制备与DNA修复因子络合的核小体核心颗粒用于冷冻电镜结构测定
Summary
该协议详细概述了使用两种用于冷冻TEM网格的样品制备方法制备核小体复合物。
Abstract
染色质背景下的DNA修复知之甚少。使用核小体核心颗粒（染色质的基本重复单位）的生化研究表明，与游离DNA相比，大多数DNA修复酶以较低的速率去除DNA损伤。关于碱基切除修复（BER）酶如何识别和去除核小体中DNA损伤的分子细节尚未阐明。然而，核小体底物的生化BER数据表明，核小体根据DNA病变和酶的位置呈现出不同的结构屏障。这表明这些酶用于去除游离DNA中DNA损伤的机制可能与核小体中使用的机制不同。鉴于大多数基因组DNA组装成核小体，需要这些复合物的结构信息。迄今为止，科学界缺乏详细的协议来对这些复合物进行技术上可行的结构研究。在这里，我们提供了两种方法来制备两种基因融合的BER酶（聚合酶β和AP核酸内切酶1）的复合物，结合到核小体入口 – 出口附近的单核苷酸间隙，用于冷冻电子显微镜（cryo-EM）结构测定。两种样品制备方法都兼容通过冷冻 对 优质网格进行玻璃化处理。该协议可用作制备具有不同BER因子，先驱转录因子和染色质修饰酶的其他核小体复合物的起点。
Introduction
真核DNA由组蛋白组织和压缩，形成染色质。核小体核心颗粒（NCP）构成了染色质的基本重复单元，其调节DNA结合蛋白的可及性，以进行DNA修复，转录和复制1。尽管NCP的第一个X射线晶体结构在二十多年前首次被解决2，并且自3，4，5，6以来已经发表了更多的NCP结构，但核小体底物中的DNA修复机制尚未被描绘出来。揭示染色质中DNA修复的分子细节将需要对参与组分进行结构表征，以了解NCP的局部结构特征如何调节DNA修复活动。这在碱基切除修复（BER）的背景下尤其重要，因为BER酶的生化研究表明核小体中独特的DNA修复机制取决于催化的酶特异性结构要求和核小体内DNA病变的结构位置7，8，9，10，11，12，13.鉴于BER是一个至关重要的DNA修复过程，人们非常有兴趣填补这些空白，同时也建立一个起点，从中可以进行涉及相关核小体复合物的其他技术上可行的结构研究。
冷冻电镜正迅速成为解决复合物三维 （3D） 结构的首选方法，其大规模制备均质样品具有挑战性。尽管与DNA修复因子（NCP-DRF）复合的NCP的设计和纯化可能需要量身定制的优化，但此处介绍的生成和冷冻稳定的NCP-DRF复合物的程序提供了有关如何优化样品和冷冻电镜网格制备的详细信息。 图 1 中所示的两个工作流（不相互排斥）以及协议中的特定详细信息确定了关键步骤，并提供了优化这些步骤的策略。这项工作将推动染色质和DNA修复领域朝着一个方向发展，即用结构研究补充生化在技术上变得可行，以更好地了解核小体DNA修复的分子机制。
Protocol
1. 通过盐渍透析组装核小体核心颗粒 注意：使用重组组蛋白进行结构研究的核小体核心颗粒的制备已被其他人详细详细描述14，15，16。按照其他人14，15描述的重组X. laevis组蛋白和组蛋白八聚体组装的纯化，并如下所述组装核小体底物。 <…
Representative Results
正确组装的NCP（图2）用于与MBP-Polβ-APE1的重组融合蛋白制备复合物（图3）。为了确定NCP与MBP-Polβ-APE1形成稳定复合物的比例，我们进行了电泳迁移率位移测定（EMSA）（图4），该测定显示NCP的单移带与5倍摩尔过量的MBP-Polβ-APE1。在优化制造这种复合物的过程中，与戊二醛交联对于防止NCP分崩离析至关重要。最初，NCP-Polβ-APE1复合物…
Discussion
纯化DNA修复因子的特定方案将取决于感兴趣的酶。然而，有一些一般性建议，包括使用重组方法进行蛋白表达和纯化18;如果感兴趣的蛋白质太小（<50 kDa），则通过冷冻电镜进行结构测定几乎是不可能的，直到最近通过使用融合系统19，纳米抗体结合支架20和优化成像策略21。
在初始阶段获得劣质网格是很?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢美国国家环境健康科学研究所冷冻电镜核心的Mario Borgnia博士和北卡罗来纳大学教堂山分校的Joshua Strauss博士在冷冻电镜网格制备方面的指导和培训。我们还感谢朱莉安娜·梅洛·达丰塞卡·雷赞德博士在该项目初期提供的技术援助。我们感谢已故Samuel H. Wilson博士及其实验室成员，特别是Rajendra Prasad博士和Joonas Jamsen博士对纯化基因融合的APE1-Polβ复合物的重要贡献和支持。该研究得到了美国国立卫生研究院，国家环境健康科学研究所的校内研究计划[批准号Z01ES050158，Z01ES050159和K99ES031662-01]的支持。
Materials
| 1 M HEPES; pH 7.5 | Thermo Fisher Scientific | 15630080 | |
| 1 M MgCl2 | Thermo Fisher Scientific | AM9530G | |
| 10x TBE | Bio-rad | 1610733 | |
| 25% glutaraldehyde | Fisher Scientific | 50-262-23 | |
| 3 M KCl | Thermo Fisher Scientific | 043398.K2 | |
| 491 prep cell | Bio-rad | 1702926 | |
| Amicon Ultra 15 centrifugal filter (MW cutoff 30 kDa) | Millipore Sigma | Z717185 | |
| Amicon Ultra 4 centrifugal filter (MW cutoff 30 kDa) | Millipore Sigma | UFC8030 | |
| AutoGrid Tweezers | Ted Pella | 47000-600 | |
| Automatic Plunge Freezer | Leica | Leica EM GP | |
| C-1000 touch thermocycler | Bio-rad | 1851148 | |
| C-clips and rings | Thermo Fisher | 6640–6640 | |
| Clipping station | SubAngostrom | SCT08 | |
| Dialysis Membrane (MW cufoff 6-8 kDa) | Fisher Scientific | 15370752 | |
| Diamond Tweezers | Techni-Pro | 758TW0010 | |
| dsDNA | Integrated DNA techonologies | N/A | |
| FEI Titan Krios | Thermo Fisher | KRIOSG4TEM | |
| FPLC purification system | AKTA Pure | 29018224 | |
| Fraction collector Model 2110 | Bio-rad | 7318122 | |
| Glow Discharge Cleaning System | Ted Pella | 91000S | |
| Grid Boxes | SubAngostrom | PB-E | |
| Grid Storage Accessory Pack | SubAngostrom | GSAX | |
| Liquid Ethane | N/A | N/A | |
| Liquid Nitrogen | N/A | N/A | |
| Minipuls 3 peristaltic two-head pump | Gilson | F155008 | |
| Nanodrop | Thermo Fisher Scientific | ND-2000 | |
| Novex 16%, Tricine, 1.0 mm, Mini Protein Gels | Thermo Fisher Scientific | EC6695BOX | |
| Pipetman | Gilson | FA10002M | |
| Pipette tips (VWR) Low Retention | VWR | 76322-528 | |
| Polyacrylamide gel solution (37.5:1) | Bio-rad | 1610158 | |
| polyethylene glycol (PEG) | Millipore Sigma | P4338-500G | |
| Pur-A-lyzer Maxi 3500 | Millipore Sigma | PURX35050 | |
| Purified recombinant DNA repair factor | N/A | N/A | |
| R 1.2/1.3 Cu 300 mesh Grids | Quantifoil | N1-C14nCu30-01 | |
| Recombinant histone octamer | N/A | N/A | |
| Spring clipping tools | SubAngostrom | CSA-01 | |
| Superdex 200 column 10/300 | Millipore Sigma | GE28-9909-44 | |
| Transmission Electron Microscope | Thermo Fisher | Talos Arctica 200 kV | |
| Tweezers Assembly for FEI Vitrobot Mark IV-I | Ted Pella | 47000-500 | |
| UltraPure Glycerol | Thermo Fisher Scientific | 15514011 | |
| Vitrobot | Thermo Fisher | Mark IV System | |
| Whatman Filter paper (55 mM) | Cytiva | 1005-055 | |
| Xylene cyanol | Thermo Fisher Scientific | 440700500 | |
| Zeba Micro Spin Desalting Columns, 7K MWCO, 75 µL | Thermo Fisher Scientific | 89877 |
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Cite This Article
Rodriguez, Y., Butay, K. J., Sharma, K., Viverette, E., Wilson, S. H. Preparation of Nucleosome Core Particles Complexed with DNA Repair Factors for Cryo-Electron Microscopy Structural Determination. J. Vis. Exp. (186), e64061, doi:10.3791/64061 (2022).