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Revealing the mechanism of replication initialization with a sequential microscopy-based approach

使用基于顺序显微镜的方法揭示复制初始化的机制

基本信息

批准号：
431471305
负责人：
Dr. Markus Götz
金额：
--
依托单位：
Centre de Biochimie Structurale
依托单位国家：
德国
项目类别：
Research Fellowships
财政年份：
2019
资助国家：
德国
起止时间：
2018-12-31 至 2020-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/431471305?language=en
关键词：
Revealing mechanism replication initialization sequential

项目摘要

Before cell division, each mitotic cell has to create an exact duplicate of its DNA to ensure genome stability and the faithful transmission of the full genetic information. Incomplete or excessive DNA replication could otherwise severely diminish the viability of the daughter cells. In eukaryotes, DNA replication takes place at specific nuclear locations during distinct time intervals. This regulation is linked to the chromatin conformation, but the exact mechanisms are yet poorly understood.Replication of eukaryotic genomes is initiated at thousands of genomic segments in parallel, so-called origins of replication. In a single cell, only a subset of these origins is used while all origins can initiate replication when looking at a population of cells. As a consequence, a large cell-to-cell variability in the origin usage exists and a study of the mechanisms controlling replication initiation would considerably profit from the use of single-cell techniques. Interestingly, despite this flexibility, replication domains emerge as 0.5-1 Mb regions of DNA duplicated at the same time and spanning several active origins. This synchronicity implies mechanisms to simultaneously activate several origins. The prevalent model predicts that the underlying chromatin conformation brings origins into spatial proximity which results in their synchronous activation. However, directly testing this model so far was not possible due to technological limitations.Here, I propose to use a super-resolution microscope (in particular 3D-STED) together with Hi‑M, a method recently developed by the host lab that permits the sequence-specific, sequential labeling and imaging of all potential origins in a replication domain (~20 in a 150-500 nm sized cluster). This unique approach will allow us to determine the position of the origins with nanometric resolution and to correlate their distribution with the overall chromatin conformation in a replication domain in mouse cells. The proposed project will permit a direct observation of the spatial organization of all potential origins in a replication domain, while origin activation takes place. Gaining a deeper insight into the role of chromatin conformation on origin organization will help to better understand the regulation of genome replication, a vital process for life.

在细胞分裂之前，每个有丝分裂细胞必须创建其DNA的精确副本，以确保基因组稳定性和完整遗传信息的忠实传输。不完全或过度的DNA复制可能会严重降低子细胞的活力。在真核生物中，DNA复制在不同的时间间隔内发生在特定的核位置。这种调控与染色质构象有关，但确切的机制尚不清楚。真核生物基因组的复制起始于数千个平行的基因组片段，即所谓的复制起点。在单个细胞中，只有这些起点的一个子集被使用，而当观察细胞群体时，所有起点都可以启动复制。因此，一个大的细胞到细胞的变异性的起源使用存在和控制复制起始的机制的研究将大大受益于使用单细胞技术。有趣的是，尽管有这种灵活性，复制结构域作为同时复制的DNA的0.5-1 Mb区域出现，并跨越几个活性起点。这种同步性意味着同时激活几个起源的机制。流行的模型预测，潜在的染色质构象带来的起源空间接近，导致它们的同步激活。然而，由于技术限制，目前还无法直接测试该模型。在这里，我建议使用超分辨率显微镜（特别是3D-STED）和Hi-M，Hi-M是宿主实验室最近开发的一种方法，可以对复制结构域中的所有潜在起点（150-500 nm大小的簇中约20个）进行序列特异性、顺序标记和成像。这种独特的方法将使我们能够以纳米分辨率确定起点的位置，并将它们的分布与小鼠细胞中复制结构域中的整体染色质构象相关联。拟议的项目将允许直接观察复制域中所有潜在原点的空间组织，而原点激活发生。深入了解染色质构象在起源组织中的作用将有助于更好地理解基因组复制的调控，这是生命的重要过程。