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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.
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Keywords:
- single-molecule fluorescence resonance energy transfer /
- magnetic tweezers /
- T7 helicase /
- stand switching
[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
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[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
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[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 物理学报 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 物理学报 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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[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 物理学报 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 物理学报 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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