Name: tools to study the role of architectural protein hmgb1 in the processing of helix distorting, site-specific dna interstrand crosslinks
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Les auteurs tiennent à remercier les membres du laboratoire Vasquez pour des discussions utiles. Ce travail a été soutenu par les Instituts nationaux de la santé / Institut national du cancer [CA097175, CA093279 à KMV]; et la prévention du cancer et de l&#39;Institut de recherche du Texas [RP101501]. Le financement des frais d&#39;accès ouvert: National Institutes of Health / National Cancer Institute [de CA093279 à KMV].

1. Préparation de ICL TFO dirigés sur le plasmide Substrats <ol><li> Incuber quantités équimolaires de plasmide pSupFG1 10,11 ADN (ADN plasmidique 5 ug est un bon point de départ) avec psoralène conjugué TFO AG30 dans 8 pi de tampon de triplex de liaison [50% de glycérol, Tris 10 mM (pH 7,6), mM MgCl 10 2] , et dH 2 O pour un volume final de 40 pi dans un tube de couleur ambre. Mélanger soigneusement par pipetage de haut en bas. Laisser incuber la réaction à 37 ° C bain d&#...

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La formation efficace des ICL TFO dirigés dépend de deux facteurs essentiels: premièrement, des composants tampons appropriée (par exemple, MgCl 2) et le temps d&#39;incubation de la TFO avec son substrat d&#39;ADN cible ( en fonction de l&#39;affinité de liaison de l&#39;TFO à sa cible recto-verso, et que les concentrations utilisées); et deuxièmement, la dose appropriée des UVA (365 nm), l&#39;irradiation pour former des reticulations de psoralène de manière efficace. la formation d&#39;...

Formant des triplex d&#39; oligonucléotides (OFT) un ADN duplex se lient d&#39;une manière spécifique d&#39; une séquence via une liaison de Hoogsteen d&#39;hydrogène pour former des structures en triple hélice 1-5. La technologie triplex a été utilisé pour interroger une variété de mécanismes biomoléculaires, tels que la transcription, la réparation des dommages de l&#39; ADN et le ciblage génique (passé en revue dans les références 6-8). OFT ont été largement utilisés pour induire des dommages spécifiques au site sur les plasmides rapporteurs 9,10. Notre laboratoire et d&#39; autres ont déjà utilisé un TFO, AG30, attaché à une molécule de psoralène pour induire l&#39; ADN interbrins réticulations spécifiques du site (de ICL) dans le gène supF sur le pSupFG1 plasmidique 5,10-12. ICL sont hautement cytotoxique que ces lésions réticulent de manière covalente les deux brins d&#39;ADN, et si elle unrepaired, peut bloquer la transcription des gènes et empêcher la réplication des machines d&#39;ADN 13,14. En raison de leur potentiel cytotoxique, des agents induisant ICL ont été utilisés comme médicaments chimiothérapeutiques dans le traitementdu cancer et d&#39; autres maladies 15. Cependant, le traitement et la réparation des ICL dans les cellules humaines ne sont pas bien comprises. Ainsi, une meilleure compréhension des mécanismes impliqués dans le traitement des ICL dans les cellules humaines peut aider à améliorer l&#39;efficacité des régimes chimiothérapeutiques à base d&#39;ICL. ICL TFO-induits et leurs intermédiaires de réparation ont le potentiel de provoquer des distorsions structurelles importantes à l&#39;hélice de l&#39;ADN. De telles distorsions sont des cibles probables pour les protéines d&#39; architecture, qui se lient à l&#39; ADN déformé avec une affinité supérieure à canonique B-forme duplex ADN 16-20. Ici, nous avons étudié l&#39;association d&#39;une protéine architecturale très abondante, HMGB1 avec ICL dans les cellules humaines par l&#39;intermédiaire d&#39;immunoprécipitation de la chromatine (ChIP) essais sur des plasmides de psoralène-réticulé et identifié un rôle pour HMGB1 dans la modulation de la topologie de l&#39;ADN plasmidique psoralène réticulé chez l&#39;homme des lysats de cellules cancéreuses. HMGB1 est une très abondante et ubiquitaire non sonton protéine architecturale qui se lie à l&#39; ADN endommagé et les substrats d&#39;ADN alternativement structurés avec une affinité supérieure canonique forme B de l&#39; ADN 17-20. HMGB1 est impliqué dans plusieurs processus ADN métaboliques, tels que la transcription, la réplication de l&#39; ADN et la réparation d&#39; ADN 16,21-23. Nous avons déjà démontré que HMGB1 se lie à ICL TFO dirigées in vitro avec une grande affinité 20. De plus, nous avons démontré que le manque de HMGB1 a augmenté le traitement mutagène de ICL TFO dirigées et HMGB1 identifié comme une réparation nucleotide excision (NER) co-facteur 23,24. Récemment, nous avons constaté que HMGB1 est associée à ICL TFO dirigées dans les cellules humaines et son recrutement à ces lésions dépend de la protéine NER, XPA 16. Surenroulement négatif de l&#39; ADN a été montré pour favoriser l&#39;élimination efficace des lésions de l&#39; ADN par NER 25, et nous avons constaté que HMGB1 induit surenroulement négatif préférentiellement sur plas ICL contenant TFO dirigéessubstrats milieu ( par rapport aux plasmides substrats non endommagés) 16, fournissant une meilleure compréhension du rôle (s) potentiel de HMGB1 comme un co-facteur de NER. Le traitement des ICL est pas entièrement comprise dans les cellules humaines; ainsi, les techniques et les analyses développées sur la base des outils moléculaires décrits ici pourraient conduire à l&#39;identification de protéines supplémentaires impliquées dans la réparation ICL, qui peut à son tour servir de cibles pharmacologiques qui peuvent être exploitées pour améliorer l&#39;efficacité des traitements de chimiothérapie du cancer. Ici, une approche efficace pour évaluer l&#39;efficacité de la formation ICL TFO dirigée dans l&#39;ADN plasmidique par électrophorèse sur gel dénaturant d&#39;agarose a été discuté. En outre, en utilisant les plasmides contenant les ICL TFO dirigées, des techniques pour déterminer l&#39;association de HMGB1 avec des plasmides ICL endommagés dans un contexte cellulaire en utilisant des essais ChIP modifiés ont été décrits. En outre, une méthode facile pour étudier les modifications topologiques introduites par ee protéine HMGB1 architecturale, en particulier sur des substrats de plasmide ICL endommagés dans les lysats de cellules humaines a été déterminée en effectuant des essais de surenroulement par électrophorèse sur gel d&#39;agarose à deux dimensions. Les techniques décrites peuvent être utilisées pour favoriser la compréhension de l&#39;implication de la réparation de l&#39;ADN et des protéines d&#39;architecture dans le traitement des lésions de l&#39;ADN ciblé sur des plasmides dans les cellules humaines. Nous décrivons des protocoles détaillés pour la formation d&#39;ICL psoralène spécifiques au site TFO dirigés sur l&#39;ADN de plasmide et le plasmide subséquent ChIP et surenroulement des dosages pour identifier des protéines qui associent les lésions et les protéines qui modifient la topologie de l&#39;ADN, respectivement. Ces dosages peuvent être modifiés pour produire avec d&#39;autres agents endommageant l&#39;ADN, OFT, des substrats de plasmide et des lignées de cellules de mammifères présentant un intérêt. En fait, nous avons montré qu&#39;il existe au moins un site potentiel TFO affinité de liaison unique et élevé au sein de chaque gène annoté dans le génome humain 26. Commentjamais, pour plus de clarté, nous avons décrit ces techniques pour l&#39;utilisation d&#39;un TFO spécifique psoralène-conjugué (pAG30) sur un plasmide mutation-rapporteur spécifique (pSupFG1) dans les cellules U2OS humaines que nous avons utilisé dans Mukherjee &amp; 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

Boîte de high mobility group 1 (HMGB1) protéine est une protéine non-histone architecturale qui est impliquée dans la régulation de nombreuses fonctions importantes dans le génome, tels que la transcription, la réplication de l&#39;ADN et la réparation d&#39;ADN. HMGB1 se lie à l&#39;ADN structurellement déformée avec une affinité supérieure à celle de l&#39;ADN-B canonique. Par exemple, nous avons constaté que la HMGB1 se lie à l&#39;ADN (reticulations interbrins CIST), qui lient de manière covalente les deux brins de l&#39;ADN, provoquent la déformation de l&#39;hélice, et si on les laisse non réparée peut provoquer la mort cellulaire. En raison de leur potentiel cytotoxique, plusieurs agents ICL-inductrices sont actuellement utilisés comme agents chimiothérapeutiques dans la clinique. Alors que les agents ICL formant montrent des préférences pour certaines séquences de base (par exemple, 5&#39;-TA-3 &#39;est le site de réticulation préféré pour psoralène), ils induisent en grande partie des dommages de l&#39; ADN d&#39;une manière aveugle. Cependant, en couplant de façon covalente l&#39;agent induisant la LSC à un oligonucléotide formant triplex (TFO), qui se lie à l&#39;ADN dans une séquence spécifiqueAinsi, des dommages de l&#39;ADN cible peut être atteint. Ici, nous utilisons un TFO conjugué de manière covalente sur &#39;extrémité à un 4&#39;-hydroxyméthyl-4,5&#39; 5 (HMT) psoralène, 8-triméthylpsoralène pour générer un ICL site spécifique sur un plasmide mutation-reporter pour l&#39;utiliser comme un outil pour étudier la modification architecturale, le traitement et la réparation des lésions de l&#39;ADN complexes par HMGB1 dans les cellules humaines. Nous décrivons les techniques expérimentales pour préparer ICL TFO dirigés sur des plasmides rapporteurs et d&#39;interroger l&#39;association de HMGB1 avec les ICL TFO dirigées dans un contexte cellulaire en utilisant des tests chromatine immunoprécipitation. En outre, nous décrivons des tests de surenroulement de l&#39;ADN pour évaluer la modification d&#39;architecture spécifique de l&#39;ADN endommagé en mesurant la quantité de tours superhélices introduits sur le psoralène réticulé plasmide par HMGB1. Ces techniques peuvent être utilisées pour étudier les rôles des autres protéines impliquées dans le traitement et la réparation des ICL TFO dirigées ou d&#39;autres dommages à l&#39;ADN ciblé dans toute lignée cellulaire d&#39;intérêt.

La formation de la LSC TFO dirigée est essentielle pour les dosages à base de plasmides, qui sont utilisées pour interroger les rôles des protéines architecturales dans le traitement ICL dans les cellules humaines. Dénaturation électrophorèse sur gel d&#39;agarose est un moyen facile de déterminer l&#39;efficacité de la formation ICL TFO dirigée. Les plasmides hébergeant ICL TFO dirigés migrent avec une mobilité plus lente à travers la matrice de gel d&#39;agarose ...

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.

Des outils pour étudier le rôle de l&#39;architecture Protein HMGB1 dans le traitement des Helix effets de distorsion, l&#39;ADN spécifique du site 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, ...

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.

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