搜索

In vitro Assay to Measure DNA Polymerase β Nucleotide Insertion Coupled with the DNA Ligation Reaction during Base Excision Repair
体外实验检测在碱基去除修复中聚合酶β联合DNA连接反应介导的核苷酸插入    

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

We previously reported that oxidized nucleotide insertion by DNA polymerase β (pol β) can confound the DNA ligation step during base excision repair (BER) (Çağlayan et al., 2017). Here, we describe a method to investigate pol β nucleotide insertion coupled with DNA ligation, in the same reaction mixture including dGTP or 8-oxo-dGTP, pol β and DNA ligase I. This in vitro assay enables us to measure the products for correct vs. oxidized nucleotide insertion, DNA ligation, and ligation failure, i.e., abortive ligation products, as a function of reaction time. This protocol complements our previous publication and describes an efficient way to analyze activities of BER enzymes and the functional interaction between pol β and DNA ligase I in vitro.

Keywords: DNA base excision repair(DNA碱基切除修复), DNA polymerase β(DNA聚合酶β), DNA ligase(DNA连接酶), Oxidative stress(氧化应激), Oxidized nucleotides(氧化核苷酸)

Background

This protocol is to observe the last two steps of the BER pathway: nucleotide insertion by pol β and DNA ligation by ligase I. Using this protocol, one measures both reactions as a function of time of incubation in the same reaction mixture in vitro. The original entity was to analyze BER enzymes pol β and DNA ligase I on the single-nucleotide gapped DNA substrate that mimics a BER intermediate. These BER enzymes bind to and function on this BER intermediate. The DNA substrate used in this protocol includes a fluorescent label at both 5’- and 3’-ends that enables us to observe single-nucleotide insertion and DNA ligation of the DNA substrate. The pol β nucleotide insertion coupled with DNA ligation mimics the hand off or channeling of the nicked DNA intermediate from nucleotide insertion step to following ligation step during BER pathway. Using this protocol, one is also able to measure ligation failure, or abortive ligation, after pol β oxidized nucleotide (8-oxo-dGTP) insertion. This is achieved by quantification of the addition of an adenylate (AMP) group at 5’-end of the BER intermediate. This protocol can also be used to measure nucleotide insertion coupled with ligation using other DNA polymerases and DNA ligases.

Materials and Reagents

  1. Eppendorf tubes (1.5 ml)
  2. Pipette tips (10 μl, 100 μl, 1,000 μl)
  3. Deoxyguanosine triphosphate (dGTP) (New England Biolabs, catalog number: N0447S )
  4. 8-oxo-2’-deoxyguanosine-5’-Triphosphate (8-oxo-dGTP) (Jena Bioscience, catalog number: NU-1117L )
  5. DNA substrate: The DNA substrate includes a fluorescent tag at both the 5’- and 3’-ends of one of the gap-containing strand in the double-stranded DNA with a single nucleotide gap opposite template base Cytosine (Çağlayan et al., 2017)
    Note: The sequence information for the DNA substrate is presented in the Notes section of the protocol.
  6. Tris-HCl (pH 7.5)
  7. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
  8. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  9. Adenosine 5’-Triphosphate (ATP) (New England Biolabs, catalog number: P0756S )
  10. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
  11. Bovine serum albumin (BSA) (New England Biolabs, catalog number: B9000S )
  12. Glycerol (Sigma-Aldrich, catalog number: G9012 )
  13. HEPES (pH 7.5)
  14. Sodium chloride (NaCl)
  15. EDTA (Sigma-Aldrich, catalog number: 93283 )
  16. Purified enzymes: Recombinant human DNA polymerase β and DNA ligase I (see Recipes and Figure 2)
  17. Formamide (Sigma-Aldrich, catalog number: F9037 )
  18. Bromophenol blue (Sigma-Aldrich, catalog number: B0126 )
  19. Xylene cyanol (Sigma-Aldrich, catalog number: X4126 )
  20. Trizma-base (Sigma-Aldrich, catalog number: T4661 )
  21. Boric acid (Promega, catalog number: H5003 )
  22. Urea (National Diagnostic, catalog number: EC-605 )
  23. Ammonium persulfate (APS) (Sigma-Aldrich, catalog number: A3678-25G )
  24. Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281-25ML )
  25. Sterile water
  26. AccuGel (40%) 19:1 Acrylamide to Bisacrylamide Stabilized Solution (National Diagnostic, catalog number: EC-850 )
  27. Full-range rainbow protein ladder (AmershamTM ECLTM RainbowTM Marker) (GE Healthcare, catalog number: RPN800E )
  28. 1x reaction buffer (see Recipes)
  29. 1x protein storage and dilution buffer (see Recipes)
  30. Enzyme mixture (see Recipes)
  31. Gel-loading dye (see Recipes)
  32. 10x TBE solution (see Recipes)
  33. Denaturing PAGE solution (15%) (see Recipes)
  34. EDTA buffer (pH 8.0) (see Recipes)

Equipment

  1. Pipettes (P2, P10, P20, P100, P200)
  2. Table-top heat block (Digital Dry Block Heater, VWR)
  3. Polyacrylamide gel electrophoresis (PAGE) apparatus (Biometra, model: Model S2 )
  4. Typhoon Phosphor Imager (GE Healthcare, model: Typhoon FLA 9500 )

Procedure

  1. Prepare 10 μl of reaction mixture (final volume) including 1 μl of 10x reaction buffer (1x final concentration), 2 μl of 1 mM dGTP or 8-oxo-dGTP (200 μM final concentration), and 1 μl of 2.5 μM DNA substrate (250 nM final concentration). Each reaction tube contains 10 µl of the reaction mixture needed for each time point. (see Recipes)
  2. Preincubate the enzyme mixture including 1 μl of 1 μM pol β and DNA ligase I (100 nM final concentration) at 37 °C for 3 min. (see Recipes)
  3. Start the reaction by addition of the enzyme mixture, prepared as above, to the reaction mixture, and pipette up and down to mix the reaction components.
  4. Incubate the reaction mixture at 37 °C for time points of 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, and 15 min.
  5. Quench the reactions with 0.3 mM EDTA. (see Recipes)
  6. Mix the reaction products with an equal volume of gel loading dye.
  7. Heat the reaction samples at 95 °C for 3 min before loading onto the gel, and load 5 μl of reaction sample.
  8. Separate the reaction products in a 15% polyacrylamide gel containing 8 M urea at 30 mA for ~2 h.
  9. Scan the gel on a Typhoon Phosphor Imager as described (Çağlayan et al., 2014; 2015 and 2017).

Data analysis

A sample gel image that shows the products of pol β nucleotide insertion, DNA ligation by DNA ligase, and ligation failure is presented below (Figure 1). The original results were published in Çağlayan et al. (2017).


Figure 1. Time course-dependent changes in the products of nucleotide insertion and DNA ligation in a coupled BER assay in vitro. Lane 1 is a minus enzyme control, lanes 2-12 are the reaction products of dGTP insertion and ligation, and lanes 13-23 are the reaction products of 8-oxo-dGTP insertion, ligation and ligation failure. The reaction time points corresponding to lanes 2-12 and 13-23 are 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, and 15 min. The sizes of DNA substrate for insertion and ligation are 18- and 16-mer, respectively. The sizes of the reaction products for dGTP or 8-oxo-dGTP insertion and ligation are 19- and 35-mer, respectively. The size of the abortive ligation product is 16-mer plus an adenylate (AMP) group (~17-mer). 

Representative gel images (Figure 2) illustrate the purity of the human recombinant enzymes, DNA polymerase β (38 kDa) and DNA ligase I (101 kDa). The procedure for the purification of these enzymes was published previously (Beard and Wilson, 1995 and Chen Representative gel images (Figure 2) illustrate the purity of the human recombinant enzymes, DNA polymerase β (38 kDa) and DNA ligase I (101 kDa). The procedure for the purification of these enzymes was published previously (Beard and Wilson, 1995 and Chen et al., 2006). 


Figure 2. Photographs of SDS-PAGE analyses for the purified enzymes used, human recombinant DNA polymerase β (A) and human recombinant DNA ligase I (B). In both panels, lane 1 represents the marker protein ladder. Lanes 2-4 (in panel A) and lanes 2-6 (in panel B) are serial dilutions of pol β and ligase I, respectively.

Notes

  1. After preincubation of the enzyme mixture including pol β and DNA ligase I, the reaction should be immediately started by mixing this enzyme mixture into the reaction tube including 1x reaction buffer, dNTP, and DNA substrate. In addition, the enzymatic activities are determined with the substrate in excess over enzyme, under steady-state conditions.
  2. The oligonucleotides (Integrated DNA Technologies, IDT) below are used to construct a double-stranded DNA substrate, including a single-nucleotide gap in one strand (Çağlayan et al., 2017). FAM indicates a fluorescent label at 5’- or 3’-end of the upstream or downstream oligonucleotide.
    Upstream oligonucleotide: FAM-5’-AACATGGGCGGCATGAAAT
    Downstream oligonucleotide: AATGCCCATCCTCACCA-3’-FAM
    Template oligonucleotide: 3’-TTGTACCCGCCGTACTTTACTTACGGGTAGGAGTGGT-5’

Recipes

  1. 1x reaction buffer
    50 mM Tris-HCl (pH 7.5)
    100 mM KCl
    10 mM MgCl2
    1 mM ATP
    1 mM DTT
    100 μg/ml BSA
    10% glycerol
  2. 1x protein storage and dilution buffer
    50 mM HEPES (pH 7.5)
    100 mM NaCl
    1 mM EDTA
    0.1% BSA
    20% glycerol
  3. Enzyme mixture
    1 mg/ml DNA polymerase β
    0.5 mg/ml DNA ligase I
    Prepare 1 μM dilutions for each enzyme in the dilution buffer above
  4. Gel-loading dye
    95% formamide
    20 mM EDTA
    0.02% bromophenol blue
    0.02% xylene cyanol
  5. 10x TBE solution
    108 g Tris-base
    55 g boric acid
    40 ml 0.5 M EDTA
    dH2O up to 1 L
  6. Denaturing PAGE solution (15%)
    40 g urea
    8 ml 10% TBE solution
    10 ml dH2O
    250 μl 10% APS
    35 μl TEMED
  7. EDTA buffer (pH 8.0)
    10 mM Tris-HCl
    1 mM disodium EDTA

Acknowledgments

This work was supported by the Intramural Research Program of the US National Institutes of Health, National Institute of Environmental Health Sciences (grants Z01 ES050158 and ES050159).

References

  1. Beard, W. A. and Wilson, S. H. (1995). Purification and domain-mapping of mammalian DNA polymerase β. Methods Enzymol 262: 98-107.
  2. Çağlayan, M., Batra, V. K., Sassa, A., Prasad, R. and Wilson, S. H. (2014). Role of polymerase beta in complementing aprataxin deficiency during abasic-site base excision repair. Nat Struct Mol Biol 21(5): 497-499.
  3. Çağlayan, M., Horton, J. K., Dai, D. P., Stefanick, D. F. and Wilson, S. H. (2017). Oxidized nucleotide insertion by pol β confounds ligation during base excision repair. Nat Commun 8: 14045.
  4. Çağlayan, M., Horton, J. K., Prasad, R. and Wilson, S. H. (2015). Complementation of aprataxin deficiency by base excision repair enzymes. Nucleic Acids Res 43(4): 2271-2281.
  5. Chen, X., Pascal, J., Vijayakumar, S., Wilson, G. M., Ellenberger, T. and Tomkinson, A. E. (2006). Human DNA ligases I, III, and IV-purification and new specific assays for these enzymes. Methods Enzymol 409: 39-52.

简介

我们以前报告说,通过DNA聚合酶β(polβ)的氧化的核苷酸插入可能会在碱基切除修复(BER)(Çağlayan等,2017)期间混淆DNA连接步骤。 在这里,我们描述了在与dGTP或8-氧代-dGTP,polβ和DNA连接酶I相同的反应混合物中研究与DNA连接相结合的polβ核苷酸插入的方法。该体外测定使得我们能够测量产物的正确性 与氧化核苷酸插入,DNA连接和连接失败,即流产结扎产物,作为反应时间的函数。 该协议补充了我们以前的出版物,并描述了一种有效的方法来分析BER酶的活性和polβ和DNA连接酶I在体外的功能相互作用。
【背景】该方案是观察BER路径的最后两个步骤:通过连接酶I通过polβ进行核苷酸插入和DNA连接。使用该方案,在体外在同一反应混合物中测量两种反应作为时间的函数。原始实体是在模拟BER中间体的单核苷酸缺口DNA底物上分析BER酶polβ和DNA连接酶I。这些BER酶与此BER中间体结合并起作用。该方案中使用的DNA底物包括在5'和3'端的荧光标记,使我们能够观察DNA底物的单核苷酸插入和DNA连接。与DNA连接偶联的polβ核苷酸插入模拟了切口DNA中间体从核苷酸插入步骤切换或引导到BER途径期间的连接步骤。使用该方案,也可以在polβ氧化的核苷酸(8-氧代-dGTP)插入后测量连接失败或者终止连接。这通过在BER中间体的5'-末端添加腺苷酸(AMP)基团进行定量来实现。该方案也可用于测量与其他DNA聚合酶和DNA连接酶连接的核苷酸插入。

关键字:DNA碱基切除修复, DNA聚合酶β, DNA连接酶, 氧化应激, 氧化核苷酸

材料和试剂

  1. Eppendorf管(1.5毫升)
  2. 移液头(10μl,100μl,1,000μl)
  3. 脱氧鸟苷三磷酸(dGTP)(New England Biolabs,目录号:N0447S)
  4. 8-氧代-2'-脱氧鸟苷-5'-三磷酸(8-氧代-dGTP)(Jena Bioscience,目录号:NU-1117L)
  5. DNA底物:DNA底物包括双链DNA中含间隙链中的一个的5'和3'端的荧光标签,其中单核苷酸间隙与模板碱基Cytosine(Çağlayanet al。 al ,,2017)
    注意:DNA底物的序列信息显示在协议的Notes部分。
  6. Tris-HCl(pH 7.5)
  7. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333)
  8. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  9. 腺苷5'-三磷酸(ATP)(New England Biolabs,目录号:P0756S)
  10. 二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:D0632)
  11. 牛血清白蛋白(BSA)(New England Biolabs,目录号:B9000S)
  12. 甘油(Sigma-Aldrich,目录号:G9012)
  13. HEPES(pH 7.5)
  14. 氯化钠(NaCl)
  15. EDTA(Sigma-Aldrich,目录号:93283)
  16. 纯化酶:重组人类DNA聚合酶β和DNA连接酶I(见配方和图2)
  17. 甲酰胺(Sigma-Aldrich,目录号:F9037)
  18. 溴苯酚蓝(Sigma-Aldrich,目录号:B0126)
  19. 二甲苯胞苷(Sigma-Aldrich,目录号:X4126)
  20. Trizma-base(Sigma-Aldrich,目录号:T4661)
  21. 硼酸(Promega,目录号:H5003)
  22. 尿素(国家诊断,目录号:EC-605)
  23. 过硫酸铵(APS)(Sigma-Aldrich,目录号:A3678-25G)
  24. 四甲基乙二胺(TEMED)(Sigma-Aldrich,目录号:T9281-25ML)
  25. 无菌水
  26. AccuGel(40%)19:1丙烯酰胺至双丙烯酰胺稳定溶液(国家诊断,目录号:EC-850)
  27. 全频彩虹蛋白梯(Amersham TM ECL TM Rainbow TM Marker)(GE Healthcare,目录号:RPN800E)
  28. 1x反应缓冲液(参见食谱)
  29. 1x蛋白质储存和稀释缓冲液(参见食谱)
  30. 酶混合物(参见食谱)
  31. 凝胶加载染料(见配方)
  32. 10x TBE溶液(参见食谱)
  33. 变性PAGE溶液(15%)(参见食谱)
  34. EDTA缓冲液(pH 8.0)(参见食谱)

设备

  1. 移液器(P2,P10,P20,P100,P200)
  2. 台式加热块(数字干式加热器,VWR)
  3. 聚丙烯酰胺凝胶电泳(PAGE)装置(Biometra,型号:S2型)
  4. 台风荧光成像仪(GE Healthcare,型号:台风FLA 9500)

程序

  1. 制备10μl含有10μl反应缓冲液(1x终浓度),2μl1mM dGTP或8-oxo-dGTP(200μM终浓度)的10μl反应混合物(终体积)和1μl2.5μMDNA底物(250nM终浓度)。每个反应管含有10μl每个时间点所需的反应混合物。 (见配方)
  2. 将包含1μl1μMPolβ和DNA连接酶I(100nM终浓度)的酶混合物在37℃预孵育3分钟。 (见配方)
  3. 通过向反应混合物中加入如上制备的酶混合物开始反应,上下移液以混合反应组分。
  4. 将反应混合物在37℃下孵育0.5分钟,1,2,3,4,5,6,8,10,12和15分钟的时间。
  5. 用0.3mM EDTA淬灭反应。 (见配方)
  6. 将反应产物与等体积的凝胶负载染料混合
  7. 将反应样品在95℃加热3分钟,然后加载到凝胶上,并加载5μl反应样品
  8. 将含有8M尿素的15%聚丙烯酰胺凝胶中的反应产物在30mA下分离〜2h
  9. 按照(Çağlayan等人,2014,2015和2017)所述扫描台风荧光成像仪上的凝胶。

数据分析

显示polβ核苷酸插入的产物,DNA连接酶的DNA连接和连接失败的样品凝胶图像如下(图1)。原始结果发表在Çağlayan等人。(2017)。


图1.在体外偶联BER测定中核苷酸插入和DNA连接产物的时间依赖性变化。泳道1是负酶对照,泳道2- 12是dGTP插入和连接的反应产物,泳道13-23是8-氧代-dGTP插入,连接和连接失败的反应产物。对应于泳道2-12和13-23的反应时间点为0.5,1,2,3,4,5,6,8,10,12和15分钟。用于插入和连接的DNA底物的大小分别为18和16聚体。 dGTP或8-氧代-dGTP插入和连接的反应产物的大小分别为19-和35-聚体。流产结扎产品的大小是16-mer加上腺苷酸(AMP)组(〜17聚体)。 

代表性的凝胶图像(图2)说明了人重组酶,DNA聚合酶β(38kDa)和DNA连接酶I(101kDa)的纯度。以前公布了这些酶的纯化方法(Beard和Wilson,1995和Chen代表性的凝胶图像(图2)说明了人重组酶,DNA聚合酶β(38kDa)和DNA连接酶I(101kDa)的纯度,以前已经公开了这些酶的纯化方法(Beard和Wilson,1995和Chen等人,2006)。 


图2.所用纯化酶的SDS-PAGE分析的照片,人重组DNA聚合酶β(A)和人重组DNA连接酶I(B)。 在两个面板中,泳道1代表标记蛋白梯。泳道2-4(在图A中)和泳道2-6(在图B中)分别是polβ和连接酶I的连续稀释。

笔记

  1. 在包含polβ和DNA连接酶I的酶混合物预温育后,应立即开始将该酶混合物混合到包含1x反应缓冲液,dNTP和DNA底物的反应管中。此外,在稳定状态条件下,酶活性用酶超过酶底物测定。
  2. 以下寡核苷酸(Integrated DNA Technologies,IDT)用于构建双链DNA底物,包括一条链中的单核苷酸间隙(Çağlayan等人,2017)。 FAM表示上游或下游寡核苷酸的5'-或3'-末端的荧光标记 上游寡核苷酸:FAM-5'-AACATGGGCGGCATGAAAT
    下游寡核苷酸:AATGCCCATCCTCACCA-3'-FAM
    模板寡核苷酸:3'-TTGTACCCGCCGTACTTTACTTACGGGTAGGAGTGGT-5'

食谱

  1. 1x反应缓冲液
    50mM Tris-HCl(pH7.5)
    100 mM KCl
    10mM MgCl 2
    1 mM ATP
    1 mM DTT
    100μg/ml BSA
    10%甘油
  2. 1x蛋白质储存和稀释缓冲液
    50 mM HEPES(pH 7.5)
    100 mM NaCl
    1 mM EDTA
    0.1%BSA
    20%甘油
  3. 酶混合物
    1毫克/毫升DNA聚合酶β
    0.5mg/ml DNA连接酶I
    为上述稀释缓冲液中的每种酶制备1μM稀释液
  4. 凝胶加载染料
    95%甲酰胺
    20 mM EDTA
    0.02%溴酚蓝
    0.02%二甲苯氰胺
  5. 10倍TBE解决方案
    108克Tris-base
    55克硼酸
    40 ml 0.5 M EDTA dH <2> O至1 L
  6. 变性PAGE溶液(15%)
    40 g尿素
    8 ml 10%TBE溶液
    10ml dH 2 O O
    250μl10%APS
    35μlTEMED
  7. EDTA缓冲液(pH 8.0)
    10 mM Tris-HCl
    1mM EDTA二钠盐

致谢

这项工作得到了美国国家卫生研究院国家卫生科学研究所校内研究计划的支持(授予Z01 ES050158和ES050159)。

参考

  1. Beard,WA and Wilson,SH(1995)。  净化和哺乳动物DNA聚合酶β的结构域映射。方法Enzymol 262:98-107。
  2. Çağlayan,M.,Batra,VK,Sassa,A.,Prasad,R。和Wilson,SH(2014)。  在基因位点碱基切除修复期间,聚合酶β在补充aprataxin缺陷中的作用。 Nat Struct Mol Biol 21(5): 497-499。
  3. Çağlayan,M.,Horton,JK,Dai,DP,Stefanick,DF and Wilson,SH(2017)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih。 gov/pubmed/28067232"target ="_ blank">通过polβ的氧化核苷酸插入在基底切除修复期间混淆结扎。 8:14045.
  4. Çağlayan,M.,Horton,JK,Prasad,R。和Wilson,SH(2015)。  通过碱基切除修复酶互补aprataxin缺陷。 核酸Res 43(4):2271-2281。
  5. Chen,X.,Pascal,J.,Vijayakumar,S.,Wilson,GM,Ellenberger,T.and Tomkinson,AE(2006)。< a class ="ke-insertfile"href ="http: .ncbi.nlm.nih.gov/pubmed/16793394"target ="_ blank">人类DNA连接酶I,III和IV纯化以及这些酶的新特异性检测。方法Enzymol 409:39-52。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Çağlayan, M. and Wilson, S. H. (2017). In vitro Assay to Measure DNA Polymerase β Nucleotide Insertion Coupled with the DNA Ligation Reaction during Base Excision Repair. Bio-protocol 7(12): e2341. DOI: 10.21769/BioProtoc.2341.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。