这项工作得到了捷克共和国的赠款机构 P302-12-G157 项目的支持。由挪威基金监督的捷克-挪威研究方案 CZ09 和捷克共和国教育、青年和体育部 (赠款号: 7F14369) 也支持实验。

<ol>
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作者声明没有利益冲突。

显微镜技术代表了研究实验室的基本工具。在这里, 简要介绍了用于研究蛋白质的招募和动力学在 DNA 损伤的方法。我们特别注意到我们的实验经验, 在当地 microirradiation 的活细胞, 我们讨论的蛋白质动力学的 FRAP 和蛋白质相互作用的 DNA 病变受体漂白烦恼28及其高级修改烦恼-电影 (图 4A-d)。这里所示的方法是真正了解 DNA 修复过程的基本工具, 特?...

dna 损伤导致 dna 病变的出现, 包括丁烷嘧啶聚 (方案), 8-氧-78-氢-2 "-脱氧, 和单或双打破1,2。γ射线是以最高的能量和高显的电离辐射的形式, 因而辐射的来源在放射疗法被广泛使用3。另一方面, 实验性的紫外线激光引起的 DNA 损伤模仿自然暴露于紫外线。UVA microirradiation, 作为一种显微方法, 代表了一个实验工具, 研究 DNA 损伤的个体活细胞。Microirradiation 40 年前第一次使用, 以揭示染色体区域的组织4,5。该技术高度依赖于共焦显微镜的功能特性或现代 nanoscopy 的技术极限。为了诱导 DNA 损伤, 细胞可以 presensitized 5 的尿 (BrdU) 或赫斯特33342之前, 紫外线照射。Bártová et al.6以前描述了 presensitization 步骤, 最近我们优化了这种 microirradiation 技术, 以避免细胞死亡或凋亡。例如, 使用 405 nm 的 UV 激光器 (不含赫斯特 33342 presensitization) 会导致53BP1 阳性双断裂 (DSBs), 其代价是丁烷嘧啶聚。另一方面, presensitization 步骤与 UV microirradiation 相结合, 会导致高水平的方案和 DSBs 同时7,8。这种方法很难应用于单 DNA 修复通路的研究。
有了 microirradiation, 就有可能分析蛋白质的招募, 动力学, 和相互作用的 DNA 损伤活细胞。Luijsterburg et al.发布了此方法的一个示例9用于异蛋白 1β, 我们最近首次显示多因子 Oct4 和与卡哈尔体 coilin 相关的蛋白质, 被吸收到紫外线诱导的 DNA 损伤6,10。蛋白质动力学在这些脱氧核糖核酸损伤也可以研究使用 FRAP (荧光恢复在漂白以后)11,12,13或烦恼 (荧光共振能量转移) 技术14 ,15。这些方法有可能揭示蛋白质在 DNA 损伤或蛋白质-蛋白质相互作用中的简单扩散。一个有用的工具, 额外的表征蛋白质是电影 (荧光寿命成像显微镜) 或它的组合与烦恼技术 (烦恼-电影)16。这些方法能够研究在稳定或瞬时表达荧光分子标记的蛋白质的活细胞中的过程17。这里, 一个指数衰减时间 (τ) 的例子, GFP 标记的 p53 蛋白及其相互作用的伙伴, mCherry 标记 53BP1, 发挥重要作用的 DNA 损伤的响应18,19显示。参数τ, 电影计算提供的荧光的寿命, 是特定的荧光染料, 其结合能力, 及其细胞环境。因此, 这种方法可以显示我们之间的蛋白质亚群之间的区别, 他们的结合能力, 和他们的功能性质后, 例如, DNA 损伤。
这里, 在我们的实验室中使用的先进的显微技术的方法学方法的概要研究在 microirradiated 染色质部位的具体的蛋白质的招募、动力学、扩散和蛋白质-蛋白质相互作用提出。本发明提供了在活细胞中诱导局部 dna 损伤的 step-by 步骤方法, 并介绍了用于研究紫外线激光引起的局部 dna 损伤相关事件的方法。

<table><tbody><tr><td>&lt;strong&gt;Cell cultivation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>HeLa</td><td>ATCC</td><td>CCL-2TM</td><td></td></tr><tr><td>ES-D3 [D3]</td><td>ATCC</td><td>CRL-11632TM</td><td></td></tr><tr><td>HeLa -Fucci cells</td><td></td><td></td><td>http://ruo.mbl.co.jp/bio/e/product/flprotein/fucci.html</td></tr><tr><td>DMEM</td><td>PAN-Biotech</td><td>P03-0710</td><td></td></tr><tr><td>DMEM high glucose</td><td>Sigma-Aldrich</td><td>D6429-500ML</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>HyClone</td><td>SV30180.03</td><td></td></tr><tr><td>ES Cell FBS</td><td>Gibco</td><td>16141-079</td><td></td></tr><tr><td>Non-Essential Amino Acids (NEAA)</td><td>Gibco</td><td>11140-035</td><td></td></tr><tr><td>mLIF (mouse Leukemia Inhibitor Factor)</td><td>Merck Millipore</td><td>ESG1107</td><td></td></tr><tr><td>MTG (1-Thioglycerol)</td><td>Sigma-Aldrich</td><td>M6145-25ML</td><td></td></tr><tr><td>Penicillin-Streptomycin Solution</td><td>Biosera</td><td>XC-A4122/100</td><td></td></tr><tr><td>Trypsin - EDTA</td><td>Biosera</td><td>XC-T1717/100</td><td>dilute with 1 &amp;times; PBS in ratio 1:6</td></tr><tr><td>Nunclon cell culture dishes</td><td>Sigma-Aldrich</td><td>P7866</td><td>cultivation of mESCs D3 cells</td></tr><tr><td>&amp;micro;-Dish 35m+A15:I35m Grid-500</td><td>Ibidi GmbH</td><td>81166</td><td>microscopic dish</td></tr><tr><td>0.2% Gelatine</td><td>Sigma-Aldrich</td><td>G1890-100G</td><td>dilute in destille water and autoclaved</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Cell transfection&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GFP-p53 plasmid</td><td>Addgene</td><td>12091</td><td></td></tr><tr><td>mCherry-PCNA plasmid</td><td></td><td></td><td>generous gift from Cristina Cardoso, Technische Universit&amp;auml;t Darmstadt</td></tr><tr><td>mCherry-53BP1 plasmid</td><td>Addgene</td><td>19835</td><td></td></tr><tr><td>Metafecetene</td><td>Biontex Laboratories GmbH</td><td>T020&amp;ndash;2.0</td><td></td></tr><tr><td>10 &amp;times; PBS</td><td>Thermo Fisher Scientific</td><td>AM9625</td><td>for transfection use 1 &amp;times; PBS diluted in nuclease-free water</td></tr><tr><td>5-bromo-2&amp;rsquo;-deoxy-uridine</td><td>Sigma-Aldrich</td><td>11296736001</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Confocal microscopy&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Microscope Leica TCS SP5</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Microscope Leica TCS SP8</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>White-light laser</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>355-nm laser</td><td>Coherent Inc.</td><td></td><td>laser power 80 mW</td></tr><tr><td>405-nm laser</td><td>Leica Microsystems</td><td></td><td>laser power 50 mW</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Immunofluorescence staining&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Coverslip</td><td>VWR International Ltd</td><td>631-1580</td><td></td></tr><tr><td>4% paraformaldehyde</td><td>Affymetrix</td><td>19943 1 LT</td><td></td></tr><tr><td>Triton X100</td><td>MP Biomedicals</td><td>2194854</td><td></td></tr><tr><td>Saponin from quillaja bark</td><td>Sigma-Aldrich</td><td>S4521</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A2153</td><td></td></tr><tr><td>53BP1</td><td>Abcam</td><td>ab21083</td><td>primary antibody</td></tr><tr><td>AlexaFluore 647</td><td>Thermo Fisher Scientific</td><td>A27040</td><td>secondary antibody</td></tr><tr><td>Vectashield</td><td>Vector Laboratories Ltd</td><td>H-1000</td><td>mounting medium</td></tr></tbody></table>

advanced confocal microscopy techniques to study protein-protein interactions and kinetics at dna lesions

地方 microirradiation 与激光代表了一个有用的工具研究 DNA 修复相关的过程中活细胞。在这里, 我们描述了一种方法来分析蛋白质动力学在 DNA 损伤的时间或蛋白质-蛋白质相互作用的局部 microirradiated 染色质。我们还展示了如何识别细胞周期的各个阶段, 使用 Fucci 细胞系统来研究 DNA 损伤时细胞周期依赖性的蛋白质动力学。本文还介绍了使用两种紫外线激光器 (355 nm 和 405 nm) 诱导不同类型 DNA 损伤的方法。只有405纳米二极管激光器 microirradiated 的细胞进行正常的有丝分裂, 没有丁烷嘧啶聚。我们还展示了如何 microirradiated 细胞可以固定在一个特定的时间点来执行免疫内源性蛋白质的利益。对于 dna 修复研究, 我们另外描述了生物物理方法的使用, 包括 FRAP (漂白后的荧光恢复) 和电影 (荧光寿命成像显微镜) 的细胞自发发生的 dna 损伤灶。我们还展示了电影 (荧光共振能量转移) 在蛋白质-蛋白质相互作用实验研究中的应用。

使用先进的共焦显微镜, 我们观察到 mCherry 标记的53BP1 和 mCherry PCNA 蛋白在 DNA 损伤的积累。对活细胞的局部 microirradiation 进行分析。为了识别 DNA 修复相关蛋白在单个细胞周期阶段的核分布模式, 我们使用了 Fucci 细胞系统, 通过它可以确定细胞周期的 G1、早期 S 和 G2 阶段 (图 1)。我们在 Suchankova et al.中发表的 Fucci 细胞模型的生物学应用<sup class=...

Watch this Scientific Journal Video about Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions at JoVE.com

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

激光 microirradiation 是研究活体细胞 DNA 修复的有效工具。一个方法的方法, 使用 UVA 激光诱导各种 DNA 病变显示。我们对维持正常细胞周期的局部 microirradiation 的方法进行了优化;因此, 辐照细胞通过有丝分裂进行。

高级共焦显微镜技术研究 DNA 损伤的蛋白质-蛋白质相互作用和动力学

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

advanced-confocal-microscopy-techniques-to-study-protein-protein

Research

JoVE Journal

Biology

10.9K 次观看.  Institute of Biophysics of the Czech Academy of Sciences. 激光 microirradiation 是研究活体细胞 DNA 修复的有效工具。一个方法的方法, 使用 UVA 激光诱导各种 DNA 病变显示。我们对维持正常细胞周期的局部 microirradiation 的方法进行了优化;因此, 辐照细胞通过有丝分裂进行。

视频: Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Atomic Force Microscopy Investigations of DNA Lesion Recognition in Nucleotide Excision Repair

AFM imaging is a powerful technique for the study of protein-DNA interactions. This single molecule method allows the simultaneous resolution of different molecules and molecular assemblies in a heterogeneous sample. In the particular context of DNA interacting protein systems, different protein complex forms and their corresponding binding positions on target sites containing DNA fragments can thus be distinguished. Here, an application of AFM to the study of DNA lesion recognition in the prokaryotic and eukaryotic nucleotide excision DNA repair (NER) systems is presented. The procedures of DNA and protein sample preparations are described and experimental as well as analytical details of the experiments are provided. The data allow important conclusions on the strategies by which target site verification may be achieved by the NER proteins. Interestingly, they indicate different approaches of lesion recognition and identification for the eukaryotic NER system, depending on the type of lesion. Furthermore, distinct structural properties of the two different helicases involved in prokaryotic and eukaryotic NER result in and explain the different strategies observed for these two systems. Importantly, these experimental and analytical approaches can be applied not only to the study of DNA repair but also very similarly to other DNA interacting protein systems such as those involved in replication or transcription processes.

Here, the study of different DNA lesion recognition approaches via single molecule AFM imaging is demonstrated with the nucleotide excision repair system as an example. The procedures of DNA and protein sample preparations and experimental as well as analytical details for the AFM experiments are described.

原子力显微镜检查DNA损伤识别核苷酸切除修复

AFM imaging is a powerful technique for the study of protein-DNA interactions. This single molecule method allows the simultaneous resolution of different ...

科研

遗传学

Single Molecule Analysis of Laser Localized Psoralen Adducts

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live cells. The DDR to helix distorting covalent DNA modifications, including interstrand DNA crosslinks (ICLs), is not as well defined. We have studied the DDR stimulated by ICLs, localized by laser photoactivation of immunotagged psoralens, in the nuclei of live cells. In order to address fundamental questions about adduct distribution and replication fork encounters, we combined laser localization with two other technologies. DNA fibers are often used to display the progress of replication forks by immunofluorescence of nucleoside analogues incorporated during short pulses. Immunoquantum dots have been widely employed for single molecule imaging. In the new approach, DNA fibers from cells carrying laser localized ICLs are spread onto microscope slides. The tagged ICLs are displayed with immunoquantum dots and the inter-lesion distances determined. Replication fork collisions with ICLs can be visualized and different encounter patterns identified and quantitated.

Lasers are frequently used in studies of the cellular response to DNA damage. However, they generate lesions whose spacing, frequency, and collisions with replication forks are rarely characterized. Here, we describe an approach that enables the determination of these parameters with laser localized interstrand crosslinks.

激光局部补骨脂素加合物的单分子分析

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live ...

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

激光微辐照在哺乳动物细胞单、双链断裂修复中的应用

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular ...

癌症研究

Immunofluorescence Microscopy of &#947;H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas including genome integrity, genotoxicity, radiation biology, aging, cancer, and drug development. In response to DSB, the histone H2AX is phosphorylated at Serine 139 in a region of several megabase pairs forming discrete nuclear foci detectable by immunofluorescence microscopy. In addition, 53BP1 (p53 binding protein 1) is another important DSB-responsive protein promoting repair of DSB by nonhomologous end-joining while preventing homologous recombination. According to the specific functions of &#947;H2AX and 53BP1, the combined analysis of &#947;H2AX and 53BP1 by immunofluorescence microscopy may be a reasonable approach for a detailed analysis of DSB. This manuscript provides a step-by-step protocol supplemented with methodical notes for performing the technique. Specifically, the influence of the cell cycle on &#947;H2AX foci patterns is demonstrated in normal fibroblasts of the cell line NHDF. Further, the value of the &#947;H2AX foci as a biomarker is depicted in x-ray irradiated lymphocytes of a healthy individual. Finally, genetic instability is investigated in CD34+ cells of a patient with acute myeloid leukemia by immunofluorescence microscopy of &#947;H2AX and 53BP1.

This manuscript provides a protocol for the analysis of DNA double-strand breaks by immunofluorescence microscopy of &#947;H2AX and 53BP1.

γH2AX 和53BP1 的免疫荧光显微镜分析 DNA 双链断裂的形成和修复

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas ...

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

检测和可视化DNA损伤诱导的蛋白质复合物悬浮细胞培养使用邻近连接测定

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

生物化学

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand genome maintenance mechanisms. Since recruitment and exchange of proteins at lesions are highly dynamic, their study requires the ability to generate DNA damage in a rapid and spatially-delimited manner. Here, we describe procedures to locally induce DNA damage in human cells using a commonly available laser-scanning confocal microscope equipped with a 405 nm laser line. Accumulation of genome maintenance factors at laser stripes can be assessed by immunofluorescence (IF) or in real-time using proteins tagged with fluorescent reporters. Using phosphorylated histone H2A.X (&#947;-H2A.X) and Replication Protein A (RPA) as markers, the method provides sufficient resolution to discriminate locally-recruited factors from those that spread on adjacent chromatin. We further provide ImageJ-based scripts to efficiently monitor the kinetics of protein relocalization at DNA damage sites. These refinements greatly simplify the study of the DDR dynamics.

Studying DNA damage repair kinetics requires a system to induce lesions at defined sub-nuclear regions. We describe a method to create localized double-stranded breaks using a laser-scanning confocal microscope equipped with a 405 nm laser and provide automated procedures to quantify the dynamics of repair factors at these lesions.

405 Nm 激光微辐照对 DNA 损伤的蛋白质招募研究

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand ...

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair complexes. This process is regulated by a myriad of proteins that promote the association and disassociation of proteins to these lesions. Thanks in large part to the ability to perform functional screens of a vast library of proteins, there is a greater appreciation of the genes necessary for the double-strand DNA break repair. Often knockout or chemical inhibitor screens identify proteins involved in repair processes by using increased toxicity as a marker for a protein that is required for DSB repair. Although useful for identifying novel cellular proteins involved in maintaining genome fidelity, functional analysis requires the determination of whether the protein of interest promotes localization, formation, or resolution of repair complexes. 
The accumulation of repair proteins can be readily detected as distinct nuclear foci by immunofluorescence microscopy. Thus, association and disassociation of these proteins at sites of DNA damage can be accessed by observing these nuclear foci at representative intervals after the induction of double-strand DNA breaks. This approach can also identify mis-localized repair factor proteins, if repair defects do not simultaneously occur with incomplete delays in repair. In this scenario, long-lasting double-strand DNA breaks can be engineered by expressing a rare cutting endonuclease (e.g., I-SceI) in cells where the recognition site for the said enzyme has been integrated into the cellular genome. The resulting lesion is particularly hard to resolve as faithful repair will reintroduce the enzyme's recognition site, prompting another round of cleavage. As a result, differences in the kinetics of repair are eliminated. If repair complexes are not formed, localization has been impeded. This protocol describes the methodology necessary to identify changes in repair kinetics as well as repair protein localization.

Repair of double-strand DNA breaks is a dynamic process, requiring not only formation of repair complexes at the breaks, but also their resolution after the lesion is addressed. Here, we use immunofluorescence microscopy for transient and long-lasting double-stranded breaks as a tool to dissect this genome maintenance mechanism.

基于免疫荧光显微镜的瞬时和持久双链 dna 损伤 dna 修复过程的表征

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.

免疫荧光DNA修复蛋白相互作用的可视化

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA ...

生物学

Laser Micro-Irradiation to Study DNA Recruitment During S Phase

DNA damage repair maintains the genetic integrity of cells in a highly reactive environment. Cells may accumulate various types of DNA damage due to both endogenous and exogenous sources such as metabolic activities or UV radiation. Without DNA repair, the cell's genetic code becomes compromised, undermining the structures and functions of proteins and potentially causing disease. 
Understanding the spatiotemporal dynamics of the different DNA repair pathways in various cell cycle phases is crucial in the field of DNA damage repair. Current fluorescent microscopy techniques provide great tools to measure the recruitment kinetics of different repair proteins after DNA damage induction. DNA synthesis during the S phase of the cell cycle is a peculiar point in cell fate regarding DNA repair. It provides a unique window to screen the entire genome for mistakes. At the same time, DNA synthesis errors also pose a threat to DNA integrity that is not encountered in non-dividing cells. Therefore, DNA repair processes differ significantly in S phase as compared to other phases of the cell cycle, and those differences are poorly understood. 
The following protocol describes the preparation of cell lines and the measurement of dynamics of DNA repair proteins in S phase at locally induced DNA damage sites, using a laser-scanning confocal microscope equipped with a 405 nm laser line. Tagged PCNA (with mPlum) is used as a cell cycle marker combined with an AcGFP-labeled repair protein of interest (i.e., EXO1b) to measure the DNA damage recruitment in S phase.

This protocol describes a non-invasive method to efficiently identify S-phase cells for downstream microscopy studies, such as measuring DNA repair protein recruitment by laser micro-irradiation.

激光微辐射研究S阶段DNA招募

DNA damage repair maintains the genetic integrity of cells in a highly reactive environment. Cells may accumulate various types of DNA damage due to both ...

Immunofluorescence Microscopy for the Analysis of DNA Double-Strand Breaks

Source: Popp, H. D., et al. <a href="https://app.jove.com/v/56617">Immunofluorescence Microscopy of &#947;H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks.</a> J. Vis. Exp. (2017)
In this video, we describe the immunofluorescence microscopy technique to detect specific DNA damage response proteins localized at the sites of DNA double-strand breaks in human mononuclear cells. The proteins are recognized by specific primary antibodies, which in turn bind to fluorophore-tagged secondary antibodies that fluoresce during microscopic visualization, enabling visualization of the proteins as distinct fluorescent foci within the cell nuclei.

用于分析 DNA 双链断裂的免疫荧光显微镜

Source: Popp, H. D., et al. Immunofluorescence Microscopy of &#947;H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks. J. ...