Activation of S-phase-promoting CDKs in late G(1) defines a ''point of no return'' after which Cdc6 synthesis cannot promote DNA replication in yeast
Activation of S-phase-promoting CDKs in late G(1) defines a ''point of no return'' after which Cdc6 synthesis cannot promote DNA replication in yeast
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DOI：
10.1101/gad.10.12.1516
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发表时间：
1996-06-15
影响因子：
10.5
通讯作者：
Nasmyth, K
中科院分区：
文献类型：
--
作者：
Piatti, S;Bohm, T;Nasmyth, K
在真核细胞中，DNA复制被限制在细胞周期的一个离散时期，并且通常直到细胞分裂后期之后才重新发生。在芽殖酵母酿酒酵母中，随着细胞退出有丝分裂（或在G（1）后期），复制前复合物（pre-RC）在未来起点的组装对于随后启动DNA复制是必需的，该复制由与B型细胞周期蛋白Clb 1-Clb 6相关的Cdc 28/Cdk 1激酶在G（1）晚期的激活触发。在G（2）和M期期间不存在前RC可以解释为什么DNA复制起点在细胞周期期间仅激发一次，即使S期促进Cdks从S期开始到M期结束都保持活性。相位前RC的形成及其在G（1）期间的维持取决于由CDC 6编码的不稳定蛋白的合成和活性。我们发现Cdc 6的合成只能在细胞周期的一个有限的窗口中促进DNA复制：在细胞分裂后期Clbs的破坏和细胞分裂后期Clb 5/Cdk 1和Clb 6/Cdk 1的激活之间。后者对应于“不归点”，在此之后Cdc 6的合成不再能促进DNA复制。Cdc 6蛋白可以在整个细胞周期中产生，并且在某些情况下，可以在G（2）和M期细胞的细胞核内积累而不诱导再复制。因此，对Cdc 6降解和/或核定位的控制对于防止起源再激发不是至关重要的。我们的数据是一致的概念，即细胞不能再纳入从头合成Cdc 6到前RC一旦Clb/Cdk 1激酶已被激活。我们发现Cdc 6p与Clb/Cdk 1激酶从晚期G（1）到后期相关联，这可能对抑制S，G（2）和M期的pre-RC组装很重要。触发起始的相同激酶对前RC组装的抑制解释了起源如何被阻止重新激发，直到后期后Clb激酶被破坏。
In eukaryotic cells, DNA replication is confined to a discrete period of the cell cycle and does not usually recur until after anaphase. In the budding yeast Saccharomyces cerevisiae, assembly of pre-replication complexes (pre-RCs) at future origins as cells exit mitosis (or later during G(1)) is necessary for subsequent initiation of DNA replication triggered by activation in late G(1) of Cdc28/Cdk1 kinases associated with B-type cyclins Clb1-Clb6. The absence of pre-RCs during G(2) and M phases could explain why origins of DNA replication fire only once during the cell cycle, even though S-phase-promoting Cdks remain active from the beginning of S phase through the end of M. phase. Formation of pre-RCs and their maintenance during G(1) depend on the synthesis and activity of an unstable protein encoded by CDC6. We find that Cdc6 synthesis can only promote DNA replication in a restricted window of the cell cycle: between destruction of Clbs after anaphase and activation of Clb5/ and Clb6/Cdk1 in late G(1). The latter corresponds to a ''point of no return,'' after which Cdc6 synthesis can no longer promote DNA replication. Cdc6 protein can be made throughout the cell cycle and, in certain circumstances, can accumulate within the nuclei of G(2) and M phase cells without inducing re-replication. Thus, control over Cdc6 degradation and/or nuclear localization is not crucial for preventing origin re-firing. Our data are consistent with the notion that cells can no longer incorporate de novo synthesized Cdc6 into pre-RCs once Clb/Cdk1 kinases have been activated. We show that Cdc6p associates with Clb/Cdk1 kinases from late G(1) until late anaphase, which might be important for inhibiting pre-RC assembly during S, G(2), and M phases. Inhibition of pre-RC assembly by the same kinases that trigger initiation explains how origins are prevented from re-firing until Clb kinases are destroyed after anaphase.