Amasino Audra, Gupta Shalini, Friedman Larry J, Gelles Jeff, Bell Stephen P
Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Department of Biochemistry, Brandeis University, Waltham, MA 02454, USA.
bioRxiv. 2023 Jan 2:2023.01.02.522488. doi: 10.1101/2023.01.02.522488.
Eukaryotic DNA replication must occur exactly once per cell cycle to maintain cell ploidy. This outcome is ensured by temporally separating replicative helicase loading (G1 phase) and activation (S phase). In budding yeast, helicase loading is prevented outside of G1 by cyclin-dependent kinase (CDK) phosphorylation of three helicase-loading proteins: Cdc6, the Mcm2-7 helicase, and the origin recognition complex (ORC). CDK inhibition of Cdc6 and Mcm2-7 are well understood. Here we use single-molecule assays for multiple events during origin licensing to determine how CDK phosphorylation of ORC suppresses helicase loading. We find that phosphorylated ORC recruits a first Mcm2-7 to origins but prevents second Mcm2-7 recruitment. Phosphorylation of the Orc6, but not of the Orc2 subunit, increases the fraction of first Mcm2-7 recruitment events that are unsuccessful due to the rapid and simultaneous release of the helicase and its associated Cdt1 helicase-loading protein. Real-time monitoring of first Mcm2-7 ring closing reveals that either Orc2 or Orc6 phosphorylation prevents Mcm2-7 from stably encircling origin DNA. Consequently, we assessed formation of the MO complex, an intermediate that requires the closed-ring form of Mcm2-7. We found that ORC phosphorylation fully inhibits MO-complex formation and provide evidence that this event is required for stable closing of the first Mcm2-7. Our studies show that multiple steps of helicase loading are impacted by ORC phosphorylation and reveal that closing of the first Mcm2-7 ring is a two-step process started by Cdt1 release and completed by MO-complex formation.
Each time a eukaryotic cell divides (by mitosis) it must duplicate its chromosomal DNA exactly once to ensure that one full copy is passed to each resulting cell. Both under-replication or over-replication result in genome instability and disease or cell death. A key mechanism to prevent over-replication is the temporal separation of loading of the replicative DNA helicase at origins of replication and activation of these same helicases during the cell division cycle. Here we define the mechanism by which phosphorylation of the primary DNA binding protein involved in these events inhibits helicase loading. Our studies identify multiple steps of inhibition and provide new insights into the mechanism of helicase loading in the uninhibited condition.
真核生物的DNA复制在每个细胞周期必须精确发生一次，以维持细胞倍性。这一结果通过在时间上分离复制性解旋酶的装载（G1期）和激活（S期）来确保。在芽殖酵母中，细胞周期蛋白依赖性激酶（CDK）对三种解旋酶装载蛋白（Cdc6、Mcm2-7解旋酶和起源识别复合物（ORC））的磷酸化作用可防止在G1期之外进行解旋酶装载。CDK对Cdc6和Mcm2-7的抑制作用已得到充分理解。在此，我们使用单分子分析来检测起源许可过程中的多个事件，以确定CDK对ORC的磷酸化如何抑制解旋酶装载。我们发现，磷酸化的ORC会将第一个Mcm2-7招募到起源位点，但会阻止第二个Mcm2-7的招募。Orc6亚基而非Orc2亚基的磷酸化会增加由于解旋酶及其相关的Cdt1解旋酶装载蛋白快速同时释放而导致的第一个Mcm2-7招募事件失败的比例。对第一个Mcm2-7环闭合的实时监测表明，Orc2或Orc6的磷酸化会阻止Mcm2-7稳定地环绕起源DNA。因此，我们评估了MO复合物的形成，MO复合物是一种需要Mcm2-7闭环形式的中间体。我们发现ORC磷酸化完全抑制MO复合物的形成，并提供证据表明这一事件是第一个Mcm2-7稳定闭合所必需的。我们的研究表明，ORC磷酸化会影响解旋酶装载的多个步骤，并揭示第一个Mcm2-7环的闭合是一个两步过程，由Cdt1释放启动，并由MO复合物形成完成。
每次真核细胞分裂（通过有丝分裂）时，它必须精确地复制其染色体DNA一次，以确保每个产生的细胞都能获得一份完整的拷贝。复制不足或过度复制都会导致基因组不稳定以及疾病或细胞死亡。防止过度复制的一个关键机制是在细胞分裂周期中，复制性DNA解旋酶在复制起点的装载与这些解旋酶的激活在时间上分离。在此，我们确定了参与这些事件的主要DNA结合蛋白的磷酸化抑制解旋酶装载的机制。我们的研究确定了多个抑制步骤，并为未受抑制条件下解旋酶装载的机制提供了新的见解。
