| 摘要: |
| 【目的】研究牛（Bos taurus）解旋酶DHX36（BtDHX36）及其突变体蛋白对含有G4结构底物结合和解旋活性的差异，为深入研究牛DHX36酶学特征和结构提供理论参考。【方法】制备底物16nt-ssDNA、Telomere G4和Telomere G4-16T，构建重组质粒pET15b-sumo-BtDHX36，通过Overlap PCR引入点突变，构建位于RSM区和OB域的突变重组质粒，分别将质粒导入大肠杆菌BL21(DE3)菌株中诱导表达；经过Ni-NTA和Hi Trap SP HP柱纯化得到目的蛋白。以解离平衡常数为判定指标，利用荧光各向异性法(FA)对酶蛋白与底物结合时的KCl浓度（0，50和100 mmol/L）、反应温度（25，30和37 ℃）、MgCl2浓度（0，2和5 mmol/L）、pH（6.5，7.5和8.5）进行优化，确定BtDHX36的体外最佳底物结合条件，比较野生型BtDHX36及其突变体对含有G4结构底物结合活性的差异。以解旋比例和速率常数为判定指标，利用快速停流 荧光共振能量转移(FRET)技术，比较野生型BtDHX36及其突变体对G4结构的解旋活性差异。【结果】成功构建了野生型BtDHX36重组质粒pET15b-sumo-BtDHX36及其RSM区突变体BtDHX36R63AI65A、BtDHX36Y69A、BtDHX36K76AN77AK78A和OB域突变体BtDHX36Y862A的重组质粒，诱导表达纯化后得到了纯度大于95%的酶蛋白。确定了野生型BtDHX36的体外最佳底物结合条件为：KCl 50 mmol/L、MgCl2 2 mmol/L、20 mmol/L Tris-HCl pH=7.5、反应温度37 ℃。各突变位点均可降低酶蛋白与Telomere G4的结合活性。OB域的Y862A突变和RSM区K76AN77AK78A突变对酶蛋白解旋Telomere G4-16T活性均无明显影响；RSM区的R63AI65A和Y69A突变对酶蛋白解旋Telomere G4-16T均有较大影响。【结论】获得了高纯度的野生型BtDHX36及突变体酶蛋白，明晰了其体外最佳底物结合条件；RSM区的R63AI65A和Y69A突变均可明显降低酶蛋白解旋Telomere G4-16T的活性。 |
| 关键词: 牛解旋酶 DHX36解旋酶 解旋酶突变体 G4底物 |
| DOI： |
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| 基金项目:国家自然科学基金项目（31370798,31870788） |
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| Binding and unbinding activity of Bos taurus DHX36 and its mutant with substrates |
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GUO Qingqing,GUO Hailei,LIU Nanü,et al
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| Abstract: |
| 【Objective】The differences in binding and unbinding activities between Bos taurus DHX36 (BtDHX36) helicase and its mutant proteins to G4-containing substrates were investigated on the enzymatic characteristics and structure of Bos taurus DHX36.【Method】Substrates 16nt ssDNA,Telomere G4 and Telomere G4-16T were prepared.The recombinant plasmid pET15b-sumo-BtDHX36 was constructed and point mutations were introduced through Overlap PCR to construct mutant recombinant plasmids located in the RSM region and OB domain.Then,the plasmids were introduced into E. coli BL21 (DE3) strains for expression and the target protein was obtained after purification with Ni-NTA and Hi Trap SP HP columns.Taking the dissociation equilibrium constant as judgment index,the fluorescence anisotropy (FA) was used to optimize concentrations of KCl (0,50 and 100 mmol/L) and MgCl2 (0,2 and 5 mmol/L), reaction temperature (25,30 and 37 ℃) and pH (6.5,7.5 and 8.5).After the best substrate binding conditions of BtDHX36 in vitro were determined,the differences in binding activities of wild type BtDHX36 and its mutants to substrates containing G4 structure were obtained.Using the unbinding ratio and rate constant as judgment index,the rapid stop flow fluorescence resonance energy transfer (FRET) technique was used to compare the differences in unwinding activities of wild type BtDHX36 and its mutants against the G4 structure.【Result】The recombinant plasmids of wild type BtDHX36 recombinant plasmid pET15b-sumo-BtDHX36 and its RSM region mutants BtDHX36R63AI65A,BtDHX36Y69A,BtDHX36K76AN77AK78A and OB domain mutant BtDHX36Y862A were successfully constructed,and the enzyme protein with purity greater than 95% was obtained after induced expression and purification.The optimal substrate binding conditions of wild-type BtDHX36 in vitro were KCl 50 mmol/L,MgCl2 2 mmol/L,20 mmol/L Tris-HCl,pH=7.5,and reaction temperature 37 ℃.Each mutation site can reduce the binding activity of the enzyme protein and Telomere G4.The enzyme protein of Y862A mutation in the OB domain and the K76AN77AK78A mutation in the RSM region had no effect on the activity of unbound Telomere G4-16T.The R63AI65A and Y69A mutations in the RSM region had greater effects on the enzyme protein unbinding Telomere G4-16T.【Conclusion】The high purity wild-type BtDHX36 and mutant enzyme proteins were obtained and the optimal substrate binding conditions in vitro were determined.R63AI65A and Y69A mutations in the RSM region can significantly reduce activity of the enzyme protein unwinding Telomere G4 16T. |
| Key words: Bos taurus helicase DHX36 helicase helicase mutant G4 substrate |
