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单分子荧光共振能量转移（smFRET）和磁镊（MT）技术目前广泛应用于研究分子马达.相较于常规技术，其具有高精度及动态观测的优点.本文研究对象为T7解旋酶，是六聚体解旋酶的典型代表.研究表明，这种解旋酶主要消耗脱氧胸苷三磷酸（dTTP）提供能量，且仅能沿着5'-3'单向进行行走和解旋工作.目前对于六聚体解旋酶的解旋和换链机制的认知仍然存在着诸多问题，因此本文主要以此作为切入点开展研究.首先通过运用smFRET技术研究T7解旋酶在不同DNA底物上的解旋现象，发现其需要3'-尾链参与到解旋工作中，但其为单链或双链结构并无明显区别；通过改变脱氧核糖核酸（DNA）序列中的GC含量，发现T7解旋酶随着序列中GC含量的升高会更容易在解旋过程中发生回退现象，导致解旋长度明显缩短；通过进一步分析发生回退先的实验数据，发现T7解旋酶除了可以瞬时回退到叉形DNA岔口或脱落外，还可以缓慢回退到叉形DNA岔口；运用MT技术研究该解旋酶，同样发现这种缓慢回退现象的存在.根据T7解旋酶解旋DNA遵循的单向性和极性，其只能沿着5'到3'方向进行行走和解旋.因此，本文推测这种缓慢回退的现象可能是解旋酶从5'-链转移至3'-链上，即发生换链过程；最后，本文提出了T7解旋酶在解旋过程中进行换链的模型，将有助于进一步理解环状六聚体解旋酶行使其功能的分子机制.
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关键词:
- 单分子荧光共振能量转移 /
- 磁镊 /
- T7解旋酶 /
- 换链
Single-molecule fluorescence resonance energy transfer (smFRET) and magnetic tweezers are widely used to study the molecular motors because of their high resolution and real-time observation. In this work, we choose these two techniques as the research means. The bacteriophage T7 helicase, as the research object, serves as a model protein for ring-shaped hexameric helicase that couples deoxythymidine triphosphate (dTTP) hydrolysis to unidirectional translocation. The DNA strand separation is 5'-3'-along one strand of double-stranded DNA. Using smFRET and magnetic tweezers to study the unwinding process of T7 helicase, we can have more in depth understanding of the unwinding and strand switching mechanisms of the ring-shaped hexameric helicases. First, by designing DNA substrates with different 3'-tail structures, we find that the 3'-tail is required for T7 helicase unwinding process, no matter whether it is single-stranded or double-stranded. These results confirm an interaction between T7 helicase and 3'-tail. Second, examining the dependence of unwinding process on GC content in DNA sequence, we find that as GC content increases, T7 helicase has higher chances to stop and slips back to the initial position by annealing stress or dissociating from DNA substrate. As the GC content increases to 100%, 79% helicases could not finish the unwinding process. Third, by further analysing the experimental data, two different slipping-back phenomena of T7 helicase are observed. One is instantaneous and the other is slow. The results from the experiment on magnetic tweezers also confirm this slow slipping-back phenomenon. This instantaneous slipping-back results from the rewinding process of unwound single-stranded DNA as studied previously. When T7 helicase cannot continue unwinding because of the high GC content in DNA sequence, it dissociates from the single-stranded DNA or slips back to the initial position very quickly because of the annealing stress. However, this slow slipping-back phenomenon cannot be explained by this reason. According to previous researches, T7 helicase can only be translocated or unwound from 5' to 3' along one strand of double-stranded DNA because of the polarity principle. We suggest that this slow slipping-back is induced by the strand switching process of T7 helicase. Through this strand switching process, T7 helicase binds to the 3'-strand and are translocated along it from 5' to 3' to the initial position, results in this slow slipping-back phenomenon. This is the first time that the slow slipping-back phenomenon has been observed, which strongly suggests the strand switching process of T7 helicase. Based on our results and previous researches, we propose the model of this strand switching process and this model may be extended to all ring-shaped hexameric helicases.-
Keywords:
- single-molecule fluorescence resonance energy transfer /
- magnetic tweezers /
- T7 helicase /
- stand switching
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[1] Dillingham M S 2011 Biochem. Soc. Trans. 39 413
[2] Jankowsky E 2011 Trends Biochem. Sci. 36 19
[3] Bernstein K A, Gangloff S, Rothstein R 2010 Annu. Rev. Genet. 44 393
[4] Zhao D Y, Liu S Y, Gao Y 2018 Acta Biochim. Biophys. Sin. 146 1093
[5] Klaue D, Kobbe D, Kemmerich F, Kozikowska A, Puchta H, Seidel R 2013 Nat. Commun. 4 2024
[6] 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
[7] Wang S, Qin W, Li J H, Lu Y, Lu K Y, Nong D G, Dou S X, Xu C H, Xi X G, Li M 2015 Nucleic Acids Res. 43 3736
[8] Dessinges M N, Lionnet T, Xi X G, Bensimon D, Croquette V 2004 Proc. Natl. Acad. Sci. USA 101 6439
[9] Sun B, Johnson D S, Patel G, Smith B Y, Pandey M, Patel S S, Wang M D 2011 Nature 478 132
[10] Johnson D S, Bai L, Smith B Y, Patel S S, Wang M D 2007 Cell 129 1299
[11] Zhao Z Y, Xu C H, Li J H, Huang X Y, Ma J B, Lu Y 2017 Acta Phys. Sin. 66 188701(in Chinese) [赵振业, 徐春华, 李菁华, 黄星榞, 马建兵, 陆颖 2017 66 188701]
[12] 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 62 168703]
[13] Lin W X, Ma J B, Nong D G, Xu C H, Zhang B, Li J H, Jia Q, Dou S X, Ye F F, Xi X G, Lu Y, Li M 2017 Phys. Rev. Lett. 119 138102
[14] Zhang H, Lee S J, Zhu B, Tran N Q, Tabor S, Richardson C C 2011 Proc. Natl. Acad. Sci. USA 108 9372
[15] Zhang H, Tang Y, Lee S J, Wei Z, Cao J, Richardson C C 2016 J. Biol. Chem. 291 1472
[16] Matson S W, Tabor S, Richardson C C 1983 J. Biol. Chem. 258 14017
[17] Ahnert P, Patel S S 1997 J. Biol. Chem. 272 32267
[18] Syed S, Pandey M, Patel S S, Ha T 2014 Cell Rep. 6 1037
[19] Donmez I, Patel S S 2008 EMBO J. 27 1718
[20] Jeong Y J, Levin M K, Patel S S 2004 Proc. Natl. Acad. Sci. USA 101 7264
[21] Patel S S, Picha K M 2000 Annu. Rev. Biochem. 69 651
[22] Morris P D, Raney K D 1999 Biochem. 38 5164
[23] Tabor S, Richardson C C 1981 Proc. Natl. Acad. Sci. USA 78 205
[24] Hacker K J, Johnson K A 1997 Biochem. 36 14080
[25] Korhonen J A, Gaspari M, Falkenberg M 2003 J. Biol. Chem. 278 48627
[26] Ahnert P, Picha K M, Patel S S 2000 EMBO J. 19 3418
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