이 작품의 체코 공화국, 프로젝트 P302-12-G157 부여 기관에 의해 지원 되었다. 실험 또한 체코 노르웨이어 연구 프로그램 CZ09, 노르웨이 자금, 그리고 교육, 청소년 및 스포츠의 체코 공화국에 의해 감독에 의해 지원 되었다 (허가 번호: 7F14369).

<ol>
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저자 아무 충돌 다는 것을 선언 합니다.

현미경 검사 법 기술 연구 실험실에서 기본 도구를 나타냅니다. 여기, 단백질 모집의 연구에 대 한 사용 하는 방법에 대 한 간략 한 설명을 하 고 속도 론 DNA 병 변에서 선물 된다. 우리는 특히 살아있는 세포의 지역 microirradiation의 분야에서 우리의 실험 경험을 언급 하 고 우리가 FRAP 및 단백질-단백질 상호 작용 수락자 표백 무서 워28 에 의해 DNA 병 변 및 그것의 첨단에 의해 단백?...

구성 된 cyclobutane pyrimidine 이합체 (CPDs), 8-oxo-7,8-dihydro-2'-deoxyguanosine, 및 단일 가닥 DNA 변의 모양을 이끌어 DNA 손상 또는 더블-스트랜드 나누기1,2. Γ 광선은 높은 에너지와 높은 penetrance 이온화 방사선의 형태, 따라서 방사선의이 소스는 방사선 치료3에서 널리 이용 된다. 다른 한편으로, 실험적으로 UV 레이저 모방 자연 UV 빛에 노출으로 인 한 DNA 손상을 유도 한다. 현미경 방법으로 UVA microirradiation은 개별 살아있는 세포에서 DNA 손상 연구 실험 도구를 나타냅니다. Microirradiation은 염색체 지구4,5의 조직 공개 40 년 전에 처음으로 사용 되었다. 이 기술은 매우 중 confocal 현미경의 기능 특성 또는 현대 nanoscopy의 기술적인 한계에 종속 됩니다. DNA 병 변을 유도 하 세포 5' bromodeoxyuridine (BrdU) 또는 Hoechst 33342 UV 조사 전에 presensitized 수 있습니다. Bártová 외. 6 이전 presensitization 단계를 설명 하 고 최근 우리 세포 죽음, 또는 apoptosis를 피하기 위하여이 microirradiation 기술에 최적화 된. 예를 들어 (Hoechst 33342 presensitization) 없이 405 nm 자외선 레이저를 사용 하 여 53BP1-긍정적인 이중 가닥 틈 (DSBs)의 유도 cyclobutane pyrimidine 이합체 (CPDs)의 비용으로 리드. 다른 한편으로, UV microirradiation와 함께 presensitization 단계 유도 CPDs과 DSBs의 매우 높은 수준을 동시에7,8. 이 방법은 단일 DNA 수 선 통로의 연구에 적용 하기가 어렵습니다.
Microirradiation, 단백질 모집, 활동, 및 살아있는 세포 병 변 DNA에서 상호 작용 분석 가능 하다. 이 방법의 예로 Luijsterburg 그 외 여러분 에 의해 출판 되었음 9 섬으로 단백질 1β, 그리고 우리에 대 한 최근 pluripotency 요소 Oct4 Cajal 몸, coilin와 관련 된 단백질 DNA UV 유도 된 병 변6,10모집은 처음으로 나타났다. 단백질이 DNA 병 변에서 활동 또한 FRAP (복구 후 Photobleaching 형광)11,,1213 를 사용 하 여 공부 될 수 있다 또는 (형광 공명 에너지 전송) 기술14 무서 워 ,15. 이러한 방법은 병 변 DNA 또는 단백질 단백질 상호 작용 단백질의 간단한 확산을 가능성이 있다. 단백질의 추가 특성에 대 한 유용한 도구가입니다 FLIM (형광 일생 화상 현미경) 또는 그것의 함께 무서 워 기술 (무서 워-FLIM)16. 이 메서드는 셀을 안정적으로 생활 또는 형광 분자17에 의해 표를 붙인 관심사의 단백질을 뚜렷이 표현 프로세스의 연구를 사용. 여기, GFP 태그 p53 단백질 및 그것의 상호 작용 협동자, mCherry 태그 53BP1, DNA 손상 응답18,19 에 있는 중요 한 역할에 대 한 지 수 감퇴 시간 (τ)의 예가 표시 됩니다. 매개 변수 τ FLIM 계산에서 제공 하는 형광 색소의 일생은 주어진된 형광 염료, 그것의 바인딩 능력 및 그것의 세포질 환경에 대 한 특정 이다. 따라서,이 방법은 표시할 수 있습니다 우리 단백질 부분 모집단, 그들의 바인딩 능력, 및 그들의 기능 속성 사이의 구분 후, 예를 들어 DNA 손상.
여기, 우리의 실험실에 시간 특정 단백질 모집, 속도 론, 확산, 및 microirradiated chromatin의 사이트에서 단백질 단백질 상호 작용을 공부 하는 데 사용 되는 고급 현미경 기술의 방법론 접근의 개요 제공 됩니다. 살아있는 세포에서 로컬 DNA 변의 유도 및 UV 레이저에 의해 발생 하는 로컬 유발된 DNA 병 변에서 DNA 손상 관련 이벤트의 연구에 대 한 유용한 방법론의 설명에 대 한 단계별 방법을 제공 합니다.

<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

레이저와 로컬 microirradiation 라이브 세포에서 DNA 복구 관련 프로세스의 연구에 대 한 유용한 도구를 나타냅니다. 여기, 우리가 로컬로 microirradiated chromatin에서 시간 또는 단백질 단백질 상호 작용을 통해 단백질 DNA 병 변에서 활동을 분석 하는 방법론 접근 방식을 설명 합니다. 우리 또한 세포 주기 세포 주기 의존적인 단백질 DNA 병 변에서 속도 론을 공부 Fucci 셀룰러 시스템을 사용 하 여 개별 단계를 인식 하는 방법을 보여 줍니다. 2 자외선 레이저를 사용 하 여의 방법론 설명 (355 nm, 405 nm) 다른 유형의 DNA 손상 유도 하는 것 또한 제공 됩니다. 만 405 nm 다이오드 레이저 셀 microirradiated 유사 분열을 통해 정상적으로 진행 하 고 cyclobutane pyrimidine 이합체 (CPDs) 없는 했다. 우리는 또한 어떻게 microirradiated 셀 관심의 내 생 단백질의 immunodetection를 수행 하기 위해 주어진된 시간에 고쳐질 수 있다 보여줍니다. 또한 생물 방법 FRAP (복구 후 Photobleaching 형광) 및 FLIM (형광 일생 화상 현미경 검사 법)를 포함 하 여 자연 발생 된 셀에서의 사용 설명 DNA 수 선 연구, DNA 손상 포커스. 우리는 또한 단백질 단백질 상호 작용의 실험 연구에 FLIM-무서 워 (형광 공명 에너지 전송)의 응용 프로그램을 보여줍니다.

고급 confocal 현미경 검사 법을 사용 하 여, DNA 병 변에서 mCherry 태그의 53BP1 및 mCherry PCNA 단백질의 축적을 관찰 합니다. 분석은 살아있는 세포의 지역 microirradiation에 의해 수행 되었습니다. 우리는 초기 S, g 1을 확인 하는 Fucci 셀룰러 시스템을 사용 하는 개별 세포 주기 단계에서 DNA 복구 관련 단백질의 핵 배포 패턴을 인식 하 고 셀의 G2 단계 주기 (그림 1</stron...

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

레이저 microirradiation는 살아있는 세포에서 DNA 수리의 연구에 대 한 유용한 도구입니다. 다양 한 DNA 병 변 유발 하 UVA 레이저의 사용에 대 한 방법론 접근 표시 됩니다. 우리 지역 microirradiation; 정상적인 세포 주기를 유지 하는 방법을 최적화합니다 따라서, 비친된 세포 유사 분열을 통해 진행합니다.

고급 단백질 단백질 상호 작용 및 DNA 병 변에서 속도 론 연구에 Confocal 현미경 검사 법 기술

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

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10.9K 조회수.  Institute of Biophysics of the Czech Academy of Sciences. 레이저 microirradiation는 살아있는 세포에서 DNA 수리의 연구에 대 한 유용한 도구입니다. 다양 한 DNA 병 변 유발 하 UVA 레이저의 사용에 대 한 방법론 접근 표시 됩니다. 우리 지역 microirradiation; 정상적인 세포 주기를 유지 하는 방법을 최적화합니다 따라서, 비친된 세포 유사 분열을 통해 진행합니다.

비디오: 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.

핵 포스 현미경 검사에 의한 DNA 절편 검사에서의 DNA 병변 인식 연구

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

연구

유전학

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.

레이저 지역화 소라렌 부가 물의 단일 분자 분석

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.

단일의 검사 및 포유류 세포에서 이중 가닥 브레이크 수리에 대 한 레이저 마이크로-방사선의 응용

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

암 연구학

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.

ΓH2AX와 53BP1 형성 및 DNA 두 배 물가 틈의 수리 분석에 대 한 면역 형광 검사 현미경 검사 법

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 Assay를 이용한 현탁 세포 배양에서의 DNA 손상 유발 단백질 복합체의 검출 및 가시화

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

생화학

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.

단백질 DNA 장애 405 Nm 레이저 마이크로 방사선 조사를 사용 하 여 채용의 조사

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.

면역 형광 검사 현미경 검사 법에 의해 변이 및 긴-지속 이중 가닥 DNA 틈에 DNA 복구 과정을 특성화

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.

면역형광에 의한 DNA 수리 단백질 상호작용의 시각화

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

생물학

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 단계 동안 DNA 모집을 연구하는 레이저 미세 조사

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 Double-Strand Break 분석을 위한 면역형광 현미경 검사

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. ...