Name: single molecule analysis of laser localized psoralen adducts
Uploaded: 2017-04-20T12:30:00.000+00:00
Duration: 11 min 46 s
Description: Watch this Scientific Journal Video about Single Molecule Analysis of Laser Localized Psoralen Adducts at JoVE.com

这项研究部分由美国国立卫生研究院，研究所院内研究老龄问题方案（Z01 AG000746-08）和范可尼贫血研究基金的支持。

1.挖-TMP的制备<ol><li> 混合50毫克（0.18毫摩尔）的4&#39;-氯甲基-4,5&#39; ，8-三甲基补骨脂素和590毫克（2.7毫摩尔）-4,7,10-三氧杂-1,13- tridecanedi胺在干燥的25mL圆在氮气下汉字烧瓶中。添加10mL的甲苯，并回流12个小时。在减压下在旋转蒸发器除去溶剂。 <ol><li>纯化在硅胶残余物通过快速柱色谱法。 （1:1:0.5 9），用氯仿，甲醇和28％氨水溶液洗脱该柱。蒸发在减压下在旋转蒸发器除去溶剂，回收纯4" - [N-（13-氨基-4,7,10- trioxatrideca）]氨甲基-4,5"，8-三甲基补骨脂素，为浅黄色粘性液体。 </li><li>的4混合5.5毫克（0.012毫摩尔）" - [N-（13-氨基-4,7,10- trioxatrideca）]氨甲基-4,5"，8-三甲基补骨脂素与5毫克（0.008毫摩尔）洋地黄毒苷NHS酯的在氮气下干燥的圆底烧瓶中。添加0.5毫升无水二甲基甲酰胺（DMF）的ð3.4μL三甲胺，并搅拌在50℃下18小时。 </li><li>在减压下在旋转蒸发器除去溶剂。 </li><li>溶于二氯甲烷中的最小量的残余物。适用于2μL斑点溶液水平地横跨用硅胶作为固定相制备性薄层板的底部。甲醇:氯仿运行制备型TLC 28％氢氧化铵（8:1:0.1）。 </li><li>屏蔽板除了垂直边缘，并与短波长UV 254nm的灯识别产品的带。从用刮刀将板刮去产品，然后使用氯仿分离纯产物:甲醇:28％氢氧化铵（8:1:0.1）的混合物。 </li><li>在减压下在旋转蒸发器除去溶剂。溶解残留的粒料在1mL 50％乙醇:H 2 O，分配到在1.5mL管中（约20个等分）和干燥的50μL等分试样在离心蒸发器，直到所有的溶剂被蒸发（〜3小时）。 </li><li>在离心蒸发器再次H 2 O和干燥:再溶解在50％EtOH中的200μL每个等分试样。在50％EtOH中的200μL再次溶解每个等分试样:H 2 O，在-20℃下长期储存干在离心蒸发器和存储粒料。 </li></ol></li><li> 为了使用，溶解于50％EtOH中的50μL的丸粒:H 2 O.准备1:100稀释溶解的挖-TMP的在H 2 O和在250nm测量OD。挖-TMP的消光系数是25,000。如果储存在-20℃下将溶液是约一个月稳定。通过在每次使用前在250nm处测量OD验证的浓度。 <ol><li>计算库存浓度:腹部×100×10 6/25000 =浓度（μM）。典型地，所述原液是约3mm。是在该范围内，以尽量减少的EtOH加入到培养基中的量是重要的。 </li></ol></li></ol> 2.激光局部地高辛-TMP的ICL <ol><li> PE与具有SRS氮激光器（337 nm）的共焦显微镜rform激光定位通过染料细胞泵送发射线的365纳米和在10赫兹烧成3个纳秒脉冲，0.7纳瓦的功率。 </li><li>使在35 mm的玻璃侧的垂直标记有底有标记笔细胞培养皿。划伤与金刚石笔培养表面上的玻璃的中心的横使得在盘侧的黑条是在交叉的一个臂的顶部。横标上的玻璃的生长表面，使得它与细胞将在相同的焦平面中。 </li><li>用70％乙醇的漂洗后标记消毒板。电镀细胞之前干燥。 </li><li>板细胞（标准实验室菌株例如HeLa或U2OS​​）在交叉之前一天或两天标记培养皿。小区应积极分裂，并在实验当天50-70％汇合。孵育24小时用1μM5-氯-2-脱氧尿苷（CldU）均匀地标签DNA。 </li><li>加挖-TMP，在50％EtOH中:H 2 O至细胞培养基至20μM的终浓度。使介质至37℃。改变在细胞上，并装在培养箱培养基（37℃，5％CO 2）30分钟，以允许该DIG TMP达到平衡。 </li><li>而将细胞培养，建立的视场，其中所述激光是活性的x / y坐标。按照该激光是由软件涉及蚀刻沿着水平和对角线镜像载玻片制造商的校准程序。两条线限定，其中该激光能够求取目标场的边界。 </li><li>放置板在环境室，在37℃下具有受控的CO 2和湿度，显微镜载物台上，并用在十字交叉的60X油浸物镜聚焦。 </li><li>引导365nm的激光束的ROI，通过在该软件的形状工具研究者成立，以形成3条带＃181; 0.6 MX微米。 ROI的轮廓由光标在位于十字交叉的附近细胞的核放置。调节Z焦平面到目标通过细胞核的激光中途。在350-370 nm范围内使用的激光器。 405nm的激光器不能诱导交联19。 注意:我们定位核仁，这可以从微分干涉对比（DIC）成像核质来区分外核质区域的投资回报率。 </li><li> 通过增加功率设置到足以抹杀一个小区（小区会变暗，然后消失）的电平确认激光的焦点和活性。这是用于通过显微镜，然后通过盖玻片并进入细胞中的激光不受阻碍光路的一个重要的诊断。 <ol><li>降低功率设置到刚好足以激活补骨脂素和诱导ICL DDR（这必须根据经验来确定对于每个激光/麦克风roscope组合）。使用激光强度设定（这里为1.7％），其不激活DDR在不存在补骨脂素（由γ-H2AX的形成监测）。 注意:这是一个重要的判定，并且如果激光源或在光路中的分量改变所述设置可能需要调整。遵循GFP的积累标记修复蛋白如XPC或FAN1，这两者都是迅速招募（秒）到激光补骨脂条纹，但不ROI暴露于单独的激光器。在DDR的一些蛋白质出现几秒钟内，而几分钟就可以要求别人。这需要进行时间过程确定每个感兴趣的蛋白质。我们还注意到，在没有暴露于激光细胞没有响应蛋白的条纹。 </li></ol></li><li>后在场内的所有单元已经有针对性的举措板以一个新的领域，力求贴近十字交叉。 </li><li>在实验中，旨在determ的INE间病灶距离的分布的细胞暴露于所述激光在20-25字段（4-5个细胞/场）围绕交叉点。为了分析在局部的ICL的附近复制图案孵育与激光器发射后10μM5-碘-2-脱氧尿苷（IDU）细胞。 注:孵化与IDU的时间是有点武断。我们使用短至20分钟，只要60分钟（见下文）。较长的脉冲（几个小时）通常导致多个复制大片聚结，从而失去的机会，以图像单叉的进度。在一些实验中CES都标有CldU 24小时，以在暴露于高辛TMP随后复制大片脉冲标记均匀地标签DNA。 </li></ol> 3.细胞收获和DNA纤维的拉伸<ol><li>从载物台取下板，除去培养基，并用磷酸盐缓冲盐水（PBS）洗涤。除去PBS溶液。将商业胰蛋白酶的10微升下降/上交叉的在玻璃表面的中间的交点EDTA溶液。 </li><li>孵育在室温下（RT）3-4分钟，然后绘制与所述分离的细胞进入吸管尖的胰蛋白酶溶液。没有必要以中和胰蛋白酶。 </li><li>放置胰蛋白酶/ EDTA的与细胞的下降在硅烷化的玻璃显微镜载片的一端。裂解细胞，并通过加入0.5％SDS溶液10μL的释放DNA，并在室温下孵育3-4分钟用吸管尖轻轻混合，使池干燥的周边。 </li><li>倾斜滑动20º，并允许液体下降到结束时运行。的DNA纤维扩展/流动液体的水动力拉伸。让载玻片风干（〜10分钟），并在3固定:1个甲醇/乙酸10分钟。从修复方案，再风干取出幻灯片。在这一点上，他们可以在-20℃下被无限期地贮存在70％EtOH中。从70％的乙醇去除允许在空气中干燥网元之前XT一步。 </li><li>在PBS洗涤玻片，然后在2.5的1M HCl变性在RT 1个小时。这种处理depurinates DNA使掺入卤化核碱基可访问的抗体。 </li><li>中和的0.4M的Tris-HCl，pH 7.4中的幻灯片。在PBS中，每次5分钟洗涤两次/ 0.5％吐温20（PBST）。 </li><li>块滑动，用5％牛血清白蛋白（BSA）和10％山羊血清的PBS中。用封口膜，以在所述滑动均匀地散布在封闭溶液并在室温下孵育1个小时轻轻覆盖滑动。 </li><li>吸取100μL的1:200稀释于阻断大鼠抗溴脱氧尿苷的溶液（特异性检测CldU）初级抗体每张幻灯片。用封口膜，以在所述滑动均匀地散布在稀释和培养在湿润的腔室，在室温1个小时轻轻覆盖滑动。除去石蜡膜和洗载玻片在PBST三次，每次5分钟。 </li><li>吸取100μL的稀释的（1:100）的山羊抗大鼠Alexa氟647在封闭液中，以每张幻灯片。覆盖用封口膜轻轻滑动并在室温下孵育45分钟。洗载玻片在PBST三次，每次5分钟。 </li><li>吸取100μL的稀释（1:40）的小鼠抗溴脱氧尿苷（检测LDU和CldU这种双特异性通过在现有孵育大鼠抗BrdU的必要CldU的阻塞。）的第一抗体和兔抗DIG抗体（1:200 ）在封闭液中，以每张幻灯片。用封口膜轻轻覆盖滑动并在湿润室中在室温下孵育1个小时。洗载玻片在PBST三次，每次5分钟。 </li><li>吸取100微升稀释的在封闭液中，以每个幻灯片的山羊抗兔探针（ 例如 ，量子点655）（1:100）的山羊抗-小鼠Alexa Fluor488的和c（5000 1）。覆盖载玻片轻轻用石蜡膜和在室温下孵育45分钟。洗载玻片在PBST三次，每次5分钟。 </li><li>排出多余的PBST到纸巾上。添加抗褪色封固剂的50μL至每个载玻片，并用盖玻片覆盖。图片或商店在-20℃下不超过48小时。 </li></ol> 4.荧光显微成像纤维<ol><li>通过63X物镜在倒置显微镜上进行图像采集具有附接的全电动滤光轮与FITC，Cy5的和Q点655检测。激励滤光器为425纳米，和发射滤波器是具有在发光峰值20中心20个纳米655纳米。 </li></ol>

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Cell. 40, 179-204 (2010).</li><li>Mori, T., Matsunaga, T., Hirose, T., Nikaido, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+monoclonal+antibody+recognizing+ultraviolet+light-induced+(6-4)+photoproducts.">Establishment of a monoclonal antibody recognizing ultraviolet light-induced (6-4) photoproducts.</a> Mutat. Res. 194, 263-270 (1988).</li><li>Poirier, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antisera+specific+for+carcinogen-DNA+adducts+and+carcinogen-modified+DNA:+applications+for+detection+of+xenobiotics+in+biological+samples.">Antisera specific for carcinogen-DNA adducts and carcinogen-modified DNA: applications for detection of xenobiotics in biological samples.</a> Mutat. Res. 288, 31-38 (1993).</li><li>Henderson, A., 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=Detection+of+G-quadruplex+DNA+in+mammalian+cells.">Detection of G-quadruplex DNA in mammalian cells.</a> Nucleic Acids Res. 42, 860-869 (2014).</li></ol>

作者什么都没有透露。

激光定位技术要求与在明亮的视野显微镜可见细胞核使用贴壁细胞。我们已经试图附加非贴壁细胞，如初级淋巴细胞，或松散粘连培养细胞如AD293，与细胞粘附制剂，如聚赖氨酸或胶原，或更复杂的混合物在玻璃表面。虽然这些治疗方法可以将细胞结合到表面，我们发现，他们一般留圆角使其很难集中到细胞核。此外，细胞通常不能存活除去生长培养基后的初始洗涤。然而，对于贴壁细胞的要求?...

DNA是从下外源性试剂如辐射，紫外线，环境毒素，燃烧产物，等。此外，还通过氧化代谢产生的内源性自由基物种攻击恒定攻击。所有这些必须化学或物理破坏DNA的完整性1的潜力。在基因组扰动可以激活DNA损伤反应（DDR），招聘和翻译后修饰级联数百，甚至数千名参与损伤修复蛋白和微RNA，细胞周期，细胞凋亡，衰老和炎症通路的调控2。 大多数的我们对DDR的信息来自于与DNA双链断裂的研究。这在很大程度上是由于技术在活细胞中导入3断裂，包括序列特异性断裂，在基因组DNA的可用性。此外，propensit断裂的y以诱导蛋白质DDR，这可以通过免疫荧光来显示的病灶，已经非常有帮助，用于识别的动力学和响应蛋白质的要求。一个用于研究DDR的关键技术是由邦纳和他的同事，谁在活细胞4的核使用激光束直接DNA双链断裂的条纹在"利益区域"（ROI）推出。实际上，他们创造了一个漫长的焦点，其中DDR的蛋白质可以通过免疫识别。这是由它们的磷酸化组蛋白H2AX的激光暴露的细胞的强条带（γ-H2AX）的示范说明。此后，激光方法已通过DNA双链断裂引起的DDR的众多研究中。虽然功能强大和流行，和戏剧性的免疫荧光图像的来源，应该指出的是，在大多数实验的激光强度进行调整，从而产生可观察到的结果，而对病变身份的关注，密度，或间距。事实上，它可能很难做这类估算。因此，它们在很大程度上忽略了，尽管由激光器5引入DNA损伤的多重性。这有助于在文献6的许多矛盾。 与此相反，以双链断裂，DNA的大多数化学修饰不刺激DDR蛋白质的分离的焦点的形成。这是我们目前的病变频率的理解之光重要的。据估计，在培养的人细胞招致每个细胞周期多达50双链断裂，在S期7，8，9很大程度上形成。形成在非增殖细胞更少。与此相反，核碱基丢失或修改事件，这是在数万每细胞/ 1天，10的数目。因此，我们最大约知道通过比较少见，而那些通过螺旋扭曲病变，合共更为常见诱发少得多的事件引起的DDR。 为了解决有关的细胞反应共价基因组DNA的修改问题，我们希望具有螺旋扭曲的DNA加合物是有内在的DDR诱导活性的工作。此外，为了方便实验设计和解释，我们感兴趣的是其引入可能对于控制时间，是适合于可视化的结构。因此，我们开发了基于补骨脂素的战略。补骨脂素被很好地表征光敏DNA嵌入有利于5&#39; TA:AT网站。不像其他的交联剂如氮芥和丝裂霉素C（MMC）它们不是DNA的反应性，除非暴露于长波紫外光（UVA）光。插层的分子与相反链上，以产生螺旋扭曲间交联胸腺嘧啶碱基（的ICL反应）11。在我们的实验中使用的三甲基补骨脂素大多数产品都是的ICL，相对少的单加成物产生（低于10％）12，以及在一条链上相邻碱基之间的内交联不形成。因为他们是强大的块复制和转录，补骨脂等交联剂，如顺铂和MMC，通常用于化疗。因此补骨脂启用研究，通过一个螺旋扭曲结构跟着DDR的激活，并且还提供了洞察到与临床重要性的化合物的细胞应答。 我们合成，其中三甲基补骨脂素是有联系的地高辛（DIG）的试剂，植物甾醇未在哺乳动物细胞中发现的，并经常用作immunotag。用于光活化的要求在活细胞中的细胞核定位允许通过补骨脂素的ICL在定义的ROI的激光（365纳米）。这些可以通过IMM显示unofluorescence反对挖标签。 DNA修复和DDR蛋白出现在激光的局部的ICL 13,14的条纹。 通过用于产生双链断裂的高激光强度激活DDR可能是由于分离的或聚集损伤15,16。因此，从这些实验结果的相关性，以天然存在的病变，本在低得多的浓度，是不确定的。为了解决有关的DNA加合物补骨脂频率和间距类似的问题，我们利用DNA纤维技术17和immunoquantum点。量子点是比荧光染料更明亮，没有被曝光漂白光。因此，它们经常用于单分子成像18，一个应用程序，其为荧光染料是不够明亮。个人DNA纤维可以上克被拉伸小姑娘滑动，并且可以通过对在细胞收获前的温育过程中掺入的核苷类似物免疫荧光来显示。我们处理的细胞与地高辛补骨脂素和露出的ROI，以激光微照射。纤维从细胞和个体辛补骨脂素加合物准备可以与immunoquantum点进行可视化。将细胞暴露于核苷类似物为相对短的时间（20-60分钟）允许复制大片在激光局部的ICL的附近的显示。

<table><tbody><tr><td>Digoxigenin NHS ester</td><td>Sigma-Aldrich</td><td>11333054001</td><td></td></tr><tr><td>Chloro-psoralen</td><td>Berry and Associates</td><td>PS 5000</td><td></td></tr><tr><td>diaminoglycol</td><td>Sigma-Aldrich</td><td>369519</td><td>4,7,10-Trioxa-1,13-tridecanediamine</td></tr><tr><td>Chloroform</td><td>Acros Organics</td><td>423550040</td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A4524</td><td></td></tr><tr><td>Ammonium solution</td><td>Sigma-Aldrich</td><td>5002</td><td></td></tr><tr><td>TLC plates</td><td>Analtech, Inc.</td><td>P02511</td><td></td></tr><tr><td>Flass glass column 24/40, 100 mL</td><td>Chemglass Life Sciences</td><td>CG-1196-02</td><td></td></tr><tr><td>Nikon T2000_E2 spinning disk confocal microscope, equipped with automated stage and environmental control chamber and plate holder</td><td>Perkin Elmer</td><td></td><td>With Volocity Software</td></tr><tr><td>Micropoint Galvo&amp;nbsp;</td><td>Andor Technologies</td><td></td><td>with a Nitrogen pulsed laser&amp;nbsp;</td></tr><tr><td>dye cell</td><td>Andor Technologies</td><td>MP-2250-2-365</td><td></td></tr><tr><td>365 dye</td><td>Andor Technologies</td><td>MP-27-365-DYE</td><td></td></tr><tr><td>IdU&amp;nbsp;</td><td>Sigma-Aldrich</td><td>17125</td><td></td></tr><tr><td>35 mm glass botomm plates 1.5 coverslip, 10 mm glass diameter, uncoated</td><td>Matek</td><td>P35G-1.5-10-C</td><td></td></tr><tr><td>microscope slides</td><td>New Comer Supply</td><td>Part # 5070</td><td>New Silane Slides</td></tr><tr><td>Mouse anti BrdU antibody (IdU)</td><td>BD Biosciences</td><td>347580</td><td>1 in 40</td></tr><tr><td>Rat anti BrdU Antibody (CldU)</td><td>Abcam</td><td>ab6326</td><td>1 in 200&amp;nbsp;</td></tr><tr><td>Rabbit anti Dig antibody</td><td>ThermoFisher Scientific</td><td>710019</td><td>1 in 200</td></tr><tr><td>Q-dot 655 goat anti Rabbit IgG</td><td>ThermoFisher Scientific</td><td>Q-11421MP</td><td>1 in 5,000</td></tr><tr><td>AF647- goat anti Rat IgG</td><td>Jackson Immunoresearch</td><td>112-605-167</td><td>1 in 100</td></tr><tr><td>AF488-goat anti mouse IgG</td><td>Jackson Immunoresearch</td><td>115-545-166</td><td>1 in 100</td></tr><tr><td>Zeiss epifluorescent microscope A200</td><td>Zeiss</td><td></td><td>&amp;nbsp;with Axiovision software</td></tr><tr><td>Q-dot 655 filter</td><td>Chroma</td><td>39107</td><td></td></tr></tbody></table>

single molecule analysis of laser localized psoralen adducts

DNA损伤反应（DDR）已经被广泛表征在活细胞中诱导激光微束照射的双链断裂（DSB的）的研究。该DDR到螺旋扭曲共价DNA修饰，包括链间交联的DNA（的ICL），没有作为很好的界定。我们研究通过的ICL刺激DDR，通过immunotagged补骨脂素的光敏激光定位，在活细胞中的细胞核。为了解决约加成分布和复制叉遇到的基本问题，我们结合与其他两种技术的激光定位。 DNA纤维经常用于显示通过在短的脉冲掺入核苷类似物的免疫荧光的复制叉的进展。 Immunoquantum点已被广泛用于单分子成像。在新方法中，从携带激光局部的ICL细胞DNA的纤维铺在显微镜载玻片上。该标记的ICL显示有immunoquantum点和日电子病变间距离来确定。复制叉的碰撞与的ICL可以可视化和不同遭遇的图案识别和定量。

激光局部地高辛TMP（ 图1A）的ICL可以通过对连接到补骨脂该DIG标记的免疫荧光来显示。尽管激光可被引导在任意轮廓的区域撞击，条纹不在细胞"自然"形状，并且合法信号可以从假象由于非特异性结合由一次或二次抗体容易区分。使用不到理想的特异性抗体时，此功能非常有用。的DDR，γ-H2AX的公知的标记物，在挖-TMP的ICL的条纹的一个例子示于图1B。</stro...

Watch this Scientific Journal Video about Single Molecule Analysis of Laser Localized Psoralen Adducts at JoVE.com

Single Molecule Analysis of Laser Localized Psoralen Adducts

激光器在对DNA损伤的细胞应答的研究经常使用的。然而，它们产生的病变，其间隔，频率，和碰撞与复制叉很少表征。在这里，我们描述了一种方法，使这些参数与激光局部间交联的决心。

激光局部补骨脂素加合物的单分子分析

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live ...

single-molecule-analysis-of-laser-localized-psoralen-adducts

Research

JoVE Journal

Genetics

6.6K 次观看.  National Institute on Aging, National Institutes of Health. 激光器在对DNA损伤的细胞应答的研究经常使用的。然而，它们产生的病变，其间隔，频率，和碰撞与复制叉很少表征。在这里，我们描述了一种方法，使这些参数与激光局部间交联的决心。 

视频: Single Molecule Analysis of Laser Localized Psoralen Adducts

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

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 ...

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 ...

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of carcinogenic processes. This covalent binding, which is considered the cause of tumorigenesis, is now evaluated as a central dogma of chemical carcinogenesis. Here, methods are described employing the reactions catalyzed by cytochrome P450 and additional biotransformation enzymes to investigate the potency of chemicals or drugs for their activation to metabolites forming these DNA adducts. Procedures are presented describing the isolation of cellular fractions possessing biotransformation enzymes (microsomal and cytosolic samples with cytochromes P450 or other biotransformation enzymes, i.e., peroxidases, NADPH:cytochrome P450 oxidoreductase, NAD(P)H:quinone oxidoreductase, or xanthine oxidase). Furthermore, methods are described that can be used for the metabolic activation of analyzed chemicals by these enzymes as well as those for isolation of DNA. Further, the appropriate methods capable of detecting and quantifying chemical/drug-derived DNA adducts, i.e., different modifications of the 32P-postlabeling technique and employment of radioactive-labeled analyzed chemicals, are shown in detail.

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Evaluating the potency of environmental chemicals and drugs, to be enzymatically bioactivated to intermediates generating covalent DNA adducts, is an important field in the development of cancer and its treatment. Methods are described for compound activation to form DNA adducts, as well as techniques for their detection and quantification.

酶活化致癌物和药物对共价 DNA 加合物的生成研究体外及其对32 P-postlabeling 的测定

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of ...

生物化学

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

单分子水平标记均匀性的全蛋白质定量

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

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 ...

Laser-free Hydroxyl Radical Protein Footprinting to Perform Higher Order Structural Analysis of Proteins

Hydroxyl Radical Protein Footprinting (HRPF) is an emerging and promising higher order structural analysis technique that provides information on changes in protein structure, protein-protein interactions, or protein-ligand interactions. HRPF utilizes hydroxyl radicals (&#9642;OH) to irreversibly label a protein&#8217;s solvent accessible surface. The inherent complexity, cost, and hazardous nature of performing HRPF have substantially limited broad-based adoption in biopharma. These factors include: 1) the use of complicated, dangerous, and expensive lasers that demand substantial safety precautions; and 2) the irreproducibility of HRPF caused by background scavenging of &#9642;OH that limit comparative studies. This publication provides a protocol for operation of a laser-free HRPF system. This laser-free HRPF system utilizes a high energy, high-pressure plasma light source flash oxidation technology with in-line radical dosimetry. The plasma light source is safer, easier to use, and more efficient in generating hydroxyl radicals than laser-based HRPF systems, and the in-line radical dosimeter increases the reproducibility of studies. Combined, the laser-free HRPF system addresses and surmounts the mentioned shortcomings and limitations of laser-based techniques.

This protocol presents a method to use inline radical dosimetry and a plasma light source to perform flash oxidation protein footprinting. This method replaces the hazardous UV laser to simplify and improve the reproducibility of fast photochemical oxidation of protein studies.

无激光羟基基蛋白足迹，对蛋白质进行更高级的结构分析

Hydroxyl Radical Protein Footprinting (HRPF) is an emerging and promising higher order structural analysis technique that provides information on changes ...

Quantification of three DNA Lesions by Mass Spectrometry and Assessment of Their Levels in Tissues of Mice Exposed to Ambient Fine Particulate Matter

DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are electrophilic, generate reactive electrophiles upon biotransformation, or induce oxidative stress. Among the oxidized nucleobases, the most studied one is 8-oxo-7,8-dihydroguanine (8-oxoGua) or 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxodGuo), a biomarker of oxidatively induced base damage in DNA. Aldehydes and epoxyaldehydes resulting from the lipid peroxidation process are electrophilic molecules able to form mutagenic exocyclic DNA adducts, such as the etheno adducts 1,N2-etheno-2&#39;-deoxyguanosine (1,N2-&#949;dGuo) and 1,N6-etheno-2&#39;-deoxyadenosine (1,N6-&#949;dAdo), which have been suggested as potential biomarkers in the pathophysiology of inflammation. Selective and sensitive methods for their quantification in DNA are necessary for the development of preventive strategies to slow down cell mutation rates and chronic disease development (e.g., cancer, neurodegenerative diseases). Among the sensitive methods available for their detection (high performance liquid chromatography coupled to electrochemical or tandem mass spectrometry detectors, comet assay, immunoassays, 32P-postlabeling), the most selective are those based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-ESI-MS/MS). Selectivity is an essential advantage when analyzing complex biological samples and HPLC-ESI-MS/MS evolved as the gold standard for quantification of modified nucleosides in biological matrices, such as DNA, urine, plasma and saliva. The use of isotopically labeled internal standards adds the advantage of corrections for molecule losses during the DNA hydrolysis and analyte enrichment steps, as well as for differences of the analyte ionization between samples. It also aids in the identification of the correct chromatographic peak when more than one peak is present.
We present here validated sensitive, accurate and precise HPLC-ESI-MS/MS methods that were successfully applied for the quantification of 8-oxodGuo, 1,N6-dAdo and 1,N2-dGuo in lung, liver and kidney DNA of A/J mice for the assessment of the effects of ambient PM2.5 exposure.

We describe here methods for sensitive and accurate quantification of the lesions 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxodGuo), 1,N6-etheno-2&#39;-deoxyadenosine (1,N6-dAdo) and 1,N2-etheno-2&#39;-deoxyguanosine (1,N2-dGuo) in DNA. The methods were applied to the assessment of the effects of ambient fine particulate matter (PM2.5) in tissues (lung, liver and kidney) of exposed A/J mice.

环境细颗粒物小鼠组织中三种 DNA 损伤的质谱定量及水平评价

DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are ...

免疫与感染

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 &#181;m, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

在联合电子能谱成像miniSOG标记的DNA修复蛋白可视化（ESI）

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...