【作者】 赵正阳；

【导师】 范三红； 奚绪光；

【作者基本信息】 西北农林科技大学 ， 生物化学与分子生物学， 2014， 硕士

【摘要】 解旋酶是生物体内广泛存在的一类参与几乎所有核酸代谢过程的分子马达，通常能结合并水解ATP，并利用水解释放的能量打开核酸双链间的氢键，为复制、转录、修复和重组等过程提供单链模板或反应中间物。Pif1家族解旋酶是一类依赖ATP的5’－3’解旋酶，属于解旋酶超家族Ⅰ（SFⅠ）的一个亚家族，广泛存在于原核、真核生物及病毒。在生物体内，Pif1解旋酶影响端粒、rDNA及线粒体DNA的复制，参与冈崎片断加工，在维持染色体和线粒体DNA稳定性方面发挥重要功能，但其结构和具体的作用机制仍不明确。本研究构建了嗜热脱铁去硫弧菌（Deferribacter desulfuricans）Pif1解旋酶DePif1的原核表达载体，探索了其在大肠杆菌表达系统中的高效表达、纯化方案，并对其DNA结合与解旋活性进行系统分析。主要获得了以下研究结果：1.在pET15b载体的基础上，通过引入SUMO促溶标签，获得了在目的蛋白DePif1的N端依次含His-SUMO双标签的重组表达载体pET15b-SUMO-DePif1。此载体导入大肠杆菌BL21(DE3)菌株，28℃诱导培养，可使融合了双标签的DePif1高效表达，并且大部分以可溶性形式存在。2.利用HisTrapTMFF柱亲和层析获得融合蛋白，SUMO蛋白酶酶切去除融合标签，再经Heparin和Ni-NTA柱分离获得无标签重组DePif1。最终每升菌液可获得9mg纯度大于95%的DePif1解旋酶，这为Pif1家族解旋酶晶体的获得奠定了基础。3.采用荧光各向异性方法，分析了DePif1与DNA底物的结合活性。结果发现，pH为6.0，NaCl浓度为50mM，是DePif1与DNA底物较为合适的结合条件。在此条件下，DePif1能很好地结合单链底物(16nt ss-DNA)、双链底物(16bp ds-DNA)和G4底物(3layers)，其结合三种底物的Km值分别为3.02±0.65、18.82±1.70、2.71±0.57。这表明，DePif1对不同底物的结合强度依次为G4-DNA>单链DNA>双链DNA。4.使用基于荧光共振能量转移的stopped-flow技术分析DePif1对DNA底物的解旋活性。结果显示，在40℃，存在ATP和Mg2+的条件下，DePif1能有效解旋双链和G4底物，且DePif1对G4-DNA的解旋活性高于双链DNA。本研究成功表达并纯化了嗜热脱铁去硫弧菌Pif1解旋酶，并证明其具有特异的G4-DNA结合和解旋能力，为Pif1家族解旋酶结构和功能的阐明奠定了基础。更多还原

【Abstract】 Helicases are a class of ubiquitous molecular motor proteins found in all organisms thatparticipate in almost all aspects of the DNA metabolism. Generally， helicases manifest anATP binding and hydrolysis activity which is depended on the presence of DNA. Bycoupling the energy of ATP hydrolysis to the breakage of hydrogen bonds between duplexoligonucleotides, helicases provide the single strand DNA as templates or reactionintermediates which are required for DNA replication, transcription, repair and recombination.Different helicases share the conserved amino acid motifs. According to the diversity of thenumber and sequence of the motifs, helicase were classified into five super family. Based onthe directivity of unwinding reaction, helicases can also be divided into two types:5’－3’ or3’－5’ direction. It has been confirmed that the mutations of helicase genes always lead toserious genetic disease, such as Werner, Bloom and Fanconi syndrome, these findings makehelicase attracting an increasing attention.Pif1helicase family is a class of ATP-dependent,5’-3’ directed helicases which belongto helicase Super FamilyⅠ, and widely exist in prokaryotic, eukaryotic and viruses. In vivo,Pif1helicases affect the replication of telomere, rDNA and mitochondrial DNA, involves inthe processing of Okazaki fragment, play an important role in maintaining the stability ofthe chromosome and mitochondrial DNA. However, the structure and mechanism of Pif1helicase family remains unclear. In this study, we constructed the expression vector of DePif1,a Pif1helicase from thermophilic Deferribacter desulfuricans, based on expression vectorpET15b. Then, we explored the high-efficiency scheme for the expression and purification ofDePif1in E. coli expression system. Finally, the DNA binding and unwinding activities ofDePif1were analyzed. The main conclusions of this study are summarized as follows:1. Based on expression vector pET15b, we obtained the recombinant plasmidpET15b-SUMO-DePif1by introducing SUMO tag to the downstream of its His-tag. So,the recombinant DePif1fused His-tag and SUMO-tag orderly at its N-terminus. Thisplasmid was introduced into E. coli BL21(DE3) strain, induced and cultured at28℃.At this condition, recombinant DePif1has an high-efficiency expression and mostly exist in soluble form.2. DePif1with His6-SUMO tag was capture by affinity chromatography with a Ni-NTAcolumn firstly. Then, the fusion tag was cleaved by SUMO protease and tag-free DePif1helicase was obtained by further purification with Heparin Sepharose Fast Flow andNi-NTA chromatography. According to this protocol,9mg DePif1with>95%puritycould be obtained per liter culture.3. The DNA binding properties of DePif1was analyzed by Fluorescence anisotropy. Wefound, pH6.0,50mM NaCl, is the appropriate condition for the binding of DePif1withDNA substrates. At this condition, DePif1can well bind to ss-DNA, ds-DNA, G4-DNA,and the Kmvalue of these three substrates is3.02±0.65、18.82±1.70、2.71±0.57,respectively. This reveals that the binding strength of DePif1with different substrates isG4-DNA> single-stranded DNA> double-stranded DNA.4. The DNA unwinding activity of DePif1was studied by stopped-flow based on FRET.The results show that at40℃, in the presence of ATP and Mg2+conditions, DePif1can unwind double-stranded DNA and G4-DNA substrates effectively. However, theunwinding activity of DePif1to G4-DNA substrate is much higher than thedouble-stranded DNA substrate.In this study, We expressed and obtained purified DePif1helicase successfully. Activityanalysis of DePif1indicates that it can bind and unwind G4-DNA specially. These will laythe foundation for the elucidating of the structure and function of Pif1family helicase更多还原