DDK regulates replication initiation by controlling the multiplicity of Cdc45-GINS binding to Mcm2-7.
DDK regulates replication initiation by controlling the multiplicity of Cdc45-GINS binding to Mcm2-7.
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DOI：
10.7554/elife.65471
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发表时间：
2021-02-22
期刊：
影响因子：
7.7
通讯作者：
Bell SP
中科院分区：
文献类型：
--
作者：
De Jesús-Kim L;Friedman LJ;Lõoke M;Ramsoomair CK;Gelles J;Bell SP
真核生物DNA复制的关键步骤发生在许可每个复制起点的Mcm 2 -7复制解旋酶对被激活时。解旋酶激活需要Cdc 45和GINS募集至Mcm 2 -7，形成Cdc 45-Mcm 2 -7-GINS复合物（CMG）。使用单分子生化测定来监测CMG形成，我们发现Cdc 45和GINS在两个阶段被招募到负载的Mcm 2 -7。最初，Cdc 45、GINS和可能的其他蛋白质以Dbf 4依赖性激酶（DDK）依赖性方式被募集到非结构化Mcm 2 -7 N末端尾部，形成Cdc 45-tail-GINS中间体（CtGs）。Mcm 2 -7尾部上多个磷酸化位点的DDK磷酸化调节每个Mcm 2 -7形成的CtG的数量。在第二个低效事件中，CtG的子集转移其Cdc 45和GINS组分以形成CMG。重要的是，较高的CtG多重性增加CMG形成的频率。我们的研究结果揭示了对DDK水平敏感的解旋酶激活的分子机制，并对复制起点效率和时间的控制产生影响。
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.