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

Die Autoren möchten die Mitglieder des Vasquez Labor für hilfreiche Diskussionen. Diese Arbeit wurde von den National Institutes of Health / National Cancer Institute unterstützt [CA097175, CA093279 zu KMV]; und die Krebsprävention und Forschungsinstitut für Texas [RP101501]. Die Finanzierung für den offenen Zugang Gebühr: National Institutes of Health / National Cancer Institute [CA093279 zu KMV].

1. Herstellung von TFO-directed ICLs auf Plasmid Substrate <ol><li> Inkubieren äquimolaren Mengen von Plasmid pSupFG1 10,11 DNA (5 ug Plasmid - DNA ist ein guter Ausgangspunkt) mit Psoralen-konjugiertes TFO AG30 in 8 ul von Triplex Bindungspuffer [50% Glycerin, 10 mM Tris (pH 7,6), 10 mM MgCl 2] und dH 2 O auf ein Endvolumen von 40 ul in einem bernsteinfarbenen Röhre. Gut mischen durch Pipettieren von oben und unten. Inkubieren der Reaktion bei 37 ° C Wasserbad für 12 Stunden. </li...

<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. 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Die effiziente Bildung von TFO-directed ICLs hängt von zwei entscheidenden Faktoren: Erstens richtigen Pufferkomponenten (zB MgCl 2) und die Zeit der Inkubation des TFO mit seiner Ziel - DNA - Substrat (abhängig von der Bindungsaffinität des TFO an seine Ziel duplex, und die Konzentrationen verwendet werden); und zweitens, die richtige Dosis an UVA (365 nm) Bestrahlung Psoralen Vernetzungen effizient bilden. Optimale Triplex-Bildung kann durch die Verwendung HPLC oder gelgereinigt TFOs erreicht we...

Triplex Forming Oligonucleotide (TFOs) bind duplex DNA in einer sequenzspezifischen Art und Weise via Hoogsteen-Wasserstoffbrückenbindung tripelhelicalen 1-5 Strukturen zu bilden. Triplex - Technologie verwendet, um eine Vielzahl von biomolekularen Mechanismen, wie Transkription, DNA - Schäden zu reparieren, und Gen - Targeting ( zusammengefasst in Referenzen 6-8) zu befragen. TFOs wurden 9,10 ausgiebig zu induzieren ortsspezifische Schäden an Reporter - Plasmide verwendet. Unser Labor und andere haben bislang auf einer TFO, AG30, gebunden an ein Psoralen Molekül 5,10-12 ortsspezifische DNA - Quervernetzungen zwischen (ICL) im supF - Gen auf dem Plasmid pSupFG1 zu induzieren. ICLs sind hochcytotoxisch wie diese Läsionen kovalente Verknüpfung der beiden DNA - Stränge zu vernetzen, und wenn nicht repariert, kann Gen - Transkription blockieren und die DNA - Replikationsmaschinerie 13,14 behindern. Wegen ihres zytotoxischen Potentials ICL-induzierende Mittel wurden als chemotherapeutische Wirkstoffe in der Behandlung verwendetvon Krebs und anderen Krankheiten , 15. Jedoch ist die Bearbeitung und die Reparatur von ICLs in menschlichen Zellen nicht gut verstanden. So kann ein besseres Verständnis der bei der Verarbeitung von ICLs Mechanismen in menschlichen Zellen helfen, die Wirksamkeit der ICL-basierten chemotherapeutischen Therapien zu verbessern. TFO-induzierte ICLs und deren Reparatur Zwischenprodukte haben das Potenzial, erhebliche strukturelle Verzerrungen bei der DNA-Helix zu verursachen. Solche Verzerrungen sind wahrscheinlich Ziele für architektonische Proteine, die mit einer höheren Affinität zu verzerrten DNA binden als an kanonischen B-Form Duplex - DNA - 16-20. Hier untersuchten wir die Assoziation eines hoch reichlich architektonischen Protein HMGB1 mit ICLs in menschlichen Zellen durch Chromatin-Immunopräzipitation (ChIP) Assays auf Psoralen netzten Plasmiden und identifiziert eine Rolle für HMGB1 in Modulieren der Topologie des Psoralen netzten Plasmid DNA in menschlichen Krebs Zelllysaten. HMGB1 ist ein sehr reichlich vorhanden und ubiquitär exprimiert nicht seinTon architektonischen Protein , das als 17-20 DNA kanonischen B-Form , um beschädigte DNA und alternativ strukturierten DNA - Substrate mit einer höheren Affinität bindet. HMGB1 ist 16,21-23 in mehreren DNA - Stoffwechselprozesse, wie beispielsweise Transkription, DNA - Replikation und DNA - Reparatur beteiligt. Wir haben zuvor gezeigt , dass HMGB1 bindet an TFO-directed ICLs in vitro mit hoher Affinität 20. Gerichtet TFO-ICLs und identifiziert HMGB1 als Nukleotidexzisionsreparatur (NER) Cofaktor 23,24 weiteren haben wir gezeigt , dass der Mangel an HMGB1 des mutagenen Verarbeitung erhöht. Vor kurzem haben wir festgestellt , dass mit HMGB1 TFO-directed ICLs in menschlichen Zellen und ihre Einstellung auf solche Läsionen auf dem NER - Protein, XPA 16 abhängig zugeordnet ist. Negative Supercoiling von DNA wurde von NER 25, und wir haben festgestellt , die effiziente Entfernung von DNA - Läsionen zu fördern gezeigt , dass HMGB1 negativen supercoiling induziert bevorzugt auf TFO-directed ICL enthaltenden plasmid Substrate (bezogen auf nicht-beschädigten Plasmiden Substrate) 16, um ein besseres Verständnis der möglichen Rolle (n) von HMGB1 als NER Cofaktor bereitstellt. Die Verarbeitung von ICLs ist nicht vollständig in menschlichen Zellen zu verstehen; Somit entwickelt die Techniken und Assays auf der Basis der molekularen Werkzeugen hierin beschrieben könnte in ICL-Reparatur beteiligt zur Identifikation von zusätzlichen Proteinen führen, die wiederum dienen als pharmakologische Ziele, die ausgenutzt werden können, um die Wirksamkeit von Krebs-Chemotherapie zu verbessern. Hierbei ist ein wirksamer Ansatz, die Effizienz der TFO-directed ICL Bildung in Plasmid-DNA zu beurteilen durch Agarosegelelektrophorese denaturierenden diskutiert. Ferner Techniken unter Verwendung der Plasmide, die die TFO-gerichtete ICLs enthält, die Assoziation von HMGB1 mit ICL geschädigten Plasmide in einem zellulären Kontext mit modifizierten ChIP Assays zu bestimmen, wurden beschrieben. Zusätzlich zu eine einfache Methode topologische Modifikationen durch th eingeführt studierene architektonischen Protein HMGB1, insbesondere on-ICL beschädigt Plasmid Substrate in menschlichen Zell-Lysaten wurde durch Durchführung supercoiling Assays über zweidimensionale Agarose-Gelelektrophorese bestimmt. Die beschriebenen Techniken können das Verständnis der Beteiligung von DNA-Reparatur und architektonischen Proteine ​​in der Verarbeitung von gezielten DNA-Schädigung auf Plasmiden in menschlichen Zellen zu fördern verwendet werden. Wir beschreiben detaillierte Protokolle für die Bildung von TFO-gerichtete ortsspezifische Psoralen ICLs auf Plasmid-DNA und anschließende Plasmid ChIP und Supercoiling Assays Proteine ​​zu identifizieren, die mit den Läsionen assoziiert, und Proteine, die die DNA-Topologie zu verändern, respectively. Diese Assays können modifiziert werden, um mit anderen DNA-schädigenden Mitteln durchzuführen, TFOs Plasmid Substrate und Säugerzelllinien von Interesse. In der Tat haben wir gezeigt , dass es mindestens eine potentielle einzigartige und hochaffinen TFO-Bindungsstelle innerhalb jedes kommentierten Gen im menschlichen Genom ist 26. Wiehaupt, aus Gründen der Klarheit beschrieben wir diese Techniken für die Verwendung eines spezifischen Psoralen-konjugiertes TFO (pAG30) auf einer spezifischen Mutation Reporterplasmid (pSupFG1) in menschlichen U2OS - Zellen , wie wir in Mukherjee &amp; Vasquez 2016 16 genutzt haben.

<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

High-Mobility-Group-Box 1 (HMGB1) Protein ein Nicht-Histon-Protein, das in Architektur Regulieren viele wichtige Funktionen im Genom, wie beispielsweise Transkription, DNA-Replikation und DNA-Reparatur beteiligt ist. HMGB1 bindet strukturell verzerrten DNA mit einer höheren Affinität als zu kanonischen B-DNA. Beispielsweise fanden wir, dass HMGB1 bindet an DNA Interstrang-Vernetzungen (ICLs), die die beiden Stränge der DNA kovalent verknüpfen, Verzerrung der Wendel verursachen und nicht reparierten wenn links kann zum Zelltod führen. Aufgrund ihrer zytotoxische Potential sind mehrere ICL auslösende Mittel, die gegenwärtig als chemotherapeutische Mittel in der Klinik eingesetzt. Während ICL bildende Mittel Präferenzen für bestimmte Basensequenzen zeigen (zB 5&#39;-TA-3 &#39;ist die bevorzugte Vernetzungsstelle für Psoralen), DNA - Schäden in einer wahllos Art und Weise sie weitgehend induzieren. Jedoch durch kovalentes Koppeln des ICL-induzierenden Mittels zu einem Triplex Forming Oligonucleotid (TFO), die in einer sequenzspezifischen DNA bindetWeise gezielte DNA-Schädigung erreicht werden. Hier verwenden wir einen TFO kovalent auf der &#39;Ende zu einem 4&#39;-Hydroxymethyl-4,5&#39; 5 konjugiert, 8-trimethylpsoralen (HMT) Psoralen eine ortsspezifische ICL auf einem mutations Reporterplasmid zu erzeugen als ein Werkzeug zu verwenden, die architektonische Modifizierung, Verarbeitung und Reparatur von DNA-Komplex Läsionen durch HMGB1 in menschlichen Zellen zu untersuchen. Wir beschreiben experimentelle Techniken TFO-gerichtete ICLs auf Reporter-Plasmide herzustellen, und die Assoziation von HMGB1 mit den TFO-gerichtete ICLs in einem zellulären Kontext mit Chromatinimmunpräzipitation Assays zu verhören. Ferner beschreiben wir DNA-Supercoiling Assays spezifische architektonische Modifikation des beschädigten DNA zu bewerten, indem die Menge der superhelikalen Windungen Messung auf dem Psoralen-vernetzt Plasmid durch HMGB1 eingeführt. Diese Techniken können verwendet werden, um die Rollen von anderen Proteinen in der Verarbeitung und Reparatur von TFO-directed ICLs oder anderen zielgerichteten DNA-Schäden in jeder Zelllinie von Interesse beteiligt zu studieren.

Bildung des TFO-directed ICL ist kritisch für die Plasmid-basierte Assays, die verwendet werden, um die Rollen von architektonischen Proteine ​​in ICL Verarbeitung in menschlichen Zellen zu befragen. denaturierender Agarosegelelektrophorese ist ein facile Weg, um die Effizienz der TFO-directed ICL Formation zu bestimmen. Die Plasmide TFO-gerichtete ICLs beherbergen wandern mit einer geringeren Mobilität durch die Agarosegelmatrix (1A, Spur 3) im Vergleich zu den ni...

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.

Tools, um die Rolle der Architektur-Protein HMGB1 zur Untersuchung bei der Verarbeitung von Helix verzerrenden, ortsspezifische DNA-Quervernetzungen zwischen

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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