Hierarchy of S-phase-promoting factors:: Yeast Dbf4-Cdc7 kinase requires prior S-phase cyclin-dependent kinase activation
Hierarchy of S-phase-promoting factors:: Yeast Dbf4-Cdc7 kinase requires prior S-phase cyclin-dependent kinase activation
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DOI：
10.1128/mcb.20.11.3795-3806.2000
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发表时间：
2000-06-01
影响因子：
5.3
通讯作者：
Schwob, E
中科院分区：
文献类型：
--
作者：
Nougarède, R;Della Seta, F;Schwob, E
在所有真核生物中，DNA合成的启动需要在复制起始处形成复制前复合体(PreRCs)，然后被两个S-T蛋白激酶、一个S期细胞周期蛋白依赖性激酶(S-cdk)和一个酵母Dbf4-cdc7同源物(Dbf4p-依赖性激酶)激活。在这里，我们发现酵母DDK活性受细胞周期调控，尽管不如S-CDKClb5-CDK1那么紧密，并在S期达到峰值，与Dbf4p水平相关。Dbf4p在整个细胞周期中都是短暂的，但它的不稳定性在G(1)期间被后期促进复合体加剧。下调DDK活性在生理上很重要，因为CDC7p和Dbf4p的联合过度表达是致命的。由于前RC的形成是一个高度有序的过程，我们询问S-CDK和DDK是否也需要以特定的顺序作用于起源的激发。我们发现这两种激酶都是独立激活的，但我们发现只有在S-CDKs被激活后，DDK才能发挥其复制DNA的功能。CDC45P是一种起始所需的蛋白质，只有在S-CDK激活后才与染色质紧密结合(邹丽红，B.Stillman，Science 280：593-596,1998)。我们发现Cac45p在体外被DDK磷酸化，提示它可能是S-CDK激活后DDK的关键底物之一。在依赖的事件序列中将起源结合的DDK与严格调控的S-CDK联系起来，可能会确保DNA复制只在正确的时间和地点开始。
In all eukaryotes, the initiation of DNA synthesis requires the formation of prereplicative complexes (pre-RCs) on replication origins, followed by their activation by two S-T protein kinases, an S-phase cyclin-dependent kinase (S-CDK) and a homologue of yeast Dbf4-Cdc7 kinase (Dbf4p-dependent kinase [DDK]). Here, we show that yeast DDK activity is cell cycle regulated, though less tightly than that of the S-CDK Clb5-Cdk1, and peaks during S phase in correlation with Dbf4p levels. Dbf4p is short-lived throughout the cell cycle, but its instability is accentuated during G(1) by the anaphase-promoting complex. Downregulating DDK activity is physiologically important, as joint Cdc7p and Dbf4p overexpression is lethal. Because pre-RC formation is a highly ordered process, we asked whether S-CDK and DDK need also to function in a specific order for the firing of origins. We found that both kinases are activated independently, but we show that DDK can perform its function for DNA replication only after S-CDKs have been activated. Cdc45p, a protein needed for initiation, binds tightly to chromatin only after S-CDK activation (L. Zou and B. Stillman, Science 280:593-596, 1998). We show that Cac45p is phosphorylated by DDK in vitro, suggesting that it might be one of DDK's critical substrates after S-CDK activation. Linking the origin-bound DDK to the tightly regulated S-CDK in a dependent sequence of events may ensure that DNA replication initiates only at the right time and place.