Antagonistic control of DDK binding to licensed replication origins by Mcm2 and Rad53
Antagonistic control of DDK binding to licensed replication origins by Mcm2 and Rad53
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DOI：
10.7554/elife.58571
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发表时间：
2020-07-23
期刊：
影响因子：
7.7
通讯作者：
Remus, Dirk
中科院分区：
文献类型：
--
作者：
Abd Wahab, Syafiq;Remus, Dirk
真核生物的复制起点是由复制DNA解旋酶Mcm 2 -7以无活性的双六聚体形式在DNA周围装载而获得许可的。随后的起源激活受到多种蛋白激酶的控制，这些蛋白激酶促进或抑制起源激活，这对于基因组维持很重要。利用重组的芽殖酵母DNA复制系统，我们发现Mcm 2的柔性N端延伸（NTE）促进Dbf 4依赖性激酶（DDK）稳定募集为Mcm 2 -7双六聚体，这反过来促进Mcm 4和-6的DDK磷酸化和随后的起始激活。相反，我们证明，检查点激酶，Rad 53，抑制DDK结合Mcm 2 -7双六聚体。出乎意料的是，该功能不依赖于Rad 53激酶活性，表明活化的Rad 53对DDK的空间抑制。这些发现确定了起源激活反应的关键决定因素，并揭示了检查点依赖性起源抑制的新机制。
Eukaryotic replication origins are licensed by the loading of the replicative DNA helicase, Mcm2-7, in inactive double hexameric form around DNA. Subsequent origin activation is under control of multiple protein kinases that either promote or inhibit origin activation, which is important for genome maintenance. Using the reconstituted budding yeast DNA replication system, we find that the flexible N-terminal extension (NTE) of Mcm2 promotes the stable recruitment of Dbf4-dependent kinase (DDK) to Mcm2-7 double hexamers, which in turn promotes DDK phosphorylation of Mcm4 and -6 and subsequent origin activation. Conversely, we demonstrate that the checkpoint kinase, Rad53, inhibits DDK binding to Mcm2-7 double hexamers. Unexpectedly, this function is not dependent on Rad53 kinase activity, suggesting steric inhibition of DDK by activated Rad53. These findings identify critical determinants of the origin activation reaction and uncover a novel mechanism for checkpoint-dependent origin inhibition.