灭癌素链霉菌中磷硫酰化修饰DNA结合结构域的功能分析  

Functional analysis of the sulfur-binding domain for specifically recognizing phosphorothioate modified DNA in Streptomyces gancidicus

作　　者：陈英 孔令新[1] 邓子新[1] 由德林[1] CHEN Ying;KONG Lingxin;DENG Zixin;YOU Delin(State Key Laboratory of Microbial Metobolism,School of Life Sciences&Biotechnology,Shanghai Jiao Tong University,Shanghai 200030,China)

机构地区：[1]上海交通大学生命科学技术学院,微生物代谢国家重点实验室,上海200030

出　　处：《微生物学报》2022年第2期543-555,共13页Acta Microbiologica Sinica

基　　金：国家自然科学基金(31630002,317700038)。

摘　　要：【目的】DNA磷硫酰化(phosphorothioation,PT)是由硫原子取代DNA骨架磷原子上的非桥联氧原子形成的一种新型DNA修饰。PT修饰除参与组成限制修饰系统外,其更为广泛的生物学功能仍有待揭示。PT修饰现有的检测方法操作复杂、成本高、耗时长,而具有操作简便、成本低、耗时短等特点的酶联免疫检测(enzyme-linked immunosorbent assay,ELISA)成为新PT检测方法开发的优选。灭癌素链霉菌(Streptomyces gancidicus)中具有天蓝色链霉菌中PT修饰依赖的IV型限制酶(ScoMcrA)的同源酶,暗示其同样含有特异性结合PT修饰DNA的硫结合结构域(sulfur binding domain,SBD)。本文对灭癌素链霉菌中的SBD(简称为Sg-SBD)与PT修饰DNA的亲和力进行定性检测与定量表征,评估其用于PT修饰ELISA方法开发的潜力。【方法】对ScoMcrA-SBD和Sg-SBD的氨基酸序列进行比对分析,利用ScoMcrA-SBD为模板进行同源建模。在大肠杆菌中异源表达Sg-SBD,利用纯化的Sg-SBD进行凝胶迁移(electrophoresis mobility shift assays,EMSA)检测。利用生物膜层干涉(biolayer interferometry,BLI)技术进行Sg-SBD结合PT修饰DNA的定量表征。【结果】生物信息学分析揭示了Sg-SBD含有结合硫原子的保守的氨基酸残基,同时表明Sg-SBD具有与ScoMcrA-SBD相似的结构。EMSA实验发现Sg-SBD可以结合PT修饰DNA形成明显的迁移带,初步证实Sg-SBD结合PT修饰DNA的活性。BLI数据显示,Sg-SBD具有比ScoMcrA-SBD更强的PT修饰DNA结合能力,其与PT修饰DNA的亲和力常数在nmol/L级别,并且Sg-SBD不结合非PT修饰DNA。【结论】Sg-SBD结合PT修饰DNA活性接近抗原-抗体的反应水平,可用于PT修饰DNAELISA快速检测方法的开发。[Objective]DNA phosphorothioation(PT)is a novel DNA modification in which a non-bridging oxygen atom on the phosphodiester bond is replaced with sulfur atom.PT modification serves as a constitute element of bacterial restriction-modification(R-M)defensive system,but more biological functions of PT modification are awaiting exploration.The methods currently used for the identification of PT-DNA are complicated,time-consuming and labor-intensive.Enzyme-linked immunosorbent assay(ELISA)has been known for its merits of easy handling,low cost and time-saving,which could be used for new PT-DNA identification method development.The homologous protein of PT-dependent REase ScoMcrA from Streptomyces coelicolor can be found in Streptomyces gancidicus,which suggested that this homologous protein presumably encodes the sulfur-binding domain(SBD)to specifically recognize sulfur atom on PT-DNA.In this study,the PT-DNA binding activity of SBD from S.gancidicus(Sg-SBD)was characterized qualitatively and quantitatively,respectively,which served as an assessment of its potential for the PT-DNA ELISA method development.[Methods]Sequence alignment of ScoMcrA-SBD and Sg-SBD was conducted.The homology modeling was used to compare the possible structure of Sg-SBD with that of ScoMcrA-SBD.ScoMcrA-SBD and Sg-SBD were heterologously expressed in E.coli and purified.Using the purified Sg-SBD,the electrophoresis mobility shift assays(EMSA)was conducted firstly.Then,the biolayer interferometry(BLI)analysis was used to confirm the binding affinity of Sg-SBD for PT-DNA quantitatively.[Results]Bioinformatics analysis revealed that Sg-SBD contains conserved amino acid residues that bind to sulfur atoms,and the structure of Sg-SBD was similar with that of ScoMcrA-SBD.The EMSA showed that Sg-SBD can bind PT-DNA and form an obvious migration band,suggesting its PT-DNA binding activity.BLI data further confirmed that Sg-SBD possessed higher binding affinity to PT-DNA than that of ScoMcrA-SBD.Moreover,Sg-SBD exhibited no affinity to non-PT-DNA.[Concl

关 键 词：DNA磷硫酰化修饰 硫结合结构域 凝胶迁移 生物膜层干涉 酶联免疫检测 

分 类 号：Q933[生物学—微生物学]