Name: tools to study the role of architectural protein hmgb1 in the processing of helix distorting, site-specific dna interstrand crosslinks
Uploaded: 2016-11-10T13:00:00
Description: Watch this Scientific Journal Video about Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks at JoVE.com

저자는 도움이 토론 바스케즈 실험실의 구성원을 감사드립니다. 이 작품은 [KMV하는 CA097175, CA093279] 건강 / 국립 암 연구소 (National Cancer Institute)의 국립 연구소에 의해 지원되었다; 암 예방 및 텍사스 연구소 [RP101501]합니다. 오픈 액세스 요금 자금 : 국립 연구소 건강 / 국립 암 연구소 [KMV에 CA093279].

플라스미드 기판에 TFO 지시 ICLS 1. 준비 <ol><li> 10,11 DNA 삼중 결합 완충액 [50 % 글리세롤, 10 mM 트리스 (PH 7.6), 10 mM의를 MgCl2 8 μL에 랄렌 공역 TFO AG30와 (5 μg의 플라스미드 DNA 좋은 출발점) 플라스미드 pSupFG1 몰량 부화 2] 호박색 튜브에서 40 μL의 최종 부피 DH 2 O. 로 pipetting 아래로 잘 섞는다. 12 시간 동안 37 ℃로 물을 욕조에서 반응을 품어. </li><li> 최대 전력 출력을 보장하기 ?...

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TFO 지시 ICLS의 효율적 형성이 중요한 요인에 따라 달라집니다 : 목표에 TFO의 결합 친화도에 따라 그 대상 DNA의 기판과의 TFO의 배양, 적절한 버퍼 구성 요소 (예를 들어,의 MgCl 2) 첫 번째 시간 (종속 이중 및 농도)를 사용; 둘째, UVA의 적절한 투여 량 (365 ㎚)을 조사 효율적 소라렌 가교 결합을 형성한다. 최적 삼중 형성 HPLC 또는 겔 정제 TFOs를 사용함으로써 달성 될 수있다. TFO 오염 불순...

삼중 나선 구조를 형성하는 1-5 훅 스틴 수소 결합을 통해 서열 - 특이 적 방식으로 올리고 뉴클레오티드 (TFOs) 바인드 듀플렉스 DNA를 플렉스가 형성. 플렉스 기술은 이러한 전사, DNA 손상을 복구하고 유전자 타겟팅 (참고 문헌 6-8에서 검토)와 같은 생체 분자 메커니즘의 다양한 심문 사용되고있다. TFOs는 리포터 플라스미드 9,10-에 부위 - 특이 적 손상을 유도하기 위해 널리 사용되어왔다. 우리의 실험실과 다른 사람은 이전에 플라스미드 pSupFG1 5,10-12에 supF 유전자에 사이트 별 DNA의 interstrand의 가교 (ICLS)을 유도하기 위해 소 랄렌 분자에 닿는 TFO, AG30을 사용하고 있습니다. ICLS 이러한 병변 공유 개의 DNA 가닥 가교로서 매우 독성이며, 수리되지두면 유전자 전사를 차단하고, DNA 복제 장치 (13, 14)을 방해 할 수있다. 때문에 독성 잠재력, ICL 유도 화제는 치료에 항암제로 사용되어왔다암 및 기타 질병 15. 그러나, 인간의 세포에서 ICLS의 처리 및 수리가 잘 이해되지 않습니다. 따라서, 인간 세포에서 ICLS의 처리에 관여하는 메카니즘에 대한 이해는 ICL 계 화학 요법의 효능을 개선하는 데 도움이된다. TFO 유도 ICLS 및 수리 중간체는 DNA의 나선 구조에 상당한 왜곡을 야기 할 가능성이있다. 이러한 왜곡은 정규 B 형 이중 DNA 16-20보다 더 높은 친화력으로 왜곡 된 DNA에 결합 건축 단백질에 대한 가능성이 대상입니다. 여기, 우리는 염색질 면역 (칩) 소 랄렌 교 플라스미드에 대한 분석을 통해 인간의 세포에서 ICLS와 매우 풍부한 건축 단백질 협회, HMGB1을 공부하고 인간의 소 랄렌 가교 플라스미드 DNA의 토폴로지를 변조에 HMGB1의 역할을 확인 암 세포 용 해물. HMGB1은 매우 풍부하고 재하 표현이 아닌 자신의정규 B 형 DNA 17-20보다 더 높은 친화력으로 손상된 DNA와 대안 구조화 된 DNA 기판에 결합 톤 건축 단백질. HMGB1 그러한 전사, DNA 복제 및 DNA 수선 16,21-23 여러 DNA 대사 과정에 관여한다. 우리는 이전에 HMGB1 높은 친 화성 (20)와 체외에서 TFO 지시 ICLS에 결합하는 것을 증명하고있다. 또한, 우리는 HMGB1의 부족 뉴클레오티드 절단 수리 (NER) 보조 인자 (23, 24)로 TFO 지시 ICLS의 돌연변이 처리 및 확인 HMGB1 증가 있음을 증명하고있다. 최근에, 우리는 HMGB1는 인간의 세포에서 TFO 지시 ICLS과 관련된 이러한 병변의 채용은 NER 단백질, XPA (16)에 의존하는 것을 발견했다. DNA의 음의 수퍼 코일은 NER (25)에 의해 DNA 병변의 효율적인 제거를 촉진하기 위해 표시되었습니다, 우리는 HMGB1는 TFO 지시 ICL 함유 플라스틱으로에 우선적으로 부정적인 수퍼 코일을 유도하는 것을 발견했다NER 공동 인자로서 HMGB1의 잠재적 인 역할 (들)에 대한 이해를 제공한다 (비 손상된 플라스미드 기판에 대해) 중간 기판 (16). ICLS의 처리는 완전한 인간 세포에서 알려져 있지 않다; 따라서, 본 명세서에 기재된 분자 도구에 기초한 기술과 개발 분석 차례로 암 화학 요법의 효능을 향상시키기 위해 이용 될 수있는 바와 같이 약물 표적을 제공 할 수있다 ICL 수리에 관련된 추가적인 단백질의 동정을 초래할 수있다. 여기서, 효과적인 방법은 논의 된 아가 로스 겔 전기 영동을 변성하여 플라스미드 DNA의 TFO 지시 ICL 형성의 효율성을 평가. 또한, TFO 지시 ICLS를 포함하는 플라스미드를 사용하는 기술이 설명되었다 변성 칩 어 세이를 이용하여 셀룰러 환경에서 ICL 손상된 플라스미드 HMGB1 연관을 결정한다. 또한, 손쉬운 방법은 제 도입 위상 수정을 공부구체적으로는 인간 세포 용 해물에서 ICL 손상된 플라스미드 기판상의 전자 구조적 단백질 HMGB1은 이차원 아가 로스 겔 전기 영동을 통해 수퍼 코일 분석을 수행함으로써 측정되었다. 설명 된 기술은 인간 세포에서 플라스미드의 타겟 DNA 손상 처리에 DNA 수리 및 구조적 단백질 관여의 이해를 촉진하기 위해 사용될 수있다. 우리는 플라스미드 DNA, 이후 플라스미드 칩 각각 DNA 토폴로지 변경 병변과 연관 단백질 및 단백질을 확인하는 분석을 수퍼 코일에 TFO 지시 부위 특이 랄렌 ICLS의 형성에 대한 상세한 프로토콜을 설명한다. 이러한 분석은 다른 DNA 손상 제, TFOs 플라스미드 기판 및 관심 포유 동물 세포 라인을 수행하도록 변형 될 수있다. 사실, 우리는 적어도 하나의 전위와 친 화성이 높은 고유 TFO 결합 부위는 인간 게놈 26의 모든 주석 유전자 내에 있다는 것을 보여 주었다. 방법우리 케르 및 바스, 2016 (16)에 이용되는 것처럼 지금까지, 명확성을 위해, 우리는 인간 U2OS 세포에서 특정 돌연변이 리포터 플라스미드 (pSupFG1)의 특정 랄렌 공역 TFO (pAG30)의 사용을위한 이러한 기술을 설명했다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5&#39;HMT Psoralen-AG30</td>
			<td>Midland Certified Reagent Co., Midland, TX</td>
			<td>Custom order</td>
			<td>HPLC (or gel) purified</td>
		</tr>
		<tr>
			<td>Simple ChIP Enzymatic Chromatin IP Kit</td>
			<td>Cell signaling Inc.</td>
			<td>9003</td>
			<td>Manufacturer suggested protocol is optimized for chromatin IP and not for plasmid IP. The control IgG and H3 antibodies as well as the Protein G beads and micrococcal nuclease are supplied by the manufacturer.</td>
		</tr>
		<tr>
			<td>GoTaq Green</td>
			<td>Promega</td>
			<td>M7122</td>
			<td>PCR master mix</td>
		</tr>
		<tr>
			<td>HMGB1 antibody</td>
			<td>Abcam</td>
			<td>Ab18256</td>
			<td></td>
		</tr>
		<tr>
			<td>Wizard Gel and PCR cleanup kit</td>
			<td>Promega</td>
			<td>A9281</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroquine-diphosphate salt</td>
			<td>Sigma</td>
			<td>C6628-50G</td>
			<td>For two-dimensional agarose gel electrophoresis</td>
		</tr>
		<tr>
			<td>EcoRI</td>
			<td>New England BioLabs</td>
			<td>R0101S</td>
			<td></td>
		</tr>
		<tr>
			<td>GenePORTER transfection reagent</td>
			<td>Genlantis</td>
			<td>T201015</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaccinia Topoisomerase I</td>
			<td>Invitrogen</td>
			<td>38042-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Blak-Ray long wave ultraviolet lamp</td>
			<td>Upland, CA</td>
			<td>B 100 AP</td>
			<td>UVA lamp</td>
		</tr>
		<tr>
			<td>EpiSonic Multi-Functional Bioprocessor 1100</td>
			<td>Epigentek</td>
			<td>EQC-1100</td>
			<td>Water bath sonicator</td>
		</tr>
		<tr>
			<td>Mylar filter</td>
			<td>GE Healthcare Life Sciences</td>
			<td>80112939</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A6111</td>
			<td></td>
		</tr>
		<tr>
			<td>BIO-RAD Chemidoc</td>
			<td>BIO-RAD</td>
			<td>XRS+ system</td>
			<td>DNA imaging system</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Fisher Scientific</td>
			<td>BP381-500</td>
			<td></td>
		</tr>
		<tr>
			<td>37% Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F8775-25ML</td>
			<td>Used for crosslinking&#160;</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>New England Biolabs</td>
			<td>P8107S</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>ThermoFisher</td>
			<td>11965092</td>
			<td>Cell culture media</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>ThermoFisher</td>
			<td>70013073</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>ThermoFisher</td>
			<td>25200056</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Bromocresol green</td>
			<td>Sigma-Aldrich</td>
			<td>114-359</td>
			<td>DNA loading dye</td>
		</tr>
		<tr>
			<td>Siliconized tubes</td>
			<td>Fisher Scientific&#160;</td>
			<td>02-681-320</td>
			<td>Low retention microfuge tube</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher Scientific</td>
			<td>BP152-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric Acid</td>
			<td>Fisher Scientific</td>
			<td>A74-1</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher Scientific</td>
			<td>BP24731</td>
			<td></td>
		</tr>
		<tr>
			<td>Photometer</td>
			<td>InternationalLight Technologies</td>
			<td>ILT1400-A</td>
			<td>UVA dose measurement</td>
		</tr>
		<tr>
			<td>Cell lines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human U2OS osteosarcoma</td>
			<td>ATCC</td>
			<td>ATCC HTB-96</td>
			<td>Cultured according to supplier&#8217;s recommendation</td>
		</tr>
		<tr>
			<td>Human cervical adenocarcinoma HeLa</td>
			<td>ATCC</td>
			<td>ATCC-CCL-2</td>
			<td>Cultured according to supplier&#8217;s recommendation</td>
		</tr>
		<tr>
			<td>Proximal forward primer</td>
			<td>5&#8242;-gcc ccc ctg acg agc atc ac</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Proximal reverse primer</td>
			<td>5&#8242;-tag tta ccg gat aag gcg cag cgg</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Distal forward primer</td>
			<td>5&#8242;-aat acc gcg cca cat agc ag</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Distal reverse primer</td>
			<td>5&#8242;-agt att caa cat ttc cgt gtc gcc</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

tools to study the role of architectural protein hmgb1 in the processing of helix distorting, site-specific dna interstrand crosslinks

높은 이동성 그룹 상자 1 (HMGB1) 단백질은 전사, DNA 복제 및 DNA 수선 등 게놈 많은 중요한 기능을 조절에 관여하는 히스톤 비 구조적 단백질이다. HMGB1은 정식 B-DNA보다 더 높은 친화력과 구조적으로 왜곡 된 DNA에 결합한다. 예를 들어, 우리는 HMGB1은 DNA가 나선의 왜곡을 야기 공유 결합 DNA의 두 가닥을 연결 가교 (ICLS)를 interstrand, 왼쪽 경우 복구되지 않은 세포 사망을 일으킬 수에 결합하는 것을 발견했다. 때문에 자신의 세포 독성 잠재력, 여러 ICL 유도 에이전트는 현재 병원에서 화학 요법 제로서 사용된다. ICL-형성 제는 특정 염기 서열 (예, &#39;- TA-3 5&#39;는 소 랄렌에 대한 선호 가교 사이트)에 대한 환경 설정을 표시하는 동안, 그들은 크게 무차별 방식으로 DNA 손상을 유도한다. 그러나, 공유 결합 서열 - 특이 적으로 DNA에 결합하는 삼중 - 형성 올리고 뉴클레오티드 (TFO)에 ICL 유도제를 연결함으로써방법은 타겟 DNA 손상이 달성 될 수있다. 여기서는 도구로 사용할 돌연변이 리포터 플라스미드의 부위 특이 ICL을 생성하는 공유 8 trimethylpsoralen (HMT) 랄렌 5 &#39;은 4&#39;- 히드 록시 메틸 4,5&#39;말단에 컨쥬 게이션 TFO를 사용 인간의 세포에서 HMGB1에 의해 복잡한 DNA 병변의 건축 변경, 처리 및 수리를 공부합니다. 우리는 실험 기술 리포터 플라스미드에 TFO 지시 ICLS을 준비하고, 염색질 면역 침전 분석법을 사용하여 휴대 맥락에서 TFO 지시 ICLS와 HMGB1의 연결을 심문에 대해 설명합니다. 또, HMGB1 의해 소라렌 가교 플라스미드 도입 superhelical 회전의 양을 측정함으로써 손상된 DNA의 특정 구조적 변형을 평가하기 위해 수퍼 코일 DNA의 분석을 설명한다. 이러한 기술은 TFO 지시 ICLS 또는 관심있는 세포주 다른 타겟 DNA 손상의 복구 처리에 관여하는 다른 단백질의 역할을 연구하기 위해 이용 될 수있다.

TFO 지시 ICL의 형성은 인간 세포에서 ICL 처리에서의 구조적 단백질의 역할을 심문하는 데 사용되는 플라스미드 기반 분석을 위해 중요하다. 아가 로스 겔 전기 영동을 변성하면 TFO 지시 ICL 형성의 효율성을 결정하기위한 용이 한 방법이다. TFO 지시 ICLS 은닉 플라스미드는 비 - 가교 결합 된 제어 플라스미드 (도 1a, 레인 2)에 비교하여, 아가 로스 겔 매트릭스 <str...

Watch this Scientific Journal Video about Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks at JoVE.com

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

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, ...

The overall goal of the modified chromatin immunoprecipitation and DNA supercoiling experiments is to study the association and subsequent topological modifications induced by architectural proteins on site-specific DNA damage induced by chemotherapeutic or carcinogenic agents in human cells. This methodology will allow us to address key questions about DNA damage and repair which should facilitate discovery of new pharmacological targets for cancer therapy. Our technology allows us to direct site-specific DNA damage, and facilitates evaluation of critical DNA repair processes relevant to human cancer.To begin, 24 hours before transfection, plate 400, 000 mammalian cells per 60 millimeter dish in DMEM supplemented with 10%FBS without antibiotics. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide overnight. In a 14-milliliter round bottom tube, combine 30 microliters of room temperature transfectionary agent with 500 microliters of 1X growth medium that has been warmed to 37 degrees Celsius.In a second tube, combine two micrograms of TFO-directed ICL-containing plasmids in 500 microliters of 1X growth medium. Incubate the tubes at room temperature for 10 minutes. Combine mix one with mix two, and pipette up and down to thoroughly mix.Then incubate the solution at room temperature for 25 to 30 minutes. Next, use warm PBS to wash the cells twice. Then, add the transfection mix in a drop-wise fashion, distributing the solution evenly throughout the plate of cells.Add one milliliter of growth medium to the plate, and bring the final volume to two milliliters. Mix well, and place the culture plates at 37 degrees Celsius and 5%carbon dioxide. Four hours later, replace the medium with three milliliters of growth medium with 10%FBS, and incubate an additional 16 hours.Following the incubation, add 80 microliters of fresh 37%formaldehyde per plate to the cells, and incubate at room temperature in the dark for 10 minutes. Quench the formaldehyde cross-linking by adding 300 microliters of chilled glycine per plate, and incubate for five minutes. Then remove the medium, and use chilled PBS to wash the plates twice.Next, after adding one millimeter of chilled PBS, collect the cells by scraping into a microcentrifuge tube on ice, keeping samples on ice at all times. Prepare cell pellets by centrifuging at 13, 400 times G, and four degrees Celsius for five minutes. Then, remove the supernatant, and place the pellets on ice.Use one milliliter of chilled Buffer A to homogeneously re-suspend the pellets, and incubate on ice for 10 minutes. Then, pellet the cells as before. Now, with one milliliter of chilled Buffer B, homogeneously re-suspend the pellets, and incubate on ice for 10 minutes with occasional mixing by inverting.Then pellet the cells as before. After re-suspending the cells again, in 200 microliters of chilled Buffer B, add two microliters of micrococcal nuclease, or MNase, and incubate the reaction at room temperature for 10 minutes. Then, stop the MNase reaction by placing the tubes on ice, and adding 40 millimolar EDTA.The level of digestion by micrococcal nuclease is very critical, because overdigestion may cause loss of signal, whereas underdigestion may cause a lot of nonspecific background. After pelleting the cells, re-suspend the pellets in 100 microliters of chromatin immunoprecipitation, or ChIP buffer, supplemented with protease inhibitor cocktail. Then, transfer the cell suspension to a thin-walled microcentrifuge tube.Sonicate the samples by floating them on a water bath sonicator for 20 seconds, followed by 20 seconds on ice. Repeat the sonication steps nine times to generate approximately 800 to 1, 200 base-pair fragments. To 20 microliters of the sonicated samples, add three microliters of five molar sodium chloride, and one microliter of RNase A.Vortex the tubes, and incubate them at 37 degrees Celsius for 30 minutes.Then, add two microliters of proteinase K, and incubate at 65 degrees Celsius for two hours. After purifying the samples, according to the text protocol, run them on 1%agarose gel, and visualize with ethidium bromide. In three separate, pre-chilled siliconized tubes, add 30 microliters of lysate, and 70 microliters of chilled 1X ChIP buffer with added protease inhibitors.Add one microgram of anti-IgG, anti-histone H3, or anti-HMGB1 antibodies to the respective tubes. Incubate overnight in a cold room with continuous rotation. In the cold room, after re-suspending Protein G beads, add four microliters of the beads to each reaction tube, and incubate in a cold room with rotation for another two to four hours.Place the tubes on a magnetic rack to pellet the beads, and remove the supernatant. Add 200 microliters of 1X ChIP buffer with protease inhibitor cocktail to the pellets, and wash in the cold with rotation for five minutes. Next, with high salt 1X ChIP buffer, perform an additional wash.Then, use 160 microliters of elution buffer to re-suspend the pellets, and incubate at 37 degrees Celsius with rotation for 30 minutes. Pellet the beads, and collect the supernatant in a fresh tube. Add six microliters of five molar sodium chloride, and two microliters of proteinase K, and incubate overnight at 65 degrees Celsius.After purifying the DNA, according to the text protocol, perform PCR reactions using primer sets to the region or regions of interest, and resolve the products on a 1%agarose gel, stained with ethidium bromide. Prior to use, thaw previously prepared DNA repair synthesis buffer on ice, and add 40 millimolar phosphocreatine, and 2.5 micrograms of creatine phosphokinase. After preparing 10X supercoiling buffer, according to the text protocol, use one microliter of vaccinia topoisomerase 1 with 1X supercoiling buffer in a 10 microliter reaction to linearize 300 nanograms of supercoiled plasmid, PCA FG1.To the relaxed plasmids, add 25 microliters of freshly-thawed HeLa cell extract, and DNA synthesis buffer. Mix well, then add one microliter of vaccinia topoisomerase 1 to the reaction, and incubate at 37 degrees Celsius for one hour. Terminate the reactions by adding 1%SDS, and 0.25 milligrams per milliliter of proteinase K, and incubate at 65 degrees Celsius for two hours.After casting a 1%agarose gel with 1X TPE buffer, add DNA-loading dye to the reactions, and load the samples directly onto the gel at least four lanes apart. Run the gel for eight to 10 hours at two volts per centimeter in 1X TBE for the first dimension at room temperature to resolve the topoisomers. Prepare two liters of 1X TBE with three micrograms per milliliter of chloroquine phosphate.Soak the gel in 200 milliliters of the solution, covered in aluminum foil for 20 minutes. To electrophorese the gel in the second dimension, turn the gel 90 degrees in a clockwise fashion, and run it for four to six hours at two volts per centimeter in chloroquine containing 1X TBE to resolve the positive and negative supercoils. The amount of chloroquine in the electrophoresis buffer, and in the gel, is very critical, as well as running the gel at a very low voltage for the second dimension is very critical to separate the positive and the negative supercoils.After staining and destaining the gel, according to the text protocol, use an imager to visualize the thermodistribution of the topoisomers. As seen in this gel, approximately 79%of the plasmids harboring TFO-directed ICLs migrate with a slower mobility through the agarose gel matrix when compared to the uncross-linked control plasmids. ChIP assays performed on lysates from human U2OS cells transfected with either control plasmid or plasmids containing TFO-directed ICLs show enrichment of HMGB1 at the P-ICL region compared to the control region when immunoprecipitated with anti-HMGB1 antibody.When HMGB1 was depleted from the cells via siRNA treatment, the P-ICL-specific enrichment was diminished. When the same samples were immunoprecipitated using an anti-IgG antibody, minimal PCR amplification was detected. Finally, in this figure, two-dimensional agarose gel electrophoresis demonstrates architectural modification of the TFO-directed ICL-containing plasmids as negative supercoiling by HMGB1 in HeLa cell extracts.Once mastered, this technique can be performed in approximately five days, if performed correctly. While attempting this procedure, it's very important to remember that all the washes should be done in the cold room, as room temperature washes may increase the background signal. Following this procedure, other methods, such as siRNA-mediated protein depletion, followed by mutation screening assays, can be performed in order to answer questions, such as, is the protein of interest involved in DNA repair?Or, does it significantly alter the mutation spectra during the process? This technique paved the way to study association between proteins and site-specific DNA damage. After watching this video, you should have a good idea of how to study the association of architectural proteins to target a DNA cross-links, and subsequently, determine the effect of such association on the topology of the DNA.Don't forget that working with ethidium bromide can be extremely hazardous, and therefore, proper protective gear should be always worn during performing this experiment.

Research

JoVE Journal

Genetics

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

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

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

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

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

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 ...

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

항생제 내성의 진화에서 다중 플라스 미드의 역할 테스트

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the ...

A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells

간단 하 고 빠른, 양적 분석 결과에 플라스 미드 DNA-단백질 Crosslinks의 측정 수리에 포유류 세포로 페

The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

미토 콘 드리 아 DNA 메 틸 화의 정확한 탐지를 위한 방법론

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

차세대 시퀀싱의 원형 염색체 구조를 사용 하 여 유전자 규제 시퀀스의 높은 처리량 Id (4 C-seq)를 캡처

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

질량 분석 기반 Proteomics 분석 소설 단백질을 공개할 OpenProt 데이터베이스를 사용 하 여 비 정식 열려있는 독서 프레임에서 번역

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

비오틴 라벨을 사용하여 단백질-핵산 상호 작용을 연구하는 생체외 생화학 적 분석을 통해

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...

A Bioinformatics Pipeline to Accurately and Efficiently Analyze the MicroRNA Transcriptomes in Plants

식물의 MicroRNA 전사체를 정확하고 효율적으로 분석하는 생물정보학 파이프라인

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in ...

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

DNA 손상의 평가를 위한 혜성 분석에서 냉동 조직의 사용

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...