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

Los autores desean agradecer a los miembros del laboratorio Vásquez útil para los debates. Este trabajo fue apoyado por los Institutos Nacionales de Salud / Instituto Nacional del Cáncer [CA097175, CA093279 a KMV]; y la Prevención del Cáncer y el Instituto de Investigación de Tejas [RP101501]. La financiación de cargo de acceso abierto: Institutos Nacionales de Salud / Instituto Nacional del Cáncer [CA093279 a KMV].

1. Preparación de ICL dirigidas-TFO en el plásmido Sustratos <ol><li> Incubar cantidades equimolares de plásmido pSupFG1 10,11 ADN (plásmido ADN 5 mg es un buen punto de partida) con psoraleno conjugado TFO GA30 en 8 l de tampón de unión triplex [glicerol al 50%, Tris 10 mM (pH 7,6), MgCl 10 mM 2] y dH 2 O hasta un volumen final de 40 l en un tubo de color ámbar. Mezcle bien con la pipeta hacia arriba y hacia abajo. Incubar la reacción a 37 ° C baño de agua durante 12 hr. </l...

<ol>
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E., Beltrame, M., Paonessa, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+recognition+of+cruciform+DNA+by+nuclear+protein+HMG1.">Specific recognition of cruciform DNA by nuclear protein HMG1.</a> Science. 243, 1056-1059 (1989).</li><li>Jung, Y., Lippard, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature+of+full-length+HMGB1+binding+to+cisplatin-modified+DNA.">Nature of full-length HMGB1 binding to cisplatin-modified DNA.</a> Biochemistry. 42, 2664-2671 (2003).</li><li>Reddy, M. C., Christensen, J., Vasquez, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+human+high+mobility+group+protein+1+and+replication+protein+A+on+psoralen-cross-linked+DNA.">Interplay between human high mobility group protein 1 and replication protein A on psoralen-cross-linked DNA.</a> Biochemistry. 44, 4188-4195 (2005).</li><li>Das, D., Scovell, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+binding+interaction+of+HMG-1+with+the+TATA-binding+protein/TATA+complex.">The binding interaction of HMG-1 with the TATA-binding protein/TATA complex.</a> J Biol Chem. 276, 32597-32605 (2001).</li><li>Little, A. J., Corbett, E., Ortega, F., Schatz, D. 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La formación eficiente de ICL dirigidas-TFO depende de dos factores cruciales: En primer lugar, los componentes del tampón adecuado (por ejemplo, MgCl 2) y el tiempo de incubación de la TFO con su sustrato de ADN diana (depende de la afinidad de unión del TFO a su diana dúplex y las concentraciones se utiliza); y segundo, la dosis apropiada de UVA (365 nm) de irradiación para formar entrecruzamientos de psoraleno de manera eficiente. la formación de triplex óptimo se puede lograr mediante el ...

Triplex de formación de oligonucleótidos (TFOs) de ADN se unen dúplex de una manera específica de secuencia a través de enlaces Hoogsteen-hidrógeno para formar estructuras de triple helicoidal 1-5. Tecnología Triplex se ha utilizado para interrogar a una variedad de mecanismos biomoleculares, tales como la transcripción, daño en el DNA de reparación, y la orientación de genes (revisado en las referencias 6-8). TFOs se han utilizado ampliamente para inducir daño de sitio específico en plásmidos informadores 9,10. Nuestro laboratorio y otros han utilizado previamente un TFO, GA30, atado a una molécula de psoraleno para inducir reticulaciones entre cadenas de ADN específicas de sitio (ICL) en el gen su p F en el plásmido pSupFG1 5,10-12. ICL son altamente citotóxicos ya que estas lesiones se entrecruzan covalentemente las dos hebras de ADN, y si se deja sin reparar, puede bloquear la transcripción de genes e impedir la maquinaria de replicación del ADN 13,14. Debido a su potencial citotóxico, agentes inductores de ICL-han sido utilizados como fármacos quimioterapéuticos en el tratamientode cáncer y otras enfermedades 15. Sin embargo, el tratamiento y la reparación de ICL en las células humanas no se entiende bien. Por lo tanto, una mejor comprensión de los mecanismos implicados en el procesamiento de ICL en las células humanas puede ayudar a mejorar la eficacia de los regímenes quimioterapéuticos basados ​​en ICL. ICL inducidos por TFO y sus intermedios de reparación tienen el potencial de causar distorsiones estructurales importantes para la hélice de ADN. Estas distorsiones son objetivos probables para las proteínas de arquitectura, que se unen a ADN distorsionada con mayor afinidad que a forma canónica B dúplex de ADN 16-20. Aquí, se estudió la asociación de una proteína de arquitectura muy abundante, HMGB1 con ICL en las células humanas a través de inmunoprecipitación de cromatina (ChIP) ensayos en plásmidos de psoraleno-reticulada y se identificaron un papel para la HMGB1 en la modulación de la topología del ADN plasmídico psoraleno reticulado en humanos lisados ​​celulares de cáncer. HMGB1 es un muy abundantes y de expresión ubicua no suarquitectónico de proteínas tono que se une al ADN dañado y sustratos de ADN, alternativamente, estructurados con mayor afinidad que forma canónica B ADN 17-20. HMGB1 participa en varios procesos metabólicos de ADN, tales como la transcripción, la replicación del ADN, y la reparación del ADN 16,21-23. Hemos demostrado previamente que la HMGB1 se une a ICL dirigidas-TFO in vitro con alta afinidad 20. Además, hemos demostrado que la falta de HMGB1 aumenta el procesamiento mutagénico de ICL dirigidas-TFO y HMGB1 identificado como una reparación por escisión de nucleótidos (NER) co-factor 23,24. Recientemente, hemos encontrado que la HMGB1 se asocia con ICL dirigidas-TFO en las células humanas y su reclutamiento a tales lesiones depende de la proteína NER, XPA 16. Superenrollamiento negativo del ADN se ha demostrado que la promoción de la eliminación eficaz de las lesiones del ADN por NER 25, y hemos encontrado que HMGB1 induce superenrollamiento negativo preferentemente en plas ICL contienen dirigidas-TFOmediados de sustratos (en relación con los plásmidos sustratos no dañado) 16, que proporcionan una mejor comprensión de la función (s) potencial de HMGB1 como un co-factor de NER. El procesamiento de ICL no se entiende completamente en las células humanas; por lo tanto, las técnicas y ensayos desarrollados en base a las herramientas moleculares descritos en este documento podrían conducir a la identificación de nuevas proteínas implicadas en la reparación ICL, que a su vez pueden servir como dianas farmacológicas que pueden ser explotados para mejorar la eficacia de los regímenes de quimioterapia del cáncer. Aquí, un enfoque eficaz para evaluar la eficacia de la formación dirigida ICL-TFO en el ADN plásmido mediante electroforesis en gel de agarosa desnaturalizante se ha discutido. Además, el uso de los plásmidos que contienen las ICL dirigidas-TFO, las técnicas para determinar la asociación de HMGB1 con plásmidos dañadas-ICL en un contexto celular utilizando ensayos de chip modificados se han descrito. Además, un método fácil para el estudio de modificaciones topológicas introducidas por THe proteína HMGB1 arquitectónico, específicamente en sustratos plásmido dañados-ICL en lisados ​​de células humanas se ha determinado mediante la realización de ensayos de superenrollamiento a través de electroforesis en gel de agarosa de dos dimensiones. Las técnicas descritas se pueden utilizar para favorecer la comprensión de la implicación de la reparación del ADN y las proteínas de arquitectura en el procesamiento de daño del ADN objetivo en los plásmidos en las células humanas. Describimos protocolos detallados para la formación de ICL de psoraleno específicas de sitio dirigidas-TFO en el ADN plásmido, y la posterior ChIP plásmido y superenrollamiento ensayos para identificar proteínas que se asocian con las lesiones, y proteínas que alteran la topología del ADN, respectivamente. Estos ensayos pueden ser modificados para llevar a cabo con otros agentes que daña el ADN, TFOs, sustratos de plásmidos, y las líneas de células de mamíferos de interés. De hecho, hemos demostrado que hay al menos un potencial afinidad único y de alta sitio TFO vinculante dentro de cada gen anotado en el genoma humano 26. Cómonunca, para mayor claridad, describimos estas técnicas para el uso de un conjugado TFO-psoraleno específico (pAG30) en un plásmido reportero mutación específica (pSupFG1) en células U2OS humanas como hemos utilizado en Mukherjee y Vasquez, 2016 16.

<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

cuadro de grupo de alta movilidad 1 proteína (HMGB1) es una proteína de arquitectura no histonas que está implicado en la regulación de muchas funciones importantes en el genoma, tales como la transcripción, la replicación del ADN, y la reparación del ADN. HMGB1 se une al ADN estructuralmente distorsionado con mayor afinidad que a B-DNA canónica. Por ejemplo, se encontró que la HMGB1 se une al ADN enlaces cruzados (ICL), que enlazan covalentemente las dos hebras del ADN, provocar una distorsión de la hélice, y si se deja sin reparar puede causar la muerte celular. Debido a su potencial citotóxico, varios agentes inductores de ICL se utilizan actualmente como agentes quimioterapéuticos en la clínica. Mientras que los agentes formadores de ICL muestran preferencias por determinadas secuencias de bases (por ejemplo, 5&#39;-TA-3 &#39;es el sitio preferido para la reticulación de psoraleno), inducen daños en el ADN en gran medida de forma indiscriminada. Sin embargo, acoplando covalentemente el agente ICL-inducir a un oligonucleótido formador de triplex-(TFO), que se une al ADN en una secuencia específicamanera, el daño del ADN objetivo se puede lograr. Aquí, nosotros usamos una TFO conjugado covalentemente en &#39;extremo a un 4&#39;-hidroximetil-4,5 &#39;5 el psoraleno trimetilpsoraleno-8, (HMT) para generar una ICL específica de sitio en un plásmido reportero mutación para su uso como una herramienta de para estudiar la modificación de arquitectura, el procesamiento, y la reparación de las lesiones del ADN complejos por HMGB1 en las células humanas. Se describen las técnicas experimentales para preparar ICL dirigidas-TFO en plásmidos informadores, y para interrogar a la asociación de HMGB1 con los dirigidos ICL-TFO en un contexto celular utilizando ensayos de inmunoprecipitación de la cromatina. Además, se describen ensayos de superenrollamiento del ADN para evaluar la modificación de arquitectura específica del ADN dañado mediante la medición de la cantidad de vueltas superhelical introducidas en el plásmido psoraleno reticulado por HMGB1. Estas técnicas se pueden utilizar para estudiar los papeles de otras proteínas implicadas en el procesamiento y la reparación de ICL dirigidas-TFO u otros daños en el ADN objetivo en cualquier línea celular de interés.

La formación de la ICL dirigida TFO es crítico para los ensayos basados ​​en plásmidos, que se utilizan para interrogar a las funciones de las proteínas de arquitectura en el procesamiento de ICL en células humanas. Electroforesis en gel desnaturalizante de agarosa es una manera fácil de determinar la eficacia de la formación ICL dirigida TFO. Los plásmidos que albergan ICL dirigidas-TFO migran con una movilidad más lenta a través de la matriz de gel de agarosa (Fig...

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.

Herramientas para estudiar el papel de la proteína HMGB1 arquitectónico en el procesamiento de la hélice causante de distorsión, de ADN específica de sitio enlaces cruzados

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

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La manipulación genética del patógeno de la planta<em&gt; Ustilago maydis</em&gt; Estudiar biología de hongos y plantas 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 ...

Investigación

Genética

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Microirradiation láser para estudiar In Vivo las respuestas celulares al daño en el ADN Simple y complejo

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

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

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Methodology for Accurate Detection of Mitochondrial DNA Methylation

Metodología para la detección exacta de la metilación del ADN mitocondrial

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

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Captura de alto rendimiento identificación de secuencias reguladoras de genes utilizando secuenciación de próxima generación de conformación Circular cromosoma (4C-seq)

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

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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

Ensayos bioquímicos in Vitro utilizando etiquetas de biotina para estudiar las interacciones entre proteínas y ácidos nucleicos

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

Un gasoducto de bioinformática para analizar de forma precisa y eficiente los transcriptomes de microRNA en las plantas

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

Uso de tejido congelado en el ensayo de cometas para la evaluación del daño causado por el ADN

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 ...