Ciz1 cooperates with cyclin-A-CDK2 to activate mammalian DNA replication in vitro
Ciz1 cooperates with cyclin-A-CDK2 to activate mammalian DNA replication in vitro
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DOI：
10.1242/jcs.059345
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发表时间：
2010-04-01
影响因子：
4
通讯作者：
Coverley, Dawn
中科院分区：
文献类型：
--
作者：
Copeland, Nikki A.;Sercombe, Heather E.;Coverley, Dawn
哺乳动物DNA复制的起始可以从分离的G1期细胞核和细胞提取液中重组，并辅以细胞周期蛋白依赖蛋白激酶(CDKs)。在这种情况下，Cyclin E支持复制前的复合体组装，而Cyclin-A相关的激酶则在晚些时候终止组装并激活DNA复制。协调这些事件的机制尚不清楚。在这里，我们证明了复制因子Ciz1通过不同的周期蛋白结合基序与周期蛋白E和A顺序相互作用。Cyclin A以依赖于其在DNA复制中所必需的功能结构域的方式取代Ciz1中的Cyclin E。此外，在无细胞检测中，重组Cyclin-A-CDK2复合体和重组Ciz1协同作用促进了G1期晚期细胞核DNA复制的启动。此外，Ciz1支持在分离的细胞核中固定细胞周期蛋白A，而RNAi耗尽Ciz1会损害固定，这表明Ciz1通过帮助将激酶靶向于特定的亚核间隔来促进启动。我们认为，Ciz1通过将Cyclin-A相关的激酶传递到Cyclin E指定的位置，帮助确保它们在相同的位置以正确的顺序执行其功能，从而协调Cyclin E和Cyclin A在细胞核中的功能。
Initiation of mammalian DNA replication can be reconstituted from isolated G1-phase nuclei and cell extracts, supplemented with cyclin-dependent protein kinases (CDKs). Under these conditions, cyclin E supports pre-replication complex assembly, whereas cyclin-A-associated kinase acts later to terminate assembly and activate DNA replication. The mechanism by which these events are coordinated is unknown. Here, we show that the replication factor Ciz1 interacts with cyclins E and A sequentially through distinct cyclin-binding motifs. Cyclin A displaces cyclin E from Ciz1 in a manner that is dependent on functional domains that are essential for its role in DNA replication. Furthermore, in cell-free assays, recombinant cyclin-A-CDK2 complexes and recombinant Ciz1 cooperate to promote initiation of DNA replication in late G1-phase nuclei. In addition, Ciz1 supports immobilization of cyclin A in isolated nuclei and depletion of Ciz1 by RNAi impairs immobilization, suggesting that Ciz1 promotes initiation by helping to target the kinase to a specific subnuclear compartment. We propose that Ciz1 acts to coordinate the functions of cyclins E and A in the nucleus, by delivering cyclin-A-associated kinase to sites that are specified by cyclin E, helping to ensure that they execute their functions in the same place and in the correct order.