Phosphorylation-dependent binding of mitotic cyclins to Cdc6 contributes to DNA replication control
Phosphorylation-dependent binding of mitotic cyclins to Cdc6 contributes to DNA replication control
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DOI：
10.1038/nature03024
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发表时间：
2004-10-28
期刊：
影响因子：
64.8
通讯作者：
Diffley, JFX
中科院分区：
文献类型：
--
作者：
Mimura, S;Seki, T;Diffley, JFX
细胞周期蛋白依赖性激酶（CDK）通过阻止芽殖酵母酿酒酵母细胞周期S、G2和M期期间复制前复合物（pre-RC）的组装，将DNA复制起点的激活限制在每个细胞周期一次（1，2）。CDK通过不同的机制抑制每个前RC组分（ORC、Cdc 6、Cdt 1/Mcm 2 -7）。我们在这里表明，有丝分裂的CDK，Clb 2/Cdc 28，紧密结合的氨基末端结构域（NTD）的Cdc 6，在这个复杂的Cdc 6是无法组装前RC。我们目前的证据表明，这种Clb 2依赖的机制有助于防止体内再复制。CDK与Cdc 6的NTD的相互作用是由细胞周期蛋白亚基Clb 2介导的，并且可以用重组Clb 2蛋白和合成的NTD肽重建。只有当NTD在CDK共有位点磷酸化时，Clb 2才能紧密结合。含有细胞周期蛋白A、B和E的人CDK也特异性结合磷酸-NTD肽。我们认为细胞周期蛋白与磷酸肽基序的直接结合可能是一种普遍的现象，有助于CDK靶向底物。
Cyclin-dependent kinases (CDKs) limit the activation of DNA replication origins to once per cell cycle by preventing the assembly of pre-replicative complexes (pre-RCs) during S, G2 and M phases of the cell cycle in the budding yeast Saccharomyces cerevisiae(1,2). CDKs inhibit each pre-RC component (ORC, Cdc6, Cdt1/Mcm2-7) by different mechanisms. We show here that the mitotic CDK, Clb2/Cdc28, binds tightly to an amino-terminal domain (NTD) of Cdc6, and that Cdc6 in this complex is unable to assemble pre-RCs. We present evidence indicating that this Clb2-dependent mechanism contributes to preventing rereplication in vivo. CDK interaction with the NTD of Cdc6 is mediated by the cyclin subunit Clb2, and could be reconstituted with recombinant Clb2 protein and synthetic NTD peptides. Tight Clb2 binding occurred only when the NTD was phosphorylated on CDK consensus sites. Human CDKs containing cyclins A, B and E also bound specifically to phospho-NTD peptides. We propose that direct binding of cyclins to phosphopeptide motifs may be a widespread phenomenon contributing to the targeting of CDKs to substrates.