この作業は、プロジェクト P302 12 G157 チェコ共和国の助成金機構によって支えられました。実験はまたノルウェーの資金によって、文部省、青少年とチェコ共和国のスポーツによって監督されているチェコ語ノルウェー語研究プログラム CZ09 によって支えられた (許可番号: 7F14369)。

<ol>
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著者は、利害の対立がないことを宣言します。

顕微鏡検査の技術は、研究室での基本的なツールを表します。ここでは、タンパク質募集の検討用の方法の簡単な説明と DNA 損傷で速度が表示されます。我々 は特に生きている細胞のローカル microirradiation の分野で我々 の実験的経験を指摘し、アクセプタ漂白フレット28による DNA 損傷とその高度な縛ると蛋白質-蛋白質の相互作用蛋白質の動力学の研究について論じるフレ?...

シクロブタン ピリミジン ダイマー (チミン)、8-oxo-7,8-dihydro-2'-deoxyguanosine と一本鎖の DNA 病変の出現につながる DNA 損傷または二重鎖切断1,2。Γ 線は高浸透の最高のエネルギーと放射線のフォーム、放射のこの源は放射線療法3に広く利用します。その一方で、紫外線を UV レーザー mimics 自然暴露によって引き起こされる DNA 損傷が実験。UVA microirradiation 顕微鏡法、個々 の細胞における DNA 損傷を研究するため実験的なツールを表します。Microirradiation は、染色体の地域4、5の組織を明らかにするために 40 年前の最初の時間に使われました。この手法は、共焦点顕微鏡の機能プロパティのどちらかまたは現代 nanoscopy の技術的な限界に大きく依存。DNA 損傷を誘発するには、細胞を 5' ブロモデオキシウリジン (BrdU) またはヘキスト 33342 UV 照射前にプレハブプレートベーすることができます。Bártováら6前述 presensitization ステップ、最近我々 は細胞死やアポトーシスを避けるためにこの microirradiation 技術を最適化します。たとえば、(ヘキスト 33342 前) なし 405 nm の紫外線レーザーの使用は、シクロブタン ピリミジン二量体 (チミン) を犠牲にして 53BP1 正二重鎖切断 (Dsb) の誘導につながります。UV microirradiation と組み合わせる presensitization の手順、他の一方で、同時にチミンと Dsb の非常に高いレベルを誘発する7,8。この方法は、単一の DNA 修復経路の研究に適用することは困難です。
Microirradiation、蛋白質の募集、速度、および細胞内 DNA 損傷における相互作用を分析することが可能です。このメソッドの例が Luijsterburgらによって出版されました。ヘテロクロマチン蛋白質 1 β と私たちの9は最近紫外線による DNA 病変6,10多能性因子 Oct4 と coilin、カハール体に関連付けられている蛋白質を募集初めて示した。これらの DNA 損傷で蛋白質の動力学 FRAP (退色後蛍光回復)11,12,13を使用して学ぶことがまたは (蛍光共鳴エネルギー移動) 技術14 フレット ,15。これらのメソッドには、DNA 損傷または蛋白質蛋白質の相互作用の蛋白質の単純な拡散を明らかにする可能性があります。蛋白質のさらに特性評価のための便利なツールは、FLIM (蛍光寿命イメージング顕微鏡) やフレット技術 (フレット FLIM)16との組み合わせです。これらのメソッドは、一過性でタグ付けされた蛍光分子17興味の蛋白質を表現する安定細胞のプロセスの研究を有効にします。ここでは、GFP 付けられた p53 タンパク質と DNA 損傷応答18,19に重要な役割を果たして相互作用パートナー、mCherry タグ 53BP1 の指数関数的減衰時間 (τ) の例を示します。パラメーター τ FLIM 計算によって提供される螢光色素の寿命は、与えられた蛍光染料、そのバインド能力とその細胞環境の特定です。したがって、このメソッド私たちを見ること蛋白質の集団、その結合能力と機能特性の違い、たとえば、DNA 損傷の後。
ここでは、時間特定の蛋白質の募集、動態、拡散、および microirradiated クロマチンのサイトでタンパク質間相互作用を検討する私たちの研究室で使用されている高度の顕微鏡検査の技術の方法論的アプローチの概要表示されます。ローカル DNA 傷害、細胞の誘導とローカル誘発 DNA 損傷紫外線レーザーによって引き起こされる DNA 損傷関連イベントの研究方法論の説明のステップバイ ステップの方法論を提供しています。

<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

レーザーでローカル microirradiation は、生きた細胞の DNA 修復関連プロセスの研究のための有用なツールを表します。ここでは、ローカル microirradiated クロマチンの時間または蛋白質蛋白質の相互作用の上で DNA 蛋白質の動力学を分析する方法論的アプローチについて述べる。また Fucci セルラ システムを使用して DNA の病変の細胞周期依存性蛋白質の動力学を研究する細胞周期の個々 の段階を認識する方法を示します。2 つの UV レーザーの使用の方法論的説明 (355 nm、405 nm) また提示異なるタイプの DNA 損傷を誘発します。405 nm 半導体レーザによる細胞 microirradiated のみ通常の有糸分裂を通って進んで行くとシクロブタン ピリミジン二量体 (チミン) を欠いています。また興味の内因性の蛋白質のバイオアセッテイを実行する指定された時点での microirradiated セルの修正方法を示します。DNA 修復研究には自発的に発生すると細胞内 FRAP (退色後蛍光回復)、FLIM (蛍光寿命イメージング顕微鏡) を含む生物物理方法の使用を記述するさらに DNA 損傷病巣。タンパク質間相互作用の実験的研究の FLIM フレット (蛍光共鳴エネルギー移動) のアプリケーションを表示します。

高度な共焦点顕微鏡を用いた DNA 損傷で mCherry タグ 53BP1 と PCNA mCherry 蛋白質の蓄積を見ました。解析は、生きている細胞のローカル microirradiation によって行われました。個々 の細胞周期段階で DNA 修復関連蛋白質の核の分布パターンを認識する我々 は G1、初期 S を決定することが、Fucci セルラ システムを使用し、セル G2 段階サイクル (図 1)。Suc...

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

レーザー microirradiation は、細胞の DNA 修復の研究のための便利なツールです。様々 な DNA 損傷を誘発する UVA レーザーの使用のための方法論的アプローチが表示されます。正常な細胞周期; を維持するローカルの microirradiation のメソッドを最適化します。したがって、照射された細胞は有糸分裂を続行します。

タンパク質-タンパク質相互作用や DNA 損傷速度論を研究する共焦点の顕微鏡検査の技術を高度な

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 ビュー.  Institute of Biophysics of the Czech Academy of Sciences. レーザー microirradiation は、細胞の DNA 修復の研究のための便利なツールです。様々 な DNA 損傷を誘発する UVA レーザーの使用のための方法論的アプローチが表示されます。正常な細胞周期; を維持するローカルの microirradiation のメソッドを最適化します。したがって、照射された細胞は有糸分裂を続行します。

ビデオ: 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.

ヌクレオチド切除修復におけるDNA損傷認識の原子間力顕微鏡検査

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

遺伝学

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.

レーザーローカライズソラレン付加物の単一分子解析

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.

マイクロ レーザーの単一の試験と哺乳類細胞における二重鎖切断修復のためのアプリケーション

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

がん研究

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、53BP1 形成と DNA 二重鎖切断の修復を分析するための蛍光顕微鏡観察

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.

近接ライゲーションアッセイを用いた懸濁細胞培養におけるDNA損傷誘発タンパク質複合体の検出と可視化

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 レーザ マイクロ照射による DNA 損傷にタンパク質募集調査

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.

蛍光顕微鏡を用いた過渡的長期的な二本鎖 DNA 切断の DNA 修復プロセスの特性

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.

免疫蛍光によるDNA修復タンパク質相互作用の可視化

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.

S期におけるDNA採用を研究するレーザーマイクロ照射

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.

DNA二本鎖切断の分析のための免疫蛍光顕微鏡

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. ...

タンパク質-タンパク質相互作用や DNA 損傷速度論を研究する共焦点の顕微鏡検査の技術を高度な

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この動画の章

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マイクロレーザーの単一の試験と哺乳類細胞における二重鎖切断修復のためのアプリケーション

ΓH2AX、53BP1 形成と DNA 二重鎖切断の修復を分析するための蛍光顕微鏡観察

近接ライゲーションアッセイを用いた懸濁細胞培養におけるDNA損傷誘発タンパク質複合体の検出と可視化

405 Nm レーザマイクロ照射による DNA 損傷にタンパク質募集調査

蛍光顕微鏡を用いた過渡的長期的な二本鎖 DNA 切断の DNA 修復プロセスの特性

免疫蛍光によるDNA修復タンパク質相互作用の可視化

S期におけるDNA採用を研究するレーザーマイクロ照射

DNA二本鎖切断の分析のための免疫蛍光顕微鏡

タンパク質-タンパク質相互作用や DNA 損傷速度論を研究する共焦点の顕微鏡検査の技術を高度な

要約

さらに動画を探す

この動画の章

ヌクレオチド切除修復におけるDNA損傷認識の原子間力顕微鏡検査

レーザーローカライズソラレン付加物の単一分子解析

マイクロ レーザーの単一の試験と哺乳類細胞における二重鎖切断修復のためのアプリケーション

ΓH2AX、53BP1 形成と DNA 二重鎖切断の修復を分析するための蛍光顕微鏡観察

近接ライゲーションアッセイを用いた懸濁細胞培養におけるDNA損傷誘発タンパク質複合体の検出と可視化

405 Nm レーザ マイクロ照射による DNA 損傷にタンパク質募集調査

蛍光顕微鏡を用いた過渡的長期的な二本鎖 DNA 切断の DNA 修復プロセスの特性

免疫蛍光によるDNA修復タンパク質相互作用の可視化

S期におけるDNA採用を研究するレーザーマイクロ照射

DNA二本鎖切断の分析のための免疫蛍光顕微鏡

マイクロレーザーの単一の試験と哺乳類細胞における二重鎖切断修復のためのアプリケーション

405 Nm レーザマイクロ照射による DNA 損傷にタンパク質募集調査