Studies to Explore DNA Replication Proteins in Functional Assemblies through Intrinsically Disordered Domains

项目总结(与父级奖励不变) 我们的长期目标是确定启动和调节DNA复制的分子机制 后生动物。细胞依赖于两个AAA+ATPase ORC和CDC6，以及第三个因子CDT1来加载潜伏的 解旋酶(McM2-7复合体)作为复制起点上的双六聚体。进入S阶段后，McM2-7 是由GINS和CDC45辅助因子激活的，融化了双链起源。由此产生的CMG (CDC45/McM2-7/GINS)组装解开亲本DNA链并通过 复制体。 最近，我们发现后生动物复制起始因子--特别是ORC的ORC1亚单位，AS 以及cdc6和cdt1-使用长的固有无序区(IDR)结合DNA并将其分配到液体中 相冷凝物(LPCS)。这一观察和其他观察使我们得到了一个新的复制功能模型，由此 启动子IDR和LPC倾向不仅控制染色质的关联，还控制McM2-7的负载，伙伴 选择和异染色质状态。在目标1中，我们将解析引发剂所依据的分子决定因素 IDR有助于凝结。在目标2中，我们将定义启动器IDR如何控制伙伴-蛋白质相互作用。在……里面 目标3，我们将建立ORC1如何利用其IDR通过相互作用与着丝粒周围异染色质相互作用 与其他LPC形成蛋白如HP1结合。预计拟议工作将产生重大成果 包括：1)定义引发剂IDR--我们已经证明它是一种新型的凝析油促进剂 元素与DNA的相互作用，2)发现与启动相关的新蛋白质 因素，以及3)解释ORC如何与基因组组织的形成和维持以及 表情。 IDR主要被认为是作为灵活的链接器，允许有序之间的流动性 结构域，或作为通过蛋白质-蛋白质经历诱导-适配过渡到折叠结构的片段 互动。最近的研究表明，IDR可以发挥另一个作用，即指定伙伴-蛋白质相互作用和 通过液相分离驱动无膜室的形成。我们的建议将 确定IDR如何能够导致与来源和来源的联合联系和潜在划分的特殊性 其他因素。我们的努力将向分子生物学领域提供使用IDR来显示相分离的信息 用于功能目的的隔室或蛋白质/核酸聚集物。据预测，约25%的蛋白质组会 由于缺乏结构化，这种洞察力的效用将广泛意义重大。

PROJECT SUMMARY (No Change from Parent Award) Our long-term goal is to define the molecular mechanisms by which DNA replication is initiated and regulated in metazoans. Cells rely on two AAA+ ATPases, ORC and Cdc6, along with a third factor, Cdt1, to load a latent helicase (the Mcm2-7 complex) as a double hexamer onto replication origins. Upon entering S-phase, Mcm2-7 is activated by the GINS and Cdc45 accessory factors, melting the duplex origin. The resultant CMG (Cdc45/Mcm2-7/GINS) assembly unwinds parental DNA strands and coordinates DNA synthesis by the replisome. Recently, we discovered that metazoan replication initiation factors – specifically the Orc1 subunit of ORC, as well as Cdc6 and Cdt1 – use long, intrinsically disordered regions (IDRs) to bind DNA and partition into liquid phase condensates (LPCs). This and other observations led us to a new functional model for replication, whereby initiator IDRs and LPC propensity controls not only chromatin association, but also Mcm2-7 loading, partner selection, and heterochromatin status. In Aim 1, we will resolve the molecular determinants by which initiator IDRs facilitate condensation. In Aim 2, we will define how initiator IDRs control partner-protein interactions. In Aim 3, we will establish how Orc1 uses its IDR to interface with pericentric heterochromatin through interactions with other LPC-forming proteins such as Hp1. Significant outcomes expected to result from the proposed work include: 1) defining how initiator IDRs – which we have shown to be a novel class of condensate-promoting element – interface with DNA and each other, 2) uncovering new proteins capable of associating with initiation factors, and 3) explaining how ORC connects to the formation and maintenance of genome organization and expression. IDRs have been predominantly thought to serve either as flexible linkers that allow mobility between ordered domains, or as segments that undergo an induced-fit transition into folded structures through protein-protein interactions. Recent work shows that IDRs can fulfill another role in specifying partner-protein interactions and driving the formation of membraneless compartments through liquid phase separation. Our proposal will establish how IDRs can lead to specificity for co-association and potential compartmentalization with origins and other factors. Our efforts will inform areas of molecular biology where IDRs are used to manifest phase-separated compartments or protein/nucleic-acid clustering for functional purposes. As ~25% of proteomes are predicted to be unstructured, the utility of such insights will be broadly significant.