我们感谢博士T. Helleday和大肠杆菌彼得曼博士与纤维的技术，以及有益的讨论实验室成员的帮助。 WN是由一名高级国际研究，国际癌症研究协会奖学金和科学研究的补助金（N301 165935）波兰国家委员会的支持。

在这里介绍的方法是使用下列出版物：Schwab等，EMBO J.，29（4）:806 - 18 5。 1。 DT40细胞培养 材料：胎牛血清（FBS），，鸡血清，青霉素/链霉素，2 -巯基乙醇的RPMI <ol><li>准备细胞培养液中添加7％FBS，3％的鸡血清，1个青霉素/链霉素和10微米2 - 巯基乙醇的RPMI培养基。 </li><li> DT40细胞生长在无菌细胞培养瓶中，在38 ° C和5％CO 2在培养箱。拆分单元格，每天密度5 × 10 5细胞/ ml 6。 </li></ol> 2， 在体内的标签 材料：IDU，CldU <ol><li>准备核苷酸类似物的股票解决方案：解散IDU在中等至2.5毫米至5毫米和CldU。热两种解决方案简要地60 ° C和旋涡，直到核苷酸类似物UES完全溶解。 </li><li> IDU添加到终浓度为25微米成倍增长DT40细胞和混合细胞悬液。细胞孵育20分钟，38 ° C和5％CO 2 。 </li><li>第一个标签的潜伏期后，添加CldU到终浓度为250微米，并视为在上一步中所描述的细胞。 </li><li>与冰冷的PBS清洗细胞，在浓度7.5 × 10 5细胞/ ml的冷PBS重悬。标记的细胞置于冰上。 </li></ol>标签过程仅仅是一个建议，可以进行修改，以解决具体问题。实验设计的更多信息，请参阅讨论部分。 3。细胞裂解和DNA的蔓延 材料：载玻片，纤维裂解液<ol><li>准备纤维裂解液：在200毫米的Tris - HCl，pH 7.5的50 mM的EDTA，0.5％SDS </li><liSPOT 2μL的细胞悬液到载玻片的一端。空气干燥5分钟或直到下降的体积大大减少，但不干燥。 </li><li>吸取7μL的细胞悬液上裂解液，轻轻搅动枪头混合的解决方案。孵育细胞裂解进行2分钟。 </li><li> 15 °倾斜的幻灯片，让纤维传播沿着幻灯片。 </li><li>一旦光纤解决方案已经达到底部的幻灯片，水平放置风干。干燥后，薄，不透明的线应沿幻灯片可见。此时，应开始拉伸纤维染色线将不可见，再后用铅笔标​​记。此标记稍后将有助于找到的纤维在显微镜下。 </li></ol> 4。免疫荧光染色 材料：甲醇，醋酸，盐酸，5％BSA的PBS，染色缸，抗BrdU抗体（鼠），抗BrdU抗体（鼠），羊抗鼠Cy3标记抗体，羊抗鼠的Alexa Fluor 488抗体，Vectashield安装介质，盖玻片，指甲油<ol><li>沉浸在甲醇/醋酸（3:1）在染色缸和10分钟的孵育幻灯片。 </li><li>在蒸馏水中H 2 O的幻灯片，然后在2.5 M盐酸浸泡80分钟洗净。 </li><li> 5分钟，洗净幻灯片在PBS的3倍。 </li><li>从染色缸中删除的幻灯片，并用纸巾收集多余的PBS。水平放置幻灯片和吸管5％BSA的每张幻灯片上。轻轻盖上盖玻片的幻灯片，传播的BSA均匀地分布在幻灯片和孵育20分钟。 </li><li>淡化在5％BSA在以下浓度的主要抗体：1​​:25抗BrdU（鼠标）和1:400抗BrdU（大鼠）。 </li><li>向下移动载玻片盖玻片，轻轻将其删除。不要使用武力，如果盖玻片坚持幻灯片。在PBS直到盖玻片变得松散一个幻灯片可水化D可以轻松删除。用纸巾收集多余的BSA和横向放置幻灯片。吸取50μL抗体溶液到每个幻灯片。盖上盖玻片，再次传播的抗体溶液均匀地分布在幻灯片和在2个小时的湿盒孵育。 </li><li>取出的盖玻片后，洗的幻灯片，在PBS三次5分钟</li><li>以下浓度稀释在5％BSA的二级抗体：1:500羊抗小鼠Cy3和1:400羊抗鼠的Alexa Fluor 488。 </li><li>应用50μL二次抗体的主要抗体所述的解决方案。防止光线和孵化1小时的幻灯片。 </li><li>取出的盖玻片后，5分钟洗幻灯片在PBS的3倍。 </li><li>添加到每个幻灯片下降Vectashield安装介质和应用盖玻片。轻压盖玻片，并围绕它用纸巾去除多余的液体。与透明的指甲城邦密封盖玻片H和让他们干。存放于-20 ° C的幻灯片</li></ol> 5。图像采集 材料：荧光显微镜，相机放置浸油下降到接近铅笔标记的幻灯片，并开始定位的纤维。通常情况下，有一个主要的纤维束，但这些纤维太纠结，以后不能分析。离主束纤维明确由对方（图2）分离领域。我们将选择的图片，只用一个颜色通道，以避免偏见。然后，我们将采取约10每个时间点，浓度或细胞株的照片。然而，图片的数量取决于多少纤维可以计算每一张图片上。沿着采取不同的图片幻灯片，幻灯片的一个区域可能无法提供有代表性的纤维长度或复制结构。 6。数据分析材料：图片分析程序，例如：ImageJ <a href="http://rsbweb.nih.gov/ij/" target="_blank">（</a> http://rsbweb.nih.gov/ij/ ） 导入的图片分析程序图片。测量长度的纤维束和/或计数不同的复制结构（图3）。只算纤维清晰可见，不扩展到画面边缘。我们通常约100纤维长度测量和计数150-200复制结构，我们将至少三次重复个别实验。 7。代表性的成果： 新复制的DNA可以可视化的抗体标记的核苷酸类似物线。在我们的实验正在进行叉代表相邻的红色和绿色信号（图3）。双标协议还允许我们定义的复制结构的四个主要类别：1）新的启动事件可以divid<html>到发射的起源，而细胞培养与第一个标签，并在第二个标签的潜伏期发射的起源。前者包括邻近的绿 - 红 - 绿信号，后者只有一条绿线。 2）终止事件表现为毗邻的红，绿，红色信号。 3）穿插的起源是在过密的起源基因组中的网站。这类网站包括连续的起源和终止信号。 4）根据实验设计，停顿/倒塌叉可以定义为一个红色的7只信号或一条红线很短的绿道8,9。 这里所描述的条件，野生型DT40细胞中有一个叉速度平均为0.4微米/分钟。我们可以探测到约63％正在进行的叉子，10％起源，16％陷入僵局叉（红色大片），8％终端和3％穿插纤维。 <html>余吕杏茜1“/&gt; 。图1（a）左侧的卡通描绘的DNA标记程序的第一步：加入IDU指数增长DT40细胞，导致容易被纳入到新合成的领先和落后的DNA链的核苷酸类似物。这可以通过使用特定的抗体进行可视化，并在显微镜下观察时，会出现一条红线。随后，同样的过程重复与CldU上图的右侧所示。第二个核苷酸类似物的潜伏期后，积极叉将作为毗邻的红，绿道可见。纤维平均长度是核苷酸类似物孵育时间成正比。 （二）从裂解DT40细胞的DNA是由重力拉伸到载玻片和注册成立的核苷酸类似物是利用特异性抗体和荧光显微镜观察。 igure 2“/&gt; 图2代表的荧光图像显示了从野生型DT40随后与IDU和CldU标记，每次20分钟的细胞纤维。纤维大多是彼此分开，因此可以很容易地分析。白条代表10微米。 <img src="/files/ftp_upload/3255/3255fig3.jpg" alt="图3" /> 图3双标纤维技术允许一个区分不同的复制结构; 1 -积极复制叉，2 -新网站的DNA复制（发射新的起源），3 -叉终止（两个汇聚成叉），4 -穿插纤维间隔紧密的起源（站点）和5 - 停滞叉（红色信号）。

<ol>
	<li>Caldwell, R. B., Fiedler, P., Schoetz, U., Buerstedde, 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=Gene+function+analysis+using+the+chicken+B-cell+line+DT40.">Gene function analysis using the chicken B-cell line DT40.</a> Methods. Mol. Biol. 408, 193-210 (2007).</li><li>Petes, T. D., Williamson, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiber+autoradiography+of+replicating+yeast+DNA.">Fiber autoradiography of replicating yeast DNA.</a> Exp. Cell. Res. 95, 103-110 (1975).</li><li>Takeuchi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+frequency+of+initiation+sites+of+DNA+replication+in+Werner's+syndrome+cells.">Altered frequency of initiation sites of DNA replication in Werner's syndrome cells.</a> Hum. Genet. 60, 365-368 (1982).</li><li>Jackson, D. A., Pombo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replicon+clusters+are+stable+units+of+chromosome+structure:+evidence+that+nuclear+organization+contributes+to+the+efficient+activation+and+propagation+of+S+phase+in+human+cells.">Replicon clusters are stable units of chromosome structure: evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells.</a> J. Cell. Biol. 140, 1285-1295 (1998).</li><li>Schwab, R. A., Blackford, A. N., Niedzwiedz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATR+activation+and+replication+fork+restart+are+defective+in+FANCM-deficient+cells.">ATR activation and replication fork restart are defective in FANCM-deficient cells.</a> EMBO. J. 29, 806-818 (2010).</li><li>Saribasak, H., Arakawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+cell+culture+conditions.">Basic cell culture conditions.</a> Subcell. Biochem. 40, 345-346 (2006).</li><li>Conti, C., Seiler, J. A., Pommier, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mammalian+DNA+replication+elongation+checkpoint:+implication+of+Chk1+and+relationship+with+origin+firing+as+determined+by+single+DNA+molecule+and+single+cell+analyses.">The mammalian DNA replication elongation checkpoint: implication of Chk1 and relationship with origin firing as determined by single DNA molecule and single cell analyses.</a> Cell. Cycle. 6, 2760-2767 (2007).</li><li>Petermann, E., Orta, M. L., Issaeva, N., Schultz, N., Helleday, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroxyurea-stalled+replication+forks+become+progressively+inactivated+and+require+two+different+RAD51-mediated+pathways+for+restart+and+repair.">Hydroxyurea-stalled replication forks become progressively inactivated and require two different RAD51-mediated pathways for restart and repair.</a> Mol. Cell. 37, 492-502 (2010).</li><li>Edmunds, C. E., Simpson, L. J., Sale, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCNA+ubiquitination+and+REV1+define+temporally+distinct+mechanisms+for+controlling+translesion+synthesis+in+the+avian+cell+line+DT40.">PCNA ubiquitination and REV1 define temporally distinct mechanisms for controlling translesion synthesis in the avian cell line DT40.</a> Mol. Cell. 30, 519-529 (2008).</li></ol>

没有利益冲突的声明。

我们描述了一种方法，允许参数的影响在单分子水平的S期DNA合成进行定量分析。在过去的十多年中，不同版本的DNA纤维fluorography技术开发的可视化的活细胞内的个体复制叉的运动。在所有这些技术的关键参数是用于实现提供良好的分离DNA纤维的玻璃表面上的DNA最好的伸展的程序。实现这一目标的一个关键步骤是显微镜幻灯片，以及溶解时间的细胞悬液的孵化时间。这些参数依赖于一个特定的实验室的温度和气流，并应凭经验确定。 DNA纤维实验的实践部分通常是在一天之内完成。然而，随后的图像采集和分析是更费时，需要实践。 标签协议可以广告艾普特解答各种科学问题：使用在第二个脉冲标记的干扰与复制代理显示器叉伸长/复制强调拖延。在这种情况下，第一个标签不需要洗第二个核苷酸是添加过量。另外，阻碍复制的药物，可以组合使用，或只是除了第一个标签后。这就要求在第一个标签，并应用到删除前的第二个核苷酸类似物药物。此过程允许一个确定的各种DNA修复因子的复制叉稳定性/复制压力的条件下（即叉拖延和/或折叠）复苏的重要性。值得注意的是，一个更详细的分析，DNA复制计划的详情，可以使用DNA的梳理和荧光原位杂交相结合的一种替代方法。然而，这在技术上更有挑战性的，需要昂贵sive设备。 能够定性和定量分析DNA复制的资料调查，在DNA复制本身的缺陷以及抑制基因组不稳定与复制叉相关的途径，提供了一个必不可少的工具。毫无疑问，这个实验将进一步帮助摆脱复制介导的DNA修复机制，使正常细胞响应/适应环境的变化以及癌细胞如何利用这些机制，以抵御针对复制叉的药物的毒性的光。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>胎牛血清金（FBS） </td><td>临时机场管理局</td><td> A15 - 151 </td><td></td></tr><tr><td>鸡血清</td><td>西格玛</td><td> C5405 </td><td></td></tr><tr><td>青霉素/链霉素</td><td>临时机场管理局</td><td> P11 - 010 </td><td></td></tr><tr><td> RPMI 1640培养</td><td> Gibco公司</td><td> 21875 </td><td></td></tr><tr><td> 5 - 碘-2&#39; - 脱氧尿苷（IDU） </td><td> Sigma - Aldrich公司</td><td> I7125 </td><td></td></tr><tr><td> 5 - 氯-2&#39; - 脱氧尿苷（CldU） </td><td>西格玛</td><td> C6891 </td><td></td></tr><tr><td>显微镜载玻片SuperFrost </td><td> VWR </td><td> 631-0910 </td><td></td></tr><tr><td>盖玻片1号</td><td>科学实验室suppplies </td><td> MIC3234 </td><td></td></tr><tr><td> Vectashield安装介质</td><td> H - 1000矢量实验室</td><td> H - 1000 </td><td></td></tr><tr><td>抗BrdU抗体（鼠） </td><td> BD公司</td><td> 347580 </td><td></td></tr><tr><td>抗BrdU抗体（鼠） </td><td> Abcam公司</td><td> ab6326 </td><td></td></tr><tr><td>羊抗小鼠Cy3标记</td><td>西格玛</td><td> C2181 </td><td></td></tr><tr><td>山羊抗大鼠的Alexa Fluor 488抗体</td><td> Invitrogen公司</td><td> A110060 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr></tbody></table>

visualization of dna replication in the vertebrate model system dt40 using the dna fiber technique

维护稳定复制叉是分裂的细胞保存的可行性和预防疾病的最重要的。涉及的流程，不仅确保在面对内源性和外源性DNA损伤的忠实的基因组的重复，又要防止基因组不稳定性，一个公认的肿瘤发展的致病因素。 在这里，我们描述了一个简单的和具有成本效益的荧光显微镜为基础的方法，可视化在禽流感的B细胞DT40的DNA复制。该细胞系提供了强大的的工具，以调查在 1脊椎动物细胞的反向遗传学蛋白质在体内的功能。缺乏一种特定的基因在DT40细胞的DNA纤维fluorography允许一个解释这种基因产品在DNA复制和基因组稳定性的功能。依靠传统的方法来分析，在脊椎动物细胞复制叉动态测量人口脉冲标记细胞DNA合成的整体失业率。这是一个定量方法和定性分析影响DNA合成的参数，不允许。相反，应遵循积极叉的运动速度可以直接使用DNA纤维技术 2-4时。在这种方法中，新生的DNA标记在体内卤化核苷酸的掺入（图1A） 。随后，个别纤维拉伸到载玻片上，并标记的DNA复制大片特异性抗体染色，荧光显微镜（图1B）可视化。开始复制以及叉的方向性是由两个不同修改的类似物的连续使用。此外，双标记的方法允许参数的定量分析，在S期DNA合成的影响，即复制结构，如正在进行的和停滞的叉，复制起始密度以及叉终端。最后，实验过程中可以完成海关在一天之内，只需要一般的实验室设备和荧光显微镜。

Watch this Scientific Journal Video about Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique at JoVE.com

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

DT40，模型脊椎动物的遗传系统，提供了强大的工具来分析蛋白质的功能。在这里，我们描述了一个简单的方法，允许在DT40细胞在单分子水平的S期DNA合成的影响参数的定性分析。

使用可视化的DNA复制DNA纤维的技术，在脊椎动物的模型系统DT40

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not ...

visualization-dna-replication-vertebrate-model-system-dt40-using-dna

Research

JoVE Journal

Biology

39.8K 次观看.  University of Oxford. DT40，模型脊椎动物的遗传系统，提供了强大的工具来分析蛋白质的功能。在这里，我们描述了一个简单的方法，允许在DT40细胞在单分子水平的S期DNA合成的影响参数的定性分析。

视频: Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

DNA Curtains Shed Light on Complex Molecular Systems During Homologous Recombination

Homologous recombination (HR) is important for the repair of double-stranded DNA breaks (DSBs) and stalled replication forks in all organisms. Defects in HR are closely associated with a loss of genome integrity and oncogenic transformation in human cells. HR involves coordinated actions of a complex set of proteins, many of which remain poorly understood. The key aspect of the research described here is a technology called "DNA curtains", a technique which allows for the assembly of aligned DNA molecules on the surface of a microfluidic sample chamber. They can then be visualized by total internal reflection fluorescence microscopy (TIRFM). DNA curtains was pioneered by our laboratory and allows for direct access to spatiotemporal information at millisecond time scales and nanometer scale resolution, which cannot be easily revealed through other methodologies. A major advantage of DNA curtains is that it simplifies the collection of statistically relevant data from single molecule experiments. This research continues to yield new insights into how cells regulate and preserve genome integrity.

<meta charset="utf8"></head><body>DNA 窗帘揭示了同源重组过程中的复杂分子系统

DNA curtains present a novel method for visualizing hundreds or even thousands of DNA-binding proteins in real-time as they interact with DNA molecules aligned on the surface of a microfluidic sample chamber.

DNA 窗帘揭示了同源重组过程中的复杂分子系统

Homologous recombination (HR) is important for the repair of double-stranded DNA breaks (DSBs) and stalled replication forks in all organisms. Defects in ...

科研

遗传学

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

可视化的单分子荧光显微镜下的DNA复制

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

生物学

<meta charset="utf8"></head><body>用于分析 DNA 复制和叉停滞事件的 2D 凝胶电泳

Electrophoretic Analysis of Replication Through Structure-Prone DNA Repeats Within the SV40-Based Human Episome

Two-dimensional neutral/neutral gel-electrophoresis (2DGE) emerged as a benchmark technique to analyze DNA replication through natural impediments. This protocol describes how to analyze replication fork progression through structure-prone, expandable DNA repeats within the simian virus 40 (SV40)-based episome in human cells. In brief, upon plasmid transfection into human cells, replication intermediates are isolated by the modified Hirt protocol and treated with the DpnI restriction enzyme to remove non-replicated DNA. Intermediates are then digested by appropriate restriction enzymes to place the repeat of interest within the origin-distal half of a 3-5 kb-long DNA fragment. The replication intermediates are separated into two perpendicular dimensions, first by size and then by shape. Following Southern blot hybridization, this approach allows researchers to observe fork stalling at various structure-forming repeats on the descending half of the replication Y-arc. Furthermore, this positioning of the stall site allows the visualization of various outcomes of repeat-mediated fork stalling, such as fork reversal, the advent of a converging fork, and recombinational fork restart.

<meta charset="utf8"></head><body>作者聚焦:表征致病性重复序列的 DNA 复制以揭示复制叉停滞和扩增的机制

Here, we outline the procedure for analyzing replication progression through pathogenic, structure-prone repeats using 2-dimensional gel electrophoresis.

通过基于 SV40 的人游离体内易结构 DNA 重复序列的电泳分析进行复制

Two-dimensional neutral/neutral gel-electrophoresis (2DGE) emerged as a benchmark technique to analyze DNA replication through natural impediments. This ...

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized &lambda; DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.

使用单分子DNA拉伸含量直接观察DNA复制的酶

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types and is widely believed to be directly associated with the development of cancer in humans. DNA damage, such as that induced by UV irradiation, severely impairs the ability of replication to duplicate the genomic template accurately. A number of gene products have been identified that are required when replication encounters DNA lesions in the template. However, a remaining challenge has been to determine how these proteins process lesions during replication in vivo. Using Escherichia coli as a model system, we describe a procedure in which two-dimensional agarose-gel analysis can be used to identify the structural intermediates that arise on replicating plasmids in vivo following UV-induced DNA damage. This procedure has been used to demonstrate that replication forks blocked by UV-induced damage undergo a transient reversal that is stabilized by RecA and several gene products associated with the RecF pathway. The technique demonstrates that these replication intermediates are maintained until a time that correlates with the removal of the lesions by nucleotide excision repair and replication resumes.

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

We present a procedure by which two-dimensional agarose-gel analysis can be used to identify the structure of replication intermediates that occur following UV irradiation.

由紫外线引起的复制中间体的可视化 E。大肠杆菌使用二维的琼脂糖凝胶电泳分析

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types ...

<meta charset="utf8"></head><body>通过 ssDNA-RPA 束生长观察到的 CMG 介导的 DNA 解旋

Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a dynamic complex of proteins termed the replisome. At the heart of the replisome is the&#160;CDC45-MCM2-7-GINS (CMG) helicase, which separates the two strands of the DNA double helix such that DNA polymerases can copy each strand. During genome duplication, replisomes must overcome a plethora of obstacles and challenges. Each of these threatens genome stability, as failure to replicate DNA completely and accurately can lead to mutations, diseases, or cell death. Therefore, it is of great interest to understand how CMG functions in the replisome during both normal replication and replication stress. Here, we describe a total internal reflection fluorescence (TIRF) microscopy assay using recombinant purified proteins, which allows for real-time visualization of surface-tethered stretched DNA molecules by individual CMG complexes. This assay provides a powerful platform to investigate CMG behavior at the single-molecule level, allowing helicase dynamics to be directly observed with real-time control over reaction conditions.

<meta charset="utf8"></head><body>作者聚焦:通过单分子可视化揭示真核 DNA 复制的动力学

This protocol demonstrates performing a single-molecule assay for live visualization of DNA unwinding by CMG helicase. It describes (1) preparing a DNA substrate, (2) purifying fluorescently labeled Drosophila melanogaster CMG helicase, (3) preparing a microfluidic flow cell for total internal reflection fluorescence (TIRF) microscopy, and (4) the single-molecule DNA unwinding assay.

通过 CMG 解旋酶对 DNA 解旋进行单分子实时可视化

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a ...

使用可视化的DNA复制DNA纤维的技术，在脊椎动物的模型系统DT40

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DNA 窗帘揭示了同源重组过程中的复杂分子系统

DNA 窗帘揭示了同源重组过程中的复杂分子系统

可视化的单分子荧光显微镜下的DNA复制

通过基于 SV40 的人游离体内易结构 DNA 重复序列的电泳分析进行复制

通过基于 SV40 的人游离体内易结构 DNA 重复序列的电泳分析进行复制

通过基于 SV40 的人游离体内易结构 DNA 重复序列的电泳分析进行复制

使用单分子DNA拉伸含量直接观察DNA复制的酶

由紫外线引起的复制中间体的可视化 E。大肠杆菌使用二维的琼脂糖凝胶电泳分析

通过 CMG 解旋酶对 DNA 解旋进行单分子实时可视化

通过 CMG 解旋酶对 DNA 解旋进行单分子实时可视化