Pif1 is a force-regulated helicase.
Pif1 is a prototypical member of the 5' to 3' DNA helicase family conserved from bacteria to human. It has a high binding affinity for DNA, but unwinds double-stranded DNA (dsDNA) with a low processivity. Efficient DNA unwinding has been observed only at high protein concentrations that favor dimerization of Pif1. In this research, we used single-molecule fluorescence resonance energy transfer (smFRET) and magnetic tweezers (MT) to study the DNA unwinding activity of Saccharomyces cerevisiae Pif1 (Pif1) under different forces exerted on the tails of a forked dsDNA. We found that Pif1 can unwind the forked DNA repetitively for many unwinding-rezipping cycles at zero force. However, Pif1 was found to have a very limited processivity in each cycle because it loosened its strong association with the tracking strand readily, which explains why Pif1 cannot be observed to unwind DNA efficiently in bulk assays at low protein concentrations. The force enhanced the unwinding rate and the total unwinding length of Pif1 significantly. With a force of 9 pN, the rate and length were enhanced by more than 3- and 20-fold, respectively. Our results imply that the DNA unwinding activity of Pif1 can be regulated by force. The relevance of this characteristic of Pif1 to its cellular functions is discussed.
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.
Jing-Hua
JH
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.
Wen-Xia
WX
Lin
College of Life Sciences, Northwest A & F University, Yangling, Shaanxi 712100, China.
Bo
B
Zhang
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.
Da-Guan
DG
Nong
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.
Hai-Peng
HP
Ju
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.
Jian-Bing
JB
Ma
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.
Chun-Hua
CH
Xu
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.
Fang-Fu
FF
Ye
College of Life Sciences, Northwest A & F University, Yangling, Shaanxi 712100, China LBPA, ENS de Cachan, CNRS, Université Paris-Saclay, F-94235 Cachan, France.
Xu Guang
XG
Xi
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.
Ming
M
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 yinglu@iphy.ac.cn.
Ying
Y
Lu
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 sxdou@iphy.ac.cn.
Shuo-Xing
SX
Dou
D000255:Adenosine Triphosphate / Q000737:chemistry; D004265:DNA Helicases / Q000737:chemistry*; D004271:DNA, Fungal / Q000737:chemistry; D007700:Kinetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*
D004271:DNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; C085463:PIF1 protein, S cerevisiae; D004265:DNA Helicases