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用单分子技术研究Sso7d与DNA的相互作用

滕翠娟 陆越 马建兵 李明 陆颖 徐春华

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用单分子技术研究Sso7d与DNA的相互作用

滕翠娟, 陆越, 马建兵, 李明, 陆颖, 徐春华

Interaction between Sso7d and DNA studied by single-molecule technique

Teng Cui-Juan, Lu Yue, Ma Jian-Bing, Li Ming, Lu Ying, Xu Chun-Hua
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  • 为了维持基因的稳定性,每种生物体都含有一套独特的染色质蛋白来保护脱氧核糖核酸(DNA)的结构,观察染色质蛋白对DNA结构的作用过程和结果,可以帮助人们了解这些蛋白的具体功能和作用机理.硫化叶菌是一种能在高温下存活的古细菌,Sso7d是硫化叶菌的一种染色质蛋白.深入地了解Sso7d和DNA链的相互作用,有助于解释硫化叶菌的DNA为何能在高温环境下保持活性,本文通过原子力显微镜(AFM)和磁镊两种单分子操作手段,研究了Sso7d与DNA的相互作用.AFM的实验结果给出了Sso7d与DNA的作用过程:结合Sso7d后,DNA首先发生弯折,然后出现loop结构,最终DNA会团聚为致密的核结构.利用磁镊装置测量了Sso7d的结合对打开DNA双链的影响,实验结果表明Sso7d的结合导致打开DNA双链的力的增大,经过数据分析,计算出Sso7d与DNA结合的结合能△G=3.1 kBT,平均每5.5个碱基对(bp)结合一个Sso7d,较高的结合密度和较大的结合能,两方面的作用结果,解释了Sso7d能够稳定DNA结构的原因.
    Each organism has its own set of chromatin proteins to protect the stable structure of DNA and thus maintain the stability of genes. Sso7d is a small nonspecific DNA-binding protein from the hyperthermophilic archaea Sulfolobus solfataricus. This protein has high thermal and acid stability. It stabilizes dsDNA and constrains negative DNA supercoils. Besides, the Sso7d binds in a minor groove of DNA and causes a sharp kink in DNA. By observing the interaction between chromatin protein and DNA structure, we can understand the function and mechanism of chromatin protein. Sulfolobus solfataricus can survive at high temperature. To understand why the DNA of Sulfolobus solfataricus retains activity at high temperature, we investigate the interaction between Sso7d and DNA by atomic force microscope (AFM) and magnetic tweezers. Atomic force microscope and magnetic tweezers are advanced single molecule experimental tools that can be used to observe the interaction between individual molecules. The experimental result of AFM reveals the process of interaction between Sso7d and DNA. The DNA structure changes at a different concentration of Sso7d and depends on reaction time. At a relatively low concentration of Sso7d, DNA strand forms a kink structure. When the concentration of Sso7d is increased, DNA loops appear. Finally, DNA becomes a dense nuclear structure at a high concentration of Sso7d. If the time of the interaction between Sso7d and DNA is increased, DNA structure tends to be more compact. These results indicate that high concentration of Sso7d is important for the compact structure of DNA. We design an experiment to find out the formation of the looped structure on DNA. Moreover, we measure the angle of kinked DNA and compared it with previous result. Through the experiment of magnetic tweezers, we measure the forces of unfolding the double-stranded DNA complexed with Sso7d at different concentrations. The experimental results show that the binding between Sso7d and DNA increases the force of unfolding the double-stranded DNA. The binding energy between Sso7d and dsDNA is 3.1kBT which is calculated from experimental data. It indicates that DNA base pairs are more stable when chromatin protein Sso7d exists. These results can explain the survival of Sulfolobus in high temperature environment.
      通信作者: 徐春华, xch@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: Xu Chun-Hua, xch@iphy.ac.cn
    • Funds: Project supported by the National Natural Science Foundation of China (Grant Nos. 11574381, 11574382).
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    Driessen R P C, Meng H, Suresh G, Shahapure R, Lanzani G, Priyakumar U D, White M F, Schiessel H, van Noort J, Dame R T 2013 Nucleic Acids Res. 41 196

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    Guo L, Feng Y, Zhang Z, Yao H, Luo Y, Wang J, Huang L 2008 Nucleic Acids Res. 36 1129

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    Guagliardi A, Napoli A, Rossi M, Ciaramella M 1997 J. Mol. Biol. 267 841

    [30]

    Dudko O K, Hummer G, Szabo A 2008 Proc. Natl. Acad. Sci. USA 105 15755

    [31]

    Pope L H, Bennink M L, van Leijenhorst Groener K A, Nikova D, Greve J, Marko J F 2005 Biophys. J. 88 3572

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    Yang W Y, Gruebele M 2003 Nature 423 193

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    Woodside M T, Behnke-Parks W M, Larizadeh K, Travers K, Herschlag D, Block S M 2006 Proc. Natl. Acad. Sci. USA 103 6190

  • [1]

    Luijsterburg M S, White M F, van Driel R, Dame R T 2008 Crit. Rev. Biochem. Mol. Biol. 43 393

    [2]

    Woese C R, Fox G E 1977 Proc. Natl. Acad. Sci. USA 74 5088

    [3]

    Grunstein M 1997 Nature 389 349

    [4]

    Luijsterburg M, Noom M, Wuite G, Dame R 2006 J. Struct. Biol. 156 262

    [5]

    Dame R T 2005 Mol. Microbiol. 56 858

    [6]

    Sandman K, Reeve J N 2005 Curr. Opin. Microbiol. 8 656

    [7]

    Sandman K, Reeve J N 2006 Curr. Opin. Microbiol. 9 520

    [8]

    Reeve J N, Bailey K A, Li W, Marc F, Sandman K, Soares D J 2004 Biochem. Soc. Trans. 32 227

    [9]

    Forterre P, Confalonieri F, Knapp S 1999 Mol. Microbiol. 32 669

    [10]

    Driessen R P C, Dame R T 2011 Biochem. Soc. Trans. 39 116

    [11]

    Mai V Q, Chen X, Hong R, Huang L 1998 J. Bacteriol. 180 2560

    [12]

    Choli T, Henning P, Wittmann-Liebold B, Reinhardt R 1988 Biochim. Biophys. Acta 950 193

    [13]

    Edmondson S P, Shriver J W 2001 Methods Enzymol. 334 129

    [14]

    White M F, Bell S D 2002 Trends Genet. 18 621

    [15]

    Lundbãck T, Hansson H, Knapp S, Ladenstein R, Hãrd T 1998 J. Mol. Biol. 276 775

    [16]

    Napoli A, Zivanovic Y, Bocs C, Buhler C, Rossi M, Forterre P, Ciaramella M 2002 Nucleic Acids Res. 30 2656

    [17]

    López-García P, Knapp S, Ladenstein R, Forterre P 1998 Nucleic Acids Res. 26 2322

    [18]

    Sun F, Huang L 2013 Nucleic Acids Res. 41 8182

    [19]

    Gera N, Hussain M, Wright R C, Rao B M 2011 J. Mol. Biol. 409 601

    [20]

    Hernandez Garcia A, Estrich N A, Werten M W T, van der Maarel J R C, LaBean T H, de Wolf F A, Cohen Stuart M A, de Vries R 2017 ACS Nano 11 144

    [21]

    Gera N, Hill A B, White D P, Carbonell R G, Rao B M 2012 PloS One 7 e48928

    [22]

    Gao Y G, Su S Y, Robinson H, Padmanabhan S, Lim L, McCrary B S, Edmondson S P, Shriver J W, Wang A H J 1998 Nat. Struct. Biol. 5 782

    [23]

    Su S, Gao Y G, Robinson H, Liaw Y C, Edmondson S P, Shriver J W, Wang A H J 2000 J. Mol. Biol. 303 395

    [24]

    Driessen R P C, Meng H, Suresh G, Shahapure R, Lanzani G, Priyakumar U D, White M F, Schiessel H, van Noort J, Dame R T 2013 Nucleic Acids Res. 41 196

    [25]

    Lou H, Duan Z, Huo X, Huang L 2004 J. Biol. Chem. 279 127

    [26]

    Guo L, Feng Y, Zhang Z, Yao H, Luo Y, Wang J, Huang L 2008 Nucleic Acids Res. 36 1129

    [27]

    Li J H, Lin W X, Zhang B, Nong D G, Ju H P, Ma J B, Xu C H, Ye F F, Xi X G, Li M, Lu Y, Dou S X 2016 Nucleic Acids Res. 44 4330

    [28]

    Agback P, Baumann H, Knapp S, Ladenstein R, Hãrd T 1998 Nat. Struct. Biol. 5 579

    [29]

    Guagliardi A, Napoli A, Rossi M, Ciaramella M 1997 J. Mol. Biol. 267 841

    [30]

    Dudko O K, Hummer G, Szabo A 2008 Proc. Natl. Acad. Sci. USA 105 15755

    [31]

    Pope L H, Bennink M L, van Leijenhorst Groener K A, Nikova D, Greve J, Marko J F 2005 Biophys. J. 88 3572

    [32]

    Yang W Y, Gruebele M 2003 Nature 423 193

    [33]

    Woodside M T, Behnke-Parks W M, Larizadeh K, Travers K, Herschlag D, Block S M 2006 Proc. Natl. Acad. Sci. USA 103 6190

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  • 收稿日期:  2018-04-09
  • 修回日期:  2018-04-21
  • 刊出日期:  2019-07-20

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