Mapping the 3D architecture of native human replisomes

摘要 DNA复制是人类基因组完整性的核心，并与大规模3D基因组体系结构密切相关 和细胞谱系特征，但我们仍然没有可靠的复制子组织图谱或任何分子 研究如何执行3D建筑的拆卸和重新组装以及如何与转录协调的工具。 我们的长期目标是在S阶段的过程中完全理解复制的3D编排 以及它与转录的协调性。此应用程序的总体目标是获得直接测量 S阶段的复制子组织，并对其3D组织进行建模。我们的中心假设是复制 起始随机地发生在几个(许多)潜在的起始点上，这些起始点在时间上非常接近3D 开始，之后叉子保持在紧密的接近，因为染色质暂时脱离转录和 相间3D相互作用。我们的理论基础是，新生DNA的高分辨率单分子3D地图将 揭示复制是如何忠实地执行并与转录协调的新的机械洞察力。 AIM1将开发一种变革性的单DNA光纤复制映射(ORM)方法，允许我们 以前所未有的吞吐量(30 GB/小时)映射单个分子上的起源和分叉极性。我们将整合 这些地图带有高分辨率的Repli-Seq和Hi-C地图，以揭示复制品是如何在时间和 太空。为了模拟个体复制体的天然3D结构，我们将开发富含复制叉的 单粒子Sprite(通过标签扩展对交互进行拆分池识别)、Hi-C和Single Cell的版本 橘子汽水。SPRITE使人们能够检测到多个同时发生的DNA和RNA相互作用 连接的和单独条码的大型染色质复合体。在AIM2中，我们将捕获包含以下内容的复合体 脉冲标记新生DNA(Repli-Sprite)以评估DNA和RNA的3D关联，包括新生RNA， 具有活跃的复制分叉(即复制体)。在AIM3中，我们将绘制非常接近活跃复制的DNA图谱 通过从Hi-C文库(Repli-Hi-C)捕获脉冲标记的新生DNA进行分叉。人口回复-Hi-C将提供 当复制分叉通过域时，高分辨率全局视图显示联系人如何不同，而单个细胞 Repli-Hi-C将启用如何在域中组织多个副本以及如何进行复制的3D模型 在每个细胞的基因组中进行时间协调。重要的是，目标2和3也将追逐被标记的 DNA在捕获前跟踪相间3D结构的动态重组。我们预计将交付一份 对人类基因组如何组织以进行DNA复制以及复制是如何进行的前所未有的看法 与3D架构和转录相协调。这一贡献将是重大的，因为它将深化我们的 了解如何协调DNA复制以保持基因组完整性和细胞类型特异性 染色质结构。这项拟议的研究具有创新性，因为它将扰乱基因组的范式 研究和DNA复制，并通过开发方法对任何组织的3D组织进行建模来开辟新的视野 涉及DNA合成的过程(例如复制、重组、修复、染色质组装)。

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).