf1 - Psf3组成的GINS （go - chi- ni - san）复合体被Cdc6和Cdt1招募到由异六聚体起源识别复合体（ORC）结合的起点上，并发挥复制解旋酶的作用。布鲁氏锥虫是一种早期分支微生物真核生物，似乎表达一种由单一Orc1/ cdc6样蛋白组成的古细菌样ORC。然而，与古细菌不同，锥虫具有真核样CMG复合物的成分，但它们是否形成活性解旋酶复合物，与ORC结合，并调节DNA复制尚不清楚。在这里，我们证明了CMG复合物在体内在锥虫体内形成，mcm2 -7解旋酶活性通过与Cdc45和体外GINS复合物的结合而被激活。Mcm2-7和GINS蛋白在整个细胞周期内被限制在细胞核内，而Cdc45在DNA复制后被输出到细胞核外，这表明Cdc45的核排斥是阻止锥虫DNA再复制的一种机制。除Mcm4、Mcm6和Psf1外，单个CMG基因的敲低会抑制DNA复制和细胞增殖。最后，我们发现了一种新的Orc1样蛋白Orc1b，作为ORC的附加成分，并表明Orc1b和Orc1/Cdc6都通过与Mcm3的相互作用与Mcm2-7结合。总之，我们确定了Cdc45.Mcm2-7。在锥虫异常的起源识别复合体中，作为与两个orc1样蛋白相互作用的复制解旋酶的GINS复合体。
Accurate DNA replication requires a complex interplay of many regulatory proteins at replication origins. The CMG (Cdc45.Mcm2-7.GINS) complex, which is composed of Cdc45, Mcm2-7, and the GINS (Go-Ichi-Ni-San) complex consisting of Sld5 and Psf1 to Psf3, is recruited by Cdc6 and Cdt1 onto origins bound by the heterohexameric origin recognition complex (ORC) and functions as a replicative helicase. Trypanosoma brucei, an early branched microbial eukaryote, appears to express an archaea-like ORC consisting of a single Orc1/Cdc6-like protein. However, unlike archaea, trypanosomes possess components of the eukaryote-like CMG complex, but whether they form an active helicase complex, associate with the ORC, and regulate DNA replication remains unknown. Here, we demonstrated that the CMG complex is formed in vivo in trypanosomes and thatMcm2-7 helicase activity is activated by the association with Cdc45 and the GINS complex in vitro. Mcm2-7 and GINS proteins are confined to the nucleus throughout the cell cycle, whereas Cdc45 is exported out of the nucleus after DNA replication, indicating that nuclear exclusion of Cdc45 constitutes one mechanism for preventing DNA re-replication in trypanosomes. With the exception of Mcm4, Mcm6, and Psf1, knockdown of individual CMG genes inhibits DNA replication and cell proliferation. Finally, we identified a novel Orc1-like protein, Orc1b, as an additional component of the ORC and showed that both Orc1b and Orc1/Cdc6 associate with Mcm2-7 via interactions with Mcm3. All together, we identified the Cdc45.Mcm2-7.GINS complex as the replicative helicase that interacts with two Orc1-like proteins in the unusual origin recognition complex in trypanosomes.