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Summary
Abstract
Introduction
Protocol
Résultats
Discussion
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Acknowledgements
Materials
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Génétique

激光 Microirradiation 研究体内细胞对简单复杂 DNA 损伤的反应

Published: January 31, 2018
doi:
10.3791/56213
, , , , , ,
1Department of Biological Chemistry, School of Medicine,University of California, Irvine, 2Beckman Laser Institute and Medical Clinic,University of California, Irvine, 3Department of Developmental and Cell Biology, School of Biological Sciences,University of California, Irvine, 4Department of Biomedical Engineering and Surgery,University of California, Irvine

Summary

该协议的目的是描述如何使用激光 microirradiation 诱导不同类型的 dna 损伤, 包括相对简单的断链和复杂的损害, 研究 dna 损伤信号和修复因子在损伤部位的组装在体内.

Abstract

DNA 损伤诱导细胞的特定信号和修复反应, 这对保护基因组完整性至关重要。激光 microirradiation 成了研究 DNA 损伤反应 (DDR)体内的有价值的实验工具。它允许实时高分辨率的单细胞分析大分子动力学, 以响应激光诱导的损伤局限于微米区域的细胞核。然而, 不同的激光条件已被使用, 不了解不同类型的损害引起的差异。因此, 损害的性质往往没有很好的特征或受到控制, 造成征聘或修改资料中明显的不一致。我们证明, 不同的辐照条件 (即, 不同的波长, 以及不同的输入功率 (度) 的飞秒 (fs) 近红外激光) 诱导不同的 DDR 和修复蛋白组件。这反映出 DNA 损伤的类型。该协议描述了如何滴定激光输入功率, 使不同数量和复杂的 DNA 损伤, 可以很容易地监测通过检测基地和交联损害, 差异聚 (ADP-核糖) (PAR) 信号,损伤部位的通路特定修复因子组件。一旦确定了损伤条件, 就有可能研究不同损伤复杂度和差异损伤信号的影响以及上游因素对任何感兴趣因素的损耗。

Introduction

在体内DNA 损伤信号不太清楚
在体内, DNA 与组蛋白和其他因素复合形成染色质纤维。染色质结构的调节对 DNA 代谢至关重要。例如, 组蛋白变体 H2AX 是由共济失调-扩张突变 (ATM) 和其他激酶后的双链断裂 (争端) 诱导磷酸化, 是重要的争端处理的损害信号放大, 以及提供一个对接地点的其他因素.损伤信号的传播和修复途径的选择似乎受到局部染色质结构的严重影响1。一些染色质重塑因子, 组蛋白的陪护, 和组蛋白修饰酶确实被招募到破坏地点, 是重要的有效的 DNA 修复, 突出了染色质调节的意义在 DDR 和修复2,3,4. 此外, 在酵母和果蝇5,6,7,8中观察到的损伤站点聚类或重新定位, 让人联想到孵育的基因座与基因调控相关的层次室9,10。最近在老鼠和人类细胞中的研究也显示了对争端修复点的动员, 这影响了维修保真度和路径选择11,12。这就增加了 DDR/修复也可能与核结构、高阶染色质组织和细胞核内的染色体动力学紧密相连的可能性。因此, 必须发展高分辨率的方法, 以研究在活细胞内核环境的背景下的 DDR 和修复过程, 以便了解 DNA 损伤的短期和长期后果。

PAR 聚合酶 (PARP) 在测量损伤部位的损伤程度和类型以及调节蛋白质组装方面的关键作用
PARP1 是 dna 尼克传感器迅速激活 dna 损伤, 在 dna 修复13中扮演关键角色。PARP1 最初被认为与 X 射线修复交叉配合 1 (XRCC1), 以促进基础切除修复 (BER), 但最近的研究显示它的作用在其他 DNA 修复途径, 包括争端修复14。活化 PARP1 使用烟酰胺腺嘌呤核苷酸 (和+) 作为基板, 以 ADP-ribosylate 多靶蛋白, 包括本身。近年来, 这种酶和其他家族成员引起了人们的广泛关注, 因为 PARP 抑制剂已经成为一种有前途的癌症治疗药物。虽然 PARP 抑制剂最初被发现是有效的乳腺癌基因 (BRCA)-突变乳腺癌细胞, 现在有大量的证据, 他们的影响, 在单一和联合治疗与 DNA 损伤的代理人/辐照广泛的癌症的突变不限于 BRCA15,16,17,1819。

在分子水平, PARP 活化被证明在组织地方染色质结构在损伤站点扮演重要角色。依赖于标准的染色质修饰酶的招募促进了争端修复, 并决定了修复路径的选择, 这表明了在损伤部位进行 par 修饰的重要脚手架作用。 13 ,21,22,,24,25,26,2728,2930,31我们最近演示了将 p53-binding 蛋白 1 (53BP1) 从损坏点排除在 PAR 32之外, 为非同源 endjoining 的53BP1 依赖 hyperactivation 提供了另一种解释 (NHEJ) 通过 PARP 抑制剂和突出 PARP 的意义在争端修复路径选择33,34。PARP1 也直接 PARylates 并影响多种 DNA 修复因子的活性14。

使用激光 Microirradiation 作为一种工具来研究 DDR/修复在体内
在 1969年35中首次描述了激光 microirradiation 在单个染色体上产生亚微米的变化, 并在1981年的36中进行了详细的回顾。几十年后, 激光 microirradiation 被证明是诱导 dna 损伤在一个明确的微米区域的细胞核, 并被证明是一个有价值的技术, 研究招募或修改各种因素的 dna 损害在体内13,37,38,39,40,41. 此方法允许检测未形成明显辐射诱发病灶 (IRIF) 在损伤部位的那些因素39,42。也可以在损伤部位和细胞核的其余部分研究染色质结构变化的时空动力学。我们仔细比较了不同激光系统诱导的 ddr 和输入功率, 以评估 DNA 损伤类型与 microirradiation 条件之间的关系32,43,44, 45。在先前的激光损伤研究中观察到了53BP1 和端重复约束因子 2 (TRF2) 的异常招聘模式, 这为激光损伤的 “非生理性” 特性的反复关注提供了依据46 ,47,48,49。我们发现, 这些明显的差异现在可以解释的差异 PARP 信号, 测量的数量和复杂的诱导损害32。我们证实: 1) 激光 microirradiated 细胞 (即使在高输入功率辐照后) 在界面中以损伤检查点控制的方式被逮捕, 并且保持可行 (至少 48 h)32,50;和 2) 修复因子的招募/修改忠实地重述那些观察与常规 DNA 损伤药物和争端处理酶32,39,42, 44、50、51、52。这些结果强烈支持研究激光损伤诱导的细胞反应的生理相关性。

Protocol

1. 碱性细胞制备 注意: 这一步是为标准免疫荧光检测内源性蛋白质的招募或修改, 并为使用细胞系稳定表达荧光标记重组蛋白。例如, Potorustridactylus (PtK2) 袋肾上皮细胞稳定表达 EGFP-53BP11220-1711或 TRF2-YFP 在我们以前的研究 (图 1)32中使用。在前一种情况下, 53BP1 (氨基酸 1220-1711) 的焦点形成区, 其中包含的齐聚域, 都铎域, 和化?…

Representative Results

使用 PtK2 细胞稳定表达 EGFP-53BP11220-1711或 TRF2-YFP, 进行激光输入功率滴定, 以确定他们的最佳招聘条件 (图 1)32。 在高输入功率激光损伤现场, 明显的 GFP-NTH1 (紫外线) 损伤引起的交联损伤, 以及专门识别基底损伤的 DNA glycosylase 的显著聚类。此外, 观察到较高的 XRCC1 和 CtIP 信号, 反映了链断裂数量的增加, 并证明在这种情况下引起复…

Discussion

使用激光 microirradiation 进行 DDR 研究的优点是:

1. 从简单的链断裂到复杂的 dna 损伤, 可以诱导不同类型和数量的 dna 损伤, 并通过调整激光辐照参数来检测 dna 损伤部位的不同修复因子。也可以在相同的细胞核中多次造成损伤, 以评估反式效果 (如图 3所示)。

2. 重要的是, 在受损地点发生的事件和发生在原子核其他地方的次要事?…

Divulgations

The authors have nothing to disclose.

Acknowledgements

我们感谢 Dr. 野在东北大学, 日本 GFP-NTH1 表达质粒, 和 Dr. 爱神 Lazzerini Denchi 在斯研究所, la, 加利福尼亚为 TRF2-YFP 和 EGFP-53BP11220-1711表达的粒。这项工作得到了空军科研办公室 (FA9550-04-1-0101) 和贝克曼激光研究所 Inc. 基金会 (m.w. B)、美国国家科学院福特基金会奖学金 (美国)、NSF MCB-1615701 和中国CRR-17-426665 (阿莫亚科)。

Materials

Ti:Sapphire NIR pulsed femtosecond laser Mira-900 Coherent Inc. Mira 900
Inverted microscope  Carl Zeiss Axiovert 200M
63X/1.4 NA objective Zeiss APOCHROMAT Ph3 
Compact Rotation Stage  Newport Corp PR50PP
Temperature Controller Warner TC-344B
Heating System Ibidi  10918
Gas Incubation System Ibidi  11920
Laser Power and Energy Meter (RoHS) Coherent FieldMaxII-TOP 
ORCA-R2 Digital CCD Camera Hamamatsu C10600
LSM 510 META Laser Scanning Microscopes Carl Zeiss
100X/1.3 NA Zeiss Plan APO Carl Zeiss
35mm Gridded Dishes  MatTek P35G-1.5-14-CGRD-D
PtK2 kidney epithelial cells ATCC CCL 56 
HeLa cells ATCC CCL-2
DMEM Life Technologies 11885-092
CO2-Independent Medium  Life Technologies 18045-088
Advanced MEM  Life Technologies 12492-013 supplemented with L-Glutamine, 4% FBS
penicillin/streptomycin Fisher Scientific 15140122
L-Glutamine Fisher Scientific 25030081
FBS Omega Scientific FB-02
Thymidine   SIGMA T9250
serum free media     (Opti-MEM I Reduced Serum Media) Fisher Scientific 11058021
Anti-Cycolbutance pyrimidine dimer (CPD) (mouse) Kamiya Biomedical Company MC-062
Anti-XRCC1 (mouse ) Gene Tex Inc GTX72311
Anti-53BP1 (rabbit) Santa Cruz Biotech sc-22760
Anti-CtIP (rabbit) Abcam ab70163
Anti-PAR polymers (mouse) Enzo Life Sciences BML-SA216-0100
Anti-PAR (rabbit) Trevigen 4336-BPC-100
Anti-TRF2 (mouse) Novus Biological  NB100-56506
Anti 6-4PP (mouse) Kamiya Biomedical
Anti-Rad21 (Rabbit)                     (for cohesin detection) generated in Yokomori (KY) lab
Anti-Rad51 (Rabbit) Santa Cruz Biotechnology SC-8349
Anti-Ku70 (mouse)  Novus Biologicals NB100-102
8-oxiguanine  Trevigen, Inc.
Anti-PARP1  (Rabbit)                     generated in KY lab ref 43
 Anti-hCAPG (Rabbit)                    (for condensin detection) generated in KY lab ref 42
Cy3 AffiniPure Donkey Anti-Rabbit IgG  Jackson ImmunoResearch Inc 711-165-152
Donkey Anti-Mouse Alexa Fluor 488 IgG Thermo Fisher Scientific A-21202
HiPerFect siRNA Transfection Reagent Qiagen 301705
Lipofectamine 2000 Transfection Reagent Thermo Fisher Scientific 11668019
GFP-SMC1 stable cell line     generated in KY lab ref 49 
GFP-NEIL2 stable cell line generated in KY lab ref 54
GFP-NTH1 stable cell line generated in KY lab ref 32
GFP-SMC1 plasmid generated in KY lab
GFP-Ku plasmid generated in KY lab
Anti-MDC1 (rabbit)  Novus Biologicals NB100–395
Anti-gH2AX (rabbit)  Millipore 07–164
EGFP-53BP1(1220-1711) PtK2 stable cell line generated in Berns lab ref 32
TRF2-YFP PtK2 stable cell line generated in Berns lab ref 32
DMSO Sigma D2650-100ML
PARG inhibitor Trevigen 4680-096-03
DNA–PKcs inhibitor  NU7026  Sigma N1537
ATM inhibitor KU55933  Calbiochem 118500
Olaparib  Apexbio Technology A4154
Paraformaldehyde  ELECTRON MICROSCOPY SCIENC MS 100503-916 (EA)
Quantity One 1-D Analysis Software Version 4.6.9 Bio-Rad SOFT-LIT-70-9600-Q1-469PC  image analysis program
Excel microsoft spreadsheet program
Triton X100 Fisher Scientific BP151-500 detergent
Dulbecco's phosphate-buffered saline (DPBS) 10X without calcium and magnesium Fisher Scientific 14200166 dilute to 1X 
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Tags

Laser MicroirradiationDNA DamageDNA RepairDNA Repair FactorsCellular ResponseReal-time AnalysisSingle-cell AnalysisFluorescently-tagged Proteins53BP1NTH1Liposome-mediated Transfection
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Kong, X., Cruz, G. M., Silva, B. A., Wakida, N. M., Khatibzadeh, N., Berns, M. W., Yokomori, K. Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage. J. Vis. Exp. (131), e56213, doi:10.3791/56213 (2018).
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