우리는 GFP NTH1 식 플라스 미드에 대 한 토 호 쿠 대학, 일본에서 야 스도 아키라 박사 및 박사 에로스 Lazzerini Denchi 스 크립 스 연구소, 라 Jolla, 캘리포니아 TRF2 YFP EGFP 53BP11220-1711 식 플라스 미드에 대 한 감사합니다. 이 작품은 공군 사무실의 과학적 연구 (FA9550-04-1-0101)와 베크만 레이저 연구소 Inc. 재단 (M.W.B), 과학 (B.A.S), 하 고 NSF MCB 1615701 CRCC 국립 아카데미에서 포드 기초 장학금에 의해 지원 되었다 음악원-17-426665 (K. Y.)에.

저자는 공개 없다.

레이저 microirradiation DDR 연구에 대 한 사용의 장점은 다음과 같습니다.
1. 그것은 가능한 다양 한 종류를 유도 하 고 양의 DNA가 복잡 한 DNA 손상, 간단한 가닥 나누기에서 손상 감지 다른 수리 DNA에서 파손 사이트 레이저 조사 매개 변수를 조정 하 여. ( 그림 3)에서 트랜스 효과 평가 하기 위해 여러 번 같은 셀 핵에에서 피해를 입힐 수 이기도 합니다....

Vivo에서 DNA 손상 신호는 잘 이해 되지 Vivo에서, DNA는 히스톤과 양식 chromatin 섬유에 다른 요인 complexed. Chromatin 구조의 규정은 DNA 물질 대사에 대 한 파라마운트의 중요성입니다. 예를 들어 히스톤 변형 H2AX 증-오점이 돌연변이 (ATM)와 다음 이중 가닥 (DSB) 유도, 휴식과 DSB 손상 신호 증폭으로 다른 도킹 사이트를 제공 하는 것이 중요 하다 다른 kinases phosphorylated은 요인입니다. 손상 신호 및 복구 통로 선택의 확산 비판적으로 피해 사이트1에서 로컬 chromatin 구조에 의해 영향을 받을 것으로 보인다. 다양 한 요인, 히스톤 보호자 및 히스톤 수정 효소를 개장 하는 chromatin 실제로 사이트 손상 모집 DDR에서 chromatin 규제의 중요성을 강조 하는 효율적인 DNA 수리를 위해 중요 한 및 복구2 , 3 , 4. 또한, 피해 사이트 클러스터링 또는 재배치 효 모와 초파리5,6,7,8, relocalization에 유전자 loci의 연상에서 관찰 되었다는 subnuclear 구획 연관 유전자 규칙9,10. 마우스와 인간 세포에 있는 최근 연구는 또한 DSB 사이트, 어느 영향 복구 충실도 및 통로 선택11,12의 동원을 공개 했다. 이 DDR/수리도 연결 될 수 있습니다 속속들이 핵 건축, 더 높은 순서 chromatin 조직과 염색체를 세포 핵에 역학 가능성 제기. 따라서, DDR을 연구 하 고 DNA 손상의 단기 및 장기 결과 이해 하기 위해서는 살아있는 세포에서 핵 생 환경에 프로세스를 복구 고해상도 방법을 개발 하기 위해 중요 하다.
파 중 합 효소 (PARP) 손상의 유형과 정도 측정 하 고 규제 피해 사이트에 단백질 조립에의 중요 한 역할 PARP1는 DNA 수리13에서 중요 한 역할을 담당 하는 DNA 손상에 의해 빠르게 활성화 DNA 닉 센서입니다. PARP1 원래 x 선 수리 크로스 보완 1 (XRCC1) 기본 절단 수 선 (BER)를 촉진 하기 위하여 함께 작동 생각 하지만 최근 연구 결과 공개 DSB 수리14를 포함 하 여 다른 DNA 복구 경로에서 역할. 자신을 포함 한 여러 대상 단백질, ADP ribosylate는 기판으로 PARP1 사용 니코틴아미드 아데닌 디뉴클레오티드 (NAD+)을 활성화. 이 효소와 다른 가족 구성원 있다 관심을 끌었다 많은 최근 몇 년 동안에서 PARP 억제제는 암에 대 한 유망 치료 약물 등장 했습니다. PARP 억제제 처음 유 방 암 유전자 (BRCA)에 효과가 있어야 발견 했다 비록-유 방 암 세포를 돌연변이 지금 에이전트/조사에 대 한 손상 DNA와 함께 모노-및 조합 치료에 그들의 효과 대 한 증거의 과다 한입니다 BRCA15,,1617,18,19,20을 돌연변이와 암의 광범위 한 스펙트럼.
분자 수준에서 PARP 활성화 조직 손상 사이트에서 로컬 chromatin 구조에 중요 한 역할을 표시 했다. Chromatin 변경 효소의 파 종속 모집 DSB 수리를 용이 하 게 하 고 지시 복구 경로 선택, 파 수정 피해 사이트에서의 중요 한 발판 역할을 제안 합니다. 13,21,22,23,,2425,26,27,28,29, 30,31 우리는 최근 손상에서 p53 바인딩 단백질 1 (53BP1)의 배제를 시연 파 32, 비 동종 endjoining (53BP1-종속 hyperactivation에 대 한 대체 설명을 제공 하 여 사이트 NHEJ) 의해 PARP 억제제 및 DSB에 PARP의 중요성을 강조 복구 통로 선택33,34. PARP1 또한 PARylates 및 활동의 여러 DNA 복구 직접 요인14.
DDR/수리에서 Vivo에서 공부 하는 도구로 서 레이저 Microirradiation를 사용 하 여 개별 염색체에 서브 미크론 변경 생산 레이저 microirradiation 196935 에 설명 된 처음 이었고 198136에 자세히 검토. 몇 십년 후, 레이저 microirradiation 세포 핵에 정의 된 submicrometer 지역에서 DNA 손상 유도 표시 했다 고 모집 또는 수정 다양 한 요인의 DNA 병 변 비보 를 연구 하는 귀중 한 기술 입증 되었다 13 , 37 , 38 , 39 , 40 , 41.이 메서드는 손상 사이트39,42에서 뚜렷한 방사선-유도 포커스 (IRIF)를 형성 하지 않습니다 그 요인의 탐지를 수 있습니다. 그것은 또한 손상 사이트에서 고 핵의 나머지에서 chromatin 구조 변화 spatiotemporal 역학 검사 가능. 우리는 신중 하 게 다른 레이저 시스템에 의해 유도 된 Ddr에 비해 DNA 손상의 유형 및 microirradiation 조건32,,4344, 사이의 관계를 평가 하는 능력을 입력 하 고 45. 53BP1 및 telomeric 탈 채용 패턴 반복 바인딩 요소 2 (TRF2) 레이저 유도 손상46의 "비 생리" 자연에 대 한 되풀이 우려에 대 한 기초를 제공 하는 이전 레이저 손상 연구에서 관찰 되었다 ,47,,4849. 우리는 양과 복잡성 유발된 손상32의 계기 신호 이러한 명백한 불일치 이제 차동 PARP에 의해 설명 될 수 있는 발견. 우리 확인: 1) (높은 입력 전력 조사) 후에 레이저 microirradiated 세포 손상 검사점 제어 종속 방식에서 interphase에 체포와 유지 가능한 (적어도 48 h까지)32,50; 그리고 2) 수리 계수 모집/수정 충실 하 게 그 endonucleases32,,3942에 의해 기존의 DNA 파괴적인 대리인을 가진 처리와 DSB 유도 관찰 정리 44,50,,5152. 이러한 결과 강하게 레이저 손상 유발 세포 응답의 생리 적인 관련성을 지원 합니다.

<table><tbody><tr><td>Ti:Sapphire NIR pulsed femtosecond laser Mira-900</td><td>Coherent Inc.</td><td>Mira 900</td><td></td></tr><tr><td>Inverted microscope&amp;nbsp;</td><td>Carl Zeiss</td><td>Axiovert 200M</td><td></td></tr><tr><td>63X/1.4 NA objective</td><td>Zeiss</td><td>APOCHROMAT Ph3&amp;nbsp;</td><td></td></tr><tr><td>Compact Rotation Stage&amp;nbsp;</td><td>Newport Corp</td><td>PR50PP</td><td></td></tr><tr><td>Temperature Controller</td><td>Warner</td><td>TC-344B</td><td></td></tr><tr><td>Heating System</td><td>Ibidi&amp;nbsp;</td><td>10918</td><td></td></tr><tr><td>Gas Incubation System</td><td>Ibidi&amp;nbsp;</td><td>11920</td><td></td></tr><tr><td>Laser Power and Energy Meter (RoHS)</td><td>Coherent</td><td>FieldMaxII-TOP&amp;nbsp;</td><td></td></tr><tr><td>ORCA-R2 Digital CCD Camera</td><td>Hamamatsu</td><td>C10600</td><td></td></tr><tr><td>LSM 510 META Laser Scanning Microscopes</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>100X/1.3 NA Zeiss Plan APO</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>35mm Gridded Dishes&amp;nbsp;</td><td>MatTek</td><td>P35G-1.5-14-CGRD-D</td><td></td></tr><tr><td>PtK2 kidney epithelial cells</td><td>ATCC</td><td>CCL 56&amp;nbsp;</td><td></td></tr><tr><td>HeLa cells</td><td>ATCC</td><td>CCL-2</td><td></td></tr><tr><td>DMEM</td><td>Life Technologies</td><td>11885-092</td><td></td></tr><tr><td>CO2-Independent Medium&amp;nbsp;</td><td>Life Technologies</td><td>18045-088</td><td></td></tr><tr><td>Advanced MEM&amp;nbsp;</td><td>Life Technologies</td><td>12492-013</td><td>supplemented with L-Glutamine, 4% FBS</td></tr><tr><td>penicillin/streptomycin</td><td>Fisher Scientific</td><td>15140122</td><td></td></tr><tr><td>L-Glutamine</td><td>Fisher Scientific</td><td>25030081</td><td></td></tr><tr><td>FBS</td><td>Omega Scientific</td><td>FB-02</td><td></td></tr><tr><td>Thymidine&amp;nbsp;</td><td>&amp;nbsp;SIGMA</td><td>T9250</td><td></td></tr><tr><td>serum free media&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (Opti-MEM I Reduced Serum Media)</td><td>Fisher Scientific</td><td>11058021</td><td></td></tr><tr><td>Anti-Cycolbutance pyrimidine dimer (CPD) (mouse)</td><td>Kamiya Biomedical Company</td><td>MC-062</td><td></td></tr><tr><td>Anti-XRCC1 (mouse )</td><td>Gene Tex Inc</td><td>GTX72311</td><td></td></tr><tr><td>Anti-53BP1 (rabbit)</td><td>Santa Cruz Biotech</td><td>sc-22760</td><td></td></tr><tr><td>Anti-CtIP (rabbit)</td><td>Abcam</td><td>ab70163</td><td></td></tr><tr><td>Anti-PAR polymers (mouse)</td><td>Enzo Life Sciences</td><td>BML-SA216-0100</td><td></td></tr><tr><td>Anti-PAR (rabbit)</td><td>Trevigen</td><td>4336-BPC-100</td><td></td></tr><tr><td>Anti-TRF2 (mouse)</td><td>Novus Biological&amp;nbsp;</td><td>NB100-56506</td><td></td></tr><tr><td>Anti 6-4PP (mouse)</td><td>Kamiya Biomedical</td><td></td><td></td></tr><tr><td>Anti-Rad21 (Rabbit)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (for cohesin detection)</td><td>generated in Yokomori (KY) lab</td><td></td><td></td></tr><tr><td>Anti-Rad51 (Rabbit)</td><td>Santa Cruz Biotechnology</td><td>SC-8349</td><td></td></tr><tr><td>Anti-Ku70 (mouse)</td><td>&amp;nbsp;Novus Biologicals</td><td>NB100-102</td><td></td></tr><tr><td>8-oxiguanine&amp;nbsp;</td><td>Trevigen, Inc.</td><td></td><td></td></tr><tr><td>Anti-PARP1&amp;nbsp; (Rabbit)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>generated in KY lab</td><td></td><td>ref 43</td></tr><tr><td>&amp;nbsp;Anti-hCAPG (Rabbit)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (for condensin detection)</td><td>generated in KY lab</td><td></td><td>ref 42</td></tr><tr><td>Cy3 AffiniPure Donkey Anti-Rabbit IgG</td><td>&amp;nbsp;Jackson ImmunoResearch Inc</td><td>711-165-152</td><td></td></tr><tr><td>Donkey Anti-Mouse Alexa Fluor 488 IgG</td><td>Thermo Fisher Scientific</td><td>A-21202</td><td></td></tr><tr><td>HiPerFect siRNA Transfection Reagent</td><td>Qiagen</td><td>301705</td><td></td></tr><tr><td>Lipofectamine 2000 Transfection Reagent</td><td>Thermo Fisher Scientific</td><td>11668019</td><td></td></tr><tr><td>GFP-SMC1 stable cell line&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>generated in KY lab</td><td></td><td>ref 49&amp;nbsp;</td></tr><tr><td>GFP-NEIL2 stable cell line</td><td>generated in KY lab</td><td></td><td>ref 54</td></tr><tr><td>GFP-NTH1 stable cell line</td><td>generated in KY lab</td><td></td><td>ref 32</td></tr><tr><td>GFP-SMC1 plasmid</td><td>generated in KY lab</td><td></td><td></td></tr><tr><td>GFP-Ku plasmid</td><td>generated in KY lab</td><td></td><td></td></tr><tr><td>Anti-MDC1 (rabbit)</td><td>&amp;nbsp;Novus Biologicals</td><td>NB100&amp;ndash;395</td><td></td></tr><tr><td>Anti-gH2AX (rabbit)</td><td>&amp;nbsp;Millipore</td><td>07&amp;ndash;164</td><td></td></tr><tr><td>EGFP-53BP1(1220-1711) PtK2 stable cell line</td><td>generated in Berns lab</td><td></td><td>ref 32</td></tr><tr><td>TRF2-YFP PtK2 stable cell line</td><td>generated in Berns lab</td><td></td><td>ref 32</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650-100ML</td><td></td></tr><tr><td>PARG inhibitor</td><td>Trevigen</td><td>4680-096-03</td><td></td></tr><tr><td>DNA&amp;ndash;PKcs inhibitor&amp;nbsp; NU7026&amp;nbsp;</td><td>Sigma</td><td>N1537</td><td></td></tr><tr><td>ATM inhibitor KU55933</td><td>&amp;nbsp;Calbiochem</td><td>118500</td><td></td></tr><tr><td>Olaparib&amp;nbsp;</td><td>Apexbio Technology</td><td>A4154</td><td></td></tr><tr><td>Paraformaldehyde&amp;nbsp;</td><td>ELECTRON MICROSCOPY SCIENC MS</td><td>100503-916 (EA)</td><td></td></tr><tr><td>Quantity One 1-D Analysis Software Version 4.6.9</td><td>Bio-Rad</td><td>SOFT-LIT-70-9600-Q1-469PC&amp;nbsp;</td><td>image analysis program</td></tr><tr><td>Excel</td><td>microsoft</td><td></td><td>spreadsheet program</td></tr><tr><td>Triton X100</td><td>Fisher Scientific</td><td>BP151-500</td><td>detergent</td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline (DPBS) 10X without calcium and magnesium</td><td>Fisher Scientific</td><td>14200166</td><td>dilute to 1X&amp;nbsp;</td></tr></tbody></table>

laser microirradiation to study in vivo cellular responses to simple and complex dna damage

DNA 손상 유도 게놈 무결성의 보호에 대 한 중요 한 셀에 특정 신호 및 복구 응답 합니다. 레이저 microirradiation 귀중 한 실험 도구는 DNA 손상 응답 (DDR) vivo에서조사 되었다. 레이저 유도 손상 세포 핵에 있는 submicrometer 지역에 대 한 응답에서 고분자 역학의 실시간 고해상도 단일 세포 분석을 수 있습니다. 그러나, 다양 한 레이저 조건 유도 된 손상의 종류에 있는 다름의 감사 없이 사용 되었습니다. 그 결과, 피해의 특성은 종종 잘 특징 또는 제어, 채용 또는 수정 프로필에 명백한 불일치를 일으키는. 우리는 다른 방사선 조건 (즉, 다른 파장으로 펨 (fs) 근처-적외선 (NIR) 레이저의 다른 입력된 파워 (irradiances)) 고유 DDR 및 복구 단백질 어셈블리 유도 시연 했다. 이 생산 하는 DNA 손상의 종류를 반영 한다. 이 프로토콜에 설명 합니다 어떻게 레이저 입력된 전력의 적정 수 다른 금액의 유도 및 자료 및 가교 손해, 신호 처리, 차동 폴 리 (ADP-ribose) (파)의 탐지에 의해 쉽게 모니터링할 수 있습니다, DNA 손상의 복잡 하 고 피해 사이트에서 경로 특정 복구 요소 어셈블리입니다. 손상을 조건 결정, 일단 관심의 어떤 요소에 업스트림 factor(s)의 고갈 뿐만 아니라 다른 손상 복잡성과 차동 손상 신호 전달의 효과 조사 가능 하다.

안정적으로 표현 EGFP 53BP11220-1711 또는 TRF2 YFP PtK2 셀을 사용 하 여, 레이저 입력된 전원 적정 실시 되었다 그들의 모집 (그림 1)32에 대 한 최적의 상태를 확인 하려면.
높은 입력 파워 레이저 피해 사이트에서 구체적으로 인식 하는 베이스 손상 GFP NTH1 DNA glycosylase 뿐만 아니라 일일 (가교 손상 자외선 (UV) 손상에 의해 일반적으로 생...

Watch this Scientific Journal Video about Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage at JoVE.com

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

이 프로토콜의 목표 레이저 microirradiation를 사용 하 여 다른 유형의 DNA 손상, 비교적 간단한 가닥 나누기 등 복잡 한 손상, DNA 손상 신호 및 복구 요소에서 어셈블리 피해 사이트 vivo에서 공부를 유도 하는 방법을 설명 하는 것입니다. .

간단 하 고 복잡 한 DNA 손상에 세포질 응답 Vivo에서 공부 레이저 Microirradiation

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

laser-microirradiation-to-study-vivo-cellular-responses-to-simple

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

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

10.2K 조회수.  University of California, Irvine. 이 프로토콜의 목표 레이저 microirradiation를 사용 하 여 다른 유형의 DNA 손상, 비교적 간단한 가닥 나누기 등 복잡 한 손상, DNA 손상 신호 및 복구 요소에서 어셈블리 피해 사이트 vivo에서 공부를 유도 하는 방법을 설명 하는 것입니다. .

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

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell&#39;s DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

We describe a robust gene replacement strategy to genetically manipulate the smut fungus Ustilago maydis. This protocol explains how to generate deletion mutants to investigate infection phenotypes. It can be extended to modify genes in any desired way, e.g., by adding a sequence encoding a fluorescent protein tag.

플랜트 병원체의 유전자 조작<em&gt; Ustilago maydis</em&gt; 곰팡이 생물과 식물 미생물 상호 작용을 연구하는

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

연구

유전학

Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, such as transcription, DNA replication, and DNA repair. HMGB1 binds to structurally distorted DNA with higher affinity than to canonical B-DNA. For example, we found that HMGB1 binds to DNA interstrand crosslinks (ICLs), which covalently link the two strands of the DNA, cause distortion of the helix, and if left unrepaired can cause cell death. Due to their cytotoxic potential, several ICL-inducing agents are currently used as chemotherapeutic agents in the clinic. While ICL-forming agents show preferences for certain base sequences (e.g., 5&#39;-TA-3&#39; is the preferred crosslinking site for psoralen), they largely induce DNA damage in an indiscriminate fashion. However, by covalently coupling the ICL-inducing agent to a triplex-forming oligonucleotide (TFO), which binds to DNA in a sequence-specific manner, targeted DNA damage can be achieved. Here, we use a TFO covalently conjugated on the 5&#39; end to a 4&#39;-hydroxymethyl-4,5&#39;,8-trimethylpsoralen (HMT) psoralen to generate a site-specific ICL on a mutation-reporter plasmid to use as a tool to study the architectural modification, processing, and repair of complex DNA lesions by HMGB1 in human cells. We describe experimental techniques to prepare TFO-directed ICLs on reporter plasmids, and to interrogate the association of HMGB1 with the TFO-directed ICLs in a cellular context using chromatin immunoprecipitation assays. In addition, we describe DNA supercoiling assays to assess specific architectural modification of the damaged DNA by measuring the amount of superhelical turns introduced on the psoralen-crosslinked plasmid by HMGB1. These techniques can be used to study the roles of other proteins involved in the processing and repair of TFO-directed ICLs or other targeted DNA damage in any cell line of interest.

Targeted DNA damage can be achieved by tethering a DNA damaging agent to a triplex-forming oligonucleotide (TFO). Using modified TFOs, DNA damage-specific protein association, and DNA topology modification can be studied in human cells by the utilization of modified chromatin immunoprecipitation assays and DNA supercoiling assays described herein.

도구 나선 왜곡, 사이트 별 DNA Interstrand 가교의 처리에 건축 단백질 HMGB1의 역할을 연구하는

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, ...

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

펩타이드 유래의 방법은 식물의 유전자와 단백질 세포 간 및 Organellar 장벽을 수송하는

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

생리 산소 조건에 노출 된 인간 세포의 캡 결합 단백질의 분석

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

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

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Here, we describe a chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation protocol to generate global epigenomic profiles from low-abundance chicken embryonic samples.

낮은 풍부 배아 샘플 chromatin Immunoprecipitation (칩) 프로토콜

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

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 ...

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.

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 ...

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 ...