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

De auteurs willen graag de leden van Vasquez laboratorium bedanken voor nuttige discussies. Dit werk werd ondersteund door de National Institutes of Health / National Cancer Institute [CA097175, CA093279 om KMV]; en de Cancer Prevention and Research Institute van Texas [RP101501]. Financiering voor open access kosten: National Institutes of Health / National Cancer Institute [CA093279 om KMV].

1. Bereiding van TFO gerichte ICLs op Plasmid Substrates <ol><li> Incubeer equimolaire hoeveelheden plasmide pSupFG1 10,11 DNA (5 ug plasmide-DNA is een goed uitgangspunt) met psoraleen geconjugeerd TFO AG30 in 8 gl triplex bindingsbuffer [50% glycerol, 10 mM Tris (pH 7,6), 10 mM MgClz 2] en dH 2 O tot een eindvolume van 40 ul in een amber buisje. Meng goed door en neer te pipetteren. Incubeer de reactie bij 37 ° C waterbad gedurende 12 uur. </li><li> Warmen de UVA lamp op volle vermo...

<ol>
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De efficiënte vorming van TFO-gerichte ICL is afhankelijk van twee belangrijke factoren: Ten eerste, goede buffercomponenten (bijv MgCl 2) en de incubatietijd van de TFO met een doel-DNA-substraat (afhankelijk van de bindingsaffiniteit van de TFO zijn doelstelling duplex, en de gebruikte concentraties); en ten tweede de juiste dosis UVA (365 nm) bestraling psoraleen verknopingen efficiënt vormen. Optimale triplex vorming kan worden bereikt door HPLC of gel gezuiverd TFOs. Onzuiverheden verontreinig...

Triplex-vormende oligonucleotiden (TFOs) binden duplex DNA in een sequentie-specifieke manier via Hoogsteen-waterstofbruggen triple helix structuren 1-5 te vormen. Triplex technologie is gebruikt om diverse biomoleculaire mechanismen, zoals transcriptie, DNA schade herstel en gene targeting (beoordeeld in referenties 6-8) ondervragen. TFOs zijn uitgebreid gebruikt voor plaatsspecifieke schade aan reporterplasmiden 9,10 induceren. Ons laboratorium en anderen hebben eerder gebruik gemaakt van een TFO, TL30, vastgemaakt aan een psoraleen molecuul naar site-specifieke DNA InterStrand crosslinks (ICL) in de supF gen op het plasmide pSupFG1 5,10-12 induceren. ICLs zijn zeer cytotoxisch die laesies covalent verknopen de twee DNA-strengen, en indien niet gerepareerd kunnen gentranscriptie blokkeren en belemmeren de DNA-replicatiemachinerie 13,14. Vanwege hun cytotoxische potentieel hebben ICL-inducerende middelen gebruikt als chemotherapeutische middelen bij de behandelingkanker en andere ziekten 15. Echter, de verwerking en reparatie van ICL in menselijke cellen niet goed begrepen. Aldus kan een beter begrip van de bij de verwerking van ICL in menselijke cellen mechanismen bijdragen tot de werkzaamheid van ICL-gebaseerde chemotherapeutische regimes verbeteren. TFO-geïnduceerde ICLs met reparatie tussenproducten kan aanzienlijke structurele verstoringen veroorzaken aan de DNA helix. Dergelijke vervormingen zijn waarschijnlijk doelstellingen voor architecturale eiwitten, die binden aan vervormde DNA met een hogere affiniteit dan aan canonieke B-vorm duplex DNA 16-20. Hier hebben we de associatie van een veel voorkomt architecturaal eiwit, HMGB1 met ICL in humane cellen via chromatine immunoprecipitatie (ChIP) assays op psoraleen-verknoopte plasmiden en die een rol HMGB1 bij het moduleren van de topologie van het psoraleen-verknoopte nucleïnezuren in humane kanker cellysaten. HMGB1 is een veel voorkomt en alomtegenwoordig tot expressie non-histone architectonische eiwit dat aan beschadigde DNA en als alternatief gestructureerde DNA substraten bindt met een hogere affiniteit dan canonieke B-vorm DNA 17-20. HMGB1 is betrokken bij diverse DNA metabole processen zoals transcriptie, DNA-replicatie, DNA herstel en 16,21-23. We hebben eerder aangetoond dat HMGB1 TFO bindt aan gerichte ICLs in vitro met een hoge affiniteit 20. Verder hebben we aangetoond dat gebrek aan HMGB1 verhoogde de mutagene behandeling van TFO-gerichte ICL en geïdentificeerd HMGB1 als NER (NER) cofactor 23,24. Onlangs hebben we gevonden dat HMGB1 is gekoppeld TFO-gerichte ICLs in humane cellen en de rekrutering van deze laesies is afhankelijk van de NER eiwit, XPA 16. Negatieve supercoiling van DNA is aangetoond dat het effectief verwijderen van DNA-laesies bevorderen door NER 25, en we hebben gevonden dat HMGB1 induceert negatieve supercoiling voorkeur op TFO-gerichte ICL-bevattende plasmid substraten (ten opzichte van niet-beschadigde plasmiden substraten) 16, een beter inzicht in de mogelijke rol (s) van HMGB1 als NER cofactor. De verwerking van ICL is niet volledig bekend in menselijke cellen; Zo kunnen de technieken en assays ontwikkeld op basis van de moleculaire methoden hierin beschreven leiden tot de identificatie van additionele eiwitten betrokken bij ICL herstel, waardoor farmacologische doelen die kunnen worden benut om de effectiviteit van chemotherapie regiems verbeteren dienen. Hier wordt een effectieve benadering om de efficiëntie van TFO-gerichte vorming ICL in plasmide DNA beoordelen aan denaturerende agarose gelelektroforese werd besproken. Verder gebruik van de plasmiden die de TFO-gerichte ICL, technieken is bij HMGB1 met ICL-beschadigde plasmiden bepalen een cellulaire context modified ChIP assays zijn beschreven. Daarnaast is er een gemakkelijke methode om topologische wijzigingen die bij th bestuderene architecturaal eiwit HMGB1, specifiek op ICL-beschadigde plasmide substraten in menselijke cellysaten werd bepaald door het uitvoeren van assays supercoiling via tweedimensionale agarosegelelektroforese. De beschreven technieken kunnen worden gebruikt voor het begrip van de rol van DNA herstel en architecturale eiwitten in de verwerking van target DNA schade aan plasmiden in menselijke cellen bevorderen. We beschrijven gedetailleerde protocollen voor de vorming van TFO-gerichte plaatsspecifieke psoraleen ICLs op plasmide-DNA, gevolgd plasmide chip en supercoiling assays voor eiwitten die associëren met de letsels en eiwitten die de DNA-topologie te veranderen identificeren, respectievelijk. Deze testen kunnen worden gemodificeerd te voeren met andere DNA beschadigende agentia, TFOs plasmide substraten en zoogdiercellijnen plaats. In feite hebben we aangetoond dat er ten minste één potentiële unieke hoge affiniteit TFO-bindingsplaats binnen elke geannoteerde genen in het menselijke genoom 26. Hoeooit, voor de duidelijkheid, beschreven deze technieken voor het gebruik van een specifieke psoraleen-geconjugeerd TFO (pAG30) op een specifieke mutatie-reporterplasmide (pSupFG1) in humane cellen als U2OS we Mukherjee &amp; Vasquez, 2016 16 hebben gebruikt.

<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

Hoge mobiliteit groep box 1 (HMGB1) eiwit is een niet-histon architectonische eiwit dat betrokken is bij het reguleren van vele belangrijke functies in het genoom, zoals transcriptie, DNA-replicatie en DNA herstel. HMGB1 bindt structureel vervormde DNA met hogere affiniteit dan canonieke B-DNA. Zo vonden we dat HMGB1 bindt aan DNA InterStrand crosslinks (ICL), die covalent verbinden de twee strengen van het DNA, veroorzaakt vervorming van de helix, en als links unrepaired celdood veroorzaken. Door hun cytotoxische reactie, worden meerdere ICL-inducerende middelen momenteel gebruikt als chemotherapeutische middelen in de kliniek. Terwijl ICL-vormende middelen tonen voorkeuren voor bepaalde basensequenties (bijvoorbeeld 5&#39;-TA-3 &#39;is de geprefereerde verknoping site voor psoraleen), zij grotendeels veroorzaken DNA-schade op een willekeurige manier. Echter, door covalente koppeling van het ICL-inducerende middel aan een triplex-vormende oligonucleotiden (TFO), dat bindt aan DNA op een sequentie-specifiekewijze kan target DNA beschadiging worden bereikt. Hier gebruiken we een TFO covalent geconjugeerd aan het 5 &#39;uiteinde een 4&#39;-hydroxymethyl-4,5&#39;, 8-trimethylpsoraleen (HMT) psoraleen een plaatsspecifieke ICL genereren op een mutatie-reporterplasmide te gebruiken als hulpmiddel de architectonische modificatie, verwerking en reparatie van complexe DNA laesies bestuderen door HMGB1 in humane cellen. We beschrijven experimentele technieken om TFO gerichte ICLs voor te bereiden op de reporter plasmiden, en de associatie van HMGB1 met de TFO gerichte ICLs ondervragen in een cellulair context met behulp van chromatine immunoprecipitatie assays. Daarnaast beschrijven we DNA supercoiling assays voor specifieke architecturale modificatie van het beschadigde DNA beoordelen door het meten van de hoeveelheid superhelische windingen ingevoerd op het psoraleen-verknoopte plasmide door HMGB1. Deze technieken kunnen worden gebruikt om de rol van andere eiwitten die betrokken zijn bij de verwerking en reparatie van TFO-gerichte ICLs of andere gerichte DNA schade in een cellijn van belang bestuderen.

Vorming van het TFO-gerichte ICL essentieel is voor het plasmide-gebaseerde testen die worden gebruikt om de rol van eiwitten in architecturale ICL verwerking ondervragen menselijke cellen. Denaturerende agarose gelelektroforese is een gemakkelijke manier om de efficiëntie van TFO-gerichte ICL formatie te bepalen. De plasmiden TFO gerichte ICLs migreren met een langzamere mobiliteit door de agarose gelmatrix (Figuur 1A, laan 3) vergeleken met de niet-verknoopte controle...

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 om de rol van de Architectural eiwit HMGB1 Bestudeer in de verwerking van Helix vervormen, site-specifieke DNA InterStrand Crosslinks

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

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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