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Enzymatic Activity Assays for Base Excision Repair Enzymes in Cell Extracts from Vertebrate Cells
脊椎动物细胞提取物的碱基切除修复酶的酶活性检测

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Abstract

We previously reported enzymatic activity assays for the base excision repair (BER) enzymes DNA polymerase β (pol β), aprataxin (APTX), and flap endonuclease 1 (FEN1) in cell extracts from Saccharomyces cerevisiae (Çağlayan and Wilson, 2014). Here, we describe a method to prepare cell extracts from vertebrate cells to investigate these enzymatic activities for the processing of the 5´-adenylated-sugar phosphate-containing BER intermediate. This new protocol complements our previous publication. The cell lines used are wild-type and APTX-deficient human lymphoblast cells from an Ataxia with Oculomotor Apraxia Type 1 (AOA1) disease patient, wild-type and APTX-null DT40 chicken B cells, and mouse embryonic fibroblast (MEF) cells. This protocol is a quick and efficient way to make vertebrate cell extracts without using commercial kits.

Materials and Reagents

  1. Cell lines used in this study
    1. The human cell lines used are the wild-type C2ABR and AOA1 L938 (Harris et al., 2009). The AOA1 cell line was derived from the peripheral blood of a Japanese AOA1 patient and has a point mutation within the HIT domain of APTX involving substitution of proline for leucine at position 206.
    2. The DT40 cell lines are the wild-type and APTX null. The APTX null cell line has the aptx gene deletion from valine 78 onwards that inactivates APTX (Ahel et al., 2006).
    3. The mouse embryonic fibroblast cell lines are pol β-/- and pol β+/+ MEFs previously developed in our laboratory (Sobol et al., 1996).
  2. RPMI 1640 medium with glutamine (Gibco, catalog number: 11875-093 )
  3. DMEM high-glucose medium (HyClone, catalog number: SH30081 )
  4. Chicken serum (Life Technologies, catalog number 16110-082 )
  5. Glutamax-1 (Gibco, catalog number: 35050-061 )
  6. Fetal bovine serum-FBS (HyClone, catalog number: SH30910 ).
  7. EDTA-free protease inhibitor cocktail tablet (Roche Applied Science, catalog number: 11836170001 )
  8. Bio-Rad Protein Dye Reagent Concentrate (Bio-Rad Laboratories, catalog number: 500-0006 )
  9. EDTA (Sigma-Aldrich, catalog number: 93283 )
  10. Potassium chloride-KCl (Sigma-Aldrich, catalog number: P9333 )
  11. Sodium chloride-NaCl (Sigma-Aldrich, catalog number: S7653 )
  12. Glycerol (Sigma-Aldrich, catalog number: G9012 )
  13. Tissue-culture grade 2-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
  14. Nonidet P 40-NP40 (Sigma-Aldrich, catalog number: 74385 )
  15. HEPES (Sigma-Aldrich, catalog number: H3375 )
  16. Magnesium chloride-MgCl2 (Sigma-Aldrich, catalog number: M8266 )
  17. Formamide (Sigma-Aldrich, catalog number: F9037 )
  18. Bromophenol blue (Sigma-Aldrich, catalog number: B0126 )
  19. Xylene cyanol (Sigma-Aldrich, catalog number: X4126 )
  20. Dithiothreitol-DTT (Sigma-Aldrich, catalog number: D0632 )
  21. Sodium borohydride-NaBH4 (Sigma-Aldrich, catalog number: 247677 )
  22. Urea (National Diagnostic, catalog number: EC-605 )
  23. Trizma-base (Sigma-Aldrich, catalog number: T4661 )
  24. Boric Acid (Promega Corporation, catalog number: H5003 )
  25. AccuGel (40%) 19:1 Acrylamide to Bisacrylamide Stabilized Solution (National Diagnostic, catalog number: EC-850 )
  26. Ammonium persulfate (Sigma-Aldrich, catalog number: A3678-25G )
  27. Tetramethylethylenediamine (Sigma-Aldrich, catalog number: T9281-25ML )
  28. Sterile water
  29. Purified enzymes: Recombinant human DNA polymerase β [purified as described Çağlayan et al. (2014)], recombinant human APTX (Fitzgerald catalog number: 80R-1256 ), and recombinant human FEN1 [purified as described Çağlayan et al. (2014)].
  30. DNA substrate: The gapped DNA substrate with a uracil base at position 17 at the 5'-end of the 3'-end FAM-labeled oligonucleotides. The sequence information for the upstream, downstream and template oligonucleotides were previously published Çağlayan et al. (2015).
  31. Lysis buffer (see Recipes)
  32. 10x reaction buffer (see Recipes)
  33. Gel-loading buffer (see Recipes)
  34. 10x TBE solution and 1x TBE solution as PAGE running buffer (see Recipes)
  35. 15% Denaturing Polyacrlamide Gel or PAGE solution (see Recipes)

Equipment

  1. Eppendorf tubes
  2. Screw cap conical tube (15 ml)
  3. Refrigerated table-top centrifuge
  4. Refrigerated Eppendorf centrifuge
  5. Table-top heat block
  6. Tissue culture CO2 incubators set at 34, 37, and 39.5 °C
  7. Cell scraper
  8. Polyacrylamide gel electrophoresis (PAGE) apparatus

Procedure

  1. Cell growth
    1. DT40 cells (wild-type and APTX-null) are maintained in RPMI 1640 medium supplemented with 10% FBS, 1% chicken serum and 2 mM L-glutamine. 2-mercaptoethanol (50 μM) is added fresh to the medium at the time of use and cells are grown in a 5% CO2 incubator at 39.5°C (Okamoto et al., 2014).
    2. The human lymphoblastoid cells (wild-type and APTX-deficient AOA1) are maintained in RPMI 1640 medium containing FBS and grown in a 5% CO2 incubator at 37°C (Harris et al., 2009).
    3. MEF cells (wild-type and pol β-deficient) are maintained in DMEM medium containing 10% FBS and 4 mM glutamax and grown in a 10% CO2 incubator at 34°C (Sobol et al., 1996).
    4. 1-2 x 150 mm dishes of MEF cells are washed twice in 10 ml cold PBS, then harvested by scraping. Cells are collected in 15 ml PBS in a 15 ml tube, then centrifuged at 1600 rpm at 4 °C for 5 min.
    5. The required volume of suspension cells (DT40 and human lymphoblasts) is centrifuged at 1600 rpm at 4 °C for 5 min, and the cell pellet are transferred to a 15 ml tube and washed twice with 10 ml PBS.
    6. Cell pellets are transferred to Eppendorf tubes in 1 ml PBS and briefly spun down. Supernatants are discarded and pellets are frozen immediately in dry ice and stored at -80 °C until use.
    7. Pellets of 20-50 million cells are used for cell extract preparation.

  2. Preparation of cell extracts
    1. The cell extracts from vertebrate cells are prepared as reported (Biade et al., 1998) and summarized below.
    2. Resuspend the cell pellet in 400 μl of ice-cold lysis buffer.
    3. Rotate the resuspended cell pellets for 1 h at 4 °C.
    4. Centrifuge the mixture at 14,000 rpm at 4 °C for 10 min to remove cell debris.
    5. Carefully transfer the supernatant fraction to a fresh Eppendorf tube. Be careful not to touch the pellet.
    6. Determine the protein concentration of the extract using Bradford assay dye reagent with BSA as standard (Bradford, 1976).

  3. Enzymatic activity assays in cell extracts
    1. Prepare 10 μl of reaction mixture (final volume) including 1x reaction buffer and 100 nM DNA substrate. The DNA substrate used includes an adenylated uracil base at the 5′ end of the 3′-FAM–labeled oligonucleotide (Çağlayan et al., 2014). For the reference reactions including purified proteins, pol β, APTX, and FEN1, the reaction mixture included a gapped DNA substrate that was pre-incubated with UDG as described (Çağlayan et al., 2014). For the reactions including cell extracts, the DNA substrate was included in the reaction mixture without UDG pretreatment.
    2. Start the reaction by adding cell extract, prepared as above, to the reaction mixture. For the reference reactions, start each reaction by adding the purified protein to the reaction mixture in final concentrations as follows: pol β (500 nM), APTX (100 nM), and FEN1 (100 nM) (Çağlayan et al., 2014 and Çağlayan et al., 2015).
    3. Incubate the reaction mixture at 37 °C for 15 min.
    4. Stabilize the reaction products by addition of 1 M freshly prepared and ice-cold NaBH4 to a final concentration of 100 mM.
    5. Incubate the reaction samples on ice for 30 min.
    6. Mix the reaction products with 10 μl of gel-loading dye.
    7. The reaction products in cell extracts from vertebrate cells are separated on a 15% polyacrylamide gel. The gel is scanned, and the data analyzed as reported (Çağlayan et al., 2014 and Çağlayan et al., 2015).

Representative data

A sample gel image of products for the pol β dRP lyase, FEN1 excision and APTX DNA deadenylation enzymatic activities in the extracts from DT40 cells is presented below (Figure 1). Migration positions of DNA substrate (line 1) and the reaction products after pol β lyase removal of the 5’-AMP-dRP group (indicated as 17-mer and shown at line 2 in reference reaction and lines 5-6 in cell extracts), APTX 5’-AMP removal (indicated as 17dRP, and shown at line 3 in reference reaction and lines 6 in cell extract), and FEN1 excision products (indicated as 15-mer and 16-mer and shown at line 4 in reference reaction and lines 5-6 in cell extracts) are presented. The original results showing enzymatic activities in the cell extracts from the vertebrate cell lines listed below were published in Çağlayan et al. (2015).


Figure 1.Pol β, APTX, and FEN1 activities in cell extracts from DT40 wild-type and APTX-null cell lines

Notes

After addition of lysis buffer to the cell pellet, the Eppendorf tube including resuspended cells should be gently mixed by tapping. Enzymatic activity in vertebrate cell extracts may be lost if samples are mixed by vortex instead of rotating at 4 °C. Typically, the enzymatic activities are determined under steady-state conditions with the substrate in excess over enzyme and under conditions of a linear time-course. In addition, the enzymatic activity should be under conditions of linearity between activity and amount of extract added to the reaction mixture.

Recipes

  1. Lysis buffer
    10 mM Tris–HCl (pH 7.8 in the final buffer solution)
    200 mM KCl
    1 mM EDTA
    20% glycerol
    0.1% NP-40
    1 mM DTT (add fresh)
    Protease inhibitor tablet
  2. 10x reaction buffer
    500 mM HEPES (pH 7.5 in the final buffer solution)
    100 mM MgCl2
    200 mM KCl
    5 mM EDTA
    20 mM DTT
  3. Gel-loading buffer
    95% formamide
    20 mM EDTA
    0.02% bromophenol blue
    0.02% xylene cyanol
  4. 10x TBE solution
    108 g Tris-base
    55 g Boric Acid
    40 ml 0.5 M EDTA
    H20 up to 1 L
  5. Denaturing PAGE solution (15%)
    40 g Urea
    30 ml 19:1 AccuGel (40%)
    8 ml 10% TBE solution
    10 ml H20
    250 μl 10% APS
    35 μl TEMED

Acknowledgments

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

References

  1. Ahel, I., Rass, U., El-Khamisy, S. F., Katyal, S., Clements, P. M., McKinnon, P. J., Caldecott, K. W. and West, S. C. (2006). The neurodegenerative disease protein aprataxin resolves abortive DNA ligation intermediates. Nature 443(7112): 713-716.
  2. Biade, S., Sobol, R. W., Wilson, S. H. and Matsumoto, Y. (1998). Impairment of proliferating cell nuclear antigen-dependent apurinic/apyrimidinic site repair on linear DNA. J Biol Chem 273(2): 898-902.
  3. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
  4. Çağlayan, M. and Wilson, S. H. (2014). Enzymatic activity assays in yeast cell extracts. Bio-protocol 4(23): e1312.
  5. Caglayan, 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.
  6. Caglayan, 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.
  7. Harris, J. L., Jakob, B., Taucher-Scholz, G., Dianov, G. L., Becherel, O. J. and Lavin, M. F. (2009). Aprataxin, poly-ADP ribose polymerase 1 (PARP-1) and apurinic endonuclease 1 (APE1) function together to protect the genome against oxidative damage. Hum Mol Genet 18(21): 4102-4117.
  8. Okamoto, S., Narita, T., Sasanuma, H., Takeda, S., Masunaga, S., Bessho, T. and Tano, K. (2014). Impact of DNA repair pathways on the cytotoxicity of piperlongumine in chicken DT40 cell-lines. Genes Cancer 5(7-8): 285-292.
  9. Sobol, R. W., Horton, J. K., Kuhn, R., Gu, H., Singhal, R. K., Prasad, R., Rajewsky, K. and Wilson, S. H. (1996). Requirement of mammalian DNA polymerase-beta in base-excision repair. Nature 379(6561): 183-186.

简介

我们先前报道了来自酿酒酵母(Saccharomyces cerevisiae)的细胞提取物中的碱基切除修复(BER)酶DNA聚合酶β(polβ),aprataxin(APTX)和瓣膜内切核酸酶1(FEN1)的酶活性测定 和Wilson,2014)。 在这里,我们描述了一种从脊椎动物细胞中制备细胞提取物以研究这些酶活性用于加工含5'-腺苷酸化糖 - 磷酸酯的BER中间体的方法。 这个新协议补充了我们以前的出版物。 所使用的细胞系是来自具有Oclomotor Apraxia 1型(AOA1)疾病患者,野生型和APTX无效DT40鸡B细胞和小鼠胚胎成纤维细胞(MEF)细胞的共济失调的野生型和APTX缺陷型人淋巴母细胞。 这个协议是一个快速和有效的方式来制作脊椎动物细胞提取没有使用商业套件。

材料和试剂

  1. 本研究中使用的细胞系
    1. 所用的人细胞系是野生型C2ABR和AOA1 L938(Harris等人,   2009)。 AOA1细胞系衍生自a的外周血 日本AOA1患者,并且在HIT结构域内具有点突变 APTX,其涉及在位置206处用脯氨酸取代亮氨酸。
    2. DT40细胞系是野生型和APTX无效。 APTX无效细胞系具有从缬氨酸78起的 aptx 基因缺失,其使APTX失活(Ahel等人,2006)。
    3. 小鼠胚胎成纤维细胞细胞系是先前在我们的实验室中开发的polβsup-和polβsup/+/+ MEFs(Sobol等人, ,1996)。
  2. 具有谷氨酰胺的RPMI 1640培养基(Gibco,目录号:11875-093)
  3. DMEM高葡萄糖培养基(HyClone,目录号:SH30081)
  4. 鸡血清(Life Technologies,目录号16110-082)
  5. Glutamax-1(Gibco,目录号:35050-061)
  6. 胎牛血清-FBS(HyClone,目录号:SH30910)
  7. 无EDTA蛋白酶抑制剂混合物片剂(Roche Applied Science,目录号:11836170001)
  8. Bio-Rad Protein Dye Reagent Concentrate(Bio-Rad Laboratories,目录号:500-0006)
  9. EDTA(Sigma-Aldrich,目录号:93283)
  10. 氯化钾-KCl(Sigma-Aldrich,目录号:P9333)
  11. 氯化钠 - NaCl(Sigma-Aldrich,目录号:S7653)
  12. 甘油(Sigma-Aldrich,目录号:G9012)
  13. 组织培养级2-巯基乙醇(Sigma-Aldrich,目录号:M3148)
  14. Nonidet P 40-NP40(Sigma-Aldrich,目录号:74385)
  15. HEPES(Sigma-Aldrich,目录号:H3375)
  16. 氯化镁-MgCl 2(Sigma-Aldrich,目录号:M8266)
  17. 甲酰胺(Sigma-Aldrich,目录号:F9037)
  18. 溴酚蓝(Sigma-Aldrich,目录号:B0126)
  19. 二甲苯Cyanol(Sigma-Aldrich,目录号:X4126)
  20. 二硫苏糖醇-DTT(Sigma-Aldrich,目录号:D0632)
  21. 硼氢化钠-NaBH 4(Sigma-Aldrich,目录号:247677)
  22. 尿素(国家诊断,目录号:EC-605)
  23. Trizma碱(Sigma-Aldrich,目录号:T4661)
  24. 硼酸(Promega Corporation,目录号:H5003)
  25. 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)
  26. 过硫酸铵(Sigma-Aldrich,目录号:A3678-25G)
  27. 四甲基乙二胺(Sigma-Aldrich,目录号:T9281-25ML)
  28. 无菌水
  29. 纯化的酶:重组人DNA聚合酶β[如描述于(2014)],重组人APTX(Fitzgerald目录号:80R-1256)和重组人FEN1 [纯化如Çağlayan et al。(2014)]。
  30. DNA底物:在3'-末端FAM-标记的寡核苷酸的5'末端的位置17处具有尿嘧啶碱基的缺口DNA底物。 上游,下游和模板寡核苷酸的序列信息先前公开了(2015)。(2015)。
  31. 裂解缓冲液(见配方)
  32. 10x反应缓冲液(参见配方)
  33. 凝胶加载缓冲液(参见配方)
  34. 10x TBE溶液和1x TBE溶液作为PAGE运行缓冲液(参见配方)
  35. 15%变性聚丙烯酰胺凝胶或PAGE溶液(见配方)

设备

  1. Eppendorf管
  2. 螺旋盖圆锥管(15ml)
  3. 冷冻式台式离心机
  4. 冷冻离心机
  5. 桌面加热块
  6. 组织培养CO 2培养箱设置在34℃,37℃和39.5℃
  7. 细胞刮刀
  8. 聚丙烯酰胺凝胶电泳(PAGE)装置

程序

  1. 细胞生长
    1. 将DT40细胞(野生型和APTX无效)保持在RPMI 1640培养基中 补充有10%FBS,1%鸡血清和2mM L-谷氨酰胺。 在该时间将2-巯基乙醇(50μM)新鲜加入到培养基中 并且细胞在39.5℃下在5%CO 2培养箱中生长(Okamoto等人,   2014)。
    2. 人类淋巴母细胞(野生型和 APTX缺陷型AOA1)保持在含有FBS的RPMI 1640培养基中 并在37℃下在5%CO 2培养箱中生长(Harris等人,2009)。
    3. MEF细胞(野生型和polβ缺陷型)保持在DMEM培养基中 含有10%FBS和4mM glutamax,并在10%CO 2培养箱中生长   34℃(Sobol等人,1996)。
    4. MEF细胞的1-2×150mm培养皿 在10ml冷PBS中洗涤两次,然后通过刮取收获。 细胞是 收集在15ml PBS中的15ml PBS中,然后在4℃以1600rpm离心   ℃5分钟。
    5. 所需体积的悬浮细胞(DT40和 人淋巴母细胞)在4℃下以1600rpm离心5分钟, 将细胞沉淀转移至15ml管中并用10℃洗涤两次   ml PBS
    6. 将细胞沉淀转移到1ml的Eppendorf管中 PBS并短暂离心。 弃去上清液,颗粒 立即在干冰中冷冻,并储存在-80℃直至使用
    7. 使用20,000-5,000,000个细胞的小球用于细胞提取物制备。

  2. 细胞提取物的制备
    1. 如所报道的那样制备来自脊椎动物细胞的细胞提取物(Biade等人,1998),并概述如下。
    2. 重悬细胞沉淀在400μl冰冷的裂解缓冲液。
    3. 将重悬的细胞沉淀在4℃下旋转1小时
    4. 在4℃下以14,000rpm离心混合物10分钟以除去细胞碎片。
    5. 小心地将上清液部分转移到新鲜的Eppendorf管中。 小心不要碰到颗粒。
    6. 使用具有BSA作为标准的Bradford测定染料试剂(Bradford,1976)确定提取物的蛋白质浓度。

  3. 细胞提取物中的酶活性测定
    1. 准备10微升反应混合物(最终体积),包括1x反应 缓冲液和100nM DNA底物。所用的DNA底物包括 腺苷酸化的尿嘧啶碱基在3'-FAM标记的5'端 寡核苷酸(Çağlayanet al。,2014)。对于参考反应 包括纯化的蛋白质,polβ,APTX和FEN1,反应混合物  包括与UDG as预孵育的缺口DNA底物 描述(Çağlayan,,2014)。对于包括细胞的反应 提取物,将DNA底物包括在反应混合物中  UDG预处理。
    2. 通过加入细胞提取物开始反应, 如上制备,加入到反应混合物中。对于参考反应,  通过将纯化的蛋白质加入到反应中开始每个反应 混合物,终浓度如下:polβ(500nM),APTX(100μM) nM)和FEN1(100nM)(Çağlayan等人,2014和Çağlayan等人, 2015)。
    3. 将反应混合物在37℃下孵育15分钟
    4. 通过加入1M新鲜制备的和冰冷的NaBH 4 4至100mM的终浓度来稳定反应产物。
    5. 在冰上孵育反应样品30分钟
    6. 将反应产物与10μl凝胶负载染料混合
    7. 来自脊椎动物细胞的细胞提取物中的反应产物是 在15%聚丙烯酰胺凝胶上分离。 凝胶被扫描,并且数据 (Çağlayan等人,2014年和Çağlayan等人,2015年)进行了分析。

代表数据

下面显示了来自DT40细胞的提取物中polβdRP裂解酶,FEN1切除和APTX DNA去腺苷化酶活性的产物的样品凝胶图像(图1)。 DNA底物(线1)和polβ裂解酶移除5'-AMP-dRP基团后的反应产物的迁移位置(指示为17聚体并显示在参考反应中的第2行和细胞提取物中的第5-6行) ,APTX5'-AMP去除(表示为17号dRP ,显示在参考反应的第3行和细胞提取物的第6行)和FEN1切除产物(表示为15聚体和16聚体并显示在参考反应的第4行和细胞提取物的第5-6行)。显示来自下面列出的脊椎动物细胞系的细胞提取物中的酶活性的原始结果公布在Çağlayan等人(2015)中。


图1.来自DT40野生型和APTX无效细胞系的细胞提取物中的Pollβ,APTX和FEN1活性

笔记

向细胞沉淀中加入裂解缓冲液后,应通过轻拍轻轻混合包括重悬细胞的Eppendorf管。如果样品通过涡旋混合,则脊椎动物细胞提取物中的酶活性可能丧失 而不是在4℃下旋转。 通常,在稳定状态条件下,在底物相对于酶过量且在线性时间过程的条件下测定酶活性。 此外,酶活性应该在添加到反应混合物中的提取物的活性和量之间的线性条件下。

食谱

  1. 裂解缓冲液
    10mM Tris-HCl(最终缓冲液中pH7.8)
    200 mM KCl
    1mM EDTA
    20%甘油 0.1%NP-40
    1 mM DTT(添加新鲜)
    蛋白酶抑制剂片剂
  2. 10x反应缓冲液
    500mM HEPES(最终缓冲溶液中pH7.5)
    100mM MgCl 2/v/v 200 mM KCl
    5 mM EDTA
    20 mM DTT
  3. 凝胶加载缓冲液
    95%甲酰胺 20 mM EDTA
    0.02%溴酚蓝
    0.02%二甲苯蓝
  4. 10x TBE解决方案
    108克Tris-碱
    55克硼酸
    40ml 0.5M EDTA
    H <2> <0>高达1 L
  5. 变性PAGE溶液(15%)
    40克尿素
    30ml 19:1 AccuGel(40%) 8ml 10%TBE溶液
    10ml H 2 O 2 250微升10%APS
    35微升TEMED

致谢

这项工作是由美国国立卫生研究院,国家环境健康科学研究院(项目号Z01 ES050158和ES050159)的校内研究计划支持。

参考文献

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  2. Biade,S.,Sobol,R.W.,Wilson,S.H.and Matsumoto,Y。(1998)。 线性DNA上的增殖细胞核抗原依赖性脱嘌呤/脱嘧啶位点修复受损。 J Biol Chem 273(2):898-902。
  3. Bradford,M.M。(1976)。 利用蛋白质染料结合原理的快速灵敏的微克量蛋白定量方法。 Anal Biochem 72:248-254。
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  5. Caglayan,M.,Batra,V.K.,Sassa,A.,Prasad,R.and Wilson,S.H。(2014)。 聚合酶β在补充aprataxin缺乏症中的作用 abasic-site base excision repair。 Nat Struct Mol Biol 21(5):497-499。
  6. Caglayan,M.,Horton,J.K.,Prasad,R。和Wilson,S.H。(2015)。 碱基切除修复酶对aprataxin缺陷的补充。核酸研究/em> 43(4):2271-2281。
  7. Harris,J.L.,Jakob,B.,Taucher-Scholz,G.,Dianov,G.L.,Becherel,O.J。和Lavin,M.F。(2009)。 Aprataxin,poly-ADP核糖聚合酶1(PARP-1)和无嘌呤核酸内切酶1(APE1)功能一起保护基因组免受氧化损伤。 Hum Mol Genet 18(21):4102-4117。
  8. Okamoto,S.,Narita,T.,Sasanuma,H.,Takeda,S.,Masunaga,S.,Bessho,T.and Tano,K。 DNA修复途径对piperlongumine在鸡DT40细胞系中的细胞毒性的影响。基因癌症 5(7-8):285-292。
  9. Sobol,R.W.,Horton,J.K.,Kuhn,R.,Gu,H.,Singhal,R.K.,Prasad,R.,Rajewsky,K.and Wilson,S.H。(1996)。 基本切除修复中哺乳动物DNA聚合酶-β的需求。自然 379(6561):183-186。
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引用:Çağlayan, M., Horton, J. K. and Wilson, S. H. (2015). Enzymatic Activity Assays for Base Excision Repair Enzymes in Cell Extracts from Vertebrate Cells. Bio-protocol 5(11): e1493. DOI: 10.21769/BioProtoc.1493.
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