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

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DNA ELISA快速检测方法的开发。

Abstract:

[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. [Conclusion] Sg-SBD could specifically bind PT-DNA, and the binding affinity could be comparable to that of antigen and antibody, which suggested the potent potential for ELISA method development for rapid detection of PT-DNA.