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Bloom综合征解旋酶(BLM)是RecQ家族DNA解旋酶中的一个重要成员,参与了DNA复制、修复、转录、重组以及端粒的维持等细胞代谢过程,在维持染色体的稳定性中具有重要作用.BLM解旋酶的突变可导致Bloom综合征.Bloom综合征是一种罕见隐性常染色体遗传疾病,患者遗传不稳定,并易患多种类型癌症.洛美沙星(LMX)可以抑制细胞内多种酶,并通过结合DNA干扰DNA代谢,从而治疗多种疾病,但是其具体的作用机理还未完全清楚.运用荧光偏振技术和自由磷检测技术,研究了LMX对BLM642～1290解旋酶DNA结合活性、解链活性和ATP酶活性的影响.运用荧光及紫外吸收光谱法研究了LMX与解旋酶结合的结合常数、结合位点数、作用力类型、结合距离等参数.结果表明,LMX与解旋酶之间能自发进行反应,两种分子有一个结合位点,通过静电引力和疏水作用力形成稳定的BLM-LMX复合物,且解旋酶的内源荧光被LMX静态猝灭,主要原因是非辐射能量转移.在这一过程中,LMX能抑制解旋酶的解链活性和ATP酶活性,而促进解旋酶的DNA结合活性.LMX对BLM解旋酶生物学活性影响的机理可能是LMX使解旋酶通过别构机制影响其ATP酶活性,并使酶的构象维持在较低解链活性的状态,通过抑制ATP催化水解与解链过程的偶联和阻止解旋酶的易位,从而抑制其解链.LMX能够促进解旋酶的DNA结合活性,可能是因为其C-6和C-7上的取代功能基团可以增加酶活力,以及增强药物、酶和DNA的结合,从而形成药物-酶-DNA复合物.这些结果为研究以DNA解旋酶为药物靶标的分子机理和理解喹诺酮类药物的作用机理奠定相关理论基础.
Bloom Syndrome Helicase (BLM), an important member of the RecQ family of DNA helicases, is involved in cellular metabolism such as DNA replication, repair, transcription, recombination and telomere maintenance and has the potential to maintain chromosomal stability An important role.BLM helicase mutations can lead to Bloom syndrome.Bloom syndrome is a rare recessive autosomal genetic disease in patients with genetic instability and susceptible to many types of cancer Lomefloxacin (LMX) can inhibit Many kinds of enzymes in the cell and interfere with DNA metabolism by DNA, so as to treat a variety of diseases, but its specific mechanism of action is not fully understood.Using fluorescence polarization and free-phosphorus detection technology, we studied the effect of LMX on the BLM642 ~ 1290 Enzyme DNA binding activity, melting activity and ATPase activity.The binding constants of LMX and helicase, the number of binding sites, the type of force, the binding distance and other parameters were studied by fluorescence and UV absorption spectroscopy.The results showed that, LMX and helicase spontaneously react between the two molecules have a binding site by electrostatic attraction and hydrophobic force to form a stable BLM-LMX complex, and helicase endogenous fluorescence was LMX static quenching, mainly due to non-radiative energy transfer.In this process, LMX can inhibit helicase unraveling activity and ATPase activity, and promote helicase DNA binding activity.LMX BLM helicase biological The mechanism of learning activity may be that LMX causes helicase to affect its ATPase activity through allosteric mechanisms and maintain the conformation of the enzyme in a state of low melting activity by inhibiting the coupling between ATP catalyzed hydrolysis and the melting process Prevents the unwinding of the helicase and thus inhibits its unwinding.LMX can promote the DNA binding activity of helicase possibly because its substituted functional groups at C-6 and C-7 can increase the enzyme activity and enhance the drug , Enzyme and DNA to form drug-enzyme-DNA complexes.These results lay the theoretical foundation for the study of the molecular mechanism of DNA helicase as a drug target and the mechanism of understanding quinolones.