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

Författarna vill tacka medlemmarna i Vasquez laboratorium för bra diskussioner. Detta arbete stöddes av National Institutes of Health / National Cancer Institute [CA097175, CA093279 att KMV]; och förebyggande av cancer och Forskningsinstitut Texas [RP101501]. Finansiering för open access laddning: National Institutes of Health / National Cancer Institute [CA093279 till KMV].

1. Framställning av TFO-riktade ICLs på plasmid substrat <ol><li> Inkubera ekvimolära mängder av plasmid-pSupFG1 10,11-DNA (5 | j, g plasmid-DNA är en bra startpunkt) med psoralen-konjugerad TFO AG30 i 8 pl av triplex bindningsbuffert [50% glycerol, 10 mM Tris (pH 7,6), 10 mM MgCb 2] och dH 2 O till en slutlig volym av 40 | il i en brunfärgad röret. Blanda noggrant genom att pipettera upp och ned. Inkubera reaktionen vid 37 ° C vattenbad i 12 h. </li><li> Värma upp UVA lampa f...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+of+DNA+lesions+associated+with+triplex-forming+oligonucleotides.">Repair of DNA lesions associated with triplex-forming oligonucleotides.</a> Mol Carcinog. 48, 389-399 (2009).</li><li>Seidman, M. M., Glazer, P. 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+potential+for+gene+repair+via+triple+helix+formation.">The potential for gene repair via triple helix formation.</a> J Clin Invest. 112, 487-494 (2003).</li><li>Vasquez, K. 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E., Ljungman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psoralen-induced+DNA+interstrand+cross-links+block+transcription+and+induce+p53+in+an+ataxia-telangiectasia+and+rad3-related-dependent+manner.">Psoralen-induced DNA interstrand cross-links block transcription and induce p53 in an ataxia-telangiectasia and rad3-related-dependent manner.</a> Mol Pharmacol. 75, 599-607 (2009).</li><li>Vare, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+interstrand+crosslinks+induce+a+potent+replication+block+followed+by+formation+and+repair+of+double+strand+breaks+in+intact+mammalian+cells.">DNA interstrand crosslinks induce a potent replication block followed by formation and repair of double strand breaks in intact mammalian cells.</a> DNA repair. 11, 976-985 (2012).</li><li>Huang, Y., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+crosslinking+damage+and+cancer+-+a+tale+of+friend+and+foe.">DNA crosslinking damage and cancer - a tale of friend and foe.</a> Transl Cancer Res. 2, 144-154 (2013).</li><li>Mukherjee, A., 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=HMGB1+interacts+with+XPA+to+facilitate+the+processing+of+DNA+interstrand+crosslinks+in+human+cells.">HMGB1 interacts with XPA to facilitate the processing of DNA interstrand crosslinks in human cells.</a> Nucleic Acids Res. 44, 1151-1160 (2016).</li><li>Bidney, D. L., Reeck, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+from+cultured+hepatoma+cells+of+two+nonhistone+chromatin+proteins+with+preferential+affinity+for+single-stranded+DNA:+apparent+analogy+with+calf+thymus+HMG+proteins.">Purification from cultured hepatoma cells of two nonhistone chromatin proteins with preferential affinity for single-stranded DNA: apparent analogy with calf thymus HMG proteins.</a> Biochem Biophys Res Commun. 85, 1211-1218 (1978).</li><li>Bianchi, M. 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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperative+recruitment+of+HMGB1+during+V(D)J+recombination+through+interactions+with+RAG1+and+DNA.">Cooperative recruitment of HMGB1 during V(D)J recombination through interactions with RAG1 and DNA.</a> Nucleic Acids Res. 41, 3289-3301 (2013).</li><li>Lange, S. S., Mitchell, D. L., 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=High+mobility+group+protein+B1+enhances+DNA+repair+and+chromatin+modification+after+DNA+damage.">High mobility group protein B1 enhances DNA repair and chromatin modification after DNA damage.</a> Proc Natl Acad Sci U S A. 105, 10320-10325 (2008).</li><li>Lange, S. S., Reddy, M. C., 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=Human+HMGB1+directly+facilitates+interactions+between+nucleotide+excision+repair+proteins+on+triplex-directed+psoralen+interstrand+crosslinks.">Human HMGB1 directly facilitates interactions between nucleotide excision repair proteins on triplex-directed psoralen interstrand crosslinks.</a> DNA repair. 8, 865-872 (2009).</li><li>Park, J. Y., Ahn, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+DNA+topology+on+plasmid+DNA+repair+in+vivo.">Effect of DNA topology on plasmid DNA repair in vivo.</a> FEBS letters. , 174-178 (2000).</li><li>Wu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-affinity+triplex-forming+oligonucleotide+target+sequences+in+mammalian+genomes.">High-affinity triplex-forming oligonucleotide target sequences in mammalian genomes.</a> Mol Carcinog. 46, 15-23 (2007).</li><li>Sheflin, L. G., Spaulding, S. 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Den effektiva bildningen av TFO-riktade ICLs är beroende av två viktiga faktorer: För det första, korrekt buffertkomponenter (t.ex. MgCl2) och tid för inkubation av TFO med dess mål-DNA-substrat (beroende på bindningsaffiniteten hos TFO till sitt mål duplex, och koncentrationerna som används); och andra, den rätta dosen av UVA (365 nm) bestrålning för att bilda psoralen tvärbindningar effektivt. Optimal triplexbildning kan uppnås genom användning av HPLC eller gel renade TFOs. Föroren...

Triplex-bildande oligonukleotider (TFOs) binder duplex-DNA på ett sekvensspecifikt sätt via Hoogsteen-vätebindning för att bilda trippelspiralstrukturer 1-5. Triplex teknik har använts för att förhöra en mängd biomolekylära mekanismer, såsom transkription, DNA-skador reparation och genmålsökning (granskas i referenserna 6-8). TFOs har använts i stor utsträckning för att framkalla platsspecifika skador på reporterplasmider 9,10. Vårt laboratorium och andra har tidigare använt en TFO, VT30, bundna till en psoralen molekyl för att framkalla platsspecifika DNA interstrand tvärbindningar (ICLs) i supF-genen på plasmiden pSupFG1 5,10-12. ICLs är mycket cytotoxiska eftersom dessa skador kovalent tvärbinda de två DNA-strängarna, och om de lämnas utan reparation, kan blockera gentranskription och hindra DNA-replikation maskiner 13,14. På grund av deras cytotoxiska potential har ICL-inducerande medel använts som kemoterapeutiska läkemedel för behandlingav cancer och andra sjukdomar 15. Emellertid är bearbetning och reparation av ICLs i humana celler inte väl förstådd. Således kan en bättre förståelse av de mekanismer som är involverade i behandlingen av ICLs i humana celler bidra till att förbättra effektiviteten hos ICL-baserade kemoterapeutiska regimer. TFO-inducerade ICLs och deras reparationsmellan har potential att orsaka betydande strukturella snedvridning av DNA-spiralen. Sådan snedvridning är troliga mål för arkitektoniska proteiner, som binder till snedvriden DNA med högre affinitet än till kanonisk B-formen duplex-DNA 16-20. Här studerade vi föreningen av en mycket riklig arkitektonisk protein, HMGB1 med ICLs i humana celler via kromatin immunoprecipitation (chip) analyser om psoralen-tvärbundna plasmider och identifierade en roll för HMGB1 vid modulering av topologin av psoralen-tvärbunden plasmid-DNA i human cancer cellysat. HMGB1 är en mycket riklig och allmänt utbrett uttryckt icke-hanstonen arkitektoniska protein som binder till skadat DNA och alternativt strukturerade DNA-substrat med högre affinitet än kanonisk B-form av DNA 17-20. HMGB1 är involverat i flera DNA metaboliska processer, såsom transkription, DNA-replikation och DNA-reparation 16,21-23. Vi har tidigare visat att HMGB1 binder till TFO-riktade ICLs in vitro med hög affinitet 20. Vidare har vi visat att bristen på HMGB1 ökade mutagen behandling av TFO-riktade ICLs och identifierade HMGB1 som en nukleotid excision reparation (NER) kofaktor 23,24. Nyligen har vi funnit att HMGB1 är associerad med TFO-riktade ICLs i humana celler och dess rekrytering till sådana lesioner är beroende av NER-proteinet, XPA 16. Negativ supercoiling av DNA har visat att främja en effektiv borttagning av DNA-skador genom NER 25, och vi har funnit att HMGB1 inducerar negativ supercoiling företrädesvis på TFO-riktade ICL-innehållande plasmitten substrat (i förhållande till icke-skadade plasmider substrat) 16, vilket ger en bättre förståelse av den potentiella roll (er) av HMGB1 som en NER co-faktor. Bearbetningen av ICLs är inte helt förstådd i humana celler; således kunde tekniker och analyser utvecklats baserat på de molekylära verktyg som beskrivs här leda till identifiering av ytterligare proteiner involverade i ICL reparation, vilket i sin tur kan tjäna som farmakologiska mål som kan utnyttjas för att förbättra effektiviteten av cancer kemoterapi. Här, för att en effektiv strategi bedöma effektiviteten av TFO-riktad ICL-bildning i plasmid-DNA genom denaturerande agarosgelelektrofores har diskuterats. Vidare, med användning av de plasmider som innehåller de TFO-riktade ICLs, tekniker för att bestämma associationen av HMGB1 med ICL-skadade plasmider i ett cellulärt sammanhang användning av modifierade ChIP-analyser har beskrivits. Dessutom, till en enkel metod att studera topologiska ändringar som infördes av the arkitektoniska protein HMGB1, särskilt på ICL-skadade plasmid substrat i humana cellysat har bestämts genom att utföra supercoiling analyser via tvådimensionell agarosgelelektrofores. De tekniker som beskrivs kan användas för att öka förståelsen av inblandningen av DNA-reparation och arkitektoniska proteiner vid bearbetning av målinriktad DNA-skada på plasmider i humana celler. Vi beskriver detaljerade protokoll för bildandet av TFO-riktade platsspecifika psoralen ICLs på plasmid-DNA, och efterföljande plasmid ChIP och supercoiling analyser för att identifiera proteiner som associerar med de lesioner, och proteiner som förändrar DNA-topologi, respektive. Dessa analyser kan modifieras för att utföra med andra DNA-skadande medel, TFOs, plasmid substrat och däggdjurscellinjer av intresse. I själva verket har vi visat att det finns åtminstone ett potentiellt unik och hög affinitet TFO-bindningsställe inom varje kommenterad genen i det humana genomet 26. Hurnågonsin, för tydlighetens skull, beskrev vi dessa tekniker för användning av ett visst psoralen-konjugerat TFO (pAG30) på en specifik mutation-reporterplasmid (pSupFG1) i humana U2OS celler som vi har använt i 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

Hög rörlighet grupprutan 1 (HMGB1) proteinet är en icke-histon arkitektoniska protein som är involverat i regleringen av många viktiga funktioner i genomet, såsom transkription, DNA-replikation och DNA-reparation. HMGB1 binder till strukturellt förvrängd DNA med högre affinitet än till kanoniska B-DNA. Till exempel, fann vi att HMGB1 binder till DNA interstrand tvärbindningar (ICLs), som kovalent länkar de två delarna av DNA vålla snedvridning av spiralen, och om de lämnas oreparerade kan orsaka celldöd. På grund av deras cytotoxiska potential, finns flera ICL-inducerande medel som för närvarande används som kemoterapeutiska medel i kliniken. Medan ICL-bildande medel visar preferenser för vissa bassekvenser (t.ex. 5&#39;-TA-3 &#39;är den föredragna tvärbindningsställe för psoralen), de i hög grad inducerar DNA-skada i en urskillningslös sätt. Men genom att kovalent koppla ICL-inducerande medel till en triplex-bildande oligonukleotid (TFO), som binder till DNA i ett sekvensspecifiktsätt, kan uppnås målinriktad DNA-skada. Här använder vi en TFO kovalent konjugerat på 5 &#39;änden till en 4&#39;-hydroximetyl-4,5&#39;, 8-trimetylpsoralen (HMT) psoralen för att generera en platsspecifik ICL på en mutation-reporterplasmid för att använda som ett verktyg att studera arkitektoniska modifiering, bearbetning, och reparation av komplexa DNA-skador av HMGB1 i humana celler. Vi beskriver experimentella tekniker för att förbereda TFO riktade ICLs på reporter plasmider och förhöra den sammanslutning av HMGB1 med TFO riktade ICLs i ett cellulärt sammanhang med hjälp av kromatin immunoprecipitation analyser. Dessutom beskriver vi DNA supercoiling analyser för att bedöma specifika arkitektoniska modifiering av den skadade DNA genom mätning av mängden superhelixvarv introduceras på psoralen-tvärbunden plasmid genom HMGB1. Dessa tekniker kan användas för att studera rollerna av andra proteiner involverade i bearbetning och reparation av TFO-riktade ICLs eller andra riktade DNA-skada i någon cellinje av intresse.

Bildandet av TFO-directed ICL är kritisk för plasmiden baserade analyser, som används för att förhöra rollerna för arkitektoniska proteiner i ICL bearbetning i humana celler. Denaturerande agarosgelelektrofores är ett enkelt sätt att bestämma effektiviteten i TFO-riktad ICL bildning. De plasmider TFO-riktade ICLs migrerar med en långsammare mobilitet genom agarosgelen matrisen (Figur 1A, bana 3) jämfört med de icke-tvärbundna styr plasmider (Figur 1...

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.

Verktyg för att studera betydelsen av Architectural Protein HMGB1 i behandlingen av Helix snedvrida, Platsspecifik DNA Interstrand tvärbindningar

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

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