Super-resolution visualization of distinct stalled and broken replication fork structures.
Super-resolution visualization of distinct stalled and broken replication fork structures.
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DOI：
10.1371/journal.pgen.1009256
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发表时间：
2020-12
期刊：
影响因子：
4.5
通讯作者：
Rothenberg E
中科院分区：
文献类型：
--
作者：
Whelan DR;Lee WTC;Marks F;Kong YT;Yin Y;Rothenberg E
由于 DNA 复制机制、转录和拓扑松弛过程之间的竞争，健康细胞中会发生内源性基因毒性应激。这会导致复制叉停滞和回归，从而进一步崩溃形成单端双链断裂 (seDSB)。超分辨率显微镜可以直接观察细胞内的复制应力和 DNA 损伤，但需要新的样品制备和分析方法。在这里，我们开发并应用多色单分子显微镜来观察拓扑异构酶 I 裂解复合物捕获产生的轻微应激下的个体复制叉，这是一种与内源性复制应激非常相似的损伤诱导。我们观察到 RAD51 和 RAD52 以及 RECQ1 是对停滞但未破坏的分叉的第一个响应蛋白，而 Ku 和 MRE11 最初被招募到 seDSB。通过实施新颖的超分辨率成像测定，我们能够辨别密切相关的复制叉应激基序及其修复途径。生物体 DNA 中的遗传密码受损可能会导致突变或细胞死亡，进而导致疾病和功能障碍。 DNA 损伤是许多人类疾病的主要原因，包括癌症、某些形式的神经退行性疾病和免疫功能障碍。 DNA 双链断裂 (DSB) 在每个复制细胞中每天都会发生几次，由于修复困难，其危害尤其严重。 DSB 的主要内源性原因是 DNA 复制叉的破坏，但越来越多的证据表明，这些复制叉的损伤和压力也可能导致中间结构未破坏，从而避免 DSB 形成，例如复制叉回归。我们开发并应用了新的检测方法，使用超分辨率显微镜来标记和可视化受损的复制叉。这使我们能够区分破损和未破损的叉，并辨别为 DSB 和回归叉修复而招募的不同蛋白质。我们的数据进一步表明，这些测定方法广泛适用于 DNA 损伤研究，并提供了一种绘制细胞内个体损伤事件时空修复图谱的新方法。
Endogenous genotoxic stress occurs in healthy cells due to competition between DNA replication machinery, and transcription and topographic relaxation processes. This causes replication fork stalling and regression, which can further collapse to form single-ended double strand breaks (seDSBs). Super-resolution microscopy has made it possible to directly observe replication stress and DNA damage inside cells, however new approaches to sample preparation and analysis are required. Here we develop and apply multicolor single molecule microscopy to visualize individual replication forks under mild stress from the trapping of Topoisomerase I cleavage complexes, a damage induction which closely mimics endogenous replicative stress. We observe RAD51 and RAD52, alongside RECQ1, as the first responder proteins to stalled but unbroken forks, whereas Ku and MRE11 are initially recruited to seDSBs. By implementing novel super-resolution imaging assays, we are thus able to discern closely related replication fork stress motifs and their repair pathways. Damage to the genetic code embedded in an organism’s DNA can result in mutation or cell death which, in turn, can lead to disease and dysfunction. DNA damage is the main cause of many human diseases including cancer, and some forms of neurodegeneration and immune dysfunction. DNA double strand breaks (DSBs), which occur a handful of times in each replicating cell each day, are especially deleterious due to the difficulty of their repair. The main endogenous cause of DSBs is the breakdown of DNA replication forks however there is increasing evidence that damage and stress at these forks can also result in unbroken intermediate structures which avoid DSB formation such as fork regression. We have developed and applied new assays for labelling and visualizing damaged replication forks using super-resolution microscopy. This has enabled us to differentiate between broken and unbroken forks and to discern the different proteins that are recruited for DSB and regressed fork repair. Our data further demonstrate that these assays are widely applicable to DNA damage research and offer a new approach to mapping the spatiotemporal repair of individual damage events inside cells.
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作者：
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通讯作者：
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Berti, Matteo;Chaudhuri, Arnab Ray;Thangavel, Saravanabhavan;Gomathinayagam, Shivasankari;Kenig, Sasa;Vujanovic, Marko;Odreman, Federico;Glatter, Timo;Graziano, Simona;Mendoza-Maldonado, Ramiro;Marino, Francesca;Lucic, Bojana;Biasin, Valentina;Gstaiger, Matthias;Aebersold, Ruedi;Sidorova, Julia M.;Monnat, Raymond J., Jr.;Lopes, Massimo;Vindigni, Alessandro
通讯作者：
Vindigni, Alessandro