RecQ Helicase-catalyzed DNA Unwinding Detected by Fluorescence Resonance Energy Transfer  

作　　者：Xing-Dong ZHANG Shuo-Xing DOU Ping XIE Peng-Ye WANG Xu Guang XI 

机构地区：[1]Laboratory of Soft Matter Physics, Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences Beijing 100080, China [2]Laboratoire de Biotechnologies et Pharmacologie Génétique Appliquée CNRS UMR 8113, Ecole Normale Supérieure de Cachan, 61 Avenue du Président Wilson, 94235 Cachan cedex, France

出　　处：《Acta Biochimica et Biophysica Sinica》2005年第9期593-600, ,共8页生物化学与生物物理学报（英文版）

基　　金：This work was supported by the grants from the National Natural Science Foundation of China (No.60025516 and No.10334100);the Innovation Project of the Chinese Academy of Sciences,and the Centre National de la Recherche Scientifique (CNRS)

摘　　要：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.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.

关 键 词：HELICASE DNA unwinding KINETICS STOPPED-FLOW fluorescence resonance energy transfer （FRET） 

分 类 号：Q939[生物学—微生物学]