支持部分由R01NS064593和R21ES016636（KV）。显微镜进行VCU - 神经生物学与解剖学显微镜设备，部分支持，资金从NIH-NINDS中心的核心授予5P30NS047463的。旋转盘共聚焦显微镜购买的与NIH-NCRR的奖（1S10RR027957）。

A.细胞制备<ol><li>正常人体主要的成纤维细胞（GM02270）卡瑞尔细胞储存库，卡姆登，新泽西州，并与hTERT 6永生。细胞生长和扩大在蜂星6-cm培养皿在媒体（4毫升）组成的MEM培养基与20％的胎牛血清（GIBCO），的non-essential/essential氨基酸，维生素，丙酮酸钠，和青霉素/链霉素（HyClone公司）。 </li><li>人胚胎肾293（HEK293）细胞，从美国典型培养物保藏中心获得的，并在蜂星6-cm培养皿中生长和扩大。中，细胞保持在介质（4毫升）中组成的DMEM培养液与10％牛胎儿血清，非必需氨基酸，L-谷氨酰胺和青霉素/链霉素。 </li><li> 53BP1 mCherry（N-MYC-53BP1 WT pLPC的普罗Addgene质粒19836）（pCLNR H2BG; Addgene质粒17735）和H2B-GFP融合基因的结构也表达嘌呤霉素或G418抗性基因，分别转（fibrobla针）或转染（HEK293）使用SuperFect（Qiagene）进入细胞，并保持在药物选择。荧光定期检查维护。 </li><li>图像采集前24-48小时，将细胞用胰蛋白酶消化成单细胞悬液，并以较低的密度接种到的3.5厘米FluroDish玻璃底板。 </li></ol> B.显微镜设置和图像采集该协议被开发使用的蔡司细胞观察SD旋转盘共聚焦显微镜配备了一个AxioObserver Z1架，双通道Yokagawa CSU-X1A 5000旋转的磁盘单元，配光QuantEM 512SC EMCCD相机，HXP 120C光纤为基础的照明灯，4激光器（一Lasos 100mW的多行，氩[458，488，514纳米，50 mW的405 nm二极管，40 mW的561 nm二极管，和30 mW的635 nm二极管），AOTF，一个Pecon的的XL multiS1阶段孵化体系，蔡司孵化模块（模块S O 2，CO 2 TempModule模块S，S，供热机组XL小号）和前个月XY torized阶段与一个NanoScanZ的压电Z插入。为了尽量减少的活细胞成像的球面像差，而在水性介质中，一个C-复消色差63x/1.20水/更正物镜和蔡司Immersol W浸没流体（具有折射率n = 1.334）的支持。对于2通道共焦成像中，RQFT 405/488/568/647分色镜和BP525/50（绿色）和BP629/62（红色）排放过滤器被使用。所用的系统软件与AV4 Multi-channel/Z/T，快速成像，生理学，MosaiX，标记和查找，双摄像头，里面4D，自动对焦和3-D反褶积模块:蔡司Axiovision（版本4.8.2.0）。 <ol><li>确保运行，CO 2气体的CO 2孵化系统的模块。如果在显微镜防振空气表支持的，打开空气供给（或N 2气体）的空气表。 </li><li>打开显微镜支架的电源开关，旋转式磁盘装置，数码相机，孵化模块，HXP照明灯，电动载物台，氩气激光，和计算机。 </li><li>经过1分钟的热身时间，将点火钥匙的氩离子激光器为"开"。 </li><li>在蔡司激光控制面板中，打开要使用的激光线的开关。 </li><li>拨动开关切换为"待机"，"激光运行"，的Lasos氩离子激光器控制器，调光控制器的最佳水平（ 即正下方的绿色指示灯会变成红色）。 </li><li>启动AxioVision软件。 注 :是专门针对特定的显微镜和组件，它控制的窗口和下拉菜单，可自定义的用户界面AxioVision。因此，每个系统都有可能是唯一的接口。因此，在随后的步骤中提供软件操作的一般说明，而不是特定的软件窗口，标签和/或下拉菜单的方向。 </li><li>成像前约1小时，在软件中，定位控制的孵化器和上部腔室的加热和镜台板的开启。把温度设置为37℃。打开的CO 2的控制和设置在5％的水平。 </li><li>选择用于成像的物镜。在这项研究中，63x/1.20 NA C-复消色差水/更正物镜使用注 :对于这样的镜头，需要浸没介质的折射率类似的水（蔡司Immersol W浸没流体）。 </li><li>如果在一个长的时间内将被采样的多个单独的位置，某些应用足够量的液浸介质中，以确保它从一个位置到另一个进行。 </li><li>放置在舞台上的菜带来物镜成与底部接触。 </li><li>在显微镜控制或软件，直接发出的光目镜，选择"适当的广角过滤器的设置荧光信号的利息（这项研究中，"红"过滤器设置53BP1和"绿色荧光蛋白"过滤器设置为H2B ）。/ P&gt; <li>查看通过眼透镜，图像聚焦，细胞中找到一个合适的领域。 </li><li>使用软件或显微镜控制，直接发射的光远离共焦旋转圆盘​​单元到端口的目镜。 </li><li>在软件中，打开相应的激光（这项研究中，561纳米激光53BP1和H2B的氩离子激光器的488 nm线）。 </li><li>对于每个通道，调整的激光的强度的声光可调谐滤波器（AOTF）控制通过调节到适当的水平。 </li><li>选择适当的的分色镜（RQFT 405/488/568/647）和发射滤波器（BP62分之62953BP1和BP五十○分之五百二十五的的H2B）。打开快门纺丝盘单元。 </li><li>选择"实时"窗口，显示当前的视野。 </li><li>在软件中，打开"相机"控制，选择相机使用（如果是双相机系统），并设定曝光时间约为100毫秒。调整％和EM获得其他埃森。 注:53BP1 mCherry结构显得有些暗淡。我们发现它有利于提高EM增益。 </li><li>在软件中，打开控制的共聚焦转盘单位和调整转盘的速度进入相机的曝光时间，捕捉到一个合适的图像（ 例如 〜100毫秒）。单击"设置"锁定的变化。 </li><li>打开"多维收购"。选择通道"选项卡，加载/选择合适的渠道。在这项研究中，我们将使用红色荧光蛋白（561 nm激光激发和BP 629/62过滤器的排放）和GFP（488 nm激光激发和BP525/50过滤器的发射线）定义的通道。 </li><li>为了确保图像配准中，一个共同的分色镜（RQFT 405/488/568/647）用于两条通道。设置软件的"自动对焦"。 注意 :工具→设置编辑器调整的MDA设置。确保有足够的激光功率设置（"足够的激光功率"是指设置，让您激发适当的荧光基团，同时最大限度地减少光漂白）。 </li><li>在"MDA"窗口中，选择"Z-Stack的标签。选择"Z-堆栈在当前的焦点位置"。设置范围〜10微米的Z-Stack，然后选择"最佳"的一些步骤，以确保奈奎斯特采样到Z 注意:确切的Z-Stack的大小将取决于你的细胞的高度。相应的调整。 </li><li> 23。单击"开始"，产生的Z-Stack的图像进行分析，以确保设置适合你调查（ 例如在这项研究中的，这是非常重要的，整个核）。 </li><li>选择"T"（时间）选项卡。对于喜树碱我们的实验中，我们设置了成像时间点之间的时间间隔为5分钟和1小时的控制（非处理的细胞）视频会议期间的整体。 注意:尽量减少光漂白细胞，实验应该被配置为使得在AOTF激光成像的时间点之间的空白。 </li><li> F多点成像几个细胞的菜，在MDA"窗口中，选择"位置"选项卡。确保"应用"设定时间点的每个位置前/后"被选中。 ，选择"Mark_Find"。使用"现场"视图，将周围的菜，并选择合适的视野。 </li><li>多维收购菜单中单击"开始"，开始进行实验，并记录控制视频（未经处理的细胞）。 </li><li>控制视频后，添加适当的处理（在这种情况下，10μM喜树碱）。我们记录了实验（药物处理的细胞）的视频在5-10分钟的时间间隔为2-4小时。 注:要考虑的是速度，一个给定的效果，治疗后出现的一个重要问题。在视频中可以看出，53BP1灶开始〜5分钟后，加入CPT。虽然一个相对容易的过程，手动添加药物的菜和重新校准的显微镜，确实需要时间。设计实验时，必须考虑到这。 </li><li>为了监测米itosis，我们的间隔设为7.5分钟，并记录为4-5小时（特定的设置取决于使用的细胞系和其细胞周期的长度）。可能需要进行调整，在较长的录音，以防止光漂白。 </li></ol> C.图像处理与分析 Volocity软件（珀金埃尔默），该协议的开发利用。获得这些数据（AxioVision）所使用的软件也有能力来处理和分析图像。我们鼓励用户利用该软件提供给他们，并查阅相关文献，对他们的应用程序。 <ol><li>打开Volocity软件。创建并命名一个新的库，并导入你的视频文件。 </li><li>要查看的文件，我们通常会发现使用"扩展聚焦方式"设定有极大的帮助。这叠加的Z-Stack片，此协议，使我们能够可视化53BP1灶在不同领域的核。 </li><li>在必要时调整的影片。 VOlocity配备了一系列的工具，以提高采集到的图像质量。很多时间通过确保在显微镜上的设置是合适的，可以被保存在编辑以后。通常情况下，它是有帮助，反卷积您的图像，调整亮度/对比度。实验的基础上，你的特定的编辑需求会有所不同。 </li><li>添加一个相对时间戳和比例尺。 </li><li>要查看单元格中的3-D，切换到"3-D不透明度"设置。这允许在空间的3-D渲染细胞的旋转，从而提供利益的结构的细胞内的多个角度。 注:这是有助益的可视化细胞在不同的平面中，以确定结构感兴趣的旅行。例如，在"扩展聚焦方式"设定，它是很难辨别53BP1不，其实，游离在有丝分裂过程中从DNA。然而，这是显而易见的，在3-D。 </li><li>动画和静止图像可以导出成多种文件类型，根据用户的喜好。 </l<html> I&gt; </ol> D.代表性的成果响应对CPT 53BP1灶形成的一个例子的图1中所示。中华映管灶的细胞暴露在5-10分钟范围内，并保持这些病灶在整个持续时间的记录。正如在图2中所示，53BP1解离染色质在有丝分裂的发病，冷凝染色体周围形成薄膜的雾度。末期发生有丝分裂结束，53BP1再次聚集成不同的病灶。虽然HEK293细胞没有接触到CPT，但它们形成丰富的，自发的53BP1所产生的内源性DNA损伤的修复病灶。这一观察结果使我们得出结论，53BP1没有形成病灶早期有丝分裂过程中，在与以前的报告显示，类似的效果后，细胞暴露于电离辐射和radiomimetic的药物5。 从图1"SRC ="/ files/ftp_upload/4251/4251fig1.jpg"/&gt; 图1。喜树碱（10μM）引起DNA双链断裂和向培养基中添加药物后的30分钟内，在骑自行车的成纤维细胞的53BP1灶形成。 <img alt="图2" src="/files/ftp_upload/4251/4251fig2.jpg" /> 图2。，53BP1并不构成灶在HEK293细胞中有丝分裂过程中，直到telophase/G1。

<ol>
	<li>Botuyan, M. V., Lee, J., Ward, I. M., Kim, J. E., Thompson, J. R., Chen, J., Mer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+the+methylation+state-specific+recognition+of+histone+H4-K20+by+53BP1+and+Crb2+in+DNA+repair.">Structural basis for the methylation state-specific recognition of histone H4-K20 by 53BP1 and Crb2 in DNA repair.</a> Cell. 127, 1361-1373 (2006).</li><li>Dimitrova, N., Chen, Y. C., Spector, D. L., de Lange, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=53BP1+promotes+non-homologous+end+joining+of+telomeres+by+increasing+chromatin+mobility.">53BP1 promotes non-homologous end joining of telomeres by increasing chromatin mobility.</a> Nature. 456, 524-528 (2008).</li><li>Feuerhahn, S., Egly, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+to+study+DNA+repair:+what's+in+the+box.">Tools to study DNA repair: what's in the box.</a> Trends Genet. 24, 467-474 (2008).</li><li>Giunta, S., Belotserkovskaya, R., Jackson, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+signaling+in+response+to+double-strand+breaks+during+mitosis.">DNA damage signaling in response to double-strand breaks during mitosis.</a> J. Cell Biol. 190, 197-207 (2010).</li><li>Giunta, S., Jackson, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Give+me+a+break,+but+not+in+mitosis:+the+mitotic+DNA+damage+response+marks+DNA+double-strand+breaks+with+early+signaling+events.">Give me a break, but not in mitosis: the mitotic DNA damage response marks DNA double-strand breaks with early signaling events.</a> Cell Cycle. 10, 1215-1221 (2011).</li><li>Golding, S. E., Morgan, R. N., Adams, B. R., Hawkins, A. J., Povirk, L. F., Valerie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pro-survival+AKT+and+ERK+signaling+from+EGFR+and+mutant+EGFRvIII+enhances+DNA+double-strand+break+repair+in+human+glioma+cells.">Pro-survival AKT and ERK signaling from EGFR and mutant EGFRvIII enhances DNA double-strand break repair in human glioma cells.</a> Cancer Biol. Ther. 8, 730-738 (2009).</li><li>Huyen, Y., Zgheib, O., Ditullio, R. A., Gorgoulis, V. G., Zacharatos, P., Petty, T. J., Sheston, E. A., Mellert, H. S., Stavridi, E. S., Halazonetis, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylated+lysine+79+of+histone+H3+targets+53BP1+to+DNA+double-strand+breaks.">Methylated lysine 79 of histone H3 targets 53BP1 to DNA double-strand breaks.</a> Nature. 432, 406-411 (2004).</li><li>Jackson, S. P., Bartek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DNA-damage+response+in+human+biology+and+disease.">The DNA-damage response in human biology and disease.</a> Nature. 461, 1071-1078 (2009).</li><li>Kanda, T., Sullivan, K. F., Wahl, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone-GFP+fusion+protein+enables+sensitive+analysis+of+chromosome+dynamics+in+living+mammalian+cells.">Histone-GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells.</a> Curr. Biol. 8, 377-385 (1998).</li><li>Massignani, M., Canton, I., Sun, T., Hearnden, V., Macneil, S., Blanazs, A., Armes, S. P., Lewis, A., Battaglia, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+fluorescence+imaging+of+live+cells+by+effective+cytosolic+delivery+of+probes.">Enhanced fluorescence imaging of live cells by effective cytosolic delivery of probes.</a> PLoS One. 5, e10459 (2010).</li><li>Nakamura, K., Sakai, W., Kawamoto, T., Bree, R. T., Lowndes, N. F., Takeda, S., Taniguchi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+vertebrate+53BP1:+a+major+role+in+non-homologous+end+joining+of+DNA+double+strand+breaks.">Genetic dissection of vertebrate 53BP1: a major role in non-homologous end joining of DNA double strand breaks.</a> DNA Repair (Amst). 5, 741-749 (2006).</li><li>Schultz, L. B., Chehab, N. H., Malikzay, A., Halazonetis, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p53+binding+protein+1+(53BP1)+is+an+early+participant+in+the+cellular+response+to+DNA+double-strand+breaks.">p53 binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks.</a> J. Cell. Biol. 151, 1381-1390 (2000).</li><li>Valerie, K., Povirk, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+mechanisms+of+mammalian+double-strand+break+repair.">Regulation and mechanisms of mammalian double-strand break repair.</a> Oncogene. 22, 5792-5812 (2003).</li><li>Wang, B., Matsuoka, S., Carpenter, P. B., Elledge, 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=53BP1,+a+mediator+of+the+DNA+damage+checkpoint.">53BP1, a mediator of the DNA damage checkpoint.</a> Science. 298, 1435-1438 (2002).</li><li>Ward, I. M., Minn, K., Jorda, K. G., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accumulation+of+checkpoint+protein+53BP1+at+DNA+breaks+involves+its+binding+to+phosphorylated+histone+H2AX.">Accumulation of checkpoint protein 53BP1 at DNA breaks involves its binding to phosphorylated histone H2AX.</a> J. Biol. Chem. 278, 19579-19582 (2003).</li></ol>

没有利益冲突的声明。

维持基因组的完整性，细胞的生存是至关重要的。如果保存的基因组会导致过早衰老，癌变，死亡8。有浓厚的兴趣，挑剔的DDR功能，源于其基础和临床研究的重要性。多年来，以帮助在细胞如何检测和修复DNA损伤的研究已开发了许多技术。传统的方法，如免疫细胞化学和西方墨点法领域的支柱，尽管最近的技术进步已经使日益复杂的方法发展。活细胞成像，在该协议中详细说明，使我们?...

<table border="1"><tbody><tr><td> 产品 </td><td> 公司 </td></tr><tr><td>陕西蜂星的培养皿</td><td>格雷纳生物一</td></tr><tr><td> FluroDish玻璃底菜</td><td>世界精密仪器公司</td></tr><tr><td> MEM媒体</td><td> GIBCO </td></tr><tr><td>非必需氨基酸</td><td> GIBCO </td></tr><tr><td>氨基酸</td><td> GIBCO </td></tr><tr><td>维生素</td><td> GIBCO </td></tr><tr><td>丙酮酸钠</td><td> Invitrogen公司</td></tr><tr><td>青霉素/链霉素</td><td>胎牛血清</td></tr><tr><td>胎牛血清</td><td> GIBCO </td></tr><tr><td> N-MYC-53BP1 WT pLPC的普罗; 质粒19836 </td><td> Addgene </td></tr><tr><td> pCLNR H2BG质粒17735 </td><td> Addgene </td></tr><tr><td> SuperFect </td><td> Qiagen公司</td></tr><tr><td>蔡司观察SD细胞成像系统</td><td>蔡司</td></tr><tr><td> AxioVision（版本4.8.2） </td><td>蔡司</td></tr><tr><td>蔡司Immersol W油</td><td>蔡司</td></tr><tr><td> Volocity软件（版本6.0） </td><td>珀金埃尔默</td></tr></tbody></table>

two- and three-dimensional live cell imaging of dna damage response proteins

双链断裂（DSBs）是最有害的DNA病变的细胞可能会遇到。如果未修复，DNA双链断裂港口的巨大潜力产生突变和染色体畸变1。为了防止这种创伤催化基因组的不稳定性，这是至关重要的，检测DNA双链断裂的细胞，激活DNA损伤反应（DDR），并修复DNA。解除武装，复员和重返社会工作的刺激时，通过触发细胞周期阻滞，以便维修发生或强制的细胞发生凋亡，以维持基因组的完整性。 DSB修复发生的主要机制通过非同源末端连接（NHEJ）和同源重组修复（HRR）（评论2例 ）。有许多的蛋白质，其活动必须精确精心策划的解除武装，复员和重返社会正常运行。这里，我们描述了一种方法，2 - 和3 - 维（D）的可视化这些蛋白质之一，53BP1。 p53结合蛋白1（53BP1）定位于地区DNA双链断裂的修饰组蛋白3,4结合，形成病灶在5-15分钟内5。组蛋白修饰和招聘53BP1和其他DDR蛋白DSB网站被认为是染色质的结构重排，以方便周边地区的损失，并有助于DNA修复6。除了 ​​直接参与修复，额外的角色被描述为53BP1在解除武装，复员和重返社会，如调节内-S检验点，G2 / M检验点，并激活下游DDR蛋白7-9。最近，人们发现，53BP1没有形成病灶在有丝分裂过程中DNA损伤，而不是等待细胞进入G1前定位附近的DNA双链断裂6。如53BP1 DDR蛋白质已被发现与有丝分裂的结构（如着丝粒）相关联的进展通过有丝分裂10期间。 在这个协议中，我们描述了使用2 - 和3-D的活细胞成像的可视化53BP1灶形成的DNA损伤剂喜树碱（CPT），以及53BP1在有丝分裂过程中的行为。喜树碱是拓扑异构酶I抑制剂，主要引起DNA双链断裂DNA复制过程中。要做到这一点，我们使用先前描述的53BP1 mCherry荧光融合蛋白结构的53BP1的蛋白质结构域的能够结合DNA双链断裂11。此外，我们使用组蛋白H2B-GFP的荧光融合蛋白的构建，能够监控整个细胞周期，但在特定的染色质的动态，在有丝分裂12。活细胞成像技术在多个方面，是一个很好的工具，以加深我们对DDR在真核细胞中的蛋白质的功能。

Watch this Scientific Journal Video about Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins at JoVE.com

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

该协议描述了一个可视化的DNA双链断裂以及其定位在有丝分裂过程中DNA损伤激活的信号蛋白的方法。

二维和三维活细胞成像的DNA损伤反应蛋白

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

two-three-dimensional-live-cell-imaging-dna-damage-response

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

14.0K 次观看.  Virginia Commonwealth University. 该协议描述了一个可视化的DNA双链断裂以及其定位在有丝分裂过程中DNA损伤激活的信号蛋白的方法。

视频: Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

激光微辐照在哺乳动物细胞单、双链断裂修复中的应用

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular ...

科研

癌症研究

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

激光 Microirradiation 研究体内细胞对简单复杂 DNA 损伤的反应

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

遗传学

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

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

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 ...

生物化学

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand genome maintenance mechanisms. Since recruitment and exchange of proteins at lesions are highly dynamic, their study requires the ability to generate DNA damage in a rapid and spatially-delimited manner. Here, we describe procedures to locally induce DNA damage in human cells using a commonly available laser-scanning confocal microscope equipped with a 405 nm laser line. Accumulation of genome maintenance factors at laser stripes can be assessed by immunofluorescence (IF) or in real-time using proteins tagged with fluorescent reporters. Using phosphorylated histone H2A.X (&#947;-H2A.X) and Replication Protein A (RPA) as markers, the method provides sufficient resolution to discriminate locally-recruited factors from those that spread on adjacent chromatin. We further provide ImageJ-based scripts to efficiently monitor the kinetics of protein relocalization at DNA damage sites. These refinements greatly simplify the study of the DDR dynamics.

Studying DNA damage repair kinetics requires a system to induce lesions at defined sub-nuclear regions. We describe a method to create localized double-stranded breaks using a laser-scanning confocal microscope equipped with a 405 nm laser and provide automated procedures to quantify the dynamics of repair factors at these lesions.

405 Nm 激光微辐照对 DNA 损伤的蛋白质招募研究

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand ...

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair complexes. This process is regulated by a myriad of proteins that promote the association and disassociation of proteins to these lesions. Thanks in large part to the ability to perform functional screens of a vast library of proteins, there is a greater appreciation of the genes necessary for the double-strand DNA break repair. Often knockout or chemical inhibitor screens identify proteins involved in repair processes by using increased toxicity as a marker for a protein that is required for DSB repair. Although useful for identifying novel cellular proteins involved in maintaining genome fidelity, functional analysis requires the determination of whether the protein of interest promotes localization, formation, or resolution of repair complexes. 
The accumulation of repair proteins can be readily detected as distinct nuclear foci by immunofluorescence microscopy. Thus, association and disassociation of these proteins at sites of DNA damage can be accessed by observing these nuclear foci at representative intervals after the induction of double-strand DNA breaks. This approach can also identify mis-localized repair factor proteins, if repair defects do not simultaneously occur with incomplete delays in repair. In this scenario, long-lasting double-strand DNA breaks can be engineered by expressing a rare cutting endonuclease (e.g., I-SceI) in cells where the recognition site for the said enzyme has been integrated into the cellular genome. The resulting lesion is particularly hard to resolve as faithful repair will reintroduce the enzyme's recognition site, prompting another round of cleavage. As a result, differences in the kinetics of repair are eliminated. If repair complexes are not formed, localization has been impeded. This protocol describes the methodology necessary to identify changes in repair kinetics as well as repair protein localization.

Repair of double-strand DNA breaks is a dynamic process, requiring not only formation of repair complexes at the breaks, but also their resolution after the lesion is addressed. Here, we use immunofluorescence microscopy for transient and long-lasting double-stranded breaks as a tool to dissect this genome maintenance mechanism.

基于免疫荧光显微镜的瞬时和持久双链 dna 损伤 dna 修复过程的表征

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

高级共焦显微镜技术研究 DNA 损伤的蛋白质-蛋白质相互作用和动力学

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

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 ...

Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is essential in revealing cellular events associated with the recruitment and assembly of DNA repair complexes at the site of DNA damage. In the last few years, several powerful tools have been developed to induce site-specific DNA damage. Moreover, novel seminal techniques allow us to study these processes at the single-cell resolution level using both fixed and living cells. Although these techniques have been used to study various biological processes, herein we present the most widely used protocols in the field of DNA repair, Fluorescence Immunostaining (IF) and Chromatin Immunoprecipitation (ChIP), which in combination with endonuclease-based site-specific DNA damage make it possible to visualize and quantify the genomic occupancy of DNA repair factors in a directed and regulated fashion, respectively. These techniques provide powerful tools for the researchers to identify novel proteins bound to the damaged genomic locus as well as their post-translational modifications necessary for their fine-tune regulation during DNA repair.

This article introduces essential steps of immunostaining and chromatin immunoprecipitation. These protocols are commonly used to study DNA damage-related cellular processes and to visualize and quantify the recruitment of proteins implicated in DNA repair.

可视化和量化基于内分泌酶的站点特定DNA损伤

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is ...

Live-Cell Imaging of Transcriptional Activity at DNA Double-Strand Breaks

DNA double-strand breaks (DSB) are the most severe type of DNA damage. Despite the catastrophic consequences on genome integrity, it remains so far elusive how DSBs affect transcription. A reason for this was the lack of suitable tools to simultaneously monitor transcription and the induction of a genic DSB with sufficient temporal and spatial resolution. This work describes a set of new reporters that directly visualize transcription in live cells immediately after the induction of a DSB in the DNA template. Bacteriophage RNA stem-loops are employed to monitor the transcription with single-molecule sensitivity. For targetting the DSB to a specific gene region, the reporter genes are engineered to contain a single recognition sequence of the homing endonuclease I-SceI, otherwise absent from the human genome. A single copy of each reporter gene was integrated into the genome of human cell lines. This experimental system allows the detection of single RNA molecules generated by the canonical gene transcription or by DNA break-induced transcription initiation. These reporters provide an unprecedented opportunity for interpreting the reciprocal interactions between transcription and DNA damage and to disclose hitherto unappreciated aspects of DNA break-induced transcription.

This protocol presents a new reporter gene system and the experimental setup to detect transcription at DNA double-strand breaks with single-molecule sensitivity.

DNA双链断裂时转录活性的活细胞成像

DNA double-strand breaks (DSB) are the most severe type of DNA damage. Despite the catastrophic consequences on genome integrity, it remains so far ...

Immunofluorescence-Based Visualization of DNA Repair Protein Interactions

Source: de la Pe&#241;a Avalos, B., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/61447">Visualization of DNA Repair Proteins Interaction by Immunofluorescence.</a> J. Vis. Exp. (2020).
In this video, we describe the immunofluorescence method to visualize the localization of the DNA repair proteins &#947;H2AX and 53BP1 at the sites of double-strand DNA breaks in irradiated cell nuclei.

基于免疫荧光的 DNA 修复蛋白相互作用可视化

Source: de la Pe&#241;a Avalos, B., et al., Visualization of DNA Repair Proteins Interaction by Immunofluorescence. J. Vis. Exp. (2020).
In this video, we ...