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DDK 通过控制 Cdc45-GINS 与 Mcm2-7 的结合倍数来调节复制起始。

DDK regulates replication initiation by controlling the multiplicity of Cdc45-GINS binding to Mcm2-7.

机构信息

Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, United States.

Department of Biochemistry, Brandeis University, Waltham, United States.

出版信息

Elife. 2021 Feb 22;10:e65471. doi: 10.7554/eLife.65471.

DOI:10.7554/eLife.65471
PMID:33616038
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7954526/
Abstract

The committed step of eukaryotic DNA replication occurs when the pairs of Mcm2-7 replicative helicases that license each replication origin are activated. Helicase activation requires the recruitment of Cdc45 and GINS to Mcm2-7, forming Cdc45-Mcm2-7-GINS complexes (CMGs). Using single-molecule biochemical assays to monitor CMG formation, we found that Cdc45 and GINS are recruited to loaded Mcm2-7 in two stages. Initially, Cdc45, GINS, and likely additional proteins are recruited to unstructured Mcm2-7 N-terminal tails in a Dbf4-dependent kinase (DDK)-dependent manner, forming Cdc45-tail-GINS intermediates (CtGs). DDK phosphorylation of multiple phosphorylation sites on the Mcm2-7 tails modulates the number of CtGs formed per Mcm2-7. In a second, inefficient event, a subset of CtGs transfer their Cdc45 and GINS components to form CMGs. Importantly, higher CtG multiplicity increases the frequency of CMG formation. Our findings reveal the molecular mechanisms sensitizing helicase activation to DDK levels with implications for control of replication origin efficiency and timing.

摘要

真核生物 DNA 复制的起始是在许可每个复制起点的 Mcm2-7 复制解旋酶对被激活时发生的。解旋酶的激活需要 Cdc45 和 GINS 募集到 Mcm2-7，形成 Cdc45-Mcm2-7-GINS 复合物（CMG）。我们使用单分子生化测定法来监测 CMG 的形成，发现 Cdc45 和 GINS 以两个阶段募集到加载的 Mcm2-7。最初，Cdc45、GINS 和可能的其他蛋白以依赖 Dbf4 依赖性激酶（DDK）的方式募集到无结构的 Mcm2-7 N 端尾部，形成 Cdc45-尾部-GINS 中间体（CtG）。Mcm2-7 尾部的多个磷酸化位点的 DDK 磷酸化调节每个 Mcm2-7 形成的 CtG 数量。在第二个低效事件中，CtG 的一部分将其 Cdc45 和 GINS 组件转移以形成 CMG。重要的是，更高的 CtG 倍数增加了 CMG 形成的频率。我们的发现揭示了使解旋酶激活对 DDK 水平敏感的分子机制，这对复制起点效率和时间的控制具有重要意义。

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