Bu eser Çek Cumhuriyeti, proje P302-12-G157 Grant ajansı tarafından desteklenmiştir. Deneyler de Norveç fon tarafından ve Milli Eğitim Bakanlığı, gençlik ve spor Çek Cumhuriyeti tarafından denetimli çek-Norveç araştırma programı CZ09 tarafından desteklenen (sayı vermek: 7F14369).

<ol>
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Yazarlar hiçbir çıkar çatışması vardır bildirin.

Mikroskopi teknikleri temel araçları araştırma laboratuarları'nda temsil eder. Burada, protein alımı için çalışma yöntemleri hakkında kısa bir açıklama kullanılan ve Kinetik DNA lezyonlar, sunulur. Biz özellikle deneysel deneyimi yaşayan hücrelerin yerel microirradiation alanında kaydetti ve sıkı BAĞLAMAK ve protein-protein etkileşim alıcısı ağartma FRET28 DNA lezyonlar ve onun gelişmiş protein Kinetik incelenmesi konuşalım değişiklik FRET-FLIM (<strong class="xf...

DNA hasar oluşan cyclobutane pirimidin dimer (CPDs), 8-oxo-7,8-dihydro-2'-deoxyguanosine ve tek iplikçikli DNA lezyonlar görünümünü açar veya iki iplikçikli1,2tatili. Γ-ışınları iyonize radyasyon yüksek enerji ve yüksek alellerle biçimidir, böylece bu kaynağı radyasyon radyoterapi3' te yaygın olarak kullanılır. Öte yandan, deneysel olarak UV lazerler taklit eder doğal maruz UV ışığı neden olduğu DNA hasar indüklenen. UVA microirradiation, mikroskobik bir yöntem olarak, bireysel yaşam hücrelerdeki DNA hasarı çalışmak için deneysel bir araç temsil eder. Microirradiation ilk kez 40 yıl önce kromozom bölgeleri4,5organizasyonu ortaya çıkarmak amacıyla kullanıldı. Bu teknik son derece işlevsel özelliklerini confocal mikroskoplar veya modern nanoscopy teknik sınırları üzerinde bağlıdır. DNA lezyonlar ikna etmek için hücreleri tarafından 5' bromodeoxyuridine (BrdU) veya Höchst 33342 UV Işınlama önce presensitized. Bártová vd. 6 presensitization adım daha önce açıklanan ve son zamanlarda biz bu microirradiation teknik hücre ölümü veya apoptosis önlemek için en iyi duruma getirilmiş. Örneğin, bir 405 nm UV lazer (olmadan Höchst 33342 presensitization) kullanımı pahasına cyclobutane pirimidin dimer (CPDs) 53BP1-pozitif iki iplikçikli kırılmalar (DSBs) indüksiyon için yol açar. Öte yandan, UV microirradiation ile kombine presensitization adımları çok yüksek düzeyde CPDs ve DSBs aynı anda neden7,8. Bu yöntem tek bir DNA onarım yol çalışma odasına uygulamak zordur.
Microirradiation ile protein alımı, kinetik ve etkileşim, canlı hücreler lezyonlarının DNA analiz etmek mümkündür. Bu yöntemi örneği Luijsterburg vd tarafından yayımlandı 9 heterochromatin protein 1β ve biz son zamanlarda pluripotency faktör Oct4 ve Aytac, Cajal organları ile ilişkili bir protein DNA UV kaynaklı lezyonlar6,10' a işe ilk kez gösterdi. Bu DNA lezyonlar, protein Kinetik da sıkı BAĞLAMAK (floresan kurtarma sonra Photobleaching)11,12,13 kullanarak okudu olabilir veya (floresan rezonans enerji transferi) teknikleri14 ÜZÜLMEK ,15. Bu yöntemler basit Difüzyon proteinlerin DNA lezyonlar veya protein-protein etkileşimleri ortaya potansiyeline sahip. Ek protein karakterizasyonu için yararlı bir araç FLIM (floresan ömür boyu Imaging mikroskobu) veya perde teknoloji (FRET-FLIM)16ile onun birlikte olduğunu. Bu yöntemler stabil olan hücreler yaşayan veya geçici protein flüoresan molekül17tarafından öğesini ilgi ifade işlemleri çalışma etkinleştirin. Burada, üstel çürüme kez (τ) GFP öğesini p53 protein ve etkileşim ortak, 53BP1, mCherry öğesini DNA hasarı yanıt18,19 ' önemli bir rol oynayan bir örneği gösterilir. Parametre τ FLIM hesaplamaları tarafından sağlanan fluorochrome ömrünü bir verilen Floresans boya, bağlama yetenekleri ve hücresel çevresi için özeldir. Bu nedenle, bu yöntem bize DNA hasar sonra örneğin, protein altgrupları, bağlama yeteneklerini ve fonksiyonel özellikleri arasındaki farklılıklar gösterebilir.
Burada, bizim laboratuvarda Saat özel protein alımı, Kinetik, difüzyon ve microirradiated Kromatin sitesinde protein-protein etkileşimleri incelemek için kullanılır bir anahat gelişmiş mikroskobu teknikleri metodolojik yaklaşımların sunulur. Canlı hücreler yerel DNA lezyonlarının indüksiyon ve metodolojileri itibariyle yerel olarak bağlı DNA lezyonlar UV lazerler tarafından neden olduğu DNA hasar ile ilgili olayların çalışmaları için yararlı bir açıklama için adım adım yöntemler sağlanır.

<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

Lazerler ile yerel microirradiation canlı hücrelerdeki DNA onarım ile ilgili süreçlerin çalışmaları için yararlı bir araç temsil eder. Burada, yerel olarak microirradiated Kromatin zaman ya da protein-protein etkileşimleri üzerinde protein Kinetik DNA lezyonlar, analiz için bir metodolojik yaklaşım tarif. Biz de tek tek hücre döngüsü bağlı protein Kinetik DNA lezyonlar, çalışmaya Fucci hücresel sistem kullanarak hücre döngüsü aşamaları tanımak gösterilmiştir. İki UV lazerler kullanımı metodolojik açıklaması (355 nm ve 405 nm) farklı DNA hasar ikna etmek için de sunulmaktadır. Yalnızca hücre microirradiated 405 nm diode lazer tarafından mitoz normalde devam etti ve cyclobutane pirimidin dimer (CPDs) yoksun olduğunu. Biz de nasıl microirradiated hücre immunodetection ilgi endojen proteinlerin gerçekleştirmek için verilen süre noktada sabit olabilir gösterir. DNA onarım çalışmaları için ayrıca sıkı BAĞLAMAK (floresan kurtarma sonra Photobleaching) ve FLIM (floresan ömür boyu Imaging mikroskobu) kendiliğinden meydana gelen hücreler dahil olmak üzere biyofiziksel yöntemlerin kullanımı tarif DNA hasarı foci. Ayrıca protein-protein etkileşimleri deneysel çalışmalarda FLIM FRET (floresan rezonans enerji transferi) bir uygulama gösterir.

Gelişmiş confocal mikroskobu kullanarak, mCherry öğesini 53BP1 ve mCherry-PCNA proteinler DNA lezyonlar adlı bir birikimi görülmektedir. Analizleri canlı hücreler yerel microirradiation tarafından gerçekleştirilmiştir. Proteinlerin DNA onarım ile ilgili nükleer dağıtım desenleri tek hücre döngüsü aşamalı olarak tanımak için biz hangi G1, erken S, belirlemek mümkündür Fucci hücresel sistem ve G2 aşama hücre döngüsü (şekil 1)...

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

Lazer microirradiation yaşayan hücrelerde DNA onarım çalışmaları için yararlı bir araçtır. Çeşitli DNA lezyonlar ikna etmek için UVA lazerler kullanımı için bir metodolojik yaklaşım gösterilir. Normal hücre döngüsü tutar yerel microirradiation için bir yöntemi optimize; Böylece, ışınlanmış hücre mitoz devam edin.

Gelişmiş protein-protein etkileşimleri ve Kinetik DNA lezyonlar, çalışmaya Confocal mikroskobu teknikleri

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 Görüntüleme.  Institute of Biophysics of the Czech Academy of Sciences. Lazer microirradiation yaşayan hücrelerde DNA onarım çalışmaları için yararlı bir araçtır. Çeşitli DNA lezyonlar ikna etmek için UVA lazerler kullanımı için bir metodolojik yaklaşım gösterilir. Normal hücre döngüsü tutar yerel microirradiation için bir yöntemi optimize; Böylece, ışınlanmış hücre mitoz devam edin.

Video: 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.

Nükleotid Eksizyon Onarımında DNA Lezyon Tanınması için Atomik Kuvvet Mikroskopi Araştırmaları

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

Genetik

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.

Lazer Lokalize Psoralen adüktlerinin Tek Molekül Analizi

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.

Lazer mikro-ile kullanma için tek incelenmesi ve çift Strand Break onarım memeli hücrelerinde

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

Kanser Araştırmaları

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 ve 53BP1 oluşumu ve DNA çift iplikli kırılmalara tamiri analiz etmek için ayirt mikroskobu

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.

Proximity Ligation Testi ile Süspansiyon Hücre Kültürlerinde DNA Hasarına Bağlı Protein Komplekslerinin Saptanması ve Görselleştirilmesi

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

Biyokimya

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.

Soruşturma 405 Nm lazer mikro-ile kullanarak DNA lezyonlar için Protein alımı

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.

DNA onarım işlemleri, Darbe Gerilimlerinden ve uzun süreli iki iplikçikli DNA sonları ayirt mikroskobu ile karakterize

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.

DNA Onarım Proteinlerinin İmmünfluoresan Etkileşiminin Görselleştirilmesi

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

Biyoloji

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 Aşamasında DNA İşe Alımını İncelemek için Lazer Mikro Işınlama

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 Çift İplikli Kırılmaların Analizi için İmmünofloresan Mikroskobu

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. ...