与抗原标记阻断病变的复制体遭遇的可视化
1Laboratory of Molecular Gerontology, National Institute on Aging,National Institutes of Health, 2Institute of Chemical Biology and Nanomedicine, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology,Hunan University, 3Department of Molecular Biology and Genetics,Cornell University, 4Department of Cellular and Molecular Medicine,University of Copenhagen, 5Horizon Discovery, 6Boston University School of Medicine
Summary
虽然与DNA加合物的复制叉碰撞可以诱导双链断裂，但对爬酶体和阻断病变之间的相互作用知之甚少。我们采用邻近连接测定来可视化这些遭遇并表征对重复体组成的影响。
Abstract
对由核酸酶、辐射和其他 DNA 破坏者诱导的双链断裂 （DSB） 的细胞反应存在相当多的见解。这在一定程度上反映了识别断裂位点的方法的可用性，以及这些序列中招募到DSB的因素的特征。然而，DSB在加工由不直接引起断裂且不在特定序列位点反应的化合物形成的DNA加合物的过程中也作为中间体出现。因此，对于大多数这些药物，允许分析与反应因子和修复蛋白的结合相互作用的技术是未知的。例如，DNA链间交联（ICL）可以在复制叉遭遇后引起断裂。虽然由被广泛用作癌症化疗药物的药物形成，但没有监测它们与复制蛋白相互作用的方法。
在这里，我们描述了我们用这些具有挑战性的加合物跟踪细胞对叉子碰撞的反应的策略。我们将类固醇抗原与补骨脂素联系起来，补骨脂素在活细胞核中形成光活化依赖性ICL。通过针对抗原标签的免疫荧光观察ICL。该标签也可以是邻近连接测定（PLA）的合作伙伴，该测定报告两种抗原的密切关联。PLA被用来区分与标记的ICL密切相关的蛋白质和不与标记ICL密切相关的蛋白质。可以定义在遇到ICL后保留的复制体蛋白质，并识别其他丢失的蛋白质。这种方法适用于任何可以通过免疫学检测的结构或DNA加合物。
Introduction
细胞对双链断裂的反应有据可查，因为一系列越来越强大的方法将断裂引导到特定的基因组位点1，2，3。位置的确定性能够明确表征在位点积累并参与DNA损伤反应（DDR）的蛋白质和其他因子，从而驱动修复断裂的非同源末端连接（NHEJ）和同源重组（HR）途径。当然，许多中断是由不攻击特定序列的辐射和化学物质等因素引入的4.但是，对于这些，有一些程序可以将末端转换为适合标记和本地化的结构5，6。生物过程也引入了断裂，例如免疫球蛋白重排，最近的技术允许它们的定位，以及7。然后可以确定响应因素与这些站点之间的关系。
断裂也作为由化合物形成的加合物的间接结果出现，这些化合物不是固有的破坏者，而是破坏DNA交易，如转录和复制。它们可能是细胞对这些障碍物的反应的一个特征，可能是在修复过程中，或者因为它们引发了易受核酸酶攻击的结构。通常，加合物、断裂以及与响应因子的关联之间的物理关系是推断性的。例如，ICL由顺铂和丝裂霉素C8等化疗药物形成，并作为非碱性位点9的反应产物。众所周知，ICL是复制叉10的有效阻断，从而阻止可以被核酸酶11切割的叉。链之间的共价连接通常通过作为中间体12，13具有专性断裂的途径来缓解，因此需要同源重组以重建复制叉14。在大多数实验中，研究者遵循感兴趣的因素对复制叉与ICL碰撞下游形成的断裂的反应。然而，由于尚无定位激发性病变的技术，因此只能假设重复体及其组成部分与ICL的接近程度。
我们已经开发了一种策略，能够分析蛋白质与非序列特异性共价加合物的关联，此处由ICL说明。在我们的系统中，这些是由补骨脂素引入的，补骨脂素是一种光活性天然产物，数千年来一直用作治疗皮肤病的方法15。我们的方法基于补骨脂素的两个重要特征。首先是它们的交联形成频率高，可以超过90%的加合物，而顺铂或丝裂霉素C8，16等流行化合物形成的不到10%。第二个是化合物在不损失交联能力的情况下进行偶联。我们已经将三甲基补骨脂素与地高辛（Dig）共价连接，地高辛是一种长期建立的免疫标签。这可以通过对 Dig 标签进行免疫染色来检测基因组 DNA 中的补骨脂素加合物，并通过常规免疫荧光17 进行可视化。
在我们以前的工作中，该试剂被应用于使用基于DNA纤维的测定法16分析与ICL的复制叉遭遇。在这项工作中，我们发现复制可以通过完整的ICL继续。这取决于ATR激酶，它被复制应激激活。考虑到 CMG 复制解旋酶的结构，复制重新启动是意外的。它由MCM异质六聚体（M）组成，其在模板链周围形成一个偏移间隙环，用于前导链合成，该链被GINS复合物（G，由PSF1，2，3和SLD5组成）和CDC45（C）18的蛋白质锁定。复制可以在回复体碰撞侧远端的ICL一侧重新开始的提议主张改变回复体的结构。为了解决在遇到ICL时哪些组件在回复中的问题，我们开发了此处描述的方法。我们利用 Dig 标签作为邻近连接测定 （PLA）19 的合作伙伴来询问 ICL 与20 爬发体蛋白质的密切关联。
Protocol
1. 细胞制备 第一天用细胞粘合剂溶液预处理 35 mm 玻璃底培养皿。 在治疗前一天将细胞铺在预处理的培养皿中。细胞应在实验当天积极分裂和50-70%汇合。注意：HeLa细胞用于本实验，使用Dulbecco修饰的鹰培养基DMEM，补充有10%胎牛血清，1x青霉素/链霉素。对贴壁细胞系没有限制。然而，在PLA分析之前，必须将非贴壁细胞离心到载玻片上并固定。 第二天?…
Representative Results
具有重复体蛋白的Dig-TMP的PLADig-TMP的结构如图 1所示。合成的细节，其中三甲基补骨脂素通过乙二醇接头与地高辛结合，之前已经讨论过17，21。将细胞与化合物孵育，然后暴露于365nm光（UVA）下，使化合物光活化并驱动交联反应。略多于 90% 的加合物是 ICL16。Dig标签可以通过免疫荧光可视化，从而?…
Discussion
尽管PLA是一种非常强大的技术，但为了获得清晰和可重复的结果，必须解决一些技术问题。抗体必须具有高亲和力和特异性。此外，尽可能减少非特异性背景信号也很重要。我们发现膜和细胞碎片有助于背景，我们已经尽可能地去除了它们。固定前用含有缓冲液的洗涤剂洗涤，固定后用甲醇洗涤有助于减少非特异性结合。需要注意的是，固定前的去垢剂处理会导致细胞脱离。我们发现，在CSK处理?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了美国国家老龄化研究所NIH校内研究计划（Z01-AG000746-08）的部分支持。J.H.得到了中国国家自然科学基金（21708007和31871365）的支持。
Materials
| Alexa Fluor 568, Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody | Invitrogen | A-10011 | 1 in 1000 |
| 35 mm plates with glass 1.5 coverslip | MatTek | P35-1.5-14-C | Glass Bottom Microwell Dishes 35mm Petri Dish Microwell |
| Alexa Fluor 488,Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody | Invitrogen | A-10001 | 1 in 1000 |
| Bovine serum albumin (BSA) | SeraCare | 1900-0012 | Blocking solution, reagents need to be stored at 4 °C |
| CDC45 antibody (rabbit) | Abcam | ab126762 | 1 in 200 |
| Cell adhesive | Life Science | 354240 | for cell-TAK solution |
| Confocal microscope | Nikkon | Nikon TE2000 spinning disk microscope | equiped with Volocity software |
| Digoxigenin (Dig) antibody (mouse) | Abcam | ab420 | 1 in 200 |
| Dig-TMP | synthesized in the Seidman Lab | ||
| Duolink Amplification reagents (5×) | Sigma-Aldrich | DUO82010 | reagents need to be stored at -20 °C |
| Duolink in situ detection reagents | Sigma-Aldrich | DUO92007 | reagents need to be stored at -20 °C |
| Duolink in situ oligonucleotide PLA probe MINUS | Sigma-Aldrich | DUO92004 | anti-mouse MINUS, reagents need to be stored at 4 °C |
| Duolink in situ oligonucleotide PLA probe PLUS | Sigma-Aldrich | DUO92002 | anti-rabbit PLUS, reagents need to be stored at 4 °C |
| Duolink in situ wash buffer A | Sigma-Aldrich | DUO82046 | Duolink Wash Buffers, reagents need to be stored at 4 °C |
| Duolink in situ wash buffer B | Sigma-Aldrich | DUO82048 | Duolink Wash Buffers, reagents need to be stored at 4 °C |
| epifluorescent microscope | Zeiss | Axiovert 200M microscope | Equipped with the Axio Vision software packages (Zeiss, Germany) |
| Formaldehyde 16% | Fisher Scientific | PI28906 | for fix solution |
| Goat serum | Thermo | 31873 | Blocking solution, reagents need to be stored at 4 °C |
| Image analysis software | open source | Cell profiler | works for analysis of single plane images |
| Image analysis software-license required | Bitplane | Imaris | Cell Biology module needed. Can quantify PLA dots/nuclei in image stacks (3D) and do 3D reconstructions |
| Ligase (1 unit/μl) | Sigma-Aldrich | DUO82029 | reagents need to be stored at -20 °C |
| Ligation reagent (5×) | Sigma-Aldrich | DUO82009 | reagents need to be stored at -20 °C |
| MCM2 antibody (rabbit) | Abcam | ab4461 | 1 in 200 |
| MCM5 antibody (rabbit monoclonal) | Abcam | Ab75975 | 1 in 1000 |
| Methanol | Lab ALLEY | A2076 | pre-cold at -20°C before use |
| phosphoMCM2S108 antibody (rabbit) | Abcam | ab109271 | 1 in 200 |
| Polymerase (10 unit/μl) | Sigma-Aldrich | DUO82030 | reagents need to be stored at -20 °C |
| Prolong gold mounting media with DAPI | ThermoFisher Scientific | P36935 | |
| PSF1 antibody (rabbit) | Abcam | ab181112 | 1 in 200 |
| RNAse A 100 mg/ml | Qiagen | 19101 | reagents need to be stored at 4 °C |
| Statistical analysis and data visualization software | open source | R studio | ggplot2 package for generation of dot plot and box plots |
| Statistical analysis and data visualization software-license required | Systat Software | Sigmaplot V13 | |
| TMP (trioxalen) | Sigma-Aldrich | T6137_1G | |
| TritonX-100 | Sigma-Aldrich | T8787_250ML | |
| Tween 20 | Sigma-Aldrich | P9416_100ML | |
| UV box | Southern New England Ultraviolet | Discontinued. See Opsytec UV test chamber as a possible replacement | |
| UV test Chamber | Opsytec | UV TEST CHAMBER BS-04 | |
| VE-821 | Selleckchem | S8007 | final concentrtion is 1µM |
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Cite This Article
Zhang, J., Huang, J., Majumdar, I., James, R. C., Gichimu, J., Paramasivam, M., Pokharel, D., Gali, H., Bellani, M. A., Seidman, M. M. Visualization of Replisome Encounters with an Antigen Tagged Blocking Lesion. J. Vis. Exp. (173), e61689, doi:10.3791/61689 (2021).