Name: atomic force microscopy investigations of dna lesion recognition in nucleotide excision repair
Uploaded: 2017-05-24T14:00:00.000+00:00
Duration: 10 min 59 s
Description: Watch this Scientific Journal Video about Atomic Force Microscopy Investigations of DNA Lesion Recognition in Nucleotide Excision Repair at JoVE.com

PUC19N, CPD-haltige Oligonukleotide und p44 wurden freundlicherweise von Samuel Wilson, Korbinian Heil und Thomas Carell und Gudrun Michels bzw. Caroline Kisker bereitgestellt. Diese Arbeit wurde durch Stipendien der Deutschen Forschungsgemeinschaft (DFG) FZ82 und TE-671/4 an IT unterstützt.

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AFM statistische Analysen von Bindungspositionen von Proteinen auf langen DNA-Fragmenten, die spezifische Zielstellen enthalten, können interessante Details über die speziellen Strategien des Proteins zeigen, um diese Stellen 2 , 3 , 4 , 5 , 6 zu erkennen. Um die daraus resultierenden Positionsverteilungen zu interpretieren, müssen die Positionen der Targe...

Atomkraftmikroskopie (AFM) ist eine leistungsfähige Technik für die Analyse der Protein-DNA-Wechselwirkungen 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 . Es erfordert nur geringe Mengen an Probenmaterial, um heterogene Proben direkt mit einer Auflösung auf Einzelmolekülebene zu visualisieren. Heterogenität kann aus verschiedenen konformationellen oder oligomeren Zuständen eines Proteins resultieren. Insbesondere können im Rahmen von Protein-DNA-Proben Proteinkomplexe unterschiedliche Stöchiometrien und / oder Konformationen aufweisen, die durch DNA-Bindung im Allgemeinen induziert werden oder an eine spezifische Zielstelle innerhalb der DNA binden. Heterogene Proben können auch zwei (oder mehrere) verschiedene Arten von Proteinen und verschiedene Proteinkomplexe enthaltenFormen ( zB bestehend aus nur einer Art von Protein gegenüber heteromeren Komplexen) können unterschiedlich mit DNA interagieren. Die hier diskutierten Studien nutzten die AFM-Bildgebung in der Luft auf statischen, getrockneten Proben von DNA-Reparaturproteinen, die an lange (~ 900 Basenpaare, bp) DNA-Fragmente gebunden sind, die eine Läsion enthalten, die ein Ziel dieser Proteine ​​darstellt. Die hohe molekulare Auflösung von AFM ermöglicht die Unterscheidung zwischen verschiedenen Arten von Proteinkomplexen und die Bestimmung der Bindungspositionen der Proteine ​​an den DNA-Fragmenten. Wichtig ist, dass die Läsionen in gut definierten Positionen in die DNA-Substrate eingebracht werden. Da die Position der Läsionsstelle in der DNA bekannt ist, liefern die Verteilungen von Proteinen, die an DNA gebunden sind, einen Einblick in (verschiedene) Läsionserkennungseigenschaften der (verschiedenen) Proteinkomplexe, zB wie gut sie eine bestimmte Art von Läsion erkennen (verglichen Zu nicht beschädigter DNA) 2 , 3 , <supClass = &quot;xref&quot;&gt; 4 , 5 , 6 . Ihre Positionen auf der DNA erlauben auch die Unterscheidung zwischen Proteinkomplexen, die spezifisch an den Läsionen und Komplexen gebunden sind, die unspezifisch anderswo an der DNA gebunden sind. Eine getrennte Charakterisierung dieser verschiedenen komplexen Typen (Komplexe, die spezifisch an der Läsion gegenüber unspezifischen Komplexen gebunden sind) können potentielle Konformationsänderungen in den Komplexen zeigen, die bei der Zielortidentifizierung induziert werden. Die DNA-Reparaturproteine, die hier konzentriert sind, sind Helicasen, die für die Läsionserkennung in der Nucleotide Exzision Reparatur (NER) Weg verantwortlich sind. In Bakterien wird NER durch die Proteine ​​UvrA, UvrB und UvrC erreicht. UvrA ist verantwortlich für die initiale Läsionserfassung in einem UvaA 2 / UvrB 2 DNA-Scan-Komplex. Nach der Läsionsüberprüfung durch UvrB wandelt sich dieser Komplex in monomeres UvrB um, das an der Läsionsstelle gebunden ist, und dieser spezifische Komplex kann dann das p rekrutierenRokaryotische NER Endonuklease UvrC. UvrC verbreitet eine kurze (12-13 nt) Strecke einzelsträngiger DNA (ssDNA), die die Läsion enthält. Die fehlende Dehnung wird dann durch DNA-Polymerase nachgefüllt. Schließlich versiegelt die DNA-Ligase die neu synthetisierte Dehnung mit der ursprünglichen DNA 9 , 10 . Bei Eukaryonten sind die meisten Proteine ​​der NER-Kaskade Teil des großen, multimeren Transkriptionsfaktors II H (TFIIH) -Komplexes. Nach der ersten Läsionserfassung über den trimeren CEN2-XPC-HR23B-Komplex wird TFIIH an die DNA-Zielstelle rekrutiert. Wenn XPD innerhalb des Komplexes die Anwesenheit einer NER-Zielläsion verifiziert, werden die eukaryotischen NER-Endonukleasen XPG und XPF rekrutiert, um eine kurze (24-32 nt) Strecke von ssDNA, die die Läsion 9 , 10 enthält , zu exzimieren. Hier wurden insbesondere die Helicasen UvrB und XPD aus prokaryotischem und eukaryotischem NER untersucht. Diese Helicases erfordern eine ungepaarte Region in derDNA (eine DNA-Blase), um auf einen der beiden DNA-Einzelstränge zu fädeln und anschließend entlang diesem durch ATP-Hydrolyse angefeuerten Strang zu translozieren. Zusätzlich zu den DNA-Läsionen wurde daher eine DNA-Blase in die Substrate eingeführt, die als Ladestelle für die Proteine ​​fungieren. Das Verfahren zur Herstellung von spezifischen Läsions-DNA-Substraten wurde zuvor beschrieben 11 . Es erfordert ein kreisförmiges DNA-Konstrukt (Plasmid) mit zwei eng beabstandeten Restriktionsstellen für eine Nickase. Im Rahmen dieser Studie wurde das Plasmid pUC19N (2729 bp) verwendet (erstellt von S. Wilsons Laboratorium, NIEHS). Dieses Plasmid enthält drei eng beabstandete Restriktionsstellen für die Nickel Nt.BstNBI, die eine 48 Nukleotid (nt) Streckung bilden. Nach der Inkubation mit der Nickel kann die Strecke der ssDNA zwischen diesen Stellen entfernt und durch ein Oligonukleotid ersetzt werden, das ein beliebiges Zielmerkmal enthält. Nach jedem Schritt wird die vollständige enzymatische Verdauung über Agarosegel getestetElektrophorese Die vernetzte zirkuläre DNA kann aufgrund ihrer geringeren elektrophoretischen Beweglichkeit gegenüber dem ursprünglichen supercoiled Plasmid unterschieden werden. Das Anhängen der DNA und das Ersetzen der entfernten Dehnung durch das spezifische Substrat-Oligonukleotid können durch Verdauung mit einem Restriktionsenzym ausgewertet werden, das das Substrat ausschließlich innerhalb des Bereichs zwischen den Nicks aufnimmt. Die Linearisierung des zirkulären Plasmids durch das Enzym wird daher für die gapped DNA unterdrückt und nach der Insertion des spezifischen Oligonukleotids wiederhergestellt. Schließlich erlauben zwei Endonuklease-Restriktionsstellen (idealerweise Einzelschneider) die Erzeugung eines linearen DNA-Substrats mit der gewünschten Länge und mit der spezifischen Zielstelle an einer definierten Position sowie einer DNA-Blase in einem Abstand von der Läsion entweder in 5 &#39;Oder 3&#39; Richtung. Die Erkennung der Läsionen durch die NER-Helicasen kann mittels AFM-Bildgebung untersucht werden . Stabile DNA-Translokation der Helicasen bei tEr Läsionsstelle ist als Peak in der Proteinpositionsverteilung auf der DNA sichtbar und zeigt eine Läsionserkennung an. Da die DNA-Translokation dieser Helicinen weiterhin mit einer 5&#39;-bis-3&#39;-Polarität gerichtet ist, zeigt die Abhängigkeit der Läsionserkennung an der Position der Ladestelle (DNA-Blase stromaufwärts oder stromabwärts der Läsion) auch an, ob die Läsion bevorzugt erkannt wird Auf dem translozierten oder auf dem entgegengesetzten, nicht translozierten ssDNA-Strang 5 , 9 . In den folgenden Abschnitten werden die angewandten Methoden eingeführt und wichtige Erkenntnisse aus diesen Experimenten werden kurz diskutiert. Wichtig ist, dass analog zur beispielhaften Arbeit zur hier beschriebenen DNA-Reparatur die AFM-Bildgebung auf die Untersuchung verschiedener DNA-interagierender Systeme wie der DNA-Replikation oder der Transkription 8 , 12 , 13 , 14 angewendet werden kann </Sup&gt;.

<table><tbody><tr><td>Molecular Force Probe (MFP) 3D</td><td>Asylum Research</td><td>N/A</td><td>atomic force microscope (AFM)</td></tr><tr><td>Precision 390</td><td>DELL</td><td>N/A</td><td>computer</td></tr><tr><td>ThermoMixer and 1.5 mL block</td><td>Eppendorf</td><td>5382000015</td><td>heat block for DNA preparation</td></tr><tr><td>Rotilabo Block-Heater H 250 &amp;amp; blocks for 0.5 mL tubes</td><td>Carl Roth GmbH</td><td>Y264.1 &amp;amp; Y267.1</td><td>heat block for protein-DNA incubations</td></tr><tr><td>Mini-Sub Cell GT</td><td>Bio-Rad Laboratories GmbH</td><td>1704467</td><td>electrophoresis chamber with gel caster and power supply</td></tr><tr><td>Power Pac Basic</td><td>Bio-Rad Laboratories GmbH</td><td>1645050</td><td>electrophoresis power supply</td></tr><tr><td>Centifuge 5415 D with rotor</td><td>Eppendorf</td><td>2262120-3</td><td>table centrifuge</td></tr><tr><td>Ultra-Lum electronic UV transillumonator MEB-15</td><td>Ultralum</td><td>900-1322-02</td><td>UV irradiation table</td></tr><tr><td>NanoDrop ND-1000</td><td>VWR International / PEQLAB Biotechnologie GmbH</td><td>N/A</td><td>UV spectrophotometer</td></tr><tr><td>TKAX-CAD with 0.2 &amp;mu;m capsule filter</td><td>Unity Lab Services</td><td>N/A</td><td>water deionization and filter unit</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;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MFP software on Igor Pro</td><td>Asylum Research</td><td>N/A</td><td>AFM software</td></tr><tr><td>ImageJ (open source Java image processing)</td><td>NIH Image</td><td>N/A</td><td>Image analysis software</td></tr><tr><td>Excel (Microsoft Office)</td><td>Microsoft Corporation</td><td>N/A</td><td>data analysis software</td></tr><tr><td>Origin9 / Origin2016</td><td>OriginLab Corporation</td><td>N/A</td><td>statistical data analysis and graphing software</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;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>OMCL-AC240TS</td><td>Olympus</td><td>OMCL-AC240TS</td><td>AFM cantilevers</td></tr><tr><td>grade V-5 muscovite</td><td>SPI Supplies</td><td>1805</td><td>mica sheets</td></tr><tr><td>Amicon Ultra 0.5 mL 50k Ultracell</td><td>Millipore Ireland Ltd.</td><td>UFC505096</td><td>centrifuge filters</td></tr><tr><td>NucleoSpin Extract II&amp;nbsp;</td><td>&amp;nbsp;Macherey-Nagel GmbH</td><td>740 609.250</td><td>Agarose gel extraction kit</td></tr><tr><td>Rotilabo cellulose paper type 111A</td><td>Carl Roth GmbH</td><td>AP59.1</td><td>AFM deposition blotting paper</td></tr><tr><td>Anatop 25 (0.02 &amp;mu;m)</td><td>Whatman GmbH</td><td>6809-2102</td><td>syringe filter</td></tr><tr><td>SSpI, BspQI</td><td>New England Biolabs (NEB)</td><td>R0132, R0712</td><td>restriction enzymes for DNA substrate preparation</td></tr><tr><td>XhoI, BglII</td><td></td><td>R0146, R0144</td><td>restriction enzymes for DNA preparation controls</td></tr><tr><td>nicking restriction enzyme Nt.BstNBI</td><td>New England Biolabs (NEB)</td><td>R0607</td><td>nickase</td></tr><tr><td>T4 DNA ligase</td><td>New England Biolabs (NEB)</td><td>M0202S</td><td>Ligase</td></tr><tr><td>Tris, HEPES</td><td>Carl Roth GmbH</td><td>4855, 9105</td><td>buffer chemicals</td></tr><tr><td>NaCl, MgCl&lt;sub&gt;2&lt;/sub&gt;, KCl, MgAcetate</td><td>Carl Roth GmbH</td><td>3957, HN03, HN02, P026</td><td>salt chemicals</td></tr><tr><td>NaAc</td><td>Sigma-Aldrich Chemie GmbH</td><td>32318</td><td>salt chemicals</td></tr><tr><td>DTT, TCEP, EDTA</td><td></td><td>6908, HN95, 8040</td><td>chemicals/reagents</td></tr><tr><td>agarose, acetic acid, HCl</td><td>Carl Roth GmbH</td><td>2267, 3738, K025</td><td>reagents</td></tr><tr><td>ATP</td><td>Carl Roth GmbH</td><td>K054</td><td>nucleotides</td></tr><tr><td>oligonucleotide #1 in Table 1</td><td>Biomers</td><td>custom</td><td>complementary DNA oligonucleotide</td></tr><tr><td>oligonucleotides #2, #3, and #6 in Table 1</td><td>Integrated DNA Technologies (IDT)</td><td>custom</td><td>fluorescein containing oligonucleotides</td></tr><tr><td>oligonucleotides #4&amp;nbsp; and #5 in Table 1</td><td>private (available from e.g. TriLink or GlenResearch)</td><td></td><td>CPD containing oligonucleotides</td></tr><tr><td>SafeSeal reaction tube 0.5 mL and 1.5 mL</td><td>Sarstedt</td><td>72.704 and 72.706</td><td>incubation tubes</td></tr><tr><td>GeneRuler 1 kb</td><td>Thermo Scientific</td><td>SM0311</td><td>DNA ladder</td></tr><tr><td>6x concentrate gel loading dye purple</td><td>New England Biolabs (NEB)</td><td>51406</td><td>DNA loading dye</td></tr><tr><td>Midori Green&amp;nbsp;</td><td>Nippon Genetics Europe GmbH</td><td>999MG28055</td><td>DNA stain</td></tr></tbody></table>

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.

Unterscheidung zwischen verschiedenen komplexen Typen auf der Basis von Protein komplexen Volumina Die Helicaseaktivität der prokaryotischen NER-Helicase UvrB wird durch DNA-Bindung 22 , 23 stimuliert. UvrB benötigt eine ungepaarte Region in der DNA (eine DNA-Blase), um korrekt auf einen der beiden einzelnen ssDNA-Stränge zu laden. In vivo wird diese DNA-Struk...

Watch this Scientific Journal Video about Atomic Force Microscopy Investigations of DNA Lesion Recognition in Nucleotide Excision Repair at JoVE.com

Rasterkraftmikroskopische Untersuchungen zur Erkennung von DNA-Läsionen bei der Reparatur von Nukleotid-Exzisionen | Genetik | Video

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

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.

Atommikroskopie Untersuchungen zur DNA-Läsionserkennung bei der 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 ...

atomic-force-microscopy-investigations-dna-lesion-recognition

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Unit 1

DNA Structure and Function

DNA Repair and Recombination

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9.4K Aufrufe.  University of Würzburg. 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.

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

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.

Anwendung von Mikro-Laserbestrahlung zur Prüfung von einzelnen und doppelt Strand Break Repair in Säugerzellen

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

Forschung

Krebsforschung

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

Laser Microirradiation In Vivo zellulären Reaktionen auf einfache und komplexe DNA-Schäden zu studieren

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

Genetik

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.

Untersuchung von Protein-Rekrutierung, DNA-Läsionen mit 405 Nm Micro-Laserbestrahlung

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.

Charakterisierung von DNA-Reparaturprozesse bei Transienten und langlebige Doppel-Strang DNA-Brüchen durch Immunfluoreszenz-Mikroskopie

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

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base excision repair (BER) molecular beacons. The substrate (beacon) is comprised of a deoxyoligonucleotide containing a single base lesion with a 6-Carboxyfluorescein (6-FAM) moiety conjugated to the 5'end and a Dabcyl moiety conjugated to the 3' end of the oligonucleotide. The BER molecular beacon is 43 bases in length and the sequence is designed to promote the formation of a stem-loop structure with 13 nucleotides in the loop and 15 base pairs in the stem1,2. When folded in this configuration the 6-FAM moiety is quenched by Dabcyl in a non-fluorescent manner via F&ouml;rster Resonance Energy Transfer (FRET)3,4. The lesion is positioned such that following base lesion removal and strand scission the remaining 5 base oligonucleotide containing the 6-FAM moiety is released from the stem. Release and detachment from the quencher (Dabcyl) results in an increase of fluorescence that is proportionate to the level of DNA repair. By collecting multiple reads of the fluorescence values, real-time assessment of BER activity is possible. The use of standard quantitative real-time PCR instruments allows the simultaneous analysis of numerous samples. The design of these BER molecular beacons, with a single base lesion, is amenable to kinetic analyses, BER quantification and inhibitor validation and is adaptable for quantification of DNA Repair activity in tissue and tumor cell lysates or with purified proteins. The analysis of BER activity in tumor lysates or tissue aspirates using these molecular beacons may be applicable to functional biomarker measurements. Further, the analysis of BER activity with purified proteins using this quantitative assay provides a rapid, high-throughput method for the discovery and validation of BER inhibitors.

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.

Quantitative, Echtzeit-Analyse der Base Excision Repair Aktivität in Zelllysaten Verwendung läsionsspezifischen Molecular Beacons

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base ...

Biologie

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 &#181;m, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

Visualisierung von miniSOG Tagged DNA Reparaturproteine ​​in Kombination mit Electron spektroskopischen Bildgebung (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

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

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

Fortgeschrittene Mikroskopiertechniken der konfokalen zur Untersuchung von Protein-Protein-Wechselwirkungen und die Kinetik bei DNA-Läsionen

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

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.

Visualisierung der DNA-Reparaturproteine Interaktion durch Immunfluoreszenz

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

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.

Laser-Mikrobestrahlung zur Untersuchung der DNA-Rekrutierung während der 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 ...

Nucleotide Excision Repair

<h4>Overview</h4>

Exposure to mutagens can damage DNA and result in bulky lesions that distort the double-helix structure or impede proper transcription. Damaged DNA can be detected and repaired in a process called nucleotide excision repair (NER). NER employs a set of specialized proteins that first scan DNA to detect a damaged region. Next, NER proteins separate the strands and excise the damaged area. Finally, they coordinate the replacement with new, matching nucleotides.

<h4>DNA distortion and damage</h4>

Cells are regularly exposed to mutagens&mdash;factors in the environment which can damage DNA and generate mutations. UV radiation is one of the most common mutagens and is estimated to introduce a significant number of changes to DNA. These include bends or kinks in the structure which can block DNA replication or transcription. If these errors are not fixed, the damage can cause mutations which in turn can result in cancer or disease depending on which sequences are disrupted.

<h4>Identification and repair of damaged regions</h4>

Nucleotide excision repair relies on specific protein complexes to recognize damaged regions of DNA and flag them for removal and repair. In prokaryotes, the process involves three proteins&mdash;UvrA, UvrB, and UvrC. The first two proteins work together as a complex, traveling along the DNA strands to detect any physical aberrations.

Once identified, the strands at the damaged location are separated, and endonuclease enzymes such as UvrC cut and excise the affected region. DNA polymerase fills the gap with new nucleotides, and then the DNA ligase enzyme seals the edges between the new and old DNA.

<h4>Mutations in NER have severe consequences</h4>

In prokaryotes, the NER complex consists of the three Uvr proteins, but in eukaryotes, more than a dozen proteins operate to regulate DNA repair. In humans, mutations in the NER pathway can cause diseases such as xeroderma pigmentosum (XP), which is associated with a 2000-fold increase in the incidence of skin cancer. Individuals suffering from XP are highly sensitive to UV exposure and can develop severe skin burns after just a few minutes of exposure to sunlight. Additionally, XP patients can show signs of premature aging and often develop neurological abnormalities. Without a properly working repair mechanism, DNA damage can accumulate and lead to abnormal cell death or potentially cancerous tumors.

DNA Damage and Nucleotide Excision Repair

Nukleotidexzisionsreparatur

Overview

Exposure to mutagens can damage DNA and result in bulky lesions that distort the double-helix structure or impede proper transcription. Damaged ...

Atommikroskopie Untersuchungen zur DNA-Läsionserkennung bei der Nucleotide Excision Repair

Zusammenfassung

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Laser Microirradiation In Vivo zellulären Reaktionen auf einfache und komplexe DNA-Schäden zu studieren

Untersuchung von Protein-Rekrutierung, DNA-Läsionen mit 405 Nm Micro-Laserbestrahlung

Charakterisierung von DNA-Reparaturprozesse bei Transienten und langlebige Doppel-Strang DNA-Brüchen durch Immunfluoreszenz-Mikroskopie

Quantitative, Echtzeit-Analyse der Base Excision Repair Aktivität in Zelllysaten Verwendung läsionsspezifischen Molecular Beacons

Visualisierung von miniSOG Tagged DNA Reparaturproteine in Kombination mit Electron spektroskopischen Bildgebung (ESI)

Fortgeschrittene Mikroskopiertechniken der konfokalen zur Untersuchung von Protein-Protein-Wechselwirkungen und die Kinetik bei DNA-Läsionen

Visualisierung der DNA-Reparaturproteine Interaktion durch Immunfluoreszenz

Laser-Mikrobestrahlung zur Untersuchung der DNA-Rekrutierung während der S-Phase

Nukleotidexzisionsreparatur

Atommikroskopie Untersuchungen zur DNA-Läsionserkennung bei der Nucleotide Excision Repair

Zusammenfassung

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Kapitel in diesem Video

Anwendung von Mikro-Laserbestrahlung zur Prüfung von einzelnen und doppelt Strand Break Repair in Säugerzellen

Laser Microirradiation In Vivo zellulären Reaktionen auf einfache und komplexe DNA-Schäden zu studieren

Untersuchung von Protein-Rekrutierung, DNA-Läsionen mit 405 Nm Micro-Laserbestrahlung

Charakterisierung von DNA-Reparaturprozesse bei Transienten und langlebige Doppel-Strang DNA-Brüchen durch Immunfluoreszenz-Mikroskopie

Quantitative, Echtzeit-Analyse der Base Excision Repair Aktivität in Zelllysaten Verwendung läsionsspezifischen Molecular Beacons

Visualisierung von miniSOG Tagged DNA Reparaturproteine ​​in Kombination mit Electron spektroskopischen Bildgebung (ESI)

Fortgeschrittene Mikroskopiertechniken der konfokalen zur Untersuchung von Protein-Protein-Wechselwirkungen und die Kinetik bei DNA-Läsionen

Visualisierung der DNA-Reparaturproteine Interaktion durch Immunfluoreszenz

Laser-Mikrobestrahlung zur Untersuchung der DNA-Rekrutierung während der S-Phase

Nukleotidexzisionsreparatur

Visualisierung von miniSOG Tagged DNA Reparaturproteine in Kombination mit Electron spektroskopischen Bildgebung (ESI)