Quantitative analysis of nuclear deformations and DNA damage foci dynamics by live-cell imaging.

4区生物学 Q4 Biochemistry, Genetics and Molecular Biology

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2023-01-09 DOI:10.1016/bs.mcb.2022.12.010

Elena Faustini, Andrea Panza, Matteo Longaretti, Francisca Lottersberger

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引用次数: 0

Abstract

The correct repair of DNA Double Strand Breaks (DSBs) is fundamental to prevent the loss of genetic information, mutations, and chromosome rearrangements. An emerging determinant of DNA repair is chromatin mobility. However, how chromatin mobility can influence DSBs repair is still poorly understood. While increased mobility is generally associated with the correct repair by Homologous Recombination (HR) of DSBs generated in heterochromatin, it promotes the mis-repair of multiple distal DSBs by Non-Homologous End Joining (NHEJ). Here we describe a method for detecting and quantifying DSBs mobility by live-cell imaging in the context of multiple DSBs prone to mis-repair by NHEJ. In addition, we discuss a set of parameters that can be used for quantitative and qualitative analysis of nuclear deformations and to discard nuclei where the deformation could affect the analysis of DSBs mobility. While this method is based on the visualization of DSBs with the mCherry-53BP1-2 fusion protein, we believe that it can also be used to analyze the mobility of nuclear foci formed by different fluorescent proteins.

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通过活细胞成像对核变形和 DNA 损伤灶动态进行定量分析。

DNA 双链断裂（DSB）的正确修复是防止遗传信息丢失、突变和染色体重排的基础。染色质流动性是 DNA 修复的一个新兴决定因素。然而，人们对染色质流动性如何影响DSB修复仍知之甚少。虽然染色质流动性的增加通常与同源重组（HR）对异染色质中产生的DSB的正确修复有关，但它会促进非同源末端连接（NHEJ）对多个远端DSB的错误修复。在此，我们介绍了一种通过活细胞成像检测和量化易被 NHEJ 误修复的多个 DSB 移动性的方法。此外，我们还讨论了一组参数，这些参数可用于核变形的定量和定性分析，并舍弃变形可能影响 DSB 移动性分析的核。虽然这种方法是基于mCherry-53BP1-2融合蛋白对DSB的可视化，但我们认为它也可用于分析不同荧光蛋白形成的核病灶的流动性。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Methods in cell biology 生物-细胞生物学

CiteScore

3.10

自引率

0.00%

发文量

125

审稿时长

3 months

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.