Recent advances in G-quadruplex (G4) studies have confirmed that G4 structures exist in living cells and may have detrimental effects on various DNA transactions. How helicases resolve G4, however, has just begun to be studied and remains largely unknown. In the present paper, we use single-molecule fluorescence assays to probe Pif1-catalysed unfolding of G4 in a DNA construct resembling an ongoing synthesis of lagging strand stalled by G4. Strikingly, Pif1 unfolds and then halts at the ss/dsDNA junction, followed by rapid reformation of G4 and ‘acrobatic’ re-initiation of unfolding by the same monomer. Thus, Pif1 unfolds single G4 structures repetitively. Furthermore, it is found that Pif1 unfolds G4 sequentially in two large steps. Our study has revealed that, as a stable intermediate, G-triplex (G3) plays an essential role in this process. The repetitive unfolding activity may facilitate Pif1 disrupting the continuously reforming obstructive G4 structures to rescue a stalled replication fork. The proposed mechanism for step-wise unfolding of G4 is probably applicable to other helicases that resolve G4 structures for maintaining genome stability.
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Research Article|
February 06 2015
Molecular mechanism of G-quadruplex unwinding helicase: sequential and repetitive unfolding of G-quadruplex by Pif1 helicase
Xi-Miao Hou;
Xi-Miao Hou
1
*College of Life Sciences, Northwest A & F University, Xi’an, Shaanxi 712100, China
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Wen-Qiang Wu;
Wen-Qiang Wu
1
*College of Life Sciences, Northwest A & F University, Xi’an, Shaanxi 712100, China
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Xiao-Lei Duan;
Xiao-Lei Duan
*College of Life Sciences, Northwest A & F University, Xi’an, Shaanxi 712100, China
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Na-Nv Liu;
Na-Nv Liu
*College of Life Sciences, Northwest A & F University, Xi’an, Shaanxi 712100, China
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Hai-Hong Li;
Hai-Hong Li
*College of Life Sciences, Northwest A & F University, Xi’an, Shaanxi 712100, China
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Jing Fu;
Jing Fu
*College of Life Sciences, Northwest A & F University, Xi’an, Shaanxi 712100, China
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Shuo-Xing Dou;
Shuo-Xing Dou
1
†Beijing National Laboratory for Condensed Matter Physics and CAS Key Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China
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Ming Li;
Ming Li
†Beijing National Laboratory for Condensed Matter Physics and CAS Key Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China
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Xu-Guang Xi
Xu-Guang Xi
*College of Life Sciences, Northwest A & F University, Xi’an, Shaanxi 712100, China
‡Laboratoire de Biologie et Pharmacologie Appliquée, Ecole Normale Supérieure de Cachan, Centre National de la Recherche Scientifique, 61 Avenue du Président Wilson, 94235 Cachan, France
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Publisher: Portland Press Ltd
Received:
August 04 2014
Revision Received:
December 02 2014
Accepted:
December 04 2014
Accepted Manuscript online:
December 04 2014
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2015 Biochemical Society
2015
Biochem J (2015) 466 (1): 189–199.
Article history
Received:
August 04 2014
Revision Received:
December 02 2014
Accepted:
December 04 2014
Accepted Manuscript online:
December 04 2014
Citation
Xi-Miao Hou, Wen-Qiang Wu, Xiao-Lei Duan, Na-Nv Liu, Hai-Hong Li, Jing Fu, Shuo-Xing Dou, Ming Li, Xu-Guang Xi; Molecular mechanism of G-quadruplex unwinding helicase: sequential and repetitive unfolding of G-quadruplex by Pif1 helicase. Biochem J 15 February 2015; 466 (1): 189–199. doi: https://doi.org/10.1042/BJ20140997
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