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Biochimie

尿嘧啶-DNA糖基化酶测定，采用基质辅助激光解吸/电离飞行时间质谱分析

Published: April 22, 2022
doi:
10.3791/63089
, , , , , , ,
1Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine,National Taiwan University, 2Department of Laboratory Medicine,National Taiwan University Hospital, 3Center for Microbial Pathogenesis, Nationwide Children’s Hospital and the Department of Pediatrics,The Ohio State University

Summary

使用MALDI-TOF质谱法开发了一种非标记，非放射性同位素方法来测定尿嘧啶-DNA糖基化酶活性，用于直接分析含阳离子/嘧啶位点的产品。该测定被证明是非常简单，特异性，快速且易于使用的DNA糖基化酶测量。

Abstract

尿嘧啶-DNA糖基化酶（UDG）是碱基切除修复途径中的关键成分，用于校正由胞嘧啶水解脱氨形成的尿嘧啶。因此，它对于基因组完整性的维持至关重要。开发了一种高度特异性，无标记，非放射性同位素的方法来测量UDG活性。UDG切割含有位点特异性尿嘧啶的合成DNA双链体，然后进行基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）分析。建立了一种方案来保存DNA中的apurinic/apyrimidinic位点（AP）产物而不会断裂。利用底物到产物的m/z值变化来评价UGG对尿嘧啶的水解作用。使用G：U底板进行UDG动力学分析，得到Km = 50 nM，Vmax = 0.98 nM /s，Kcat = 9.31 s-1。将该方法应用于尿嘧啶糖基化酶抑制剂（UGI）测定，IC50值为7.6 pM。在单链和双链DNA底物中不同位置使用尿嘧啶的UDG特异性显示出不同的切割效率。因此，这种简单，快速和多功能的MALDI-TOF MS方法可以成为各种单功能DNA糖基化酶的极好参考方法。它还具有作为DNA糖基化酶抑制剂筛选工具的潜力。

Introduction

虽然尿嘧啶是RNA中的正常碱基，但它是基因组DNA中常见且高度致突变的病变。尿嘧啶可由脱氧胞苷的自发/酶解脱氨引起。在每个活细胞中，这种脱氨在生理条件下每天发生100-500次1，2。如果这些改变没有得到修复，DNA序列组成可能会发生变化，从而导致突变。由于DNA中的尿嘧啶在复制过程中更喜欢与dATP配对，如果胞嘧啶脱氨为尿嘧啶，在两个复制事件中，在一半的后代DNA3中将出现新的G：C到A：T过渡突变。

在维持遗传稳定性的细胞策略中，碱基切除修复（BER）是修复DNA4中受损碱基（如尿嘧啶）的重要机制。BER是一个高度进化保守的过程。有两种常见的 BER 通路：短贴片通路，通向单个核苷酸的修复束，以及产生至少两个核苷酸修复束的长贴片通路5。BER 是一种协调机制，分几个步骤进行。BER的第一步是通过损伤特异性DNA糖基酶水解受损的核苷酸碱基，以产生阳极/嘧啶（AP）中间位点6。随后，通过核酸内切酶在AP位点切割糖磷酸主链，通过裂解酶清除DNA末端，通过DNA聚合酶填充间隙，并通过连接酶密封最终的缺口5。

尿嘧啶-DNA糖基化酶（UDG）从含有尿嘧啶的DNA中水解尿嘧啶，用于大肠杆菌中的BER。使用放射性标记DNA的传统UDG测定涉及不同的分离技术6，7，8，9，10，11，12，13通常耗时，劳动密集型，标记试剂昂贵，程序复杂，并且需要强化培训和实践以降低暴露于放射性物质的风险。除了分子信标和Förster共振能量转移技术15，16，17，18，19，20之外，荧光定量寡核苷酸测定已被开发出来作为放射性同位素标记的替代品14。但是，上述所有方法都需要特定的标签。最近开发了无标记生物传感器测定法21，22，23和基于G-四链体24，25，26形成的比色方法。然而，探针中的多个A：U对或专门设计的序列使酶单元定义复杂化。

MALDI-TOF MS是一种在DNA分析中可能非常有用的技术。开发的应用包括单核苷酸多态性基因分型27，28，修饰核苷酸分析29和DNA修复中间体鉴定30，31，32，33，34。应易于用于DNA糖基化酶分析的MALDI-TOF MS，以检测含AP位点的DNA产物。然而，在许多实验条件下，DNA中的AP位点容易断裂33。这里介绍了一种UDG测定，使用MALDI-TOF MS直接测量AP位点的产生，没有明显的断链噪声。这种无标记方法易于使用，并且在DNA糖基化酶抑制剂筛选的药物应用中具有很高的潜力。

Protocol

1. 基材/模板准备 双相区域设计 G+C 含量平衡至 50 ± 10% 且最低熔化温度为 50 °C 的 uracil 基板/模板双相。注：18 nt底物和19 nt模板之间的一个核苷酸差异（表1 和 图1）有助于更好的MS信号解释和适当的退火。模板链可作为互补DNA以产生A-U或G-U不匹配（表1），但也可以用作MS测量中的参考信号。HPLC纯化的合成寡核苷酸的使用对本?…

Representative Results

模板和基材以以U为中心（U+9）的合成寡核苷酸与G模板配对为例（图1A），对模板和含尿嘧啶的底物的等摩尔量的空白控制可用于合成寡核苷酸纯度的质量控制（图1B;信号与指定的m/ z和低背景噪声相匹配）。对于MS数据分析，测量了峰高（图2）。预计19 nt模板DNA在糖基化酶水解后保持不变;因此，该信号可以作为AP产…

Discussion

本文提供了使用UDG MALDI-TOF MS测定方法直接检测含AP的DNA产物的详细程序。该方法的主要优点是含尿嘧啶的底物无需标记，可扩展，易于使用，并在基底设计中提供更大的灵活性。

UDG供应商推荐的苯酚/氯仿提取能够使酶失活，以防止产品DNA降解。然而，苯酚提取方案涉及危险化学品的繁琐相分离。另一种酸终止方法使用HCl将反应pH降低到2±0.5也有效地灭活了UDG。随后用Tris碱基?…

Divulgations

The authors have nothing to disclose.

Acknowledgements

我们感谢NCFPB功能基因组学综合核心设施（台湾台北）和NRPB药物基因组学实验室（台湾台北）的技术支持。这项工作得到了台湾科学技术部的支持，R.O.C[批准号MOST109-2314-B-002 -186至K.-Y.S.，MOST 107-2320-B-002-016-MY3至S.-Y.C，MOST 110-2320-B-002-043至W.-h.F.]。H.-L.C.是国立台湾大学博士奖学金的获得者。开放获取收费资金：科学技术部，R.O.C。

Materials

2-Amino-2-hydroxymethyl-propane-1,3-diol (Tris base) J.T baker Protocol 1,2
Autoclaved deionized water MILLIPORE Protocol 1,2
EDTA J.T Baker Protocol 1,2
Gloves AQUAGLOVE Protocol 1,2,3
Hydrochloric acid (HCl) SIGMA Protocol 1,2
Ice bucket Taiwan.Inc Protocol 2
Low retention pipette tips(0.5-10 µL) extra gene Protocol 1,2
Low retention pipette tips(1,250 µL) national scientific supply co, Inc. Protocol 1,2
Low retention pipette tips(200 µL) national scientific supply co, Inc. Protocol 1,2
MassARRAY  Agena Bioscience, CA Protocol 4, 5
Mass spectrometry control programs include Typer Chip Linker, SpectroACQUIRE, and Start RT Process.
MassARRAY Nanodispenser AAT Bioquest, Inc. RS1000 Protocol 3
Microcentrifuge Kubota Protocol 2
Microcentrifuge Clubio Protocol 2
Microcentrifuge tube (1.5 mL) National scientific supply co, Inc. Protocol 2
Microcentrifuge tube rack Taiwan.Inc Protocol 1,2
Micropipette  (P1000) Gilson Protocol 1,2
Micropipette  (P2) Gilson Protocol 1,2
Micropipette (P10) Gilson Protocol 1,2
Micropipette (P100) Gilson Protocol 1,2
Micropipette (P200) Gilson Protocol 1,2
Micropipette (SL2) Rainin Protocol 1,2
Oligonucleotides Integrated DNA Technologies (Singapore) Protocol 1,2
Quest Graph IC50 Calculator (v.1) AAT Bioquest, Inc. Fig. 4
https://www.aatbio.com/tools/ic50-calculator-v1
Sodium hydroxide (NaOH) WAKO Protocol 2
SpectroCHIP array  Agena Bioscience, CA #01509 Protocol 3, 5
Timer Taiwan.Inc Protocol 2
Typer 4.0 software  Agena Bioscience, CA #10145 Protocol 6
Typer 4.0 consists four programs including Assay Designer, Assay Editor, Plate Editor, and Typer Analyzer.
UDG Reaction Buffer (10x) New England Biolabs, MA B0280S Protocol 2
Uracil Glycosylase Inhibitor New England Biolabs, MA M0281S Protocol 2
Uracil-DNA Glycosylase New England Biolabs, MA M0280L Protocol 2
UV-VISBLE spectrophotometer UV-1601 SHIMADZU Protocol 1
Water bath ZETA ZC-4000 (Taiwan.Inc) Protocol 2
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Tags

Keywords: Uracil-DNA GlycosylaseMALDI-TOF Mass SpectrometryDNA RepairAP ProductLabel-freeEnzyme Activity AssayGuanine-uracil BaseReaction BufferSubstrate PreparationReaction Termination
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Chang, H., Su, K., Goodman, S. D., Cheng, W., Lin, L., Yang, Y., Chang, S., Fang, W. Uracil-DNA Glycosylase Assay by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry Analysis. J. Vis. Exp. (182), e63089, doi:10.3791/63089 (2022).
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