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RecQ家族解旋酶是DNA解旋酶中高度保守的一个重要家族,参与DNA复制、修复、重组、转录及维持端粒稳定等细胞代谢过程,在维持染色体稳定性与完整性中起着重要作用.甲磺酸培氟沙星(pefloxacin mesylate,PFM)是一种新型氟喹诺酮类抗菌药物,对一些革兰氏阴性菌具有明显的杀菌效果,临床上已广泛使用.本研究利用荧光偏振、自由磷检测技术研究PFM对大肠杆菌RecQ解旋酶的DNA结合活性、解链活性、ATPase活性的影响.结果表明,低浓度PFM可促进大肠杆菌RecQ解旋酶与ssDNA、dsDNA结合,达到一定量后PFM则抑制酶与DNA底物的结合,这种影响与DNA底物有关;PFM对RecQ解旋酶的DNA解链活性和ATP酶活性都具有抑制作用,但其抑制的效果有极显著差异(P<0.01):比较PFM对两种活性抑制的Ci值(对解链活性抑制的Ci值为(1.5±0.2)μmol/L,对ATP酶活性抑制的Ci值为(0.010±0.005)μmol/L)可知,PFM对大肠杆菌RecQ解旋酶ATPase活性的抑制强于其解链活性.这些结果可为研究以DNA解旋酶为药物靶标的分子机理奠定相关理论基础.
RecQ family helicases are a highly conserved family of DNA helicases that are involved in cell metabolism such as DNA replication, repair, recombination, transcription, and telomere stabilization and play an important role in maintaining chromosomal stability and integrity Pefloxacin mesylate (PFM) is a novel fluoroquinolone antibacterial drug which has obvious bactericidal effect on some Gram-negative bacteria and has been widely used clinically.In this study, we used fluorescence polarization, Phosphorus detection technology to study the effect of PFM on DNA-binding activity, chain-splitting activity and ATPase activity of Escherichia coli RecQ helicase.The results showed that low concentration PFM could promote the combination of Escherichia coli RecQ helicase and ssDNA and dsDNA, PFM inhibits the binding of enzymes to DNA substrates, which is related to DNA substrates. PFM has an inhibitory effect on DNA cleavage activity and ATPase activity of RecQ helicase, but its inhibitory effect is significantly different ( (P <0.01): The Ci values of PFM inhibition against two activities (Ci value of (1.5 ± 0.2) μmol / L for inhibition of melting activity and (0.010 ± 0.005) μmol / L), PFM unrolled Escherichia coli RecQ The inhibition of enzyme ATPase activity is stronger than its melting activity.These results can lay a theoretical foundation for the study of the molecular mechanism of DNA helicase as a drug target.