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
Summary
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。
Abstract
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>在体内</em>。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>在体内</em>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。
Protocol
1。增长和紫外线照射。 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。 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?…
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| An example of the Southern analysis of the 2D gel probed | GE | G15T8 | Yellow Lighting | |
| 15 μwatt germicidal lamp | Sylvania | F20T12/GO | UV Lamp | |
| Blak-Ray UV Intensity Meter 254nm | Daigger | EF28195T | UVC photometer | |
| 0.025 μm pore disks | Whatman | VSWP04700 | Floating dialysis disks | |
| PvuII | Fermentas | ER0632 | Restriction Endonuclease | |
| Nick-translation kit | Roche Diagnostics | 976776 | To make 32P-labeled probe | |
| Blotting Paper | Whatman | 3030-704 | For Southern transfer | |
| Nylon membrane | GE Healthcare | RPN203S | For Southern transfer |
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Tags
UV-induced Replication IntermediatesE. ColiTwo-dimensional Agarose-gel AnalysisInaccurate ReplicationDNA DamageCellular RearrangementsMutagenesisCancer DevelopmentGene ProductsReplication Encounters DNA LesionsReplicating PlasmidsUV-induced DNA DamageReplication ForksRecARecF PathwayNucleotide Excision Repair
Cite This Article
Jeiranian, H. A., Schalow, B. J., Courcelle, J. Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis. J. Vis. Exp. (46), e2220, doi:10.3791/2220 (2010).