title: Potent in vitro interference of fleroxacin in DNA-binding, unwinding and ATPase activities of Bloom helicase.
abstract: OBJECTIVE
  To study the effect of fleroxacin (FLRX) on biological properties of Bloom (BLM) helicase catalytic core (BLM642-1290 helicase) in vitro and the molecular mechanism of interaction between the two molecules.
  
  
  METHODS
  DNA-binding and unwinding activities of BLM642-1290 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 BLM642-1290 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.
  
  
  CONCLUSIONS
  FLRX may affect the biological activities and conformation of BLM642-1290 helicase, and DNA helicase may be used as a promising drug target for some diseases.
authors.affiliation: Key Laboratory of Animal Genetics, Breeding and Reproduction in Plateau Mountainous Region, Ministry of Education, College of Animal Science, Guizhou University, Guiyang 550025, Guizhou, China.
authors.forename: Heng
authors.initials: H
authors.lastname: Luo
authors.forename: Hou Qiang
authors.initials: HQ
authors.lastname: Xu
authors.forename: Xiang
authors.initials: X
authors.lastname: Chen
authors.forename: Mei
authors.initials: M
authors.lastname: Ding
authors.forename: Qi Xin
authors.initials: QX
authors.lastname: Yang
authors.forename: Kun
authors.initials: K
authors.lastname: Li
mesh_terms: D004247:DNA / Q000378:metabolism; D016576:Fleroxacin / Q000494:pharmacology*; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology*; D053484:RecQ Helicases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
chemical_list: D019384:Nucleic Acid Synthesis Inhibitors; D004247:DNA; C097151:Bloom syndrome protein; D053484:RecQ Helicases; D016576:Fleroxacin
keywords: BLM helicase; Biological activity; Fleroxacin; Interaction mechanism