Name: characterizing dna repair processes at transient and long-lasting double-strand dna breaks by immunofluorescence microscopy
Uploaded: 2018-06-08T13:30:00
Description: Watch this Scientific Journal Video about Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy at JoVE.com

我们感谢乔尔 Sanneman 和 Philine 博士 Wangemann 的共焦显微镜核心, 由堪萨斯州立大学兽医学院资助, 他们支持的努力开发这种技术。pCBASceI 是玛丽亚 Jasin (Addgene 质粒号 26477) 30 的礼物。U2OS DR-GFP 细胞是玛丽亚 Jasin18的一种礼物。

请注意, 本协议是为包含 SceI 识别站点18的 U2OS 单元格编写的。细胞不需要 U2OS, 但必须包含 SceI 的网站。根据所使用的细胞类型, 可能需要对该协议进行调整 (例如, 种子细胞数和潜伏期)。
1. 定义争端修复复杂地层的动力学
<ol><li>在10厘米的组织培养板上生长 U20S-DRGFP 细胞, 直到85–90% 汇合。</li>
	<li>去除培养基, 在室温下 (RT) 在3毫升 EDTA 中孵育2分钟?...

<ol>
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dna 损伤修复的一般分析和双链 DNA 断裂的修复具体来说是一个活跃的研究领域, 因为它的后果跨越肿瘤发生到基本生物学6,20。本手稿详述了一种方法, 准确地剖析了 RAD51 和γ H2AX 蛋白对 DSBs 通过 HR 的分辨率的贡献. 展望未来, 此方法可用于阐明修复蛋白在 DSBs14中的附加功能。修复动力学的比较和对 SceI 诱导的 DSBs 与细胞之间的控制细胞...

每天, 人体内的每一个细胞都被轰炸, 估计有1万的 DNA 病变1。这种存在的威胁使我们面临突变、肿瘤以及细胞死亡的风险。为了保护基因组的保真度, 哺乳动物细胞进化成了一系列复杂的蛋白质联想和修饰对 DNA 损伤的反应。这种反应被组织成多个途径, 统称为 DNA 损伤反应 (DDR)2,3。复员方案包括 dna 修复蛋白在 dna 损伤的积累, 在世俗和空间上协调。DDR 经常诱发细胞周期骤停, 以避免在复制受损 DNA2、4、5时可能发生的损伤传播或强化。反过来, 细胞活力也必须在修复完成后关闭解除修复复合体的细胞周期停止。
在各种类型的 DNA 损伤中, DSBs 是最有害的。未能修复 DSBs 可能导致染色体重排或大规模删除, 如失去整个染色体武器。DSBs 的修复分为两个路径6,7,8。同源重组 (HR) 要求姐妹染色体作为 DNA 模板使用, 因而被限制在细胞周期9,10的晚 S 和 G2/M 阶段。非同源端连接 (NHEJ) 没有这些限制, 但在修复 DSBs11、12时可能会导致小的删除。
特别报告人修复和复员方案一般是积极的调查领域。尽管被组织成很方便的分离通路, 却有大量的冗余。事实上, 许多蛋白质 (例如 BRCA1、BRCA2 和 RPA 复合体) 都涉及多个路径13、14、15、16。一条通路修复病变, 可导致损伤中间体, 必须由另一通路14修复。这些途径交织在一起, 结合他们的复杂任务, 将正确的蛋白质在正确的地方适当地为必要的时间, 需要一个多层次的管理过程。
最近的一份报告强调了复员方案错综复杂的情况, 表明修复复杂的形成、解决和定位可以分别被削弱17。下面的协议的总目标是彻底解剖细胞修复争端的能力。利用免疫荧光显微镜, 在 DSBs 诱导后的代表性时间点可以可视化损伤部位的修复蛋白积累。
这种技术对常用的方法有几个优点。通常, 在单个时间点进行修复, 不能代表修复配合物的装配和离解的动态过程。从初始激活到完全分辨率, 观察整个修复范围, 可确保延迟修复不认错为完全抑制。相反, 它保证了无法停用的修复响应的诱导, 不认错为正常或过度激活。
然而, 延迟蛋白复合物的形成和修复蛋白的错误定位可能与这种方法没有明确的区别。为了确定修复蛋白在定位时是否与延迟有关, 可以通过酶解细胞 DNA 来引入 "长效" 的分裂分子。所产生的病变是裁每次修复时, 导致一个明显的大核修复重点和消除时间限制, 从招聘。这可以通过修改现有的方法, 使用罕见的切割限制性 (如SceI) 来诱导一个长期的争端解决办法来实现。DSBs 的寿命使免疫荧光显微镜能直观地显示出难以捉摸的修复蛋白。增强的丰度还可以改善可视化, 当检测受到限制的抗体质量, 这一特点, 可能是有用的, 当较小的研究蛋白质被认为对 DNA 修复的影响。
值得注意的是, 我们提供了一个自由图像处理和分析软件 (如ImageJ) 的明确指令。这就消除了图像分析中的一个主要的金融障碍, 打开了对更广泛的受众进行 DNA 损伤修复的审问。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12mm Coverslips</td>
			<td>VWR</td>
			<td>89015-725</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde (PFA)</td>
			<td>ThermoFisher Scientific</td>
			<td>28908</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>VWR</td>
			<td>82050-892</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well glass bottom plate</td>
			<td>Cellvis</td>
			<td>P96-1.5H-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-H2AX Alexa488</td>
			<td>EMD Millipore</td>
			<td>05-636-AF488</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Rad51 (D4B10)&#160;</td>
			<td>Cell Signaling Technology</td>
			<td>8875S</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-formats plugin for ImageJ</td>
			<td>National Institute of Health (NIH)&#160;</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/plugins/index.html</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>VWR</td>
			<td>97061-416</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>ThermoFisher Scientific</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, High Glucose</td>
			<td>ThermoFisher Scientific</td>
			<td>12100046</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Invitrogen</td>
			<td>15576-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>VWR</td>
			<td>89510-194</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit IgG Alexa594</td>
			<td>ThermoFisher Scientific</td>
			<td>&#160;A-11012&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide</td>
			<td>sigma-Aldrich</td>
			<td>216763-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ Software</td>
			<td>National Institute of Health (NIH)&#160;</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/</td>
		</tr>
		<tr>
			<td>I-SceI Expression Vector</td>
			<td>Addgene</td>
			<td>26477</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail Polish- Insta Dri</td>
			<td>Sally and Hansen</td>
			<td>Clearly Quick (103)</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Bio Basic</td>
			<td>PD8117</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Reagent</td>
			<td>Life Technologies</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>T4049-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>TurboFect Transfection Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>R0531</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterizing dna repair processes at transient and long-lasting double-strand dna breaks by immunofluorescence microscopy

在 DNA 中修复双链断裂 (DSBs) 是一个高度协调的过程, 它有必要形成和解决多蛋白修复复合体。这一过程是由无数的蛋白质, 促进蛋白质的联想和解除这些病变。在很大程度上, 由于能够执行一个巨大的蛋白质库功能屏幕, 有一个更大的赞赏的基因, 双链 DNA 断裂修复。经常敲除或化学抑制剂屏幕识别参与修复过程中的蛋白质, 使用增加的毒性作为一个标记的蛋白质, 这是需要的争端处理修复。功能分析虽然对于确定涉及维持基因组保真度的新的细胞蛋白很有用, 但需要确定感兴趣的蛋白质是否促进修复复合体的定位、形成或解决。
通过免疫荧光显微镜可以很容易地检测到修复蛋白的积累。因此, 这些蛋白在 dna 损伤部位的联想和解除可以通过在诱导双链 DNA 断裂后观察这些核病灶来获得。这种方法还可以识别错误本地化的修复因子蛋白, 如果修复缺陷不同时发生, 修复不完全延迟。在这种情况下, 长时间的双链 DNA 断裂可以通过表达一个罕见的切割限制性 (例如, I-SceI) 在细胞中, 该酶的识别站点已经集成到细胞基因组中来设计。由于忠实的修复将重新引入酶的识别部位, 引发另一轮的分裂, 因此导致的病变特别难以解决。因此, 消除了修复动力学的差异。如果未形成修复配合物, 则会阻碍局部化。本协议描述了确定修复动力学的变化以及修复蛋白质定位所需的方法。

图 1描述了使用 ImageJ 对最大值/焦点量化的正确噪声判别的选择。DAPI 的合并图像和感兴趣的修复蛋白在左面板上。图 1A显示了90的噪声判别, 并标记了正确的焦点数目。在定量过程中不计算边缘上的原子核 (用粉红色箭头描绘) 和原子核外的病灶 (用黄色箭头描绘)。图 1B描述了低噪音公差50。在原子核外和?...

Watch this Scientific Journal Video about Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy at JoVE.com

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

双链 DNA 断裂的修复是一个动态过程, 不仅需要在断裂时形成修复配合物, 而且要解决病变后的解决方法。在这里, 我们使用免疫荧光显微镜的短暂和长期的双滞留断裂作为一个工具来解剖这个基因组维持机制。

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 ...

Research

JoVE Journal

Cancer Research

Cell-Free DNA Integrity Analysis in Urine Samples

无细胞DNA完整性分析尿样中

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of ...

科研

癌症研究

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

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 ...

Evaluating In Vitro DNA Damage Using Comet Assay

Evaluating In Vitro DNA Damage Using Comet Assay

利用彗星试验评价体外DNA 损伤

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer ...

Immunofluorescence Microscopy of &#947;H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

Immunofluorescence Microscopy of ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â³H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

γH2AX 和53BP1 的免疫荧光显微镜分析 DNA 双链断裂的形成和修复

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas ...

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

用医用金属丝捕获循环肿瘤细胞的肿瘤细胞: 基于免疫荧光和 DNA 的3D 方法

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

Evaluation of Colorectal Cancer Risk and Prevalence by Stool DNA Integrity Detection

通过大刀DNA完整性检测评估结肠直肠癌风险和患病率

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

患者生物流体中肿瘤相关循环DNA的检测与监测

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

人类结肠癌细胞DNA损伤与修复福西的免疫荧光成像

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

癌症患者血浆样本中无细胞DNA检测

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis ...