笔者想感谢瓦斯奎兹实验室的成员，有益的讨论。这项工作是由健康/美国国家癌症研究所的国家机构[CA097175，CA093279到KMV]的支持;与癌症预防和得克萨斯研究院[RP101501。资助为开放式接入费:健康/美国国家研究院国家癌症研究所[CA093279到KMV。

1.质粒基板TFO定向ICLS的制备<ol><li>孵育等摩尔量的质粒pSupFG1 10,11- DNA（5微克质粒DNA是一个很好的起点）与补骨脂共轭TFO AG30在8微升三缸结合缓冲液[50％甘油，10毫摩尔Tris（pH值7.6），10mM的氯化镁的2]和DH 2 O至40微升在琥珀色管的终体积。通过上下吹打调匀。在37℃水浴12小时的反应。 </li><li>预热UVA灯，以保证全功率输出。将石蜡膜三缸反应，然后将石蜡膜的UVA（365纳米）灯下冰。放置在灯和反应之间的聚酯薄膜过滤器。 </li><li> UVA照射为1.8焦耳/ cm 2的总剂量。储存样品于4℃用于进一步使用。 注意:穿适当的防护眼镜和服装的搭配UVA照射。 </li><li>线性200ng的上述制备的质粒与重striction酶生态 R1在使用制造商20μl总体积10倍提供缓冲。热变性，在65℃下进行20分钟的酶。旋转样品10分钟以10,000 xg离心并在4℃下储存。 </li><li>制备50倍的碱性缓冲液[1.5M的氢氧化钠，50毫乙二胺四乙酸（EDTA）]。 0.5毫克琼脂糖加入至50ml卫生署2 O.煮沸以溶解琼脂糖，并将其放置在50℃的水浴中。 </li><li>溶解琼脂糖的温度下降到50℃后，加入1毫升50X碱性缓冲液，混合，然后倒入凝胶进入凝胶托盘。等待一段时间，凝胶在使用前室温固化。 </li><li>加入4微升的0.5M EDTA至20微升线性DNA样品的孵育在室温下10分钟。 </li><li>添加6倍的碱性凝胶加载缓冲液（300毫氢氧化钠，6毫摩尔EDTA，18％（重量/体积）的高分子量的多糖，0.06％溴甲酚绿在卫生署2 O）与样品和为1 kb的DNA梯。 注:这是使用6X碱性凝胶缓冲液很重要，请讨论。 </li><li>负载样品到在冷室的凝胶，并在每平方厘米3.2伏运行过夜在1×的碱性缓冲液（12-16小时）。一旦样品已进入凝胶，放置在凝胶顶部的玻璃板，以防止其浮动。 </li><li>通过浸泡在中和缓冲液[的1M Tris-Cl（上pH值7.6），1.5M氯化钠，3M乙酸钠（pH 5.2）]，在室温下45分钟的凝胶中和样品。 </li><li>在50毫升水中染色用于DNA的凝胶用4微升1％溴化乙锭（EtBr）的在室温下1小时。用水脱色15分钟。通过使用成像系统观察DNA。 注意:乙锭是一种强效的诱变剂，并且可以通过皮肤被吸收。避免与溴化乙锭直接接触。 </li></ol>在人类细胞中TFO定向含ICL-的质粒转染2.免疫沉淀和<ol><li>板400000哺乳动物细胞（ 例如，U2OSØsteosarcoma细胞）每60mm培养皿中的Dulbecco改进的Eagle培养基（DMEM），补充有10％胎儿牛血清（FBS）的无抗生素的转染之前24小时。孵育细胞过夜，在37℃，5％的CO 2。 </li><li>在转染前，温热的生长培养基和磷酸盐缓冲盐水（PBS），以37℃的水浴中。暖转染试剂至室温。 </li><li>孵育30微升转染试剂用1×生长培养基的500微升（混合物1）和2 TFO定向含ICL-于500μl1X生长培养基（混合物2）的质粒中分离14毫升圆底管微克在10分钟室内温度。 </li><li>添加混合1混合2，吹打上下拌匀。孵育在室温下25-30分钟。 </li><li>用温PBS洗涤细胞两次。添加转染混合物中逐滴的方式在整个细胞的板均匀分布。 1毫升生长培养基添加到板，并使终体积至2ml。我们混合二。放置在37℃培养箱，用5％ 的 CO 2的培养板4小时。 </li><li>替换用补充有10％FBS3毫升生长介质的介质，进一步孵育16小时。不要使用抗生素。 </li><li>治疗细胞用80微升每板37％的新鲜的甲醛至大约1％的终浓度，并在不存在光的在室温下孵育10分钟。 注意:甲醛是有毒的，并应根据其安全指示进行处理。甲醛接触可造成伤害皮肤，眼睛和呼吸道。 </li><li>通过加入300微升每板冷却甘氨酸（在市售试剂盒提供）和温育5分钟，骤冷甲醛交联。删除媒体并用冷PBS洗涤两次，然后通过在1ml冷却（4℃）PBS刮入离心管收集细胞。保持冰样本在任何时候。 </li><li>通过在4℃下离心5分钟制备细胞沉淀T使用台式冷冻离心机13400 XG。吸管出上清液，并放置在冰上的细胞沉淀。 </li><li>均匀悬浮颗粒在1ml冷却（4℃）的缓冲液A（在市售试剂盒提供），并在冰上孵育10分钟。通过倒转该管偶尔混合。颗粒细胞作为前（参见上面的步骤2.9）。 </li><li>均匀悬浮在1ml冷冻沉淀（4℃）缓冲液B（在市售试剂盒提供），并通过反相在冰上孵育10分钟，偶尔搅拌。离心沉淀细胞像以前一样（参见上面的步骤2.9）。 </li><li>在200μl冷却缓冲液B再次重悬细胞中加入2微升微球菌核酸酶（MNase）中，并在室温下孵育10分钟。轻弹试管混合反应偶见。通过放置在冰上的管中并加入40毫摩尔EDTA停止MNase反应。颗粒细胞作为前（参见上面的步骤2.9）。 </li><li>悬浮细胞在100μl1×染色质immunoprecipitation缓冲液（芯片）缓冲（以市售试剂盒提供），辅以蛋白酶抑制剂的鸡尾酒。 </li><li>放置悬浮细胞在薄壁离心管。由浮动它们水浴超声器，持续20秒，随后在冰上20秒温育超声处理的样品。重复步骤超声9倍，产生〜800-1,200 bp的片段。 <ol><li>向20微升超声处理的样品添加3微升的5M的NaCl和1μlRNA酶A.混合均匀通过涡旋并在37℃下孵育30分钟。随后，加入2微升蛋白酶K，并在65℃孵育2小时。纯化使用PCR纯化试剂盒的样品。在1％琼脂糖凝胶运行的样本及与溴化乙锭可视化。 注:所产生的DNA的最大强度应等于或低于1,000基点。 </li></ol></li><li>在三个单独的试管中，添加30微升溶胞产物在一预冷硅化试管中，然后加70微升冷却的1×片外缓冲器的添加了蛋白酶抑制剂。加入1μ克抗免疫球蛋白（IgG），抗组蛋白H3（作为阳性对照），或抗HMGB1抗体与相应的管子。在冷室中连续旋转孵育过夜。 </li><li>悬浮G蛋白珠均匀在冷室。加入4微升G蛋白珠每管和孵化在寒冷的房间与其它旋转2-4小时。 </li><li>用磁力架，沉淀珠子和除去上清液。加入200μl1X片外缓冲器与蛋白酶抑制剂混合物混合以沉淀并在冷室与旋转洗5分钟。重复洗两次澡。 </li><li>执行与高盐沉淀1X缓冲液（含70毫米氯化钠）一个额外的清洗。 </li><li>悬浮用160微升的洗脱缓冲液（在市售试剂盒提供）的粒料，并在37℃用旋转温育30分钟。 </li><li>颗粒珠子收集上清在新管。添加5M的NaCl 6微升和2μl蛋白酶K，并在65孵育过夜C。 </li><li>纯化使用PCR产物纯化试剂盒的DNA和洗脱使用50微升的dh 2 O该DNA </li><li>进行PCR使用的引物组对感兴趣的区域（多个）16的反应，并解决与的EtBr染色的1％琼脂糖凝胶的产品。 </li></ol> 3.超螺旋测定和二维琼脂糖凝胶电泳<ol><li>制备DNA修复合成缓冲液（45毫摩尔的Hepes-KOH，pH值7.8; 70毫氯化钾; 7.4毫MgCl 2的0.9毫摩尔DTT; 0.4毫摩尔EDTA; 2毫摩尔ATP，20μM的的dNTPs，3.4％甘油和18微克牛血清白蛋白） 。存储在-80℃。在冰上解冻之前使用添加40毫米磷酸和2.5微克肌酸磷酸激酶。 </li><li>制备10倍超螺旋缓冲液[500毫摩尔Tris（pH值8.0），500mM的氯化钠，25mM的MgCl 2的，1毫摩尔EDTA]，并储存在室温下。 </li><li>超螺旋线性化质粒DNA pSupFG1 300纳克完全使用1微升牛痘拓扑异构酶I（5-15 U /微升）用1X在10微升反应超螺旋缓冲区。孵育在37℃下该混合物1小时。 </li><li>在冰上解冻的HeLa无细胞提取液24。到完全放松的质粒，加入25微升的HeLa无细胞提取物和DNA修复合成缓冲器。拌匀。添加额外的1微升牛痘拓扑异构酶I的的组合，并在37℃孵育1小时。 </li><li>加入十二烷基硫酸钠（SDS）（1％终浓度）和蛋白酶K（0.25毫克/毫升终浓度）终止反应。孵育在65℃下2小时。 </li><li>投与1倍的Tris硼酸EDTA（TBE）缓冲液中的1％琼脂糖凝胶。添加DNA上样染料和直接加载反应凝胶上间隔至少4车道。在2伏/厘米在1×TBE（尺寸1）在室温下运行8-10小时的凝胶，以解决topoisomers。 注:通过添加54克Tris碱的准备1升的dh 2 O的5倍TBE原液。 27.5克硼酸和20ml的0.5M EDTA（pH 8.0）中的。 </li><li>准备2升1倍的TBE与3微克/毫升氯喹的磷酸盐。浸泡在200毫升该溶液20分钟的凝胶。用铝箔覆盖凝胶托盘。 </li><li>到electrophorese在第二维凝胶，转动凝胶90°以顺时针方式和在含有1x TBE氯喹2伏/厘米运行4-6小时，以解决的正和负超螺旋。 </li><li>染色用的EtBr凝胶用于在摇臂1小时。脱色与卫生署2 O的凝胶15分钟，随后可视使用的成像器的topoisomers的热分布。 </li></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=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. 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-frequency+intrachromosomal+gene+conversion+induced+by+triplex-forming+oligonucleotides+microinjected+into+mouse+cells.">High-frequency intrachromosomal gene conversion induced by triplex-forming oligonucleotides microinjected into mouse cells.</a> Proc Natl Acad Sci U S A. 97, 9003-9008 (2000).</li><li>Vasquez, K. M., Wang, G., Havre, P. A., Glazer, P. 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The authors declare no conflict of interest.

TFO定向ICLS的有效形成取决于两个关键因素:第一，适当的缓冲液成分（ 如 ， 氯化镁 ），并用其靶DNA底物的TFO的孵育时间（取决于TFO的与其靶的结合亲和力双工和浓度使用）;第二，UVA的适当剂量（365 nm）的照射下有效地形成补骨脂交联。最佳三缸形成可以通过使用HPLC或凝胶纯化TFOs来实现。杂质污染的TFO可能会导致不希望的交联产物，其可以在实验结果进一步复杂化。以增加TFOs的...

通过Hoogsteen碱基-氢键形成三寡核苷酸（TFOs）结合双链DNA的序列特异性的方式，以形成三重螺旋结构1-5。三缸技术已被用于询问各种生物分子机制，如转录，DNA损伤修复和基因定位（在参考文献6-8中综述）。 TFOs已广泛用于诱导对报告质粒9,10-位点特异性破坏。我们的实验室和其他人以前用过的一个TFO，AG30，拴补骨脂素分子诱导特异性位点DNA间交联（ICLS）在质粒pSupFG1 5,10-12的supF基因。 ICLS是高度细胞毒性，因为这些病变共价交联的两条DNA链，并且如果左未修理，可阻断基因转录和阻碍DNA复制机械13,14。因为它们的细胞毒性潜力，ICL诱导剂已经被用作化学治疗药在治疗的癌症和其他疾病15。然而，ICLS的人类细胞中的处理和修复还不是很清楚。因此，更好地理解在人类细胞参与ICLS的处理的机制可能有助于改善基于ICL-化疗方案的效力。 TFO诱导ICLS及其修复中间体具有引起显著结构扭曲到DNA螺旋的潜力。这种失真是用于建筑的蛋白质，其结合到扭曲的DNA比对规范B型双螺旋DNA 16-20更高的亲和力可能的目标。这里，我们通过染色质免疫沉淀（ChIP）上补骨脂交联的质粒分析研究了高丰度的建筑蛋白的关联，HMGB1与人体细胞ICLS和在调节人体补骨脂交联的质粒DNA的拓扑确定为HMGB1作用肿瘤细胞裂解物。 HMGB1是一种高度丰富和广泛表达非他音建筑蛋白结合到受损的DNA和可替换的结构的DNA底物，比典型的B-型DNA 17-20更高的亲和力。 HMGB1参与几个DNA的代谢过程，如转录，DNA复制和DNA修复16,21-23。我们以前表明，HMGB1结合的TFO定向ICLS 体外以高亲和性20。此外，我们已经证明，缺乏的HMGB1增加TFO定向ICLS的诱变处理和识别的HMGB1的核苷酸切除修复（NER）辅因子23,24。最近，我们已发现，HMGB1是在人细胞中具有TFO定向ICLS相关联，并且其招募这种病变取决于NER蛋白质，XPA 16。的DNA的负超螺旋已经显示由NER 25以促进高效去除DNA损伤的，我们已经发现，HMGB1优先诱导负超螺旋上的TFO定向含ICL-纤溶酶中间基板（相对于非破坏质粒基板）16，提供了一个更好的理解的HMGB1作为NER辅因子的潜在作用（多个）。 ICLS的处理未完全在人类细胞中理解;因此，根据本文所描述的分子工具的技术和开发的实验可能导致涉及ICL修理额外的蛋白质，这反过来又可以起到可以被利用来改善癌症化疗方案的效力作为药理学靶的鉴定。 这里，一个有效的方法，以通过变性琼脂糖凝胶电泳进行了讨论评估TFO定向的ICL形成的质粒DNA的效率。另外，使用含有TFO定向ICLS质粒，技术来使用改性ChIP实验确定在蜂窝上下文HMGB1和ICL-损坏质粒的关联已经描述。此外，一个浅显的方法来研究由日推出拓扑修改Ë建筑HMGB1蛋白质，特别是对人的细胞裂解物ICL-损坏质粒基材已被通过二维凝胶电泳进行超螺旋化验确定。所描述的技术可用于促进DNA修复和建筑的蛋白质中的目标DNA损伤处理的参与对人体细胞的质粒的理解。 我们描述了TFO定向的位点特异性补骨脂ICLS对质粒DNA，和随后的质粒沉淀和超螺旋测定法分别以确定与病变相关联该改变的DNA拓扑蛋白，和蛋白，形成详细协议。这些测定法可以修改与其他DNA损伤剂，TFOs，质粒底物，和感兴趣的哺乳动物细胞系进行。事实上，我们已经表明，至少有一个潜在的独特和高亲和力的TFO结合位点在人基因组26每注释基因内。怎么样以往，为清楚起见，我们在人U2OS细胞的特定突变报道质粒（pSupFG1）描述了这些技术的使用特定的补骨脂素-缀合的TFO（pAG30），因为我们在慕克吉＆Vasquez的，2016年16已经利用。

<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

高迁移率族蛋白1（HMGB1）蛋白是参与调节基因组中的许多重要的功能，如转录，DNA复制和DNA修复的非组蛋白的建筑蛋白。 HMGB1结合具有较高的亲和力结构扭曲的DNA，而不是典型的B-DNA。例如，我们发现，HMGB1结合DNA链间交联（ICLS），其共价连接的DNA的两条链，导致螺旋的畸变，并且如果左未修理可引起细胞死亡。由于它们的细胞毒性潜力，几个ICL诱导剂目前用作在临床化疗剂。而ICL形成剂显示某些碱基序列（如 5&#39;-TA-3"是首选的交联网站补骨脂）的喜好，他们在很大程度上引起不加区别的时尚DNA损伤。然而，由ICL诱导剂共价偶联到三链形成性寡核苷酸（TFO），其结合的DNA序列中的特异性方式，可以实现目标DNA损伤。这里，我们使用一个TFO共价结合于5&#39;末端到4&#39;-羟甲基4,5&#39;，8三甲基补骨脂（HMT）补骨脂上产生一个突变报告质粒一个位点特异性的ICL作为工具使用通过在人类细胞HMGB1研究建筑修改，处理和复杂的DNA损伤的修复。我们描述的实验技术编写有关报告质粒TFO定向ICLS，并使用染色质免疫测定法来询问蜂窝背景与TFO定向ICLS HMGB1的关联。此外，我们描述的DNA超螺旋测定法通过测量由HMGB1补骨脂交联的质粒引入超螺旋匝的数量，以评估受损的DNA的特定的架构的修改。这些技术可以用于研究参与TFO定向ICLS或在任何感兴趣的细胞系其他靶向DNA损伤的处理和修复的其它蛋白质的作用。

在TFO定向的ICL的形成是用于基于质粒测定法，其用于询问在人类细胞中的建筑蛋白在ICL处理的作用是至关重要的。变性琼脂糖凝胶电泳是确定TFO定向的ICL形成的效率的便利途径。窝藏TFO定向ICLS质粒迁移通过时相比，未交联的对照质粒（ 图1A，泳道2）的琼脂糖凝胶基质（ 图1A，泳道3）较慢迁移率。在泳道2和3（ 图1A）的条带的密度量?...

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.

工具来研究蛋白质建筑HMGB1的作用，螺旋窜改，站点特定的DNA间交联处理

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

tools-to-study-role-architectural-protein-hmgb1-processing-helix

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Genetics

8.2K 次观看.  The University of Texas at Austin. 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. 

视频: Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell&#39;s DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

We describe a robust gene replacement strategy to genetically manipulate the smut fungus Ustilago maydis. This protocol explains how to generate deletion mutants to investigate infection phenotypes. It can be extended to modify genes in any desired way, e.g., by adding a sequence encoding a fluorescent protein tag.

植物病原体的遗传操作<em&gt;玉米黑粉菌</em&gt;研究真菌生物学和植物微生物相互作用

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

科研

遗传学

A Method to Study the C924T Polymorphism of the Thromboxane A2 Receptor Gene

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in atherogenesis progression, ischemia, and myocardial infarction. Here we present a methodology of patient genotyping to investigate the post-transcriptional role of the C924T polymorphism (rs4523) situated at the 3&#39; region of the TBXA2 receptor gene. This method relies on DNA extraction from whole blood, polymerase chain reaction (PCR) amplification of the TBXA2 gene portion containing the C924T mutation, and identification of wild type and/or mutant genotypes using a restriction digest analysis, specifically a restriction fragment length polymorphism (RFLP) on agarose gel. In addition, the results were confirmed by sequencing the TBXA2R gene. This method features several potential advantages, such as high efficiency and the rapid identification of the C924T polymorphism by PCR and restriction enzyme analysis. This approach allows a predictive study for plaque formation and atherosclerosis progression by analyzing patient genotypes for the TBXA2R C924T polymorphism. Application of this method has the potential to identify subjects who are more susceptible to atherothrombotic processes, in particular subjects in a high-risk, aspirin-treated group.

In the present study, we describe a methodology to analyze the C924T genotype. The protocol consists of three phases: DNA extraction, amplification by polymerase chain reaction (PCR), and analysis of the restriction fragment length polymorphism (RFLP) on agarose gel.

血栓烷 A2 受体基因 C924T 多态性的研究方法

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in ...

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the increase in gene copy number per cell provided by multicopy plasmids could accelerate the evolution of plasmid-encoded genes. In this work, we present an experimental system to test the ability of multicopy plasmids to promote gene evolution. Using simple molecular biology methods, we constructed a model system where an antibiotic resistance gene can be inserted into Escherichia coli MG1655, either in the chromosome or on a multicopy plasmid. We use an experimental evolution approach to propagate the different strains under increasing concentrations of antibiotics and we measure survival of bacterial populations over time. The choice of the antibiotic molecule and the resistance gene is so that the gene can only confer resistance through the acquisition of mutations. This &#34;evolutionary rescue&#34; approach provides a simple method to test the potential of multicopy plasmids to promote the acquisition of antibiotic resistance. In the next step of the experimental system, the molecular bases of antibiotic resistance are characterized. To identify mutations responsible for the acquisition of antibiotic resistance we use deep DNA sequencing of samples obtained from whole populations and clones. Finally, to confirm the role of the mutations in the gene under study, we reconstruct them in the parental background and test the resistance phenotype of the resulting strains.

Here we present an experimental method to test the role of multicopy plasmids in the evolution of antibiotic resistance.

多拷贝产质粒在抗生素耐药性演变中的作用试验

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the ...

A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells

The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in mammalian cell lines. Rather than globally assaying removal of xenobiotic-induced or spontaneous chromosomal DPC removal, this assay examines the repair of a homogeneous, chemically defined lesion specifically introduced at one site within a plasmid DNA substrate. Importantly, this approach avoids the use of radioactive materials and is not dependent on expensive or highly-specialized technology. Instead, it relies on standard recombinant DNA procedures and widely available real-time, quantitative polymerase chain reaction (qPCR) instrumentation. Given the inherent flexibility of the strategy utilized, the size of the crosslinked protein, as well as the nature of the chemical linkage and the precise DNA sequence context of the attachment site can be varied to address the respective contributions of these parameters to the overall efficiency of DPC repair. Using this method, plasmids containing a site-specific DPC were transfected into cells and low molecular weight DNA recovered at various times post-transfection. Recovered DNA is then subjected to strand-specific primer extension (SSPE) using a primer complementary to the damaged strand of the plasmid. Since the DPC lesion blocks Taq DNA polymerase, the ratio of repaired to un-repaired DNA can be quantitatively assessed using qPCR. Cycle threshold (CT) values are used to calculate percent repair at various time points in the respective cell lines. This SSPE-qPCR method can also be used to quantitatively assess the repair kinetics of any DNA adduct that blocks Taq polymerase.

The goal of this protocol is to quantify the repair of defined DNA-protein crosslinks on plasmid DNA. Lesioned plasmids are transfected into recipient mammalian cell lines and low-molecular weight harvested at multiple time points post-transfection. DNA repair kinetics are quantified using strand-specific primer extension followed by qPCR.

一种简便、快速、定量的测定 DNA 蛋白 Crosslinks 在转染哺乳动物细胞中的研究

The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

线粒体 DNA 甲基化的精确检测方法

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

酵母神经退行性蛋白病模型中组蛋白后转化修饰改变的表征

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame (ORF) annotation impose two arbitrary criteria: a minimum length of 100 codons and a single ORF per transcript. However, a growing number of studies report expression of proteins from allegedly non-coding regions, challenging the accuracy of current genome annotations. These novel proteins were found encoded either within non-coding RNAs, 5' or 3' untranslated regions (UTRs) of mRNAs, or overlapping a known coding sequence (CDS) in an alternative ORF. OpenProt is the first database that enforces a polycistronic model for eukaryotic genomes, allowing annotation of multiple ORFs per transcript. OpenProt is freely accessible and offers custom downloads of protein sequences across 10 species. Using OpenProt database for proteomic experiments enables novel proteins discovery and highlights the polycistronic nature of eukaryotic genes. The size of OpenProt database (all predicted proteins) is substantial and need be taken in account for the analysis. However, with appropriate false discovery rate (FDR) settings or the use of a restricted OpenProt database, users will gain a more realistic view of the proteomic landscape. Overall, OpenProt is a freely available tool that will foster proteomic discoveries.

OpenProt is a freely accessible database that enforces a polycistronic model of eukaryotic genomes. Here, we present a protocol for the use of OpenProt databases when interrogating mass spectrometry datasets. Using OpenProt database for analysis of proteomic experiments allows for discovery of novel and previously undetectable proteins.

基于质谱的蛋白质组学分析, 利用 OpenProt 数据库揭开从非规范开放阅读框架翻译的新蛋白质

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental methods to study protein-nucleic acid interactions require the use of fluorescent tags, radioactive isotopes, or other labels to detect and analyze specific target molecules. Biotin, a non-radioactive nucleic acid label, is commonly used in electrophoretic mobility shift assays (EMSA) but has not been regularly employed to monitor protein activity during nucleic acid processes. This protocol illustrates the utility of biotin labeling during in vitro enzymatic reactions, demonstrating that this label works well with a range of different biochemical assays. Specifically, in alignment with previous findings using radioisotope 32P-labeled substrates, it is confirmed via biotin-labeled EMSA that MEIOB (a protein specifically involved in the meiotic recombination) is a DNA-binding protein, that MOV10 (an RNA helicase) resolves biotin-labeled RNA duplex structures, and that MEIOB cleaves biotin-labeled single-stranded DNA. This study demonstrates that biotin is capable of substituting 32P in various nucleic acid-related biochemical assays in vitro.&#160;

Presented here are protocols for in vitro biochemical assays using biotin labels that may be widely&#160;applicable for studying protein-nucleic acid interactions.

使用生物素标签研究蛋白质-核酸相互作用的体外生化分析

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...

A Bioinformatics Pipeline to Accurately and Efficiently Analyze the MicroRNA Transcriptomes in Plants

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in regulating gene expression at the post-transcriptional level. Sequencing sRNA libraries by Next Generation Sequencing (NGS) methods has been widely employed to identify and analyze miRNA transcriptomes in the last decade, resulting in a rapid increase of miRNA discovery. However, two major challenges arise in plant miRNA annotation due to increasing depth of sequenced sRNA libraries as well as the size and complexity of plant genomes. First, many other types of sRNAs, in particular, short interfering RNAs (siRNAs) from sRNA libraries, are erroneously annotated as miRNAs by many computational tools. Second, it becomes an extremely time-consuming process for analyzing miRNA transcriptomes in plant species with large and complex genomes. To overcome these challenges, we recently upgraded miRDeep-P (a popular tool for miRNA transcriptome analyses) to miRDeep-P2 (miRDP2 for short) by employing a new filtering strategy, overhauling the scoring algorithm and incorporating newly updated plant miRNA annotation criteria. We tested miRDP2 against sequenced sRNA populations in five representative plants with increasing genomic complexity, including Arabidopsis, rice, tomato, maize and wheat. The results indicate that miRDP2 processed these tasks with very high efficiency. In addition, miRDP2 outperformed other prediction tools regarding sensitivity and accuracy. Taken together, our results demonstrate miRDP2 as a fast and accurate tool for analyzing plant miRNA transcriptomes, therefore a useful tool in helping the community better annotate miRNAs in plants.

A bioinformatics pipeline, namely miRDeep-P2 (miRDP2 for short), with updated plant miRNA criteria and an overhauled algorithm, could accurately and efficiently analyze microRNA transcriptomes in plants, especially for species with complex and large genomes.

生物信息学管道，用于准确、高效地分析植物中的微RNA转录

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in ...