摘要
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 helicase family is one of the important members of highly conservative DNA helicases. RecQ helicase participates in the cell metabolism processes including DNA replication, repair, recombination, transcription, telomere maintenance, and plays key roles in maintaining stability and integrity of chromosome. Pefioxacin mesylate （PFM） is a new quinolone antibacterial agent, displays the distinct bactericidal effect on some Gram-negative bacteria and has been widely applied in clinic. In thisstudy, the effects of PFM on DNA-binding activity, unwinding activity and ATPase activity of E. coli RecQ helicase were detected by fluorescence polarization, free phosphorus assay. Results showed that the lower concentration of PFM could promote the binding of E. coli RecQ helicase to ssDNA or dsDNA, however, the binding of the helicase with DNA substrates was inhibited by PFM after the concentration of PFM reached to a certain amount, and the effects of PFM on DNA-binding of the helicase was related with the DNA substrates. PFM could inhibit the DNA unwinding and ATPase activities of the RecQ helicase, but the inhibition showed a significant difference （P 〈 0. 01 ） by comparing the Ci values of PFM on two biochemical activities （ as for unwinding activity, Civalue was （ 1.5 ± 0. 2）μmol/L; as for ATPase activity, Ci value was （0. 010± 0. 005 ） μmol/L）. The inhibition of PFM on the ATPase activity was stronger than that of the DNA unwinding activity. The results may provide the relevant theoretical foundations for studying the molecular mechanism of DNA helicase as the drug targets.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2012年第12期1133-1140,共8页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家重点基础研究发展计划(973计划,No.2010CB534912)
教育部博士点基金(No.200806570003)
贵州省优秀人才省长资金(No.200822)
贵州省国际合作计划项目(No.黔科合外G字[2011]7008号)
贵州大学研究生创新基金(No.校研农2011010)资助~~
