Mapping the 3D architecture of native human replisomes

绘制天然人类复制体的 3D 架构

• 批准号: 10461210
• 负责人: David M Gilbert
• 金额: $ 56.02万
• 依托单位: SAN DIEGO BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-02 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10461210
• 关键词: 3-Dimensional; Address; Architecture; Bar Codes; Cell Lineage; Cells; Chromatin; Chromatin Modeling; Chromosomes; Complement; Complex; Cytology; DNA; DNA Probes; DNA biosynthesis; DNA replication fork; DNA-Directed RNA Polymerase; Defect; Detection; Enhancers; Environment; Event; Fire - disasters; Frequencies; Genetic; Genetic Transcription; Genome; Genome Mappings; Genomic Instability; Goals; Health; Heterogeneity; Hi-C; Human; Human Chromosomes; Human Genome; Individual; Interphase; Kinetics; Knowledge; Label; Libraries; Ligation; Maps; Measurement; Measures; Methods; Mission; Modeling; Molecular; National Human Genome Research Institute; Nuclear; Optics; Outcome; Physiologic pulse; Population; Process; Public Health; RNA; Replication Initiation; Replication Origin; Replicon; Research; Resolution; S phase; Sister; Site; Source; Testing; Thinking; Time; Work; cell type; crosslink; genome integrity; human DNA; imaging approach; innovation; insight; novel; novel strategies; optical fiber; particle; preservation; promoter; recombinational repair; single molecule; spatiotemporal; stem cells; success; three dimensional structure; three-dimensional modeling; tool

ABSTRACT DNA replication is central to human genome integrity and is intimately tied to large-scale 3D genome architecture and cell lineage specification, yet we still do not have reliable maps of replicon organization nor any molecular tools to study how dismantling and re-assembly of 3D architecture is executed and coordinated with transcription. Our long-term goal is a complete understanding of the 3D choreography of replication over the course of S phase and its coordination with transcription. The overall objective of this application is to obtain direct measurements of replicon organization during S phase and model their 3D organization. Our central hypothesis is that replication initiation occurs stochastically at several (of many) potential origins that are in close 3D proximity at the time of initiation, after which forks remain in close proximity as chromatin transiently disengages from transcription and interphase 3D interactions. Our rationale is that high resolution single molecule 3D maps of nascent DNA will uncover novel mechanistic insights into how replication is faithfully executed and coordinated with transcription. AIM1 will develop a transformative single DNA fiber optical replication mapping (ORM) method, permitting us to map origins and fork polarities on single molecules with unprecedented throughput (30Gb/hr). We will integrate these maps with high resolution Repli-seq and Hi-C maps to reveal how replicons are organized in time and space. To model the native 3D structure of individual replisomes, we will develop replication fork-enriched versions of single-particle SPRITE (split pool recognition of interactions by tag extension), Hi-C and single cell Hi-C. SPRITE enables detection of multiple simultaneously occurring DNA and RNA interactions within cross- linked and individually bar-coded large chromatin complexes. In AIM2, we will capture complexes containing pulse-labeled nascent DNA (Repli-SPRITE) to assess 3D association of DNA and RNA, including nascent RNA, with active replication forks (i.e. replisomes). In AIM3, we will map DNA in close proximity to active replication forks by capturing pulse-labeled nascent DNA from Hi-C libraries (Repli-Hi-C). Population Repli-Hi-C will provide a high resolution global view of how contacts differ as replication forks pass through domains, while single cell Repli-Hi-C will enable 3D models of how multiple replicons are organized within domains and how replication is temporally coordinated across the genome in each cell. Importantly, AIMs 2 and 3 will also chase the labeled DNA before capture to track the dynamic re-assembly of interphase 3D structures. We expect to deliver an unprecedented view of how the human genome is organized for DNA replication and how replication is coordinated with 3D architecture and transcription. This contribution will be significant because it will deepen our understanding of how DNA replication is orchestrated to preserve genome integrity and cell-type specific chromatin architectures. The proposed research is innovative because it will disrupt paradigms in genome research and DNA replication, and open new horizons by developing methods to model 3D organization of any process involving DNA synthesis (e.g. replication, recombination, repair, chromatin assembly).

抽象的 DNA复制是人类基因组完整性的核心，与大规模3D基因组结构密切相关 和细胞谱系规范，但是我们仍然没有可靠的复制品组织图或任何分子 研究3D体系结构的拆卸和重新组装的工具如何通过转录执行和协调。 我们的长期目标是对S阶段的3D复制编排的完全理解 及其与转录的协调。该应用程序的总体目的是获得直接测量 在阶段的复制组织组织和建模其3D组织。我们的中心假设是复制 启动在几个（许多）潜在的起源中随机发生，在几个（许多）潜在的起源中，在3D 启动，之后，由于染色质瞬时脱离转录和 相间3D相互作用。我们的理由是，高分辨率的单分子3D新生DNA将会 发现了新的机械性洞察力，即如何忠实地执行和与转录协调复制。 AIM1将开发一个变换性的单DNA光纤复制映射（ORM）方法，使我们能够 具有前所未有的吞吐量（30GB/hr）的单分子上的地图起源和叉子极性。我们将整合 这些具有高分辨率Replosi-seq和Hi-C地图的地图，以揭示复制子的及时组织方式和 空间。为了建模单个复制的天然3D结构，我们将开发复制叉富含 单粒子精灵的版本（按标签扩展对相互作用的分裂识别），HI-C和单个单元格 嗝。 Sprite可以在交叉中同时检测多个发生的DNA和RNA相互作用 链接和单独编码的大染色质复合物。在AIM2中，我们将捕获包含的复合物 脉冲标记的新生DNA（repli-sprite）评估DNA和RNA的3D关联，包括新生RNA， 带有主动复制叉（即重新分析）。在AIM3中，我们将密切映射DNA，靠近主动复制 通过从HI-C库（Repli-Hi-C）捕获脉冲标记的新生DNA来分叉。人口repli-hi-c将提供 高分辨率的全球视图，即触点如何随着复制叉通过域而有所不同，而单个单元格 repli-hi-c将启用3D模型，以了解多个复制子在域内的组织方式以及复制的方式 在每个细胞中的基因组上进行时间协调。重要的是，目标2和3也将追逐标签 捕获前的DNA跟踪相间3D结构的动态重新组装。我们希望提供 关于人类基因组如何组织DNA复制以及复制方式的前所未有的观点 与3D体系结构和转录协调。这项贡献将是重要的，因为它将加深我们 了解如何策划DNA复制以保持基因组完整性和细胞类型特异性 染色质体系结构。拟议的研究具有创新性，因为它会破坏基因组的范例 研究和DNA复制，并通过开发方法来建模任何方法 涉及DNA合成的过程（例如复制，重组，修复，染色质组装）。