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

项目概要（与母公司奖相同） 我们的长期目标是确定DNA复制启动和调控的分子机制， 后生动物。细胞依赖于两种AAA+ ATP酶ORC和Cdc 6，沿着第三种因子Cdt 1， 解旋酶（Mcm 2 -7复合物）作为双六聚体连接到复制起点上。进入S相后，Mcm 2 -7 由GINS和Cdc 45辅助因子激活，使双链体起源熔化。生成的CMG （Cdc 45/Mcm 2 -7/GINS）组装解开亲本DNA链并协调DNA合成。 复制体 最近，我们发现后生动物的复制起始因子-特别是ORC的Orc 1亚基， 以及Cdc 6和Cdt 1-使用长的内在无序区（IDR）结合DNA并分配到液体中 相冷凝物（LPC）。这一点和其他观察使我们产生了一种新的复制功能模型， 启动子IDR和LPC倾向不仅控制染色质缔合，而且控制Mcm 2 -7加载、配偶体 选择和异染色质状态。在目标1中，我们将解决引发剂的分子决定因素 IDR有助于浓缩。在目标2中，我们将定义起始IDRs如何控制伴侣-蛋白质相互作用。在 目的3，我们将建立Orc 1如何使用其IDR通过相互作用与臂间异染色质相互作用 与其他LPC形成蛋白如Hp 1。预期拟议工作将产生的重大成果 包括：1）定义引发剂IDR--我们已经证明是一类新的促进缩合物的 元件-与DNA和彼此的界面，2）发现能够与起始相关联的新蛋白质 因素，以及3）解释ORC如何连接到基因组组织的形成和维持， 表情 IDR主要被认为是作为柔性接头，允许有序连接之间的流动性， 结构域，或作为通过蛋白质-蛋白质间的诱导配合转变为折叠结构的片段 交互.最近的工作表明，IDR可以在指定伴侣蛋白质相互作用中发挥另一个作用， 通过液相分离驱动无膜室的形成。我们的建议将 确定IDR如何导致与起源的共同关联和潜在区室化的特异性， 其他因素我们的努力将告知分子生物学领域，其中IDR用于显示相分离 区室或蛋白质/核酸簇用于功能目的。据预测，约25%的蛋白质组 如果没有结构化，这种见解的效用将具有广泛的意义。

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.