Potent in vitro Interference of Fleroxacin in DNA-binding,Unwinding and ATPase Activities of Bloom Helicase  

作　　者：LUO Heng XU Hou Qiang CHEN Xiang DING Mei YANG Qi Xin LI Kun 

机构地区：[1]Key Laboratory of Animal Genetics,Breeding and Reproduction in Plateau Mountainous Region,Ministry of Education,College of Animal Science,Guizhou University [2]Guizhou Key Laboratory of Animal Genetics,Breeding and Reproduction,College of Animal Science,Guizhou University [3]Provincial Key Laboratory for Agricultural Pest Management of Mountainous Region,Guizhou University

出　　处：《Biomedical and Environmental Sciences》2013年第4期231-242,共12页生物医学与环境科学（英文版）

基　　金：supported by the National Basic Research Program of China (No.2010CB534912);the Doctoral Program of the Ministry of Education (No.200806570003);the Governor of Guizhou Province Talents Fund (No.200822);Guizhou University innovation fundsfor graduate student (No. Nongke 2012027)

摘　　要：Objective To study the effect of fleroxacin （FLRX） on biological properties of Bloom （BLM） helicase catalytic core （BLM 642-2290 helicase） in vitro and the molecular mechanism of interaction between the two molecules. Methods DNA-binding and unwinding activities of BLM 642-1290 helicase were assayed by fluorescence polarization and gel retardation assay under conditions that the helicase was subjected to different concentrations of FLRX. Effect of FLRX on helicase ATPase activity was analyzed by phosphorus-free assay based on a colorimetric estimation of ATP hydrolysis-produced inorganic phosphate. Molecular mechanism of interaction between the two molecules was assayed by ultraviolet and fluorescence spectra. Results The DNA unwinding and ATPase activities of BLM 642-1290 helicase were inhibited whereas the DNA-binding activity was promoted in vitro. A BLM-FLRX complex was formed through one binding site, electrostatic and hydrophobic interaction force. Moreover, the intrinsic fluorescence of the helicase was quenched by FLRX as a result of non-radioactive energy transfer. The biological activity of helicase was affected by FLRX, which may be through an allosteric mechanism and stabilization of enzyme conformation in low helicase activity state, disruption of the coupling of ATP hydrolysis to unwinding, and blocking helicase translocation on DNA strands. Conclusion FLRX may affect the biological activities and conformation of BLM 642-1290 helicase, and DNA helicase may be used as a promising drug target for some diseases.Objective To study the effect of fleroxacin （FLRX） on biological properties of Bloom （BLM） helicase catalytic core （BLM 642-2290 helicase） in vitro and the molecular mechanism of interaction between the two molecules. Methods DNA-binding and unwinding activities of BLM 642-1290 helicase were assayed by fluorescence polarization and gel retardation assay under conditions that the helicase was subjected to different concentrations of FLRX. Effect of FLRX on helicase ATPase activity was analyzed by phosphorus-free assay based on a colorimetric estimation of ATP hydrolysis-produced inorganic phosphate. Molecular mechanism of interaction between the two molecules was assayed by ultraviolet and fluorescence spectra. Results The DNA unwinding and ATPase activities of BLM 642-1290 helicase were inhibited whereas the DNA-binding activity was promoted in vitro. A BLM-FLRX complex was formed through one binding site, electrostatic and hydrophobic interaction force. Moreover, the intrinsic fluorescence of the helicase was quenched by FLRX as a result of non-radioactive energy transfer. The biological activity of helicase was affected by FLRX, which may be through an allosteric mechanism and stabilization of enzyme conformation in low helicase activity state, disruption of the coupling of ATP hydrolysis to unwinding, and blocking helicase translocation on DNA strands. Conclusion FLRX may affect the biological activities and conformation of BLM 642-1290 helicase, and DNA helicase may be used as a promising drug target for some diseases.

关 键 词：Biological activity BLM helicase FLEROXACIN Interaction mechanism 

分 类 号：R965[医药卫生—药理学]