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G-四联体（G-quadruplex，G4）是广泛存在于细胞基因组中的一种DNA结构，在DNA的代谢如复制、转录、同源重组等过程中起重要作用.G4解旋酶近年来受到广泛研究，其中Bloom（BLM）解旋酶的研究已经相当丰富，但仍有一些基本问题不清楚.我们应用全内反射瞬逝场照明磁镊对BLM解旋G4的动力学过程进行了深入研究，观察到了BLM解旋G4的分步过程.相对于单分子荧光共振能量转移技术而言，借助磁镊的长时间观测性能，我们在近饱和三磷酸腺苷（ATP）浓度的实验体系中观察到BLM长时间反复解开G4或者长时间维持G4于打开状态的两种作用方式.最后，使用相同的实验条件做了单分子荧光共振能量转移实验，确定了加载2–3 pN的外力对BLM解旋G4没有显著影响.G-quadruplex (G4) is a DNA structure which commonly exists in human genome, and it is considered as an important structure in DNA metabolism such as replication, transcription and homologous recombination. The G-quadruplex helicases have been widely investigated these years. Of them, the Bloom (BLM) helicase is most thoroughly studied. However, there are some basic problems that are still unclear. Most of previous studies of G4 are performed by single molecule fluorescence resonance energy transfer technique. The G4 is in a free state in these experiments, which is different from the physiological environment in cells. The traditional magnetic tweezers have a limitation of spatial resolution in a low force circumstance. Thus here we use high resolution magnetic tweezer under the illumination of total internal reflection fluorescence to study the process of BLM resolving G4. Our modification of magnetic tweezer is to separate the measurements of force and distance of magnetic tweezer in order to improve the spatial resolution, which allows us to observe the unfolding process of G4. With a 2-3 pN force we find that the process of BLM unfolding G4 in low ATP concentration is stepwise, and the G4 is mainly in the state between G-quadruplex and G-triplex. We also find that the BLM could interact with G4 for a long time. Our apparatus is also able to obtain the long time observation results compared with the single molecule fluorescence technique. So we perform experiments with a nearly saturated ATP concentration. We find that the BLM has two ways to maintain G4 dissolution in this condition. The BLM could unfold the G4 repetitively in a long period and it could also keep the G4 in unfolding state for a long time after it has opened the G4. Finally, we also perform single molecule fluorescence resonance energy transfer experiment in the same condition, and we find that the 2-3 pN force in magnetic tweezers has a rare influence on the process of BLM interacting with G4. The results of single molecule fluorescence resonance energy transfer experiments are corresponding to the results of magnetic tweezer in the same conditions. All of our experimental results show that ATP dependent BLM has a high affinity with G4 and BLM has a different way to resolve G4 in high ATP concentration. These results could provide new ideas of the mechanism of BLM resolving G4. Our modified magnetic tweezer shows its capacity in G4 single molecule study, and it could be a useful tool in the future single molecule studies.
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Keywords:
- Bloom helicase /
- G-quadruplex /
- total internal reflection /
- magnetic tweezer
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[1] Phan A T 2010 FEBS J. 277 1107
[2] Maizels N, Gray L T 2013 PLoS Genet. 9 1003468
[3] Noer S L, Preus S, Gudnason D, Aznauryan M, Mergny J L, Birkedal V 2016 Nucl. Acids Res. 44 464
[4] Lim K W, Amrane S, Bouaziz S, Xu W X, Mu Y G, Patel D J, Luu K N, Phan A T 2009 J. Am. Chem. Soc. 131 4301
[5] Hansel R, Lohr F, Trantirek L, Dotsch V 2013 J. Am. Chem. Soc. 135 2816
[6] Li W, Hou X M, Wang P Y, Xi X G, Li M 2013 J. Am. Chem. Soc. 135 6423
[7] Koirala D, Mashimo T, Sannohe Y, Yu Z, Mao H, Sugiyama H 2012 Chem. Commun. 48 2006
[8] Balasubramanian S, Neidle S 2009 Curr. Opin. Chem. Biol. 13 345
[9] Huppert J L, Balasubramanian S 2005 Nucl. Acids Res. 33 2908
[10] Todd A K, Johnston M, Neidle S 2005 Nucl. Acids Res. 33 2901
[11] Croteau D L, Popuri V, Opresko P L, Bohr V A 2014 Annu. Rev. Biochem. 83 519
[12] Cheok C F, Bachrati C Z, Chan K L, Ralf C, Wu L, Hickson I D 2005 Biochem. Soc. Trans. 33 1456
[13] Goto M 2000 Clin. Exp. Rheumatol. 18 760
[14] Lindor N M, Furuichi Y, Kitao S, Shimamoto A, Arndt C, Jalal S 2000 Am. J. Med. Genet. 90 223
[15] German J, Sanz M A, Ciocci S, Ye T Z, Ellis N A 2007 Hum. Mutat. 28 743
[16] Ellis N A, Groden J, Ye T Z, Straughen J, Lennon D J, Ciocci S, Proytcheva M, German J 1995 Cell 83 655
[17] Wu L, Hickson I D 2003 Nature 426 870
[18] Xu Y N, Bazeille N, Ding X Y, Lu X M, Wang P Y, Bugnard E, Grondin V, Dou S X, Xi X G 2012 Nucl. Acids Res. 40 9802
[19] Sun H, Karow J K, Hickson I D, Maizels N 1998 J. Biol. Chem. 273 27587
[20] Budhathoki J B, Ray S, Urban V, Janscak P, Yodh J G, Balci H 2014 Nucl. Acids Res. 42 11528
[21] Chatterjee S, Zagelbaum J, Savitsky P, Sturzenegger A, Huttner D, Janscak P, Hickson I D, Gileadi O, Rothenberg E 2014 Nat. Commun. 5 5556
[22] Tippana R, Hwang H, Opresko P L, Bohr V A, Myong S 2016 Proc. Natl. Acad. Sci. USA 113 8448
[23] Wu W Q, Hou X M, Li M, Dou S X, Xi X G 2015 Nucl. Acids Res. 43 4614
[24] 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]
[25] Boncina M, Lah J, Prislan I, Vesnaver G 2012 J. Am. Chem. Soc. 134 9657
[26] Ambrus A, Chen D, Dai J X, Bialis T, Jones R A, Yang D Z 2006 Nucl. Acids Res. 34 2723
[27] Manosas M, Xi X G, Bensimon D, Croquette V 2010 Nucl. Acids Res. 38 5518
[28] Roy R, Hohng S, Ha T 2008 Nat. Meth. 5 507
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