Sophie T B Morgan, Donna R Whelan, Ashley M Rozario
{"title":"Visualizing DNA damage and repair using single molecule super resolution microscopy.","authors":"Sophie T B Morgan, Donna R Whelan, Ashley M Rozario","doi":"10.1016/bs.mcb.2023.02.004","DOIUrl":null,"url":null,"abstract":"<p><p>Single molecule super resolution microscopy overcomes the diffraction limit by separating individual fluorophore emissions over time, resulting in spatial resolutions that are far superior to epifluorescence microscopy. This allows for DNA damage response (DDR) events to be investigated in greater detail. A variety of DNA damaging drugs can be used on S-phase synchronized immortalized cell lines alongside 5-ethynyl-2'-deoxyuridine (EdU) pulse labelling to ultimately visualize DNA repair pathways at distinct time points and quantify colocalizations between nascent DNA and immunolabeled DDR proteins. This chapter will outline super resolution microscopy assays to interrogate the spatiotemporal organization of DNA repair proteins at damaged foci during DDR events within immortalized cell lines.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Methods in cell biology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1016/bs.mcb.2023.02.004","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2023/8/21 0:00:00","PubModel":"Epub","JCR":"Q4","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
引用次数: 0
Abstract
Single molecule super resolution microscopy overcomes the diffraction limit by separating individual fluorophore emissions over time, resulting in spatial resolutions that are far superior to epifluorescence microscopy. This allows for DNA damage response (DDR) events to be investigated in greater detail. A variety of DNA damaging drugs can be used on S-phase synchronized immortalized cell lines alongside 5-ethynyl-2'-deoxyuridine (EdU) pulse labelling to ultimately visualize DNA repair pathways at distinct time points and quantify colocalizations between nascent DNA and immunolabeled DDR proteins. This chapter will outline super resolution microscopy assays to interrogate the spatiotemporal organization of DNA repair proteins at damaged foci during DDR events within immortalized cell lines.
单分子超分辨显微镜通过分离单个荧光团的发射时间，克服了衍射极限，其空间分辨率远远高于外荧光显微镜。这样就能更详细地研究 DNA 损伤反应（DDR）事件。在 S 期同步永生化细胞系上使用各种 DNA 损伤药物，同时进行 5- 乙炔基-2'-脱氧尿苷（EdU）脉冲标记，最终可观察不同时间点的 DNA 修复途径，并量化新生 DNA 与免疫标记的 DDR 蛋白之间的共定位。本章将概述超分辨显微镜检测方法，以探究永生化细胞系中发生 DDR 事件期间 DNA 修复蛋白在受损病灶上的时空组织。
期刊介绍：
For over fifty years, Methods in Cell Biology has helped researchers answer the question "What method should I use to study this cell biology problem?" Edited by leaders in the field, each thematic volume provides proven, state-of-art techniques, along with relevant historical background and theory, to aid researchers in efficient design and effective implementation of experimental methodologies. Over its many years of publication, Methods in Cell Biology has built up a deep library of biological methods to study model developmental organisms, organelles and cell systems, as well as comprehensive coverage of microscopy and other analytical approaches.
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