RecQ helicase-catalyzed DNA unwinding detected by fluorescence resonance energy transfer.
A fluorometric assay was used to study the DNA unwinding kinetics induced by Escherichia coli RecQ helicase. This assay was based on fluorescence resonance energy transfer and carried out on stopped-flow, in which DNA unwinding was monitored by fluorescence emission enhancement of fluorescein resulting from helicase-catalyzed DNA unwinding. By this method, we determined the DNA unwinding rate of RecQ at different enzyme concentrations. We also studied the dependences of DNA unwinding magnitude and rate on magnesium ion concentration. We showed that this method could be used to determine the polarity of DNA unwinding. This assay should greatly facilitate further study of the mechanism for RecQ-catalyzed DNA unwinding.
Zhang
Xing-Dong
XD
Laboratory of Soft Matter Physics, Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100080, China.
Dou
Shuo-Xing
SX
Xie
Ping
P
Wang
Peng-Ye
PY
Xi
Xu Guang
XG
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D004247:DNA / Q000187:drug effects*; D004265:DNA Helicases / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D007700:Kinetics; D008274:Magnesium / Q000008:administration & dosage
D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D008274:Magnesium