Guikema实验室的研究由荷兰科学研究组织（VIDI授权016126355）和"Stichting Kinderen Kankervrij"KiKA（项目252）的创新研究奖励计划资助。

1.细胞治疗和DNA损伤诱导<ol><li>在补充有20％FCS，50μMβ-巯基乙醇，2mM L-谷氨酰胺，100U / mL青霉素和100μg/ mL链霉素的IMDM中培养人BCR-ABL + B细胞急性成淋巴细胞细胞系BV173或SUP-B15在5％CO 2的气氛中37℃。在6孔板中以5mL /孔计数细胞并以2×10 6细胞/ mL平板。 注意:任选地，可以使用各种来源的悬浮细胞系。 </li><li> 为了阻止细胞周期G1期的BCR-ABL + B-ALL细胞，加入5μM的伊马替尼甲磺酸盐（STI571），并孵育过夜。 <ol><li>任选地，当研究整个细胞周期或BCR-ABL阴性细胞类型中的蛋白质 - 蛋白质相互作用时，省略此步骤。 </li></ol></li><li> 通过向培养物中加入50ng / mL新卡那霉素（NCS）诱导DNA损伤，并孵育2小时。 <ol><li>或者，通过使用放射源[ 137 Cs]以0.5Gy / min以5Gy的剂量照射细胞来诱导DNA损伤。照射后，让细胞在37℃，5％CO 2气氛中恢复2小时。 </li></ol></li><li>任选地，为了评估ATM激酶活性对DNA损伤的反应，在加入NCS或照射治疗之前加入5μM的ATM激酶抑制剂KU55933。 </li><li>通过在烧杯或锥形烧瓶中的50mL 1x PBS的顶部层叠5g级分的V BSA，制备1×PBS + 10％BSA，并放置在室温下直到BSA溶解。不要摇晃或搅拌，因为会引起大量的起泡。缓慢过滤0.22μm过滤器并保持在冰上。 </li><li>收获细胞，用冰冷的1x PBS洗涤两次。计数细胞并在1×PBS + 10％BSA中以2×10 6 / mL重悬。保持细胞在冰上。 </li></ol>细胞离心，固定和渗透<ol><li>准备4％多聚甲醛溶液1倍PBS新鲜。加入2 g PFA至50 mL 1x PBS，并在55°C振荡水浴中孵育，直至溶液澄清。通过0.22μm过滤器过滤溶液，并在RT存储下使用。 注意:任选地，4％PFA固定剂可以在-20°C下冷冻保存。冻结后，解冻一次即可使用。否则，PBS中的4％PFA溶液可以直接购买。 </li><li>仔细研磨76毫米×26毫米的显微镜幻灯片与无绒组织，并通过将其放置在Coplin罐中的96％乙醇中5分钟来脱脂。让显微镜空气干燥10分钟。 </li><li>将显微镜载玻片放入面积为6 mm的细胞离心/细胞学漏斗中。通过将50μL1×PBS + 10％BSA移液到每个漏斗中预先涂覆载玻片，并以500rpm离心1分钟。 </li><li>向每个漏斗加入100μL细胞悬浮液（等于0.2×10 6个细胞），并以500rpm离心5分钟。对于每种抗体组合，需要ast 4制剂（双抗体实验;两个单抗体对照;无抗体对照）。 </li><li> 小心地拆卸漏斗，并确保不要触摸点。立即进行样品固定。 <ol><li>任选地，将载玻片风干至少30分钟。风干后，将薄片单独包裹在铝箔中，储存于-20°C直至使用。当使用冷冻制剂时，请确保解冻用纸巾包裹的载玻片，以防止幻灯片上露出冷凝。 </li></ol></li><li>使用PAP笔式液体阻塞剂（5mm尖端）在斑点周围绘制一个圆圈（大约直径15-20 mm）。 </li><li>向每个斑点加入50μL的4％PFA固定溶液，并在室温下孵育30分钟。 </li><li>在轻轻搅拌下，在室温下，在Coplin罐中用1x PBS洗涤细胞3次5分钟。 </li><li>使用无绒毛组织将斑块周围的斑块干燥，并使用pI从尽可能多的液体中除去尽可能多的液体宠物没有触摸现场，或通过倾斜幻灯片。向每个斑点加入50μL的0.25％Triton-X在1×PBS中，并在室温下不搅拌孵育10分钟。 </li><li>在轻轻搅拌下，在Coplin罐中，在TBS（TBS-T）中的约50-60mL的0.05-60ml的Tween-20中洗涤载玻片3次5分钟。可以以这种方式同时处理10个幻灯片。 </li></ol> 3.阻断和一级抗体孵育<ol><li>点击TBS-T，用无棉绒纸巾围绕斑点干燥。使用移液管尽可能多地从现场清除液体，而不必接触该位置，或通过倾斜滑块。短暂旋转阻塞溶液，并向每个点加入一滴（约40μL）。在预热的加湿室中在37℃下孵育1小时。 </li><li>在商业抗体稀释剂，涡旋抗体稀释剂使用前，以1:100混合山羊抗ATM和1:100的兔抗磷酸化Ser15-p53，制备抗体混合物。用于控制实验制备仅含有山羊抗ATM和仅抗兔抗磷酸化Ser15-p53抗体（单抗体对照）的抗体稀释液。 </li><li>点击阻塞溶液，但注意不要让细胞在加入抗体稀释液之前干燥。加入30μL每种抗体混合液稀释液，并在4℃下在加湿室中孵育过夜。 </li><li> 通过将一袋缓冲液A和缓冲液B混合物的内容物溶解在最终体积为1000mL的水中来原位洗涤缓冲液A和洗涤缓冲液B。 <ol><li>或者，通过将8.8g NaCl，1.2g Tris-碱和0.5mL Tween-20溶解在800mL水中来制备洗涤缓冲液A。使用HCl将pH调节至7.4，加入最终体积为1000 mL。 </li><li>或者，通过将5.84g NaCl，4.24 Tris-碱和26.0g Tris-HCl溶于500mL水中制备洗涤缓冲液B.使用HCl将pH调节至7.5。加入水至最终体积为1000 mL。 </li><li>过滤两种溶液通过0.22μm过滤器。 </li></ol></li><li>用轻轻搅拌的室温下，在Coplin罐中用1x 原位洗涤缓冲液A洗涤载玻片2次5分钟。 </li></ol> 4. PLA探针，连接和扩增<ol><li>通过在商业抗体稀释液中稀释1:5的抗山羊PLUS和抗兔MINUS来制备PLA探针。从幻灯片中抽出1x 原位洗涤缓冲液A，并向每个斑点加入30μLPLA探针混合物。在预热的加湿室中37℃孵育1小时。 注意:只要所应用的一抗在不同的物种中升高，PLA二级抗体PLUS和MINUS PLA探针的组合也可以使用（抗小鼠，抗兔，抗山羊）包含一个PLUS探针和一个MINUS探针。 </li><li>在轻轻摇动的同时，在Coplin罐中用〜50-60mL 1x 原位洗涤缓冲液A洗涤载玻片2次5分钟。/ LI&gt; <li>通过向39μL的连接溶液中加入1μL的连接酶，在冰上准备连接连接酶溶液，并向每个点加入40μL的连接连接酶溶液。在预热的加湿室中在37℃下孵育30分钟。 </li><li>在轻轻搅拌下，在Coplin罐中用〜50-60mL 1x 原位洗涤缓冲液A洗涤载玻片2次5分钟。 </li><li>将放大聚合酶混合物在冰上通过在水中1:5稀释扩增储备溶液来制备。然后向每个斑点的39.5μL的1x扩增溶液中加入0.5μL的聚合酶。 注意:扩增试剂是光敏感的，应防止直接照射。扩增试剂有四种不同的颜色（RED:激发594nm，发射624nm; FarRED:激发644nm，发射669nm; ORANGE:激发554nm，发射576nm; GREEN:激发501nm，发射523nm）确保显微镜设置可用适当的激发特性和滤镜来对这些颜色进行成像。 </li><li>点击1x 原位洗涤缓冲液A，每个点加入40μL的Amplification-Polymerase混合物。在预热的加湿室中在37℃下孵育100分钟。 </li></ol> 5.安装和成像<ol><li>通过向99 mL水中加入1 mL 1x洗涤缓冲液B来制备0.01x 原位洗涤缓冲液B。 </li><li>点击放大聚合酶混合物，并用轻轻搅拌的RT在Coplin罐中用1x 原位洗涤缓冲液B洗涤载玻片2次10分钟。 </li><li>在0.01x洗涤缓冲液B中洗涤载玻片1分钟</li><li>分离多余的0.01x洗涤缓冲液B，并用无绒纸巾将斑块周围的玻片干燥。使用移液管尽可能多地从现场清除液体，而不必接触该位置，或通过倾斜滑块。 </li><li>在不含DAPI的安装介质中稀释含DAPI的安装介质1:5，以避免核的过度染色。 </li><li>将25-30μL含有DAPI的1:5 DAPI安装介质添加到24 mm x 24 mm的盖玻片上，并用滑块拾起盖板，施加轻微的压力，以确保没有被困的气泡。用指甲油密封盖玻片，并在成像前等待至少15分钟。 </li><li> 使用（共焦）荧光显微镜分析载玻片。 <ol><li>在至少4个获取的图像场中计数每个细胞的点状PLA信号的数目（20x目标，基于使用处理的细胞中7±5个信号的预期平均值的功率计算，每个条件或抗体组合计数至少20个细胞，和未经处理的细胞中的2±2个信号，I型误差的概率为0.05，功率为80％，如前所述15 ）。 注意:最佳分辨率是通过使用软件包（ 例如 ，ImageJ，Leica Acquisition Suite（LAS））获取Z-stack和去卷积来实现的。信号在DAPI染色定义的核周期内，ls应该很容易识别。 </li><li>不要计数出现在细胞核外的信号。通常，NCS处理或γ照射导致每个细胞约5-10个phosho-p53 / ATM PLA信号。 "单抗体"对照可能会产生一些背景信号，但每20个细胞中每1个不得超过1-2个PLA信号。 </li><li>出版和演示目的，使用40X或63X浸油目标拍摄图像。幻灯片可以在4°C储存，并应防止曝光。 </li></ol></li></ol>

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作者没有什么可以披露的。

在本报告中，证明PLA可用于确定和可视化悬浮细胞培养物中蛋白质之间的特异性相互作用。值得注意的是，这里描述的方案不限于DNA修复复合物的研究，而且还适用于可视化和定量悬浮细胞培养物中的其他蛋白质 - 蛋白质相互作用。显示当暴露于DNA损伤诱导剂时，ATM激酶与G1-阻滞的BCR-ABL + B-ALL细胞中的磷酸化p53相互作用。以前，我们使用PLA技术来表明ATM和FOXO1转录因子在BCR-ABL + B-ALL细胞中相互作?...

DNA损伤触发高度受限制的一系列事件，涉及蛋白质 - 蛋白质相互作用和翻译后修饰，可确保DNA的有效快速修复，从而保护基因组完整性1 。通常，通过（共聚焦）荧光显微镜监测所谓的电离辐射诱导病灶（IRIF）的形成，研究暴露于电离辐射的细胞中的DNA修复。许多DNA修复和DNA损伤感应蛋白形成IRIF，其代表在染色质位点成核的蛋白质复合物，其维持DNA损伤2,3 。随着时间的推移，IRIF的位置和分辨率为DNA修复的时空组织提供了重要的见解，并且可能表明不同DNA修复途径的参与。损伤的DNA损伤和细胞周期阶段的性质决定了哪个DNA修复途径被激活。佛<html> r实例，在积极参与DNA复制（S期）的细胞中，同源重组（HR）是显性DNA修复途径，而在细胞周期的G1-或G2 / M期细胞中，同源末端连接（NHEJ）修复途径占优势。 DNA损伤后的最早事件之一是激活DNA损伤感应激酶共济失调血管扩张 - 突变蛋白（ATM），其主要在细胞周期的G1-和G2 / M期活跃并调节NHEJ，和共济失调血管扩张症和Rad3相关蛋白（ATR），其通过激活HR作用于S期。 ATM和ATR都是非常多效的激酶，磷酸化参与DNA修复，细胞死亡和存活的许多蛋白质4 。已经显示两种激酶在暴露于基因毒性应激后磷酸化并激活肿瘤抑制蛋白p53，表明这些激酶是关键肿瘤抑制信号轴的上游介质"xref"&gt; 5，6 。 IRIF的形成和组成通常通过使用双色免疫荧光染色和显微镜测定不同蛋白质的共定位来评估，然而，并非所有作为修复蛋白复合物的蛋白质形成IRIF，这限制了该方法的适用性。此外，（共焦）免疫荧光显微镜受光衍射性质的限制，导致约200-300nm的相当差的空间分辨率，超过大多数亚细胞结构的大小，其基本上禁止蛋白质 - 蛋白质相互作用的直接询问分子水平。因此，通过（共焦）荧光显微镜检测的免疫荧光染色模式的共定位不一定表示直接的蛋白质 - 蛋白质相互作用。最近，已经开发了新的超分辨率技术，如三维结构照相显微镜（3D-SIM） 7 ，成功应用于纳米尺度细胞研究53BP1和BRCA1 IRIF形成，揭示了共聚焦激光扫描显微镜无法检测到的这些蛋白质的空间分布特征。 可以使用其他几种方法来检测体内的蛋白质 - 蛋白质相互作用，例如共免疫沉淀，下拉法和酵母双杂交筛选方法。然而，这些技术相当繁琐，需要大量的细胞或蛋白质或涉及蛋白质的过表达，其引入实验工件。最近，已经开发了一种新技术，其允许原位 （ 即在细胞和组织中）的蛋白质 - 蛋白质相互作用的可视化和定量，称为邻近连接测定（PLA） 9,10 。镨通过与寡核苷酸（所谓的PLA探针）缀合的二级抗体检测识别感兴趣的两种蛋白质的重要​​抗体。如果两种不同的二级抗体由于被第一抗体识别的蛋白质之间的相互作用而足够接近，则缀合的寡核苷酸杂交并连接形成封闭的环状DNA底物。随后通过滚环扩增扩增此圆形底物，并用荧光染料缀合的互补寡核苷酸显色。使用PLA，蛋白质 - 蛋白质相互作用的亚细胞定位被保留，因为荧光标记的滚环扩增产物保持附着于PLA探针。基于抗体直径约为7-10nm 11的发现，该测定法的分辨率&lt;50nm。只有两对抗体（primary + secon）才能进行滚环扩增dary）在由其大小（10 + 10 + 10 + 10 = 40nm）定义的周界内物理相互作用。信号放大步骤增加了PLA测定的灵敏度，并且能够检测到几乎不表达的蛋白质的相互作用。 PLA产生可以在每个细胞基础上量化的点状，类似焦点的信号模式，由此可以评估蛋白质 - 蛋白质相互作用中的细胞内和细胞间变化。 DNA修复复合物和IRIFs的形成和组成主要在贴壁细胞系如人骨肉瘤上皮细胞系U2OS，人胚胎肾细胞系HEK293和视网膜色素上皮细胞系RPE-1中进行研究，生长和易转染。使用诸如淋巴样和骨髓细胞系的悬浮细胞培养物不太频繁，因为它们不易于转染，并且通常不粘附到盖玻片上，因此需要额外的/替代的eps成像。然而，DNA损伤的解决方案在淋巴样和骨髓恶性肿瘤的情况下非常相关，因为DNA损伤反应经常受到这些肿瘤中的基因组（驱动）畸变的影响，在正常淋巴和骨髓的恶性转化中起关键作用（祖细胞） 12,13,14 。 该协议描述了如何在悬浮细胞培养物中诱导DNA损伤后，如何使用PLA来评估和定量蛋白质 - 蛋白质相互作用。在这里，执行PLA以确定和可视化在诱导经历G1期细胞周期停滞的人B细胞白血病细胞中的DNA损伤时ATM和p53之间的相互作用。值得注意的是，本文提出的方案并不限于研究在G1停滞的白血病细胞中的ATM和p53相互作用，而且还可以用于其他蛋白质 - 蛋白质相互作用在各种细胞类型和悬浮细胞培养物中。

<table><tbody><tr><td>BV173 cell line</td><td>DSMZ</td><td>AC-20</td><td>BCR-ABL+ B-ALL cell line</td></tr><tr><td>SUP-B15 cell line</td><td>DSMZ</td><td>ACC-389</td><td>BCR-ABL+ B-ALL cell line</td></tr><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium (IMDM)</td><td>Gibco (Life Technologies)</td><td>21980-032</td><td></td></tr><tr><td>Fetal Calf Serum</td><td>Sigma Aldrich</td><td>F7524</td><td>lot #: 064M3396</td></tr><tr><td>L-glutamine</td><td>Gibco (Life Technologies)</td><td>25030-024</td><td></td></tr><tr><td>penicillin/streptomycin</td><td>Gibco (Life Technologies)</td><td>15140-122</td><td></td></tr><tr><td>imatinib methanesulfonate</td><td>LC Laboratories</td><td>I-5508</td><td>Dissolve in DMSO, prepare 10 mM stock solution</td></tr><tr><td>neocarzinostatin</td><td>Sigma Aldrich</td><td>N9162</td><td>Mutagenic/teratogenic, handle with care</td></tr><tr><td>KU55933</td><td>Selleckchem</td><td>S1092</td><td>Dissolve in DMSO, prepare 5 mM stock solution</td></tr><tr><td>Starfrost Microscopy Slides</td><td>Waldemar Knittel</td><td>VA11200 003FKB</td><td></td></tr><tr><td>PAP pen liquid blocker</td><td>Sigma Aldrich</td><td>Z377821-1EA</td><td></td></tr><tr><td>Cytospin funnel</td><td>Q Path Labonord SAS</td><td>003411324</td><td></td></tr><tr><td>Duolink In Situ Red Starter Kit Goat/Rabbit</td><td>Sigma Aldrich</td><td>DUO92105</td><td>Available for different species/combinations, also available in FarRED, Orange and Green</td></tr><tr><td>goat-anti-ATM</td><td>Bethyl Laboratories</td><td>A300-136A</td><td>PLA-grade; we succesfully used lot#A300-136A-1 in our studies</td></tr><tr><td>rabbit-anti-phospho-Ser15-p53</td><td>Cell Signaling Technology</td><td>9284</td><td>We succesfully used lot #9284-4 in our studies</td></tr><tr><td>Vectashield antifading mounting medium with DAPI</td><td>Vector Labs</td><td>H-1200</td><td></td></tr><tr><td>Vectashield antifading mounting medium</td><td>Vector Labs</td><td>H-1000</td><td></td></tr><tr><td>4% paraformaldehyde in PBS</td><td>Santa Cruz Biotechnology</td><td>sc-281692</td><td>Also available from various other vendors</td></tr></tbody></table>

detection and visualization of dna damage-induced protein complexes in suspension cell cultures using the proximity ligation assay

DNA损伤反应协调自发发生的DNA损伤的修复，由遗传毒性应激引起，或出现在淋巴细胞中程序性DNA断裂的上下文中。共济失调 - Telangiectasia突变激酶（ATM），ATM-和Rad3相关激酶（ATR）和DNA依赖性蛋白激酶（DNA-PKcs）的催化亚基是首先在DNA损伤诱导时被激活，并且是控制DNA修复，凋亡和细胞存活的网络的中央调节剂。作为肿瘤抑制途径的一部分，ATM和ATR通过磷酸化激活p53，从而调节p53的转录活性。 DNA损伤也导致所谓的电离辐射诱导灶（IRIF）的形成，其代表DNA损伤传感器和在DNA损伤部位积聚的修复蛋白质的复合物，其通过荧光显微镜显现。然而，蛋白质在IRIF中的共定位并不一定意味着d直接的蛋白质 - 蛋白质相互作用，因为荧光显微镜的分辨率是有限的。 原位接近连接测定（PLA）是一种新颖的技术，可以以前所未有的特异性和灵敏度直接显示细胞和组织中的蛋白质 - 蛋白质相互作用。该技术基于与感兴趣的蛋白质结合的特异性抗体的空间接近。当询问的蛋白质在〜40nm内时，扩增反应由与抗体缀合的寡核苷酸引发，扩增产物通过荧光标记显现，产生对应于相互作用蛋白质的亚细胞位置的信号。使用ATM和p53之间建立的功能相互作用作为示例，在此证明PLA如何可用于悬浮细胞培养物中以研究作为DNA损伤反应的组成部分的蛋白质之间的直接相互作用。

残留Ser15上p53的磷酸化显示依赖于ATM激酶活性16 。为了证明和证实PLA技术对悬浮细胞培养物细胞分裂素制剂的特异性，表明通过在细胞周期G1期停滞的BCR-ABL + B-ALL细胞2小时NCS处理诱导DNA损伤导致ATM和磷酸化Ser15-p53之间的特异性相互作用，如预期的那样。在用NCS处理的大多数细胞的细胞核中观察到点状PLA信号（ 图1B ），每个细胞平均?...

Watch this Scientific Journal Video about Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay at JoVE.com

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

在这里，证明了原位邻近连接测定（PLA）如何可以用于检测和可视化暴露于基因毒性应激的悬浮细胞培养物中ATM和p53之间的直接蛋白质 - 蛋白质相互作用。

检测和可视化DNA损伤诱导的蛋白质复合物悬浮细胞培养使用邻近连接测定

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

detection-visualization-dna-damage-induced-protein-complexes

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

9.9K 次观看.  Academic Medical Center, University of Amsterdam, Lymphoma and Myeloma Center Amsterdam (LYMMCARE). 在这里，证明了原位邻近连接测定（PLA）如何可以用于检测和可视化暴露于基因毒性应激的悬浮细胞培养物中ATM和p53之间的直接蛋白质 - 蛋白质相互作用。 

视频: Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc) 7-9. This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments 10. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one 12. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage12.
Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks)11,12, fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO4 blunt-ended break (DNase I-type breaks)11,12, if T4 DNA ligase is present in the solution 13,14 . When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO4 blunt-ended breaks, the ligase reacts with 5'PO4 DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO4 blunt-ended DNA breaks.
This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.

在检测DNA损伤的体外半人工分子机器，其使用的大会

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

科研

生物工程

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 ...

遗传学

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in understanding how such pathways are organized and how they function. There are many methods to detect protein-protein interactions in cells, but relatively few can be used to detect interactions between endogenous proteins. One such method, the proximity ligation assay (PLA), has several advantages to recommend its use. Compared to other common methods of protein-protein interaction analysis, PLA has relatively high sensitivity and specificity, can be performed with minimal cell manipulation, and, in the protocol described herein, requires only two target-specific antibodies derived from different species (e.g., from mouse and rabbit) and one specialized reagent: a set of secondary antibodies that are covalently linked to specific oligonucleotides that, when brought in close proximity of one another, create an amplifiable platform for in situ PCR or rolling circle amplification. In this presentation, we show how to apply the PLA technique to visualize changes in MST1 and MST2 proximity in fixed cells. The technique described in this manuscript is particularly applicable for the analysis of cell signaling studies.

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.

原位近接结扎法检测蛋白异构体的 Heterodimerization

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in ...

生物化学

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by homologous recombination (HR). In addition, HR represents an important mechanism of replication fork rescue following stalling or collapse. The regulation of the many reversible and irreversible steps of this complex pathway promotes its fidelity. The physical analysis of the recombination intermediates formed during HR enables the characterization of these controls by various nucleoprotein factors and their interactors. Though there are well-established methods to assay specific events and intermediates in the recombination pathway, the detection of D-loop formation and extension, two critical steps in this pathway, has proved challenging until recently. Here, efficient methods for detecting key events in the HR pathway, namely DNA double-stranded break formation, D-loop formation, D-loop extension, and the formation of products via break-induced replication (BIR) in Saccharomyces cerevisiae are described. These assays detect their relevant recombination intermediates and products with high sensitivity and are independent of cellular viability. The detection of D-loops, D-loop extension, and the BIR product is based on proximity ligation. Together, these assays allow for the study of the kinetics of HR at the population level to finely address the functions of HR proteins and regulators at significant steps in the pathway.

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

The D-loop capture (DLC) and D-loop extension (DLE) assays utilize the principle of proximity ligation together with quantitative PCR to quantify D-loop formation, D-loop extension, and product formation at the site of an inducible double-stranded break in Saccharomyces cerevisiae.

通过邻近连接和定量PCR检测酿酒酵母同源重组中间体

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

检测<em&gt;原位</em&gt;蛋白质 - 蛋白质复合物的<em&gt;果蝇</em&gt;幼虫神经肌肉接头使用接近结扎分析

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

神经科学

Proximity Ligation Assay to Study Oncogene-Derived Transcription-Replication Conflicts

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. Conflicts between the replication and transcription machinery, termed transcription-replication conflicts (TRCs), pose a considerable risk to genome stability, a critical factor in cancer development. While several factors regulating these collisions have been identified, pinpointing primary causes remains difficult due to limited tools for direct visualization and clear interpretation. In this study, we directly visualize TRCs using a proximity ligation assay (PLA), leveraging antibodies specific to PCNA and phosphorylated CTD of RNA polymerase II. This approach allows precise measurement of TRCs between replication and transcription processes mediated by RNA polymerase II. The method is further enhanced through DNA primers conjugated covalently to these antibodies, coupled with PCR amplification using fluorescent probes, providing a highly sensitive and specific means of detecting endogenous TRCs. Fluorescence microscopy enables the visualization of these conflicts, offering a powerful tool to study genome instability mechanisms associated with cancer. This technique addresses the gap in direct TRC visualization, allowing for a more comprehensive analysis and understanding of the underlying processes driving genome instability in cells.

Here, we present a sensitive and specific method to detect oncogene-induced transcription-replication conflicts (TRCs) using the proximity ligation assay (PLA). This approach leverages specific antibodies targeting PCNA and phospho-CTD RNAPII, enabling the assessment of TRC prevalence between RNA polymerase II transcription and DNA replication machinery.

用于研究癌基因衍生的转录-复制冲突的邻位连接测定

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. ...

癌症研究

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

细菌、酵母和人类细胞中瞬态形成的分子茶龙组件的原位监测

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

生物学

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.

免疫荧光DNA修复蛋白相互作用的可视化

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA ...

<meta charset="utf8"></head><body>评估 DNA 损伤中的蛋白质相互作用和定位

Proximity Ligand Assay to Localize Proteins in DNA Damage Sites

The DNA damage response is a genetic information safeguard that protects cells from perpetuating damaged DNA. The characterization of the proteins that cooperate in this process allows the identification of alternative targets for therapeutic intervention in several diseases, such as cancer, aging-related diseases, and chronic inflammation. The Proximity Ligand Assay (PLA) emerged as a tool for estimating interaction between proteins as well as spatial proximity among organelles or cellular structures and allows the temporal localization and co-localization analysis under stress conditions, for instance. The method is simple because it is similar to conventional immunofluorescence and allows the staining of an organelle, cellular structure, or a specific marker such as mitochondria, endoplasmic reticulum, PML bodies, or DNA double-strand marker, yH2AX simultaneously. The phosphorylation of the S139 at Histone 2A variant, H2AX, then referred to as yH2AX, is widely used as a very sensitive and specific marker of DNA double-strand breaks. Each focus of yH2AX staining corresponds to one break in DNA that occurs a few minutes after the damage. The analysis of changes in yH2AX foci is the most common assay for studying if the protein of interest is implicated in DNA damage response (DDR). Whether a direct role in the DNA damage site is expected, fluorescence microscopy is used to verify the colocalization of the protein of interest with yH2AX foci. However, except for the new super-resolution fluorescence methods, to conclude, the local interaction with DNA damage sites can be a little subjective. Here, we show an assay to evaluate the localization of proteins in the DDR pathway using yH2AX as a marker of the damage site. This assay can be used to characterize the temporal localization under different insults that cause DNA damage.

<meta charset="utf8"></head><body>作者聚焦：将邻位配体检测与 γ-H2AX 染色相结合以表征 DNA 损伤反应中的蛋白质相互作用

Here, we present a simple and rapid protocol for the detection of protein interaction at DNA damage sites.

邻位配体检测，用于定位 DNA 损伤位点的蛋白质

The DNA damage response is a genetic information safeguard that protects cells from perpetuating damaged DNA. The characterization of the proteins that ...

检测和可视化DNA损伤诱导的蛋白质复合物悬浮细胞培养使用邻近连接测定

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检测

用于研究癌基因衍生的转录-复制冲突的邻位连接测定

细菌、酵母和人类细胞中瞬态形成的分子茶龙组件的原位监测

免疫荧光DNA修复蛋白相互作用的可视化

邻位配体检测，用于定位 DNA 损伤位点的蛋白质

邻位配体检测，用于定位 DNA 损伤位点的蛋白质