嗜热脱铁去硫弧菌Pif1解旋酶生物学活性的分子特征分析

已知Pif1解旋酶在维持基因组稳定性方面发挥重要作用，且不同生物Pif1解旋酶具有不同的生物学活性；然而迄今为止，嗜热细菌Pif1解旋酶的生物学活性分子特征的研究尚未见报道。本文运用生物化学与生物物理学前沿技术，系统地研究了嗜热脱铁去硫弧菌Pif1解旋酶（Defe.Pif1）结合活性与解旋活性的分子特征。通过原核表达纯化系统，本研究获得纯度95%以上、无标签的Defe.Pif1蛋白。利用荧光偏振技术研究Defe.Pif1结合反应的底物特异性，揭示出Defe.Pif1优先结合ssDNA与G4 DNA，并对含3′-尾链的部分双链底物有较强亲和力，其结合反应底物特异性为：ssDNA > G4 DNA > 3′-ssDNA-dsDNA≈Y-structure > Other substrates。通过快速停留检测技术研究Defe.Pif1的解旋活性，明确其最适解旋温度为50℃，最佳反应溶液为10 mmol/L NaCl、3 mmol/L DTT、3 mmol/L MgCl2及1 mmol/L ATP；进一步的解旋动力学特征分析结果显示，Defe.Pif1可以高效解旋含G4结构的DNA底物，其解旋5′-G4 dsDNA底物时的解旋幅度超过90%，解旋速率也与其解旋5′-ss-dsDNA底物的速率相近，提示Defe.Pif1解旋G4 DNA更接近Bs.Pif1的单体反应模式。此外，本研究还发现Defe.Pif1解旋不同类型复制叉/复制泡底物时拥有独特的解旋倾向性：与解旋其它复制中间体DNA的低效性不同，Defe.Pif1解旋12nt bubble底物的速率与幅度均较高，暗示12nt bubble结构很可能是该解旋酶复制中间体的天然底物。上述结果证明，Defe.Pif1不仅具有Pif1解旋酶家族成员共同的结合与解旋G4 DNA等活性特征；而且作为嗜热细菌的解旋酶，它还具有独特的反应条件与解旋底物特异性。本研究为研究Pif1解旋酶家族其它成员的分子特征与生物功能提供了潜在的研究策略，为阐明此类Pif1解旋酶的分子作用机制奠定实验基础。

Molecular Characteristic Analysis of Biological Activity of Pif1 Helicase from Thermophile Deferribacter desulfuricans

The Pif1 helicases exert important biological function in the maintenance of genome stability, and the helicases in different species have different biologic activities. However, the molecular characteristics of biological activity of Pif1 helicase from thermophilic bacteria have not been reported so far. In this study, we applied a series of new biochemical and biophysical techniques to systematically analyze the molecular characteristics of binding and unwinding activities of Deferribacter desulfuricans Pif1 helicase. For this purpose we obtained no-tag Defe.Pif1 protein with above 95% purity by optimizing the expression in E.coli and protein purification system. Subsequently, the DNA binding properties of Defe.Pif1 were determined by fluorescence polarization method, which showed priority of Defe.Pif1 helicase bound with ssDNA and G4 DNA, and displayed relatively higher binding affinities for dsDNA substrates with 3′-overhang, which indicated that the substrates specificity for its binding reaction was ssDNA > G4 DNA > 3′-ssDNA-dsDNA≈Y-structure > Other substrates. To probe the unwinding activity of Defe.Pif1 helicase by stopped flow FRET assay, we first confirmed the optimal unwinding temperature was 50℃, and the best reaction buffer contained 10 mmol/L NaCl, 3 mmol/L DTT, 3 mmol/L MgCl2 and 1 mmol/L ATP. The further unwinding kinetic assays indicated that the unwinding amplitude of Defe.Pif1 with 5′-G4-dsDNA was more than 90%, and the reaction rate was similar with 5′-ss-dsDNA, which meant this helicase could effectively unwind G4 structure more likely through the way as the monomer response model of Bs.Pif1. Moreover, the unwinding reactions of Defe.Pif1 with different DNA replication intermediates revealed that the 12 nt bubble substrate showed much higher unwinding rate and amplitude, rather than other forks or bubbles, implied the 12 nt bubble could be a natural replication intermediate substrate for this helicase. Our results suggest that Defe.Pif1 not only shares the same biological functions with other Pif1 helicases, but also has special reaction conditions and substrate specificity. Our data also provide aid in the study of the molecular features and biological function of the Pif1 helicase-family,which is perhaps significant for elucidating the molecular mechanisms of these Pifl helicases.