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: 10576326
• 负责人: JAMES M BERGER
• 金额: $ 53.78万
• 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/10576326
• 关键词: ATP phosphohydrolase; Address; Area; Automobile Driving; Behavior; Binding; Biochemical; Biochemistry; Biology; Biophysical Process; Cell Cycle Regulation; Cell Proliferation; Cells; Chromatin; Chromosome Structures; Chromosomes; Complex; DNA; DNA Binding; DNA biosynthesis; Data; Defect; Development; Disease; Dwarfism; Elements; Eukaryota; Event; Exclusion; Gene Expression; Gene Silencing; Genetic; Genome; Goals; Heterochromatin; Image; In Vitro; Intervention; Knowledge; Laboratory Finding; Lead; Liquid substance; Maintenance; Malignant Neoplasms; Membrane; Modeling; Molecular; Molecular Biology; Mutagenesis; Nucleic Acids; ORC1L gene; Outcome; Pathology; Pathway interactions; Peptide Initiation Factors; Phase; Physical condensation; Probability; Prokaryotic Initiation Factor-3; Property; Proteins; Proteome; Reagent; Replication Initiation; Replication Origin; Role; S phase; Signal Transduction; Sorting; Specific qualifier value; Specificity; Structure; System; Testing; Work; cellular imaging; experience; flexibility; helicase; human disease; imaging approach; in vivo; insight; interest; melting; novel; origin recognition complex; programs; prospective; protein protein interaction; recruit; structural determinants; tumorigenesis

PROJECT SUMMARY 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 我们的长期目标是定义启动和调节DNA复制的分子机制 后生动物。单元格依靠两个AAA+ ATPases，ORC和CDC6以及第三个因素CDT1加载潜在 解旋酶（MCM2-7复合物）作为复制起源的双重六聚体。进入S期后，MCM2-7 被杜松子和CDC45附件因子激活，从而融化双工来源。最终的CMG （CDC45/MCM2-7/杜松子 复制体。 最近，我们发现后生复制倡议因素 - 特别是ORC的ORC1亚基，如 以及CDC6和CDT1 - 使用长，本质上无序的区域（IDR）结合DNA并分成液体 相冷凝（LPC）。这个和其他观察结果使我们建立了一个新的功能模型，以进行复制 引发剂IDR和LPC倾向控制不仅染色质关联，还可以加载MCM2-7。 选择和异染色质状态。在AIM 1中，我们将解决启动器的分子确定词 IDRS最喜欢的冷凝。在AIM 2中，我们将定义启动器IDRS如何控制伴侣 - 蛋白质相互作用。在 AIM 3，我们将建立ORC1如何使用其IDR通过相互作用与洋关染色质接口 与其他LPC形成的蛋白（如HP1）。预计拟议工作的重大结果 包括：1）定义启动器IDR-我们已经证明是新颖的凝结物促进 元素 - 与DNA和彼此的接口，2）发现能够与启动相关的新蛋白 因素，以及3）解释ORC如何与基因组组织的形成和维护以及 表达。 IDR被认为是允许在有序之间移动的灵活的链接器 域，或作为通过蛋白质蛋白进行诱导拟合过渡到折叠结构的段 互动。最近的工作表明，IDR可以在指定合作伙伴 - 蛋白质互动和 通过液相分离来驱动膜隔室的形成。我们的建议将 确定IDR如何导致与起源和潜在隔离的特异性 其他因素。我们的努力将为分子生物学领域提供信息，以表现出相位分离 用于功能目的的隔室或蛋白质/核酸聚类。由于预计约有25％的蛋白质 保持非结构化，这种见解的实用性将是广泛的。