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

通过本质无序结构域探索功能组装中 DNA 复制蛋白的研究

• 批准号: 10579065
• 负责人: JAMES M BERGER
• 金额: $ 5.61万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10579065
• 关键词: ATP phosphohydrolase; Area; Automobile Driving; Award; Cells; Chromatin; Chromosomes; Complex; DNA; DNA Binding; DNA biosynthesis; Defect; Development; Disease; Dwarfism; Elements; Genetic; Genome; Goals; Heterochromatin; Lead; Liquid substance; Maintenance; Malignant Neoplasms; Modeling; Molecular; Molecular Biology; Nucleic Acids; ORC1L gene; Outcome; Parents; Peptide Initiation Factors; Phase; Physical condensation; Prokaryotic Initiation Factor-3; Proteins; Proteome; Replication Initiation; Replication Origin; Role; S phase; Specific qualifier value; Specificity; Structure; Testing; Work; flexibility; helicase; human disease; insight; melting; novel; protein protein interaction

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.

项目摘要（与家长奖没有变化） 我们的长期目标是确定 DNA 复制启动和调节的分子机制 后生动物。细胞依靠两种 AAA+ ATP 酶 ORC 和 Cdc6 以及第三个因子 Cdt1 来加载潜在的 解旋酶（Mcm2-7 复合物）作为双六聚体到达复制起点。进入S期后，Mcm2-7 被 GINS 和 Cdc45 辅助因子激活，熔化双链体起源。由此产生的CMG (Cdc45/Mcm2-7/GINS) 组装解开亲代 DNA 链并协调 DNA 合成 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 亚基，如 以及 Cdc6 和 Cdt1 – 使用长的、本质上无序的区域 (IDR) 来结合 DNA 并分配到液体中 相凝聚体（LPC）。这一观察结果和其他观察结果使我们得出了一种新的复制功能模型，其中 引发剂 IDR 和 LPC 倾向不仅控制染色质关联，还控制 Mcm2-7 负载、伙伴 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 有助于冷凝。在目标 2 中，我们将定义引发剂 IDR 如何控制伴侣-蛋白质相互作用。在 目标 3，我们将确定 Orc1 如何利用其 IDR 通过相互作用与中心周异染色质结合 与其他 LPC 形成蛋白（例如 Hp1）。拟议工作预计将产生重大成果 包括：1) 定义引发剂 IDR——我们已经证明这是一类新型的凝析油促进剂 元素 – 与 DNA 以及彼此之间的界面，2) 发现能够与起始相关的新蛋白质 因素，以及 3) 解释 ORC 如何与基因组组织的形成和维护联系起来 表达。 IDR 主要被认为可以作为灵活的连接器，允许有序之间的移动性 结构域，或作为通过蛋白质-蛋白质经历诱导拟合转变为折叠结构的片段 互动。最近的工作表明，IDR 可以在指定伴侣蛋白相互作用和 通过液相分离驱动无膜室的形成。我们的建议将 确定 IDR 如何导致与起源和潜在的共关联和潜在划分的特异性 其他因素。我们的努力将为分子生物学领域提供信息，在这些领域中，IDR 用于表现相分离 用于功能目的的区室或蛋白质/核酸簇。预计约 25% 的蛋白质组 由于是非结构化的，因此此类见解的效用将具有广泛的意义。