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

著者らは、有用な議論のためにバスケスの研究室のメンバーに感謝したいと思います。この作品は、健康/国立癌研究所【KMVにCA097175、CA0​​93279]の国立研究所によってサポートされていました。がん予防テキサス州の研究所[RP101501]と。健康/国立がん研究所の国立研究所[KMVにCA093279]：オープンアクセスチャージのための資金。

プラスミド基板上TFO指向のICLの調製<ol><li> 10,11 DNA三重結合緩衝液[50％グリセロール、10mMトリス（pHは7.6）、10 mMの塩化マグネシウムの8μlのソラレン共役TFO AG30と（5μgのプラスミドDNAが良い出発点である）プラスミドpSupFG1の等モル量をインキュベート2]とのdH 2 O琥珀色のチューブで40μlの最終体積に。ピペッティングにより完全に混和します。 12時間37℃の水浴で反応?...

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E., Beltrame, M., Paonessa, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+recognition+of+cruciform+DNA+by+nuclear+protein+HMG1.">Specific recognition of cruciform DNA by nuclear protein HMG1.</a> Science. 243, 1056-1059 (1989).</li><li>Jung, Y., Lippard, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature+of+full-length+HMGB1+binding+to+cisplatin-modified+DNA.">Nature of full-length HMGB1 binding to cisplatin-modified DNA.</a> Biochemistry. 42, 2664-2671 (2003).</li><li>Reddy, M. C., Christensen, J., Vasquez, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+human+high+mobility+group+protein+1+and+replication+protein+A+on+psoralen-cross-linked+DNA.">Interplay between human high mobility group protein 1 and replication protein A on psoralen-cross-linked DNA.</a> Biochemistry. 44, 4188-4195 (2005).</li><li>Das, D., Scovell, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+binding+interaction+of+HMG-1+with+the+TATA-binding+protein/TATA+complex.">The binding interaction of HMG-1 with the TATA-binding protein/TATA complex.</a> J Biol Chem. 276, 32597-32605 (2001).</li><li>Little, A. J., Corbett, E., Ortega, F., Schatz, D. 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. W. <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+1+preferentially+conserves+torsion+in+negatively+supercoiled+DNA.">High mobility group protein 1 preferentially conserves torsion in negatively supercoiled DNA.</a> Biochemistry. 28, 5658-5664 (1989).</li><li>Betous, R., 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=Role+of+TLS+DNA+polymerases+eta+and+kappa+in+processing+naturally+occurring+structured+DNA+in+human+cells.">Role of TLS DNA polymerases eta and kappa in processing naturally occurring structured DNA in human cells.</a> Mol Carcinog. 48, 369-378 (2009).</li><li>Luo, Z., Macris, M. A., Faruqi, A. F., Glazer, P. 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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=Chromosomal+mutations+induced+by+triplex-forming+oligonucleotides+in+mammalian+cells.">Chromosomal mutations induced by triplex-forming oligonucleotides in mammalian cells.</a> Nucleic Acids Res. 27, 1176-1181 (1999).</li><li>Puri, N., 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=Minimum+number+of+2'-O-(2-aminoethyl)+residues+required+for+gene+knockout+activity+by+triple+helix+forming+oligonucleotides.">Minimum number of 2'-O-(2-aminoethyl) residues required for gene knockout activity by triple helix forming oligonucleotides.</a> Biochemistry. 41, 7716-7724 (2002).</li><li>Christensen, L. A., Finch, R. A., Booker, A. J., Vasquez, K. 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そのターゲットに依存TFOの結合親和性に応じて、その標的DNA基質（とまず、適切な緩衝成分（ 例えば 、 塩化マグネシウム）とTFOのインキュベーション時間：TFO指向のICLの効率的な形成は、二つの重要な要因に依存していますデュプレックス、および使用される濃度）。第二、UVA（365 nm）の照射の適切な用量は、効率的にソラレン架橋を形成します。最適な三重らせん形成は?...

三重らせん構造1-5を形成するためのトリプレックス形成オリゴヌクレオチドフーグスティーン水素結合を介した配列特異的様式で（のTFO）結合二本鎖DNA。三重技術は、転写、DNA損傷修復、ならびに遺伝子ターゲティング（参考文献6-8に概説）などの生体分子メカニズムの多様を調べるために使用されています。 TFOは、レポータープラスミド9,10-の部位特異的な損傷を誘導するために広く使用されています。私たちの研究室などは以前に、プラスミドpSupFG1 5,10-12にsupF遺伝子に部位特異的なDNAの間架橋（のICL）を誘導するためにソラレン分子につながTFO、AG30を、使用しています。これらの病変は、共有結合で2本のDNA鎖を架橋として、および未修復のままの場合のICLは、遺伝子の転写を阻止し、DNA複製機構13,14を妨げることができ、非常に細胞毒性です。なぜなら、それらの細胞毒性ポテンシャルを、ICL誘導剤は、治療において化学療法剤として使用されています癌やその他の病気15の。しかしながら、ヒト細胞中でのICLの加工及び修復は十分に理解されていません。したがって、ヒト細胞中のICLの処理に関与するメカニズムのより良い理解は、ICLベースの化学療法レジメンの有効性を改善するのに役立ち得ます。 TFO誘発性のICLとその修復中間体は、DNAヘリックスに有意な構造的歪みを引き起こす可能性を持っています。このような歪みは、標準的なB型二重鎖DNA 16-20よりも高い親和性で歪んだDNAに結合建築タンパク質、その考えられる標的です。ここでは、クロマチン免疫沈降（チップ）ソラレン架橋のプラスミド上のアッセイによって、ヒト細胞中でのICLと非常に豊富な建築タンパク質の結合、HMGB1を学び、ヒトにおいてソラレン架橋プラスミドDNAのトポロジーを調節することでHMGB1の役割を同定し癌細胞溶解物。 HMGB1は非常に豊富かつ普遍的に発現された非彼損傷したDNAに結合するトーン建築タンパク質および標準的なB型DNA 17-20よりも高い親和性を持つ代わりに構造化されたDNA基質。 HMGB1は、転写、DNA複製、DNA修復16,21-23などのいくつかのDNA代謝過程に関与しています。我々は以前HMGB1は高親和性20でインビトロで TFO指向のICLに結合することを実証しています。さらに、我々は、HMGB1の欠如はTFO指向のICLの変異原処理を増加し、ヌクレオチド除去修復（NER）補因子23,24としてHMGB1を同定していることを実証しました。最近、我々は、HMGB1がヒト細胞においてTFO向けのICLに関連付けられており、このような病変への募集はNERタンパク質、XPA 16に依存していることを発見しました。 DNAの負の超らせんをNER 25によりDNA損傷の効率的な除去を促進することが示されており、我々は、HMGB1はTFO指向ICL含有のPLAに優先的に負の超らせん形成を誘導することを発見しました（非損傷プラスミド基質に対して）、中間基板16、NER補因子としてHMGB1の潜在的な役割（複数可）のより良い理解を提供します。 ICLの処理は、完全ヒト細胞では理解されていません。このように、本明細書中に記載の分子ツールに基づいて開発された技術およびアッセイは、順番に、がん化学療法レジメンの有効性を改善するために利用することができる薬理学的標的として役立つ可能性がある、ICL修復に関与する追加のタンパク質の同定につながる可能性があります。 ここでは、アガロースゲル電気泳動を変性させることによって、プラスミドDNAでTFO指向ICL形成の効率を評価するための効果的なアプローチが検討されています。さらに、TFO指向のICLを含むプラスミドを用いて、修飾のChIPアッセイを用いて細胞の文脈でICL損傷プラスミドでHMGB1の結合を決定するための技術が記載されています。また、容易な方法は、番目によって導入されたトポロジカル修正を研究します特にヒト細胞溶解物中のICL損傷プラスミド基板上の電子建築タンパク質HMGB1は、二次元のアガロースゲル電気泳動を介して、スーパーコイルアッセイを実施することによって決定されています。説明される技術は、ヒト細胞中でのプラスミドの標的DNA損傷の処理において、DNA修復および建築タンパク質の関与の理解を促進するために使用することができます。 我々は、プラスミドDNA、及びその後のプラスミドチップそれぞれ、DNAのトポロジーを改変病変と会合するタンパク質、およびタンパク質を同定するためのアッセイをスーパーコイルのTFO-部位特異的ソラレンのICLを形成するための詳細なプロトコルを記述する。これらのアッセイは、他のDNA損傷剤、のTFO、プラスミド基質、および目的の哺乳動物細胞株を使用して実行するように変更することができます。実際に、我々はヒトゲノム26内のすべての注釈付き遺伝子内の少なくとも一つの潜在的な固有かつ高親和性のTFO結合部位が存在することを示しています。どうやって我々はムカジー＆バスケス、2016年16で利用してきたように、これまで、分かりやすくするために、我々は人間のU2OS細胞に特異的な変異レポータープラスミド（pSupFG1）上の特定のソラレン共役TFO（pAG30）を使用するためにこれらの技術を説明しました。

<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

高移動度グループボックス1（HMGB1）タンパク質は、転写、DNA複製、DNA修復などのゲノム中の多くの重要な機能を調節することに関与する非ヒストン建築タンパク質です。 HMGB1は、標準的なB-DNAへのより高い親和性を有する構造的に歪んだDNAに結合します。例えば、我々は、HMGB1がDNAに結合することがわかった共有結合、DNAの2本鎖をリンクらせんの歪みを引き起こし、未修復のままの場合は細胞死を引き起こす可能性が架橋（のICL）を、インター。それらの細胞毒性ポテンシャルを、いくつかのICL誘導剤は、現在臨床での化学療法剤として使用されます。 ICL形成剤は、特定の塩基配列の環境（ 例えば、5&#39;-TA-3 &#39;ソラレンのための好ましい架橋部位である）を示すが、それらは主に無差別様式でDNA損傷を誘発します。しかし、共有結合的に配列特異的にDNAに結合する三重鎖形成オリゴヌクレオチド（TFO）、にICL誘導剤を結合することにより、方法は、標的DNAの損傷を達成することができます。ここでは、ツールとして使用するために変異レポータープラスミド上のサイト固有のICLを生成するために、共有結合、8トリメチルソラレン（HMT）ソラレン5 &#39;、4&#39;-ヒドロキシメチル-4,5-へ&#39;末端に結合させTFOを使用しますアーキテクチャの変更、処理、およびヒト細胞におけるHMGB1による複雑なDNA損傷の修復を研究します。私たちは、レポータープラスミドにTFO指向のICLを調製するための、およびクロマチン免疫沈降アッセイを用いて、細胞の状況にTFO指向のICLとHMGB1の関連を調べるために実験技術について説明します。また、我々はHMGB1によってソラレン架橋プラスミドに導入超らせん巻きの量を測定することにより、損傷を受けたDNAの特定の建築変更を評価するために、DNAの超らせんのアッセイが記載されています。これらの技術は、TFO指向のICLまたは目的の任意の細胞株における他の標的DNA損傷処理および修復に関与する他のタンパク質の役割を研究するために使用することができます。

TFO指向ICLの形成はヒト細胞でICL処理の建築タンパク質の役割を調べるために使用されているプラ​​スミドに基づくアッセイ、のために重要です。変性アガロースゲル電気泳動は、TFO指向ICL形成の効率を決定するための容易な方法です。非架橋対照プラスミド（ 図1A、レーン2）と比較した場合TFO指向のICLを保有するプラスミドをアガロースゲルマトリッ...

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.

ツールは、Helix歪曲、部位特異的DNA鎖間架橋の処理に建築タンパク質HMGB1の役割を研究するために

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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