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Determination of Adeno-associated Virus Rep DNA Binding Using Fluorescence Anisotropy
使用荧光各向异性度测定腺相关病毒的Rep DNA结合   

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Abstract

Quantitative measurement of proteins binding to DNA is a requisite to fully characterize the structural determinants of complex formation necessary to understand the DNA transactions that regulate cellular processes. Here we describe a detailed protocol to measure binding affinity of the adeno-associated virus (AAV) Rep68 protein for the integration site AAVS1 using fluorescent anisotropy. This protocol can be used to measure the binding constants of any DNA binding protein provided the substrate DNA is fluorescently labeled.

Keywords: Adeno-associated virus(腺相关病毒), Rep proteins(Rep蛋白), Fluorescence(荧光), Anisotropy(各向异性), Protein-DNA binding(蛋白质-DNA结合)

Background

Fluorescence polarization anisotropy has become one of the most popular methods to measure interaction of proteins with a large variety of ligands including small molecules, nucleic acids, peptides and other proteins. The method is quick, inexpensive and can be modified to be used in plate readers equipped with fluorescence detectors. The technique is based on the principle that when a fluorescent molecule is excited with plane polarized light, the emitted light remains polarized in the same plane if the molecule is stationary or if it rotates slowly. In contrast, if the molecule rotates rapidly (due to small size), the light is emitted in a different plane. These changes can be quantified by the normalized differences in parallel and perpendicular intensities. Polarization is defined as P = (I= - I)/(I= + I), where I= is the parallel intensity and I is the perpendicular intensity. An alternative way is to define the anisotropy, A = (I= - I)/(I= + 2I). Both parameters can be used interchangeably to describe the changes in polarization. Thus, when a small fluorescent DNA molecule binds a protein, the larger complex will rotate more slowly than the DNA molecule, changing the plane of the polarized light and increasing the anisotropy value. We have used this technique to measure the binding affinity of AAV Rep68 for different DNA substrates (Yoon-Robarts et al., 2004; Musayev et al., 2015; Bardelli et al., 2016). The non-structural AAV Rep proteins carry out most of the DNA transactions that are required to complete the virus life cycle. These include DNA replication, transcriptional regulation, site-specific integration and packaging of DNA into preformed capsids (Im and Muzyczka, 1990; Weitzman et al., 1994; Wonderling et al., 1995). The large AAV Rep proteins (Rep78/Rep68) contain an N-terminal origin binding domain (OBD) that specifically binds the Rep binding sites (RBS) and displays nuclease activity (Hickman et al., 2004; Musayev et al., 2015). The RBS sites consist of two or more 5’-GCTC-3’ repeats and are found at the viral origin of replication, in several promoters and at the AAVS1 integration site (Weitzman et al., 1994; McCarty et al., 1994). In addition, a C-terminus SF3 helicase domain is required for high affinity binding and DNA unwinding (James et al., 2003; Mansilla-Soto et al., 2009).The protocol described here can be modified to fit any protein-DNA system or any other instrument such as plate readers.

Materials and Reagents

  1. Pipette tips  
  2. 15 ml conical tubes (USA scientific, catalog number: 1475-1611 )
  3. Black 1.5 ml Eppendorf tubes (Argos Technologies, catalog number: T7456-001 )
  4. 16 gauge needle (BD, catalog number: 305197 )
  5. Fluorescein labeled AAVS1 sense DNA strand with the following sequence: 5’-TGGCGGCGGTTGGGGCTCGGCGCTCGCTCGCTCGCTGGGCG-3’
    AAVS1 anti-sense strand with the sequence: 5’-CGCCCAGCGAGCGAGCGA GCGCCGAGCCCCAACCGCCGCCA-3’
    Note: DNA can be synthesized using any synthesis facility such as IDT services at a 100 nanomole scale. The sense strand can be labeled at the 5’ end with fluorescein (6-FAM).
  6. Purified recombinant AAV Rep68 protein was expressed in E.coli and purified using Ni-NTA affinity column followed by a gel filtration column as described previously (Musayev et al., 2015)
  7. Sodium hydroxide (NaOH)
  8. Sodium chloride (NaCl) (Fisher Scientific, catalog number: BP358 )
  9. 2-Amino-2-(hydroxymethyl)propane-1,3-diol (Tris) (Sigma-Aldrich, catalog number: T1503 )
  10. Ethylendiaminetetraacetic acid (EDTA) (Gold Bio, catalog number: E-210-1 )
  11. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Gold Bio, catalog number: H-401-500 )
  12. Tris(2-carboxyethyl)phosphine (TCEP) (Gold Bio, catalog number: TECP )
  13. Double distilled water
  14. Q1 buffer (see Recipes)
  15. Q2 buffer (see Recipes)
  16. TES buffer (see Recipes)
  17. Binding buffer (see Recipes)

Equipment

  1. Pipettes  
  2. MonoQ anion-exchange column (GE Healthcare, catalog number: 17-5166-01 )
  3. HiTrap 5 ml desalting column (GE Healthcare, catalog number: 11-0003-29 )
  4. GE Healthcare AKTA purifier
  5. Labconco Freezone 2/5 Benchtop lyophilizer
  6. Thermo Scientific NanoDrop ND-2000c spectrophotometer (Thermo Fisher Scientific, model: NanoDropTM 2000/2000c )
  7. Denville IncuBlock heating block
  8. ISS PC1 fluorimeter (ISS, model: PC1TM )

Software

  1. Microsoft Excel
  2. GraphPad Prism 7TM
  3. Vinci Instrument control and data acquisition software from ISS

Procedure

  1. Preparation of fluorescein labeled AAVS1 DNA
    1. Fluorescent labeled DNA can be purified by HPLC or using preparative acrylamide gel electrophoresis (PAGE) directly by the synthesis facility or can be purified by anion-exchange chromatography as described next. DNA is dissolved in 500 μl of Q1 buffer and injected into a MonoQ anion-exchange column. DNA was purified using a linear gradient from buffer Q1 (100 mM NaCl) to 100% Q2 buffer (1 M NaCl).
    2. Fractions are desalted using a 5 ml GE HiTrap desalting column pre-equilibrated with water. DNA elutes in the void volume.
    3. Collected DNA fractions are placed in a 15 ml conical tube, frozen and placed in a Labcono Freezone 2.5 lyophilizer overnight. To allow drying of the sample, the lid of the 15 ml conical tube should be pierced with a needle 4-5 times. The DNA should be fibrous and dry by the next day.
    4. After lyophilization, resuspend the DNA in 100 μl of TES buffer. Measure the DNA concentrations using a Thermo Scientific NanoDrop ND-2000c spectrophotometer using the calculated extinction coefficients of the DNA oligonucleotides at a wavelength of 260 nm.
    5. To prepare the double-stranded DNA, combine the two DNA strands in a ratio where the non-labeled strand is in 1.1 molar excess with respect to the fluorescein-labeled strand. Place the DNA strands in a black 1.5 ml microcentrifuge tube. Using a Denville IncuBlock heating block, heat the DNA at 99 °C for 3 min. Then, turn off the heating block and leave the DNA cooling to room temperature.
    6. The final DNA concentration is calculated using the number of moles of the labeled-DNA strand and the final volume after mixing the two strands.

  2. DNA binding studies with Rep68
    Rep68 concentration is determined by absorbance at 280 nm using the extinction coefficient calculated from the Rep68 amino acid sequence using EXPASY Protparam site (http://web.expasy.org/protparam/). Prepare different concentrations of Rep68 in binding buffer and mix with DNA allowing the binding reaction to reach equilibrium at 20 °C for 20-30 min. For other proteins a preliminary experiment needs to be performed to obtain a rough estimate of the concentrations to be used. Titrations are carried out using a final 5 nM DNA concentration in binding buffer. A typical binding experiment is performed as follows:  
    1. It is recommended that the PC-1 fluorimeter is turned on the night before the experiment to stabilize the instrument (Figure 1). Start the Vinci software on the computer. Access instrument control and set the excitation and emission filters to 492 nm and 528 nm, respectively.
    2. Turn-on the lamp of the PC1 fluorimeter and let it warm up for at least 1 h.
    3. Calculate the amount of Rep68 stock solution needed to achieve the following final concentrations in the binding reactions: 0, 10, 25, 50, 75, 100, 150, 200, 300, 400 and 500 nM.
      Generally, three replicates are done for each concentration.
    4. Mix the different Rep68 concentrations with 5 nM DNA in a final volume of 300 μl. Incubate samples for 20-30 min away from light.
    5. Measure the anisotropy value of each concentration point using the ‘Single point Polarization’ function in the Vinci software and record 10 measurements per concentration.


      Figure 1. PC1 fluorimeter from ISS

Data analysis

  1. Open all the data in Microsoft Excel and for each concentration calculate the average anisotropy value from the 10 measurements.
  2. Make a new Graphpad project using the Rep68 concentration as the x values and the average anisotropy as the y value.
  3. Subtract the DNA anisotropy value from all concentrations.
  4. Using Prism 7TM (GraphPad), perform a nonlinear fitting using a single specific binding model. Prism uses the following equation:


    Where,
    Amax is the maximum anisotropy value at saturation,
    Cx is the concentration of Rep68,
    Kd is binding constant.
  5. To calculate fraction of DNA bound, the maximum value obtained from the fitting is used as the anisotropy value at saturation when all DNA is bound (Amax). Fraction of DNA bound at each concentration is the anisotropy at each concentration (Ax) divided by Amax. This is shown in Figure 2A. Anisotropy values (Ax) at each concentration (Group A) were converted to fraction bound (Group B).
  6. Below is an example of a typical experiment.


    Figure 2. Data analysis. A. Anisotropy data and correction for fraction bound; B. Nonlinear Fitting of the data to specific binding model.

Notes

  1. It is recommended that during the incubation step, samples be kept away from light and incubated at the temperature of the instrument by means of a water bath or heating/cooling block.
  2. Protein stocks are stored at high concentration (~0.1-0.5 mM) at -80 °C and are diluted in binding buffer to the required concentrations. Generally, a series of dilutions from 100 μM to 100 nM are prepared to cover the concentration range of the titration curve.
  3. If incubation time is not known, this can be determined by incubating sample and reading anisotropy every five minutes. Equilibrium is estimated when the measured anisotropy remains constant.

Recipes

  1. Q1 buffer
    10 mM NaOH
    100 mM NaCl (pH 12.0)
  2. Q2 buffer
    10 mM NaOH
    1 M NaCl (pH 12.0)
  3. TES buffer
    10 mM Tris
    100 mM NaCl
    1 mM EDTA (pH 8.0)
  4. Binding buffer
    25 mM HEPES
    200 mM NaCl
    1 mM TCEP (pH 7.0)

Acknowledgments

This work was supported by NIH grant R01-GM092854. The authors declare no conflict of interests.

References

  1. Bardelli, M., Zarate-Perez, F., Agundez, L., Linden, R. M., Escalante, C. R. and Henckaerts, E. (2016). Identification of a functionally relevant AAV Rep68 oligomeric interface. J Virol 90(15): 6612-24.
  2. Hickman, A. B., Ronning, D. R., Perez, Z. N., Kotin, R. M. and Dyda, F. (2004). The nuclease domain of adeno-associated virus rep coordinates replication initiation using two distinct DNA recognition interfaces. Mol Cell 13(3): 403-414.
  3. Im, D. S. and Muzyczka, N. (1990). The AAV origin binding protein Rep68 is an ATP-dependent site-specific endonuclease with DNA helicase activity. Cell 61(3): 447-457.
  4. James, J. A., Escalante, C. R., Yoon-Robarts, M., Edwards, T. A., Linden, R. M. and Aggarwal, A. K. (2003). Crystal structure of the SF3 helicase from adeno-associated virus type 2. Structure 11(8): 1025-1035.
  5. Mansilla-Soto, J., Yoon-Robarts, M., Rice, W. J., Arya, S., Escalante, C. R. and Linden, R. M. (2009). DNA structure modulates the oligomerization properties of the AAV initiator protein Rep68. PLoS Pathog 5(7): e1000513.
  6. McCarty, D. M., Pereira, D. J., Zolotukhin, I., Zhou, X., Ryan, J. H. and Muzyczka, N. (1994). Identification of linear DNA sequences that specifically bind the adeno-associated virus Rep protein. J Virol 68(8): 4988-4997.
  7. Musayev, F. N., Zarate-Perez, F., Bishop, C., Burgner, J. W., 2nd and Escalante, C. R. (2015). Structural insights into the assembly of the adeno-associated virus type 2 Rep68 protein on the integration site AAVS1. J Biol Chem 290(46): 27487-27499.
  8. Weitzman, M. D., Kyostio, S. R., Kotin, R. M. and Owens, R. A. (1994). Adeno-associated virus (AAV) Rep proteins mediate complex formation between AAV DNA and its integration site in human DNA. Proc Natl Acad Sci U S A 91(13): 5808-5812.
  9. Wonderling, R. S., Kyostio, S. R. and Owens, R. A. (1995). A maltose-binding protein/adeno-associated virus Rep68 fusion protein has DNA-RNA helicase and ATPase activities. J Virol 69(6): 3542-3548.
  10. Yoon-Robarts, M., Blouin, A. G, Bleker, S., Kleinmschmidt, J. A., Aggarwal, A. K., Escalante, C. R. and Linden, R. M. (2004). Residues within the B’ motif are critical for DNA binding by the superfamily 3 helicase Rep40 of adeno-associated virus type 2. J Biol Chem 279(48): 50472-50481.

简介

与DNA结合的蛋白质的定量测量是完全表征理解调节细胞过程的DNA交易所必需的复合物形成结构决定簇的必要条件。在这里,我们描述了使用荧光各向异性来测量腺相关病毒(AAV)Rep68蛋白与整合位点AAVS1的结合亲和力的详细方案。如果底物DNA被荧光标记,则该方案可用于测量任何DNA结合蛋白的结合常数。

背景 荧光偏振各向异性已经成为测量蛋白质与大分子,核酸,肽和其他蛋白质等多种配体相互作用的最流行方法之一。该方法快速,便宜,可以修改为配备荧光检测器的读卡器。该技术基于以下原理:当荧光分子被平面偏振光激发时,如果分子是静止的或者如果其旋转缓慢,发射的光在同一平面内保持极化。相反,如果分子快速旋转(由于体积小),则光在不同的平面中发射。这些变化可以通过平行和垂直强度的归一化差异量化。极化被定义为P =(I⊥⊥⊥⊥⊥⊥⊥⊥⊥⊥⊥⊥⊥⊥⊥⊥⊥⊥⊥⊥⊥⊥⊥⊥⊥⊥⊥⊥⊥⊥⊥⊥⊥⊥⊥⊥)其中I = 是平行强度,I⊥是垂直强度。一种替代方式是定义各向异性,A =(I⊥⊥⊥⊥⊥⊥⊥⊥⊥< /sub>)。两个参数可以互换使用来描述极化的变化。因此,当小荧光DNA分子结合蛋白质时,较大的复合物将比DNA分子旋转得更慢,改变偏振光的平面并提高各向异性值。我们已经使用这种技术来测量AAV Rep68对不同DNA底物的结合亲和力(Yoon-Robarts等人,2004; Musayev等人,2015; Bardelli 等人,2016)。非结构AAV Rep蛋白进行完成病毒生命周期所需的大部分DNA交易。这些包括DNA复制,转录调节,位点特异性整合以及将DNA包装到预先形成的衣壳中(Im和Muzyczka,1990; Weitzman等人,1994; Wonderling等人 ,1995)。大的AAV Rep蛋白(Rep78 / Rep68)含有特异性结合Rep结合位点(RBS)并显示核酸酶活性的N末端起始结合结构域(OBD)(Hickman等,2004; Musayev等人,2015)。 RBS位点由两个或更多个5'-GCTC-3'重复组成,并且在病毒复制起点,几个启动子和 AAVS1 整合位点(Weitzman等, ,1994; McCarty等人,1994)。此外,需要C末端SF3解旋酶结构域用于高亲和力结合和DNA解卷(James等人,2003; Mansilla-Soto等人,2009)这里描述的方案可以修改为适合任何蛋白质DNA系统或任何其他仪器,如读板器。

关键字:腺相关病毒, Rep蛋白, 荧光, 各向异性, 蛋白质-DNA结合

材料和试剂

  1. 移液器提示
  2. 15毫升锥形管(美国科学,目录号:1475-1611)
  3. 黑色1.5ml Eppendorf管(Argos Technologies,目录号:T7456-001)
  4. 16号针(BD,目录号:305197)
  5. 荧光素标记的AAVS1有义DNA链,具有以下序列:5'-TGGCGGCGGTTGGGGCTCGGCGCTCGCTCGCTCGCTGGGCG-3'
    AAVS1 具有序列的反义链:5'-CGCCCAGCGAGCGAGCGA GCGCCGAGCCCCAACCGCCGCCA-3'
    注意:可以使用任何合成设备(如IDT服务)以100纳摩尔量表合成DNA。有义链可以在5'末端用荧光素(6-FAM)标记。
  6. 纯化的重组AAV Rep68蛋白在大肠杆菌中表达,并使用Ni-NTA亲和柱纯化,随后如先前所述的凝胶过滤柱(Musayev等人,2015)
  7. 氢氧化钠(NaOH)
  8. 氯化钠(NaCl)(Fisher Scientific,目录号:BP358)
  9. 2-氨基-2-(羟甲基)丙烷-1,3-二醇(Tris)(Sigma-Aldrich,目录号:T1503)
  10. 乙二胺四乙酸(EDTA)(Gold Bio,目录号:E-210-1)
  11. 4-(2-羟乙基)-1-哌嗪乙磺酸(HEPES)(Gold Bio,目录号:H-401-500)
  12. 三(2-羧乙基)膦(TCEP)(Gold Bio,目录号:TECP)
  13. 双蒸水
  14. Q1缓冲区(见配方)
  15. Q2缓冲(见配方)
  16. TES缓冲区(见配方)
  17. 绑定缓冲区(见配方)

设备

  1. 移液器
  2. MonoQ阴离子交换柱(GE Healthcare,目录号:17-5166-01)
  3. HiTrap 5 ml脱盐柱(GE Healthcare,目录号:11-0003-29)
  4. GE Healthcare AKTA净化器
  5. Labconco Freezone 2/5台式冻干机
  6. Thermo Scientific NanoDrop ND-2000c分光光度计(Thermo Fisher Scientific,型号:NanoDrop TM 2000/2000c)
  7. Denville IncuBlock加热块
  8. ISS PC1荧光计(ISS,型号:PC1 TM

软件

  1. Microsoft Excel
  2. GraphPad Prism 7 TM
  3. 来自ISS的Vinci Instrument控制和数据采集软件

程序

  1. 荧光素标记的AAVS1 DNA的制备
    1. 荧光标记的DNA可以通过HPLC纯化或使用制备型丙烯酰胺凝胶电泳(PAGE)直接由合成设备纯化,或者如下所述通过阴离子交换层析纯化。将DNA溶解于500μlQ1缓冲液中并注入到MonoQ阴离子交换柱中。使用从缓冲液Q1(100mM NaCl)到100%Q2缓冲液(1M NaCl)的线性梯度纯化DNA。
    2. 使用预先用水预平衡的5ml GE HiTrap脱盐塔将馏分脱盐。 DNA在空隙体积中洗脱。
    3. 将收集的DNA级分置于15ml锥形管中,冷冻并置于Labcono Freezone 2.5冻干机中过夜。为了允许样品干燥,15毫升锥形管的盖子应用针刺4-5次。 DNA应在第二天纤维化并干燥。
    4. 冷冻干燥后,将DNA重悬于100μlTES缓冲液中。使用Thermo Scientific NanoDrop ND-2000c分光光度计测量DNA浓度,使用在260nm波长的DNA寡核苷酸的计算的消光系数。
    5. 为了制备双链DNA,以相对于荧光素标记的链为1.1摩尔过量的未标记链的比例组合两个DNA链。将DNA链放入黑色的1.5 ml微量离心管中。使用Denville IncuBlock加热块,将DNA在99℃加热3分钟。然后,关闭加热块,使DNA冷却至室温
    6. 使用标记的DNA链的摩尔数和混合两股之后的最终体积计算最终的DNA浓度。

  2. 用Rep68进行DNA结合研究
    Rep68浓度通过使用EXPASYPrepparam位点( http://web.expasy.org/protparam/)。在结合缓冲液中制备不同浓度的Rep68,并与DNA混合,使结合反应在20℃达到平衡20-30分钟。对于其他蛋白质,需要进行初步实验以获得要使用的浓度的粗略估计。使用结合缓冲液中最终的5nM DNA浓度进行滴定。典型的绑定实验如下执行:  
    1. 建议在实验前一天晚上打开PC-1荧光计以稳定仪器(图1)。在计算机上启动Vinci软件。访问仪器控制并将激发和发射滤波器分别设置为492 nm和528 nm
    2. 打开PC1荧光计的灯泡,让其预热至少1小时。
    3. 计算在结合反应中达到以下终浓度所需的Rep68储备溶液的量:0,10,25,50,75,100,150,200,300,400和500 nM。
      一般来说,每个浓度都会进行三次重复
    4. 将不同的Rep68浓度与最终体积为300μl的5nM DNA混合。将样品孵育20-30分钟,避光。
    5. 使用Vinci软件中的"单点极化"功能测量每个浓度点的各向异性值,并记录每个浓度的10次测量。


      图1.来自ISS的PC1荧光计

数据分析

  1. 打开Microsoft Excel中的所有数据,每个浓度计算10次测量的平均各向异性值。
  2. 使用Rep68浓度作为x值和平均各向异性作为y值制作新的Graphpad项目。
  3. 减去所有浓度的DNA各向异性值
  4. 使用Prism 7 TM (GraphPad),使用单个特定的绑定模型执行非线性拟合。棱镜使用以下公式:


    哪里,
    最大最大值是饱和度下的最大各向异性值,
    C x 是Rep68的浓度,
    K d 是绑定常数。
  5. 为了计算DNA结合的分数,从拟合得到的最大值用作所有DNA结合时饱和时的各向异性值(A max max)。在每个浓度下结合的DNA的分数是每个浓度(A x x x)除以Amax的各向异性。这在图2A中示出。将各浓度(A组)的各向异性值(A x )转化为结合部分(B组)。
  6. 以下是典型实验的一个例子

    图2.数据分析。 A.各向异性数据和分数结合的校正; B.数据对特定绑定模型的非线性拟合

笔记

  1. 建议在孵育步骤中,将样品远离光线,并在仪器的温度下通过水浴或加热/冷却块进行温育。
  2. 蛋白质库存在-80℃下以高浓度(〜0.1-0.5mM)储存,并在结合缓冲液中稀释至所需浓度。通常,制备100μM至100nM的一系列稀释液以覆盖滴定曲线的浓度范围。
  3. 如果不知道孵化时间,这可以通过每五分钟温育样品和阅读各向异性来确定。当测量的各向异性保持不变时,估计平衡

食谱

  1. Q1缓冲区
    10 mM NaOH
    100mM NaCl(pH 12.0)
  2. Q2缓冲区
    10 mM NaOH
    1M NaCl(pH 12.0)
  3. TES缓冲区
    10 mM Tris
    100 mM NaCl
    1mM EDTA(pH8.0)
  4. 绑定缓冲区
    25 mM HEPES
    200 mM NaCl
    1mM TCEP(pH 7.0)

致谢

这项工作得到NIH授权R01-GM092854的支持。作者声明没有利益冲突。

参考文献

  1. Bardelli,M.,Zarate-Perez,F.,Agundez,L.,Linden,RM,Escalante,CR and Henckaerts,E.(2016)。  识别功能相关的AAV Rep68寡聚体界面。 90(15):6612 -24。
  2. Hickman,AB,Ronning,DR,Perez,ZN,Kotin,RM和Dyda,F。(2004)。  腺相关病毒的核酸酶结构域使用两个不同的DNA识别界面协调复制起始。分子细胞 13(3):403 -414。
  3. Im,DS and Muzyczka,N。(1990)。  AAV起始结合蛋白Rep68是具有DNA解旋酶活性的ATP依赖性位点特异性内切核酸酶。细胞 61(3):447-457。
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引用:Zarate-Perez, F., Santosh, V., Bardelli, M., Agundez, L., Linden, R., Henckaerts, E. and Escalante, C. R. (2017). Determination of Adeno-associated Virus Rep DNA Binding Using Fluorescence Anisotropy. Bio-protocol 7(6): e2194. DOI: 10.21769/BioProtoc.2194.
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