PUC19N、CPD含有オリゴヌクレオチド、およびp44は、Samuel Wilson、Korbinian HeilおよびThomas Carell、およびGudrun MichelsおよびCaroline Kiskerによりそれぞれ提供された。この研究は、ドイツForschungsgemeinschaft（DFG）FZ82およびTE-671/4からITへの助成金によって支えられました。

<ol>
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著者は何も開示することはない。

特定の標的部位を含む長いDNA断片上のタンパク質の結合位置のAFM統計分析は、これらの部位2,3,4,5,6を認識するためにタンパク質が用いる特定の戦略に関する興味深い詳細を明らかにすることができる。得られた位置分布を解釈するために、DNA中の標的の位置を正確に知る必要がある。これは、明確な配列位置に特異的部位を環状プラスミドDNAに導入し、この特異的部位を含むDNAの断片を制限?...

原子間力顕微鏡（AFM）は、タンパク質-DNA相互作用1,2,3,4,5,6,7,8,9の解析に強力な技術です。単一分子レベルの分解能で異種試料を直接視覚化するには、少量の試料材料しか必要としない。異種性は、タンパク質の異なる立体配座またはオリゴマー状態から生じ得る。特に、タンパク質-DNA試料に関しては、タンパク質複合体は、一般にDNA結合によって誘導されるか、またはDNA内の特定の標的部位に結合する異なる化学量論および/またはコンホメーションを示すことができる。異種試料はまた、2種類（またはそれ以上）の異なる種類のタンパク質および異なるタンパク質複合体形態（ 例えば 、1つのタイプのタンパク質対ヘテロメリック複合体のみからなる）はDNAと異なって相互作用することができる。ここで論じた研究は、これらのタンパク質の標的を表す病変を含む長い（約900塩基対、bp）DNA断片に結合したDNA修復タンパク質の静的乾燥サンプルの空気中でのAFMイメージングを利用する。 AFMの高い分子分解能は、異なるタイプのタンパク質複合体の間の区別を可能にし、DNA断片上のタンパク質の結合位置を決定する。重要なことに、病変は、明確な位置でDNA基質に導入される。 DNA中の病変部位の位置は既知であるので、DNAに結合したタンパク質の分布は、（異なる）タンパク質複合体の（異なる）病変認識特性、 例えばそれらが特定のタイプの病変をどれくらいよく認識するか（ 例えば 、非損傷DNAに対する） <supclass = &quot;xref&quot;&gt; 4,5,6。 DNA上のそれらの位置はまた、病変部に特異的に結合したタンパク質複合体とDNA上の他の場所に非特異的に結合した複合体との区別を可能にする。これらの異なる複合型（病変部と非特異的複合体に特異的に結合した複合体）の別個の特徴付けは、標的部位の同定に誘導された複合体における立体配座変化を明らかにする可能性がある。 ここで焦点を当てたDNA修復タンパク質は、ヌクレオチド切除修復（NER）経路における病変認識の原因となるヘリカーゼである。細菌では、NERはタンパク質UvrA、UvrBおよびUvrCによって達成される。 UvrAは、UvaA 2 / UvrB 2 DNAスキャン複合体における初期病変感知の原因である。 UvrBによる病変確認の際、この複合体は病変部位で結合した単量体UvrBに変換され、この特定の複合体はp原核生物NERエンドヌクレアーゼUvrC。 UvrCは、病変を含む一本鎖DNA（ssDNA）の短い（12-13nt）ストレッチを切除する。失われたストレッチは、DNAポリメラーゼによって再充填される。最後に、DNAリガーゼは新しく合成されたストレッチを元のDNA 9,10 で密封する。真核生物では、NERカスケードの大部分のタンパク質は、大きな多量体転写因子II H（TFIIH）複合体の一部である。三量体CEN2-XPC-HR23B複合体による初期病変感知の後、TFIIHをDNA標的部位に補充する。複合体内のXPDがNER標的病変の存在を確認すると、真核生物のNERエンドヌクレアーゼXPGおよびXPFが病変を含むssDNAの短い（24〜32nt）ストレッチを切除するために補充される9,10 。ここでは、具体的には、原核生物および真核生物NER由来のヘリカーゼUvrBおよびXPDをそれぞれ研究した。これらのヘリカーゼは、2つのDNA一本鎖のうちの1つに糸を通し、続いてATP加水分解によってこの鎖に沿って転位するDNA（DNAバブル）。したがって、DNA損傷に加えて、DNAバブルが、タンパク質の負荷部位として機能する基質に導入された。 特異的病変DNA基質の調製手順は以前に記載されている11 。それは、ニカナーゼのための2つの密接に隔てられた制限部位を有する環状DNA構築物（プラスミド）を必要とする。この研究の文脈では、プラスミドpUC19N（2729bp）を使用した（S. Wilsonの実験室、NIEHSによって作製された）。このプラスミドは、48ヌクレオチド（nt）のストレッチを構成するニカナーゼNt.BstNBIの3つの密接に隔てられた制限部位を含む。ニカナーゼとのインキュベーション後、これらの部位間のssDNAのストレッチを除去し、任意の標的特性を含むオリゴヌクレオチドで置き換えることができる。各工程の後、完全な酵素消化をアガロースゲル電気泳動。ニッキングされた環状DNAは、元のスーパーコイルプラスミドと比較してその電気泳動移動度が低いために区別することができる。 DNAのギャップ付けおよび除去されたストレッチの特定の基質オリゴヌクレオチドによる置換は、基質のみをニックの間の領域内で切断する制限酵素による消化によって評価することができる。したがって、酵素による環状プラスミドの直線化は、ギャップを有するDNAについて抑制され、特異的オリゴヌクレオチドの挿入後に回復する。最後に、2つのエンドヌクレアーゼ制限部位（理想的には単一のカッター）は、線状DNA基質の生成を可能にし、長さは所望の通りであり、特定の標的部位と定義された位置にDNA泡があり、 &#39;または3&#39;方向。 NERヘリカーゼによる病変の認識は、AFMイメージングによって調べることができる。 tにおけるヘリカーゼのDNA転位の停止病変部位は、DNA上のタンパク質位置分布のピークとして見え、病変認識を示す。これらのヘリカーゼのDNA転位はさらに5 &#39;から3&#39;の極性で方向性があるため、装填部位（病変の上流または下流のDNAバブル）の位置に対する病変認識の依存性も病変が優先的に認識されるかどうかを示す転位されたまたは反対の、非転座のssDNA鎖5,9。以下のセクションでは、使用される方法が紹介され、これらの実験からの主要な所見が簡単に議論される。重要なことに、ここに示すDNA修復の例示的な研究に類似して、AFMイメージングは​​、DNA複製または転写などの異なるDNA相互作用系の研究に適用することができる8,12,13,14 </。

<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.

タンパク質複合体の体積に基づいて異なる複合型を区別する原核生物のNERヘリカーゼUvrBのヘリカーゼ活性は、DNA結合によって刺激される22,23 。 UvrBは、2つの単一のssDNA鎖のうちの1つに正確にロードするために、DNA（DNAバブル）中に不対領域を必要とする。 インビボでは、このDNA構造は、NERカスケー?...

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

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研究 |遺伝学 |ビデオ

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.

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 ...

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 ビュー.  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.

ビデオ: 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.

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

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.

生体内で単純および複雑な DNA 損傷に対する細胞応答を研究するレーザー Microirradiation

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

遺伝学

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 ...

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.

病変の特異的分子ビーコンを活用した細胞ライセート中の塩基除去修復活性の定量、リアルタイム分析

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

生物学

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.

電子分光イメージングと組み合わせたminiSOGタグ付きのDNA修復タンパク質の可視化（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.

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 ...

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 ...

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 ...

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

ヌクレオチド切断修復

Overview

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

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

要約

さらに動画を探す

この動画の章

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

生体内で単純および複雑な DNA 損傷に対する細胞応答を研究するレーザー Microirradiation

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

病変の特異的分子ビーコンを活用した細胞ライセート中の塩基除去修復活性の定量、リアルタイム分析

電子分光イメージングと組み合わせたminiSOGタグ付きのDNA修復タンパク質の可視化（ESI）

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

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

ヌクレオチド切断修復

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

要約

さらに動画を探す

この動画の章

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

生体内で単純および複雑な DNA 損傷に対する細胞応答を研究するレーザー Microirradiation

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

病変の特異的分子ビーコンを活用した細胞ライセート中の塩基除去修復活性の定量、リアルタイム分析

電子分光イメージングと組み合わせたminiSOGタグ付きのDNA修復タンパク質の可視化（ESI）

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

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

ヌクレオチド切断修復

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