405 Nm 激光微辐照对 DNA 损伤的蛋白质招募研究
Summary
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。
Abstract
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。
Introduction
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。
Protocol
1. 细胞的预敏化 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?…
Representative Results
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?…
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Olympus FLUOVIEW FV3000 resonant laser scanning confocal microscope | Olympus | ||
| FluoView FV3000 software (version 2.1) | Olympus | ||
| 405 nm laser | Coherent | Radiation source | |
| Microscope incubator AIO-UNO-OLYUS-1 | Okolab | OKO-UNO-1 | |
| 60X /1.4 Objective | Olympus | Oil immersion objective | |
| 8-well culture slides on coverglass (X-well) | Sarstedt | 94.6190.802 | |
| U2OS osteosarcoma human cell line | ATCC | HTB-96 | Stable cell lines |
| 4’,6-Diamidino-2-Phenylindole (DAPI) | ThermoFisher | D1306 | Caution toxic |
| 5-bromo-2′-deoxyuridine (BrdU) | Sigma-Aldrich | 59-14-3 | |
| Paraformaldehyde | Sigma-Aldrich | 30525-89-4 | Caution toxic |
| Bisbenzimide H 33342 (Hoechst 33342) | ThermoFisher | 62249 | |
| Bovine serum albumin (BSA) | ThermoFisher | BP1600-100 | |
| Trypsin EDTA 0.1% | ThermoFisher | 15400-054 | |
| Triton X-100 | BioShop | TRX777.100 | |
| Tween-20 | ThermoFisher | BP337-500 | |
| Sucrose | BioShop | SUC507 | |
| JetPRIME transfection reagent | Polyplus | 114-07 | |
| ProLong Diamond Antifade mountant with DAPI | ThermoFisher | P36962 | |
| DMEM 1X, + phenol red | Wisent | 319-005-CL | |
| DMEM 1X, – phenol red | Wisent | 319-051-CL | |
| Fetal bovine serum (FBS) | Wisent | 080-150 | |
| Mouse anti-RPA32 antibody | Abcam | ab2175 | 1:500 for IF |
| Rabbit anti-γ-H2A.X antibody | Cell Signaling | 9718S | 1:500 for IF |
| Rabbit Anti-53BP1 antibody | Cell Signaling | 4937S | 1:500 for IF |
| Rabbit Anti-PRP19 antibody | Abcam | ab27692 | 1:500 for IF |
| Goat anti-rabbit IgG Alexa Fluor 647 | Cell Signaling | 4414S | 1:250 for IF |
| Goat anti-mouse IgG Alexa Fluor 488 | Cell Signaling | 4408S | 1:250 for IF |
| Fiji image analysis open-source software | https://fiji.sc | ||
| 1918-K Power meter with a 918D-SL-0D3 sensor | Newport |
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Cite This Article
Gaudreau-Lapierre, A., Garneau, D., Djerir, B., Coulombe, F., Morin, T., Marechal, A. Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation. J. Vis. Exp. (133), e57410, doi:10.3791/57410 (2018).