Objective Bloom’s syndrome is an autosomal recessive disorder characterized by genomic instability and a predisposition to many cancers. Mutations of the BLM gene （encoding a BLM helicase） may form a structure of the etiology of this disease. As a global pollutant, mercury poses a major threat to human health. The current study was conducted to elucidate the effects of Hg^2＋ on the structure and activity of BLM642‐1290 recombinant helicase, and to further explore the molecular mechanisms of mercury toxicity to the DNA helicase. Methods The effects of Hg^2＋ on biological activity and structure of BLM642‐1290 recombinant helicase were determined by fluorescence polarized, ultraviolet spectroscopic, and free‐phosphorus assay technologies, respectively. Results The helicase activity, the DNA‐binding activity, and the ATPase activity of BLM642‐1290 recombinant helicase were inhibited by Hg^2＋ treatment. The LMCT （ligand‐to‐metal charge transition） peaks of the helicase were enhanced with the increase of the Hg^2＋ level. The LMCT peaks of the same concentration of helicase gradually increased over time. Conclusions The biological activity of BLM642‐1290 recombinant helicase is inhibited by Hg^2＋ treatment. The conformation of the helicase is significantly altered by Hg^2＋ . There exist two binding sites between Hg^2＋ and the helicase, which are located in the amino acid residues 1063‐1066 and 940‐944 of the helicase, respectively.
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.
LUO HengXU Hou QiangCHEN XiangDING MeiYANG Qi XinLI Kun
