Name: investigation of protein recruitment to dna lesions using 405 nm laser micro-irradiation
Uploaded: 2018-03-20T15:00:00
Description: Watch this Scientific Journal Video about Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation at JoVE.com

这项工作得到了加拿大自然科学和工程研究委员会 (5026 至 a. m) 和由来自癌症研究协会的下一代科学家奖学金 (21531 至 a. m) 的支持。我们感谢吉恩-弗朗索瓦 Lucier 为斐济 Python 脚本提供了卓越的质量控制, 并对统计分析提出了建议。我们感谢斯蒂芬妮博士 a Yazinski 的固定溶液配方。我们感谢保罗-朱利 Karsenti 的帮助与激光功率测量。

1. 细胞的预敏化
<ol><li>24 h 在微辐照实验之前, 4万 U2OS 细胞每井在 1x DMEM 包含10% 个血清在8井养殖幻灯片与170µm 厚实盖玻片象玻璃或聚合物底部保证最大透射率405毫微米激光光和卓越的成像激光扫描倒置共焦显微镜。如果使用8井 microslide, 则可以使用500µL 的介质或解决方案, 为以后的所有处理和清洗该协议的步骤。 注意: 由于其扁平的形态, 良好的粘附性和较大的细胞核, U2OS 细胞有助于检测在?...

<ol>
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使用上面概述的方法, 405 纳米激光微辐照的细胞预敏与 BBET 或 BrdU 允许局部生成 DNA 病变, 包括 DSBs 在细胞核内黏附人细胞。DDR 在这些 DSBs 的动力学类似于在真核细胞中生成的其他方法9,18,19。在微辐照部位进行的肿瘤切除术可导致 ssDNA 的产生, 可以采用 RPA32-targeting 抗体或 RPA32-GFP 融合。与染色质相关的因素, 另一方面, 可以定?...

细胞不断暴露在 DNA 损伤的内源和外源, 威胁着基因组的完整性。DDR 是信号通路的集合, 用于检测、信号和修复 DNA 病变以维持基因组的稳定性。在 DNA 双链断裂 (DSBs), DDR 主要发生在两个互补平台上: γ H2A. X 标记染色质和切除的单链 DNA (ssDNA) 区域, 通常涂有 ssDNA 绑定的复杂 RPA1,2。
用胸苷模拟 5-溴-2 ' 脱氧尿苷 (BrdU) 或 bisbenzimide 乙醇三盐酸盐 (BBET, 赫斯特 33342) 对细胞进行预敏化的紫外激光微照射, 产生 dna 损伤的混合体, 包括单链断裂 (办学团体) 和 DSBs, 它在染色质和 ssDNA 平台上引出局部 DDR 3, 4.以前的研究表明, 在争端解决的这两个截然不同的平台上, 对基因组维护因素的招募可以用高能紫外线 (335-365 nm) 和如果2、5的组合来区分。配备此类激光器的显微镜成本高昂, 因为它们需要高能激光和专用紫外线--发射目标, 使它们远低于激光扫描共聚焦显微镜与405纳米激光的普及程度。线.在微辐照部位进行蛋白质的招募和交换的研究, 也被用来描述基因组维持因子的动态行为所需要的费力的人工图像分析所排除。
在这里, 我们表明, 对 BrdU 或 BBET 核酸染色的细胞进行预敏化, 然后用 405 nm 激光在共焦显微镜上进行微辐照, 可以监测 DNA 损伤基因组维持因子动态。使用伽玛 H2A X 或 RPA 复合子单元作为平台标记, 加上 z 堆叠, 以获得更大的景深和反褶积以提高分辨率, 使实验者能够区分当地招募到 DSBs 的因素, 从传播到早期病灶周围的大染色质域。这种分分类根据不同的核内隔间, 有助于改善 uncharacterized 蛋白的潜在作用, 招募到微辐照的网站。此外, 我们提供方便的协议和优化的管道, 以快速分析基因组维护因素的动态, 使用开放源码的软件斐济 (一个 ImageJ 分发)6,7,8。这些对目前微辐照方法的改进使对复员方案的研究在几乎任何实验室环境中都可能发生。

<table border="0" cellpadding="0" cellspacing="0" width="1022">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Olympus FLUOVIEW FV3000 resonant laser scanning confocal microscope</td>
			<td style="width:167px;">Olympus</td>
			<td style="width:171px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">FluoView FV3000 software (version 2.1)</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">405 nm laser</td>
			<td>Coherent</td>
			<td></td>
			<td>Radiation source</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Microscope incubator AIO-UNO-OLYUS-1</td>
			<td>Okolab</td>
			<td>OKO-UNO-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">60X /1.4 Objective</td>
			<td>Olympus</td>
			<td></td>
			<td>Oil immersion objective</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">8-well culture slides on coverglass (X-well)</td>
			<td>Sarstedt</td>
			<td>94.6190.802</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">U2OS osteosarcoma human cell line</td>
			<td>ATCC</td>
			<td>HTB-96</td>
			<td>Stable cell lines&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">4&#8217;,6-Diamidino-2-Phenylindole (DAPI)</td>
			<td>ThermoFisher</td>
			<td>D1306</td>
			<td>Caution toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">5-bromo-2&#8242;-deoxyuridine&#160;(BrdU)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>59-14-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>30525-89-4</td>
			<td>Caution toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Bisbenzimide H 33342 (Hoechst 33342)</td>
			<td>ThermoFisher</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Bovine serum albumin (BSA)</td>
			<td>ThermoFisher</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Trypsin EDTA 0.1%&#160;</td>
			<td>ThermoFisher</td>
			<td>15400-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Triton X-100&#160;</td>
			<td>BioShop</td>
			<td>TRX777.100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Tween-20&#160;</td>
			<td>ThermoFisher</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Sucrose&#160;</td>
			<td>BioShop</td>
			<td>SUC507</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">JetPRIME transfection reagent&#160;</td>
			<td>Polyplus</td>
			<td>114-07</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">ProLong Diamond Antifade mountant with DAPI&#160;</td>
			<td>ThermoFisher</td>
			<td>P36962</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">DMEM 1X, + phenol red</td>
			<td>Wisent&#160;</td>
			<td>319-005-CL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">DMEM 1X, - phenol red</td>
			<td>Wisent</td>
			<td>319-051-CL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Fetal bovine serum (FBS)</td>
			<td>Wisent&#160;</td>
			<td>080-150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Mouse anti-RPA32 antibody</td>
			<td>Abcam</td>
			<td>ab2175</td>
			<td>1:500 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Rabbit anti-&#947;-H2A.X antibody</td>
			<td>Cell Signaling&#160;</td>
			<td>9718S</td>
			<td>1:500 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Rabbit Anti-53BP1 antibody</td>
			<td>Cell Signaling&#160;</td>
			<td>4937S</td>
			<td>1:500 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Rabbit Anti-PRP19 antibody</td>
			<td>Abcam</td>
			<td>ab27692</td>
			<td>1:500 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Goat anti-rabbit IgG Alexa Fluor 647</td>
			<td>Cell Signaling&#160;</td>
			<td>4414S</td>
			<td>1:250 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Goat anti-mouse&#160; IgG Alexa Fluor 488&#160;</td>
			<td>Cell Signaling&#160;</td>
			<td>4408S</td>
			<td>1:250 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Fiji image analysis open-source software&#160;</td>
			<td></td>
			<td></td>
			<td>https://fiji.sc</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">1918-K Power meter&#160; with a 918D-SL-0D3 sensor</td>
			<td>Newport</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

investigation of protein recruitment to dna lesions using 405 nm laser micro-irradiation

dna 损伤反应 (DDR) 使用过多的蛋白质来检测、信号和修复 dna 损伤。描述这种反应对于了解基因组维护机制至关重要。由于在病变中的蛋白质的招募和交换是高度动态的, 他们的研究需要有能力以快速和空间分隔的方式产生 DNA 损伤。在这里, 我们描述的程序, 以当地诱发 DNA 损伤的人类细胞使用一个常用的激光扫描共聚焦显微镜配备405纳米激光线。基因组维持因子在激光条纹上的积累可以用免疫荧光 (IF) 或实时使用荧光记者标记的蛋白质来评估。使用磷酸化组蛋白 H2A。x (γ H2A x) 和复制蛋白 A (RPA) 作为标记, 该方法提供了足够的分辨率, 以区分当地征聘的因素, 从传播到相邻的染色质。我们进一步提供基于 ImageJ 的脚本, 以有效地监测 DNA 损伤部位蛋白质孵育由的动力学。这些改进大大简化了对复员方案动力学的研究。

在微辐照后, 根据基因组维护蛋白1、12的性质, 允许细胞在特定时间段内恢复。DSBs 将被核酸, 最广泛的过程中的 S 和 G2 阶段的细胞周期, 以创建一个有限的区域 ssDNA, 这是迅速覆盖的爱国军和其他基因组维护因素9。这个 ssDNA 地区四周都是染色质, 在复员方案期间进行了广泛的修改, 以管制其他复员方案因素的招?...

Watch this Scientific Journal Video about Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation at JoVE.com

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

研究 DNA 损伤修复动力学需要一个系统来诱发定义的亚核区域的病变。我们描述了一个方法来创建本地化的双绞合, 使用激光扫描共聚焦显微镜配备405纳米激光, 并提供自动化程序, 以量化的修复因素在这些病变的动态。

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 ...

Research

JoVE Journal

Genetics

RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing

角膜上皮伤口愈合过程中的热休克蛋白27的功能为基础的RNA干扰研究

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a ...

科研

遗传学

Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks

工具来研究蛋白质建筑HMGB1的作用，螺旋窜改，站点特定的DNA间交联处理

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, ...

Protocols for C-Brick DNA Standard Assembly Using Cpf1

使用Cpf1的C-Brick DNA标准装配的方案

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this ...

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

与ChEC-seq的蛋白质-DNA相互作用的全基因组映射<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

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 ...

Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation

用 5-Bromouridine 标记和免疫沉淀 RNA 合成的研究

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in ...

A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells

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 ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

基于纠错 DNA 和 RNA 测序的稀有事件检测方法

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

基于质谱的蛋白质组学分析, 利用 OpenProt 数据库揭开从非规范开放阅读框架翻译的新蛋白质

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

使用生物素标签研究蛋白质-核酸相互作用的体外生化分析

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...