段晓雷,许欢,姚淼,曾洁,申丽,刘娜女,张涛,肖代敏
DUAN Xiaolei,XU Huan,YAO Miao,ZENG Jie,SHEN Li,LIU Nanv,ZHANG Tao,XIAO Daimin

• 1:遵义医学院检验医学院
• 2:西北农林科技大学生命科学学院

摘要(Abstract)：

目的:探究嗜热厌氧杆菌的Ana. Pif1解旋酶核心结构域蛋白(Ana. Pif1-HD)与DNA底物结合的反应特性。方法:利用DNAman、Cluxtal X等软件,对Ana. Pif1的核心结构域蛋白进行系统进化树分析与同源序列比对; Ana. Pif1-HD表达载体构建后转入大肠杆菌进行原核表达,并通过Ni-NTA、SUMO酶切、S200分子筛等层析柱纯化获得Ana. Pif1-HD蛋白;利用Stopped-flow FRET技术监测Ana. Pif1-HD蛋白的解旋活性,利用荧光偏振检测技术分析Ana. Pif1-HD与不同长度、不同结构DNA底物的结合反应各向异性值,并进行拟合分析与统计。结果:Ana. Pif1的核心结构域蛋白为靠近N-端的448氨基酸,通过原核表达与纯化获得纯度为97%、浓度为5 g/L的Ana. Pif1-HD蛋白,并利用Stopped-flow FRET与荧光偏振等技术验证其具有Ana. Pif1全长蛋白90%的解旋活性与95%的结合活性; Ana. Pif1-HD的最佳结合反应条件是42℃在pH 6. 0含20 mmol/L Na Cl及3 mmol/L MgCl2的溶液中进行; Ana. Pif1-HD与不同长度ss DNA底物结合时的解离速率常数递增,并明确其ssDNA的binding-size为10. 34 nt;研究首次揭示Ana. Pif1-HD与不同复制中间体DNA结合反应的底物特异性ssG4 DNA> G4 DNA> 3'-ssDNA-dsDNA≈Y-型>其它底物。结论:成功表达纯化具有全长活性的Ana. Pif1解旋酶核心结构域蛋白,首次揭示该解旋酶核心蛋白与各类不同DNA底物的结合反应特性。
Objective: To explore the characteristics of binding reactions of Anaerobaculum hydrogeniforman Pif1 helicase's core domain(Ana. Pif1-HD). Methods: The conserved Ana. Pif1-HD was analyzed through homology sequence alignment and phylogenetic tree by bioinformatics software DNAman and CluxtalX. The constructed Ana. Pif1-HD was inducible expressed in E. coli,and purified through Ni-NTA,SUMO protease cleaved,Superdex 200 gel filtration chromatography. Stopped-flow FRET was used to detect the unwinding activity of Ana. Pif1-HD,and the characteristics of such helicase binding reactions were mainly measured by fluorescence polarization technique. Result: The Ana. Pif1-HD protein was confirmed as 448 aa in the N-terminal region. By inducible expression and purification,Ana. Pif1-HD protein with 97% purity and 5 mg/m L concentration was obtained,and this purified product was confirmed with above 90% unwinding activity and 95% binding activity. The optimum binding conditions of Ana. Pif1-HD were pH = 6. 0 PBS buffer contains 20 mmol/L NaCl and3 mmol/L MgCl2 under 42 ℃. Our research showed the dissociation rate constant(Kd) gradually increased with the increase in length of ss DNA,and the binding-size of Ana. Pif1-HD with ss DNA was10. 34 nt. Furthermore,the DNA-binding substrates specificity of Ana. Pif1-HD was revealed that ssG4 DNA > G4 DNA > 3'-ssDNA-dsDNA≈Y-structure > Other substrates. Conclusions: The Ana. Pif1-HD with Ana. Pif1-full protein's activity is successfully purified,and this research systematically analyzes the binding characteristics of the Ana. Pif1 helicase for the frist time. It is helpful in providing good experimental base for the further elucidation of the molecular mechanism of thermophiles' helicases.

关键词(KeyWords)： Pif1解旋酶;嗜热厌氧棒菌;荧光偏振技术;结合反应
Pif1 helicase;Anaerobaculum hydrogeniforma;fluorescence polarization method;binding reaction

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(31660241);; 贵州省科技合作基础研究项目[黔科合基础(2018)1196];; 遵义医学院博士科研启动资金[院字(2017)05号]

作者(Author): 段晓雷,许欢,姚淼,曾洁,申丽,刘娜女,张涛,肖代敏
DUAN Xiaolei,XU Huan,YAO Miao,ZENG Jie,SHEN Li,LIU Nanv,ZHANG Tao,XIAO Daimin

DOI: 10.19367/j.cnki.1000-2707.2018.11.006

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