在我们的实验室的工作是事业奖由美国国家科学基金会和NIGMS，美国国立卫生研究院授予R15GM86839区MCB0551798支持。

1。增长和紫外线照射。 <ol><li> 200μL新鲜隔夜生长在戴维斯中型1辅以葡萄糖0.4％，0.2％casamino酸，和10微克/毫升胸腺嘧啶（DGCthy中型）和100μg/ mL氨苄青霉素质粒pBR322的文化沉淀。然后，细胞沉淀悬浮于200μLDGCthy缺乏氨苄青霉素介质和使用的预防针DGCthy中等20毫升。 </li><li>文化是生长没有氨苄青霉素选择一个摇床中于37 ° C为0.5外径600（〜5 × 10 8细胞/毫升）。没有氨苄青霉素增长避免了对异常或非生产性的复制中间体中可能出现的一些突变体的选择。此外，如果用紫外线光诱导损伤，从媒体清除氨苄青霉素是必要的，因为它吸收了强烈的细胞在这些波长和盾牌，减少紫外线的有效剂量。 </li><li>黄色的灯光下工作，文化是一个躁动的旋转平台上放置在直径15厘米的培养皿。我们的文化是放置在一个15瓦杀菌灯产生的〜1 J/m2/sec，这是使用紫外线光度计测量的照射量率的距离。文化是照射50 J/m2然后立即放回晃动在实验期间的37℃培养箱中培养。此剂量产生的，平均1环丁烷嘧啶二聚体每4.5 kb的单链DNA。黄色灯光防止光解环丁烷嘧啶二聚体的光复活逆转。 </li></ol> 2。 DNA的提取。 <ol><li>在复制中间体进行审查的时候，文化0.75毫升等分放入0.75毫升冰冷NET30缓冲区（pH值8.0，100毫米氯化钠，10毫米三，30毫米EDTA），并置于冰上，直到结束日久。我们通常运行90分钟的时间当然，研究样本为0，15，30，45，60和90分钟。 EDTA和低温的服务，以有效地制止复制和核苷酸切除修复。 </li><li>然后每个样品颗粒，悬浮于150μL1.5 mg / ml的溶菌酶和0.2 mg / ml的TE RNaseA（10毫米三，pH值8.0，1 mM EDTA的），并溶解在37 ° C为20分钟。此时，加入10μl蛋白酶K（10mg/ml的），并加入10μl20％sarkosyl的添加和孵化允许持续1小时。蛋白酶K和sarkosyl有助于释放酚抽提发生之前，可能与膜蛋白相关的DNA片段。由于积极DNA复制蛋白或膜复合物往往是必然的，这有助于增加产量恢复的复制片段。 </li><li>每个样品中加入2卷苯酚和管轻轻倒5分钟，然后提取样品。然后氯仿/异戊醇（24 / 1）2卷管轻轻再次倒立5分钟。 </li><li>在14000 RPM离心5分钟，每个样品的水相在离心样品被删除，并放置到一个新的管。然后添加4卷氯仿/异戊醇（24 / 1），管轻轻倒5分钟，再离心5分钟14,000转。 </li><li>顶部，每个样本中的水相，然后透析1小时，发现一个47毫米的Whatman 0.025微米孔径的硬盘，250毫升的0.1X TE漂浮在一个烧杯，每个样品100μL。由于更容易受到剪切，复制与单链地区或分支点的结构是我们通常切割用刀片枪头口更宽，并尽量减少剪切力。在一般情况下，移液应保持到最低限度，直到后的DNA用限制性内切酶消化。 </li><li>每个样本，然后消化PvuII（New England Biolabs公司）线性化质粒的复制起点的下游。 </li><li>在琼脂糖凝胶，100μL氯仿和6X样染料含有溴酚蓝和二甲苯蓝20μL加载之前被添加到每个样品和混合。然后，水样染料相40μl装入凝胶。为了最大限度地减少结构性偏差，结构构件和DNA剪切，这个分离过程中不包括任何的步骤，以丰富单链地区，沉淀，或集中的DNA样本，下面的细胞裂解。 </li></ol> 3。双向凝胶电泳和Southern杂交分析。 <ol><li> 2维的琼脂糖凝胶电泳分析，修改3。第一维，限制的DNA样本是运行在1V/cm 1X TBE通过0.4％琼脂糖凝胶。一公升的10X TBE原液包含: <ul><li> 108克三基地</li><li> 55克硼酸</li><li> 40毫升0.5M EDTA（pH值8.0） </li></ul></li><li>一个lambda的HindⅢ大小标记是在第一线加载，然后装入样品在每一个其他车道。我们通常运行的第一ðimension〜12-15小时。跳绳车道，使得更容易切片车道投中第二个维度。低电压和低％琼脂糖凝胶电泳分离的DNA片段的大小主要取决于服务。 </li><li>对于第二个层面，切胶道第一维凝胶使用一个大的屠刀。第一线包含的lambda的HindⅢ标记可以用溴化乙锭染色。为指导，作物使用的lambda的HindⅢ标记和丢弃凝胶低于线性质粒，预计运行的地区。在这些条件下，我们发现，线性pBR322的运行略高于二甲苯蓝染色。 </li><li>投第二个维度，每个泳道是横放跨越了一个空的凝胶施法的顶部。准备一个解决方案的1.0％琼脂糖1X TBE并冷却至55 ° C。此时，凝胶溶液倒在投第二个层面，以确保完全覆盖凝胶片。一旦凝胶，它是在6.5V/cm运行在电泳单位，允许循环缓冲区。我们通常运行的第二个维度〜5.5-7小时。有效的高电压和高％琼脂糖凝胶电泳分离的DNA片段根据其形状，以及大小。通过第二个维度，非线性形状运行更慢。 </li><li>以下电泳，凝胶，然后在H 2 O的冲洗，洗两次在0.25米盐酸400毫升15分钟，与H 2 O冲洗，然后两次在400毫升0.4M的NaOH冲洗30分钟。酸清洗服务成较小的片段，更有效地转移部分缺口的DNA分子。这一步是必要的，因为大尺寸和不同寻常的形状DNA复制中间体。 </li><li>凝胶的DNA，然后转移到一个Hybond ñ +尼龙膜 4 。我们用0.4M氢氧化钠碱向下传输系统。简单地说，两片在0.4M NaOH溶液中浸泡吸墨纸放在大栈的纸巾。尼龙膜浸泡在0.4M氢氧化钠和放在吸墨纸上。在此之上，然后仔细分层凝胶的NaOH溶液中浸湿，另一块吸墨纸。最后，一​​个长片湿面纸是分层的顶部和其两端放置一个包含1 L 0.4M NaOH溶液作为灯芯的菜肴。我们通常让DNA转移为6-12小时。 </li><li>尼龙膜被删除，在5X SSC缓冲液洗20-30秒，并在10.5毫升预杂交溶液杂交辊瓶放置。然后，膜孵育在42℃，旋转超过6小时。预杂交的解决方案: <ul><li> 5.0毫升甲酰胺</li><li> 0.5毫升，20％SDS </li><li> 2.5毫升20X SSC * </li><li> 2.0毫升50X登哈特* </li><li> 0.5毫升鲑鱼精DNA（10mg/ml的） * SSC和登哈特的食谱中可以找到5 </li></ul></li><li>在预杂交期间，1微克质粒质粒缺口平移标记由罗氏制药公司使用标有[32 P] - dCTPα-提供的协议与32P。放射性标记的探针（100μL）是变性孵化在98 ° C为10分钟一个螺丝帽的离心管中，然后立即放在冰上。 </li><li>变性的探针加入5.9毫升的杂交液，其中包含: <ul><li> 3.0毫升甲酰胺</li><li> 1.5毫升南南合作</li><li> 1.0毫升H 2 O </li><li> 0.15毫升50X登哈特的</li><li> 0.10毫升20％的SDS </li><li> 0.15毫升10mg/ml的鲑鱼精DNA </li></ul></li><li>预杂交的解决方案是泼出去的滚瓶，滚瓶，然后返回到42℃的孵化器和旋转超过12小时以上，杂交液浇入。 </li><li>杂交液是泼出去的滚瓶。然后，印迹洗20分钟〜150毫升一洗0.5X SSC，0.1％SDS在42 ° C的解决方案与旋转的辊瓶的4倍。 </li><li>膜是最后一次洗涤后，放在纸巾上，直到液体已明显消失。然后将滤膜聚氯乙烯保鲜膜包裹在接触到phosphorimager屏幕。我们可视化和量化的放射性，使用风暴840及其相关ImageQuant软件。 </li></ol> 4。代表性的成果: <img src="/files/ftp_upload/2220/2220fig1.jpg" alt="图1" /> 图1。质粒复制中间体的存在和由紫外线引起的DNA损伤的情况下观察。PvuII迁移模式消化质粒质粒2D -琼脂糖凝胶电泳分析观察图解。非复制的线性质粒作为一个线性的4.4 kb片段运行。复制的质粒，比非复制的片段，由于其尺寸较大的迁移速度较慢并没有形成Y形结构nlinear形状。这种迁移模式，形成一道弧线，从线性区域扩展以及对。紫外线照射后，双Y或X形分子观察锥地区的迁移速度比Y形分子的弧线。用32P标记的pBR322的探测的二维凝胶南分析的一个例子是细胞紫外线照射和紫外线照射（从出版商的许可复制）后15分钟后立即显示。

<ol>
	<li>Davis, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Isolation+of+Biochemically+Deficient+Mutants+of+Bacteria+by+Means+of+Penicillin.">The Isolation of Biochemically Deficient Mutants of Bacteria by Means of Penicillin.</a> Proc Natl Acad Sci U S A. 35, 1-10 (1949).</li><li>Courcelle, J., Donaldson, J. R., Chow, K. H., Courcelle, C. T. <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-Induced+Replication+Fork+Regression+and+Processing+in+Escherichia+coli.">DNA Damage-Induced Replication Fork Regression and Processing in Escherichia coli.</a> Science. 299, 1064-1067 (2003).</li><li>Friedman, K. L., Brewer, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+replication+intermediates+by+two-dimensional+agarose+gel+electrophoresis.">Analysis of replication intermediates by two-dimensional agarose gel electrophoresis.</a> Methods Enzymol. 262, 613-627 (1995).</li><li>Spivak, G., Hanawalt, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+damage+and+repair+in+specific+DNA+sequences.">Determination of damage and repair in specific DNA sequences.</a> METHODS: A Companion to Methods in Enzymology. 7, 147-161 (1995).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning - A laboratory manual. , (2001).</li><li>Donaldson, J. R., Courcelle, C. T., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RuvABC+is+required+to+resolve+holliday+junctions+that+accumulate+following+replication+on+damaged+templates+in+Escherichia+coli.">RuvABC is required to resolve holliday junctions that accumulate following replication on damaged templates in Escherichia coli.</a> J Biol Chem. 281, 28811-28821 (2006).</li><li>Donaldson, J. R., Courcelle, C. T., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RuvAB+and+RecG+Are+Not+Essential+for+the+Recovery+of+DNA+Synthesis+Following+UV-Induced+DNA+Damage+in+Escherichia+coli.">RuvAB and RecG Are Not Essential for the Recovery of DNA Synthesis Following UV-Induced DNA Damage in Escherichia coli.</a> Genetics. 166, 1631-1640 (2004).</li><li>Chow, K. H., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RecO+Acts+with+RecF+and+RecR+to+Protect+and+Maintain+Replication+Forks+Blocked+by+UV-induced+DNA+Damage+in+Escherichia+coli.">RecO Acts with RecF and RecR to Protect and Maintain Replication Forks Blocked by UV-induced DNA Damage in Escherichia coli.</a> J Biol Chem. 279, 3492-3496 (2004).</li><li>Belle, J. J., Casey, A., Courcelle, C. T., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+the+DnaB+helicase+leads+to+the+collapse+and+degradation+of+the+replication+fork:+a+comparison+to+UV-induced+arrest.">Inactivation of the DnaB helicase leads to the collapse and degradation of the replication fork: a comparison to UV-induced arrest.</a> J Bacteriol. 189, 5452-5462 (2007).</li><li>Chow, K. H., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RecBCD+and+RecJ/RecQ+initiate+DNA+degradation+on+distinct+substrates+in+UV-irradiated+Escherichia+coli.">RecBCD and RecJ/RecQ initiate DNA degradation on distinct substrates in UV-irradiated Escherichia coli.</a> Radiat Res. 168, 499-506 (2007).</li><li>Al-Hadid, Q., Ona, K., Courcelle, C. T., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RecA433+cells+are+defective+in+recF-mediated+processing+of+disrupted+replication+forks+but+retain+recBCD-mediated+functions.">RecA433 cells are defective in recF-mediated processing of disrupted replication forks but retain recBCD-mediated functions.</a> Mutat Res. 645, 19-26 (2008).</li></ol>

图1显示了典型的从野生型细胞的存在和紫外线损伤的情况下取得的结果。 〜1％的总质粒DNA损害的情况下，可以发现，在Y弧，当细胞迅速增长指数相。照射后，在Y形分子的瞬时增加观察堵塞的复制叉积累损坏的网站。 X形复制的中​​间体，也瞬时积累和坚持，直到一段时间，当病变修复相关。 南分析，可以砸出复制中间体的凝胶，用塑料吸管，纯化，并直接通过电子显微?...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>An example of the Southern analysis of the 2D gel probed</td>
<td>&nbsp;</td>
<td>GE</td>
<td>G15T8</td>
<td>Yellow Lighting</td>
</tr>
<tr>
<td>15 &mu;watt germicidal lamp</td>
<td>&nbsp;</td>
<td>Sylvania</td>
<td>F20T12/GO</td>
<td>UV Lamp</td>
</tr>
<tr>
<td>Blak-Ray UV Intensity Meter 254nm</td>
<td>&nbsp;</td>
<td>Daigger</td>
<td>EF28195T</td>
<td>UVC photometer</td>
</tr>
<tr>
<td>0.025 &mu;m pore disks</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>VSWP04700</td>
<td>Floating dialysis disks</td>
</tr>
<tr>
<td>PvuII</td>
<td>&nbsp;</td>
<td>Fermentas</td>
<td>ER0632</td>
<td>Restriction Endonuclease</td>
</tr>
<tr>
<td>Nick-translation kit</td>
<td>&nbsp;</td>
<td>Roche Diagnostics</td>
<td>976776</td>
<td>To make 32P-labeled probe</td>
</tr>
<tr>
<td>Blotting Paper</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>3030-704</td>
<td>For Southern transfer</td>
</tr>
<tr>
<td>Nylon membrane</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>RPN203S</td>
<td>For Southern transfer</td>
</tr>		</tbody>
		</table>
		</div>

visualization of uv-induced replication intermediates in e. coli using two-dimensional agarose-gel analysis

在DNA损伤的存在不准确的复制是负责大部分的细胞重排，并观察在所有类型的细胞，是人们普遍认为，直接与人类的癌症发展相关的突变。如紫外线照射引起的的DNA损伤，严重损害了精确重复的基因组模板的复制能力。已经确定的基因产物，需要复制时，在模板中遇到的DNA损伤。然而，剩下的挑战已确定在复制过程中如何将这些蛋白质的过程中病变<em&gt;在体内</em&gt;。利用大肠杆菌作为模型系统，我们描述了二维琼脂糖凝胶电泳分析程序，可以用来识别复制的质粒上出现的结构性中间体<em&gt;在体内</em&gt;下面的紫外线引起的DNA损伤。使用这个程序已经证明，阻止紫外线损伤的复制叉经过一个短暂的逆转，是由RecA和几个基因相关产品与RecF通路稳定。这些复制中间体保持，直到时间的病灶切除核苷酸切除修复和复制恢复相关的技术演示。

Watch this Scientific Journal Video about Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis at JoVE.com

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

我们目前可以使用其中的一个两维的琼脂糖凝胶电泳分析，以确定紫外线照射后发生的复制中间体的结构的过程。

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 ...

visualization-uv-induced-replication-intermediates-e-coli-using-two

Research

JoVE Journal

Biology

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

10.6K 次观看.  Portland State University. 我们目前可以使用其中的一个两维的琼脂糖凝胶电泳分析，以确定紫外线照射后发生的复制中间体的结构的过程。

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

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate detailed models for the function of many types of DNA-binding proteins, yet, the exact mechanisms of these processes and their organization in the complex environment of the living cell remain far less understood. We recently introduced a method for quantifying DNA-repair activities in live Escherichia coli cells using Photoactivated Localization Microscopy (PALM) combined with single-molecule tracking. Our general approach identifies individual DNA-binding events by the change in the mobility of a single protein upon association with the chromosome. The fraction of bound molecules provides a direct quantitative measure for the protein activity and abundance of substrates or binding sites at the single-cell level. Here, we describe the concept of the method and demonstrate sample preparation, data acquisition, and data analysis procedures.

Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.

可视化的活细菌细胞蛋白-DNA相互作用使用光活化的单分子跟踪

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome ...

科研

免疫与感染

Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli

Conjugation represents one of the main mechanisms facilitating horizontal gene transfer in Gram-negative bacteria. This work describes methods for the study of the mobilization of naturally occurring conjugative plasmids, using two naturally-occurring plasmids as an example. These protocols rely on the differential presence of selectable markers in donor, recipient, and conjugative plasmid. Specifically, the methods described include 1) the identification of natural conjugative plasmids, 2) the quantification of conjugation rates in solid culture, and 3) the diagnostic detection of the antibiotic resistance genes and plasmid replicon types in transconjugant recipients by polymerase chain reaction (PCR). The protocols described here have been developed in the context of studying the evolutionary ecology of horizontal gene transfer, to screen for the presence of conjugative plasmids carrying antibiotic-resistance genes in bacteria found in the environment. The efficient transfer of conjugative plasmids observed in these experiments in culture highlights the biological relevance of conjugation as a mechanism promoting horizontal gene transfer in general and the spread of antibiotic resistance in particular.

Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli

Conjugation mediates horizontal gene transfer by mobilizing plasmid DNA across two different cells, facilitating the spread of beneficial genes. This work describes a widely-used method for the efficient detection of conjugative plasmid transfer, based on the use of differential markers in the conjugative plasmid, donor, and recipient to detect transconjugation.

大肠杆菌中天然偶联质粒介导的水平基因转移检测

Conjugation represents one of the main mechanisms facilitating horizontal gene transfer in Gram-negative bacteria. This work describes methods for the ...

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 ...

Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor

The exposure of living organisms to environmental and cellular stresses often causes disruptions in protein homeostasis and can result in protein aggregation. The accumulation of protein aggregates in bacterial cells can lead to significant alterations in the cellular phenotypic behavior, including a reduction in growth rates, stress resistance, and virulence. Several experimental procedures exist for the examination of these stressor-mediated phenotypes. This paper describes an optimized assay for the extraction and visualization of aggregated and soluble proteins from different Escherichia coli strains after treatment with a silver-ruthenium-containing antimicrobial. This compound is known to generate reactive oxygen species and causes widespread protein aggregation. 
The method combines a centrifugation-based separation of protein aggregates and soluble proteins from treated and untreated cells with subsequent separation and visualization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining. This approach is simple, fast, and allows a qualitative comparison of protein aggregate formation in different E. coli strains. The methodology has a wide range of applications, including the possibility to investigate the impact of other proteotoxic antimicrobials on in vivo protein aggregation in a wide range of bacteria. Moreover, the protocol can be used to identify genes that contribute to increased resistance to proteotoxic substances. Gel bands can be used for the subsequent identification of proteins that are particularly prone to aggregation.

Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor

This protocol describes the extraction and visualization of aggregated and soluble proteins from Escherichia coli after treatment with a proteotoxic antimicrobial. Following this procedure allows a qualitative comparison of protein aggregate formation in vivo in different bacterial strains and/or between treatments.

用蛋白毒性应激器治疗大 肠杆菌 后蛋白质聚合物的提取和可视化

The exposure of living organisms to environmental and cellular stresses often causes disruptions in protein homeostasis and can result in protein ...

生物化学

<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 ...