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磁镊结合DNA发夹的方法在RecA蛋白介导的同源重组机制研究中的潜在应用

张宇微 颜燕 农大官 徐春华 李明

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磁镊结合DNA发夹的方法在RecA蛋白介导的同源重组机制研究中的潜在应用

张宇微, 颜燕, 农大官, 徐春华, 李明

Combination of magnetic tweezers with DNA hairpin as a potential approach to the study of RecA-mediated homologous recombination

Zhang Yu-Wei, Yan Yan, Nong Da-Guan, Xu Chun-Hua, Li Ming
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  • 同源序列识别与链交换过程是同源重组领域的重要研究方向.RecA蛋白作为重组酶家族的重要成员而一直被广泛研究.利用smFRET以及传统磁镊、光镊等技术,人们对同源重组过程的分子机制有了较深入的了解,然而,这些技术无法同时兼顾大量程与高精度的需求.本文提出一种传统磁镊结合DNA发夹结构的研究方案,并以大肠杆菌中的RecA介导的同源重组过程为例来阐述该方法的优点.使用本实验方案,我们实时观察到以下过程:1) RecA介导的链交换平均速度与已有结果一致,但并非匀速,而是以台阶式的跳变进行;2)直接观察到RecA第二结合位点与被置换链的动态相互作用过程,测量到第二结合位点与被置换链之间的结合力为3.0 pN,与光镊结合磁镊测量出的结果相符;3)能够区分链交换的方向性并观察到按照不同方向进行链交换的反应细节.本文提供了一个可以兼顾精度和测量范围的实验方法,并以RecA蛋白为例设计实验验证了其可靠性.磁镊结合DNA发夹结构的方法具备用于研究RecA或其他同源重组蛋白工作机理的潜质.因此,本文的工作有望成为单分子生物学领域研究同源重组过程的一个重要方法.
    Homologous recombination(HR) is essential for maintaining the genome fidelity and generating genetic diversity. As a prototypical member of the recombinases, RecA from Escherichia coli has been extensively studied by using single-molecule FRET(smFRET), magnetic tweezers, optical tweezers, etc. However, these methods cannot meet the needs of wide-ranged observations nor high spatial resolution at the same time. For sequence comparison, the average base-to-base distance of the homologous dsDNA will be stretched from 0.34 nm to 0.51 nm. The increment for per base pair is 0.17 nm, which is far beyond the spatial resolution of magnetic tweezers so that it cannot be directly measured. As a high-resolution technique, the smFRET enables us to observe more details of reactions. However, its valid measuring distance is 3-8 nm, which limits the observation range. Here, we propose an approach by combining magnetic tweezers with DNA hairpin, which may solve the problem effectively in the study of HR. In this paper, one end of the DNA molecule with a 270 bp hairpin is immobilized onto the surface of the flow cell, while a magnetic bead is attached to the other end. An external magnetic force is applied to the magnetic bead by placing a permanent magnet above the flow cell. The first 90 bp(from the junction of the hairpin) of the hairpin is homologous to the ssDNA within the ssDNA-RecA filament. Thus, the filament searches for homology along the hairpin, and incorporates into the homologous segment for strand exchange. After that, the displaced strand can be opened by pulling at a force of ~7 pN, and each opened base pair results in a 0.82 nm increase in DNA extension. By using this approach, we show that 1) RecA-mediated strand exchange proceeds in a stepwise manner and the average speed is ~7.6 nt/s, which is in accordance with previous result; 2) the dynamic interaction between the second DNA-binding site(SBS) and the displaced strand can be observed in real-time, and the binding force is calculated accurately through the x-dimensional fluctuations; 3) the processes of strand-exchange in different directions can be observed, and the directions are distinguishable through the reaction patterns. The results suggest that the combination of magnetic tweezers with DNA hairpin is a potential approach to the study of RecA or other recombinases. Therefore, our design can be an important single-molecule approach to the research of HR mechanism.
      通信作者: 李明, mingli@iphy.ac.cn
    • 基金项目: 国家自然科学基金(批准号:11574381,11574382)资助的课题.
      Corresponding author: Li Ming, mingli@iphy.ac.cn
    • Funds: Project supported by the National Natural Science Foundation of China(Grant Nos. 11574381, 11574382).
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    Wang S, Zheng H Z, Zhao Z Y, Lu Y, Xu C H 2013 Acta Phys. Sin. 62 168703(in Chinese)[王爽, 郑海子, 赵振业, 陆越, 徐春华2013物理学报62 168703]

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    Smith S B, Cui Y, Bustamente C 1996 Science 271 795

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    Lieber M R 2010 Annu. Rev. Biochem. 79 181

    [2]

    Kowalczykowski S C, Dixon D A, Eggleston A K, Lauder S D, Rehrauer W M 2008 Nature 453 463

    [3]

    Di Capua E, Engel A, Stasiak A, Koller T 1982 J. Mol. Biol. 157 87

    [4]

    Dombroski D, Scraba D, Bradley R, Morgan A 1983 Nucleic Acids Res. 11 7487

    [5]

    Chen Z, Yang H, Pavletich N P 2008 Nature 453 489

    [6]

    Ragunathan K, Joo C, Ha T 2011 Structure 19 1064

    [7]

    Cox M M 2007 Nat. Rev. Mol. Cell Biol. 8 127

    [8]

    Lee J Y, Terakawa T, Qi Z, Steinfeld J B, Redding S, Kwon Y, Gaines W A, Zhao W, Sung P, Greene E C 2015 Science 349 977

    [9]

    Danilowicz C, Yang D, Kelley C, Prevost C, Prentiss M 2015 Nucleic Acids Res. 43 6473

    [10]

    Qi Z, Redding S, Lee J Y, Gibb B, Kwon Y, Niu H, Gaines W A, Sung P, Greene E C 2015 Cell 160 856

    [11]

    Ragunathan K, Liu C, Ha T 2008 Mol. Cell 30 530

    [12]

    de Vlaminck I, van Loenhout M T, Zweifel L, den Blanken J, Hooning K, Hage S, Kerssemakers J, Dekker C 2012 Mol. Cell 46 616

    [13]

    Roy R, Hohng S, Ha T 2008 Nat. Meth. 5 507

    [14]

    Xu Y, Chen H, Qu Y J, Efremov A K, Li M, Ouyang Z C, Liu D S, Yan J 2014 Chin. Phys. B 23 068702

    [15]

    Zhu C L, Li J 2015 Chin. Phys. Lett. 32 108702

    [16]

    Wang S, Zheng H Z, Zhao Z Y, Lu Y, Xu C H 2013 Acta Phys. Sin. 62 168703(in Chinese)[王爽, 郑海子, 赵振业, 陆越, 徐春华2013物理学报62 168703]

    [17]

    Smith S B, Cui Y, Bustamente C 1996 Science 271 795

    [18]

    Lantsov V 1997 Proc. Natl. Acad. Sci. 94 11935

    [19]

    Mossa A, Manosas M, Forns N, Huguet J M, Ritort F 2009 J. Stat. Mech. Theory E 2009 2060

    [20]

    Mazin A V, Kowalczykowski S C 1996 Proc. Natl. Acad. Sci. 93 10673

    [21]

    Gosse C, Croquette V 2002 Biophys. J. 82 3314

    [22]

    Bustamante C, Smith S B, Liphardt J, Smith D 2000 Curr. Opin. Struct. Biol. 10 279

    [23]

    Zheng H Z, Nong D G, Li M 2013 Chin. Phys. Lett. 30 118702

    [24]

    Cox M M, Lehman I 1981 Proc. Natl. Acad. Sci. 78 6018

    [25]

    Kim J I, Cox M, Inman R 1998 Proc. Natl. Acad. Sci. 95 9843

    [26]

    Lee J, Lee S, Ragunathan K, Joo C, Ha T, Hohng S 2010 Angew. Chem. 122 10118

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出版历程
  • 收稿日期:  2016-07-22
  • 修回日期:  2016-08-12
  • 刊出日期:  2016-11-05

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