野猪DHX36解旋酶酶学特性及保守性分析

Enzymatic Characteristics and Analysis of the Sus Scrofa DHX36 Helicase

DHX36解旋酶(DEAH-box helicase 36)在生物体内广泛存在,可作为外源核酸传感器广泛参与机体免疫反应,因其对G-四链体具有高度亲和性和解旋活性而备受关注。目前已报道了3个物种DHX36解旋酶的三维结构,但其酶学活性的保守性研究鲜见报道。本文以哺乳动物野猪(Sus scrofa)DHX36解旋酶(SsDHX36)为研究对象,通过生物化学与生物物理研究技术系统研究了其酶学性质,并对比其与低等无脊椎动物黑腹果蝇(Drosophila melanogaster)DHX36解旋酶(DmDHX36)的活性,探究DHX36在物种间的功能保守性。本研究通过原核表达系统,分离纯化得到了纯度大于95%的SsDHX36解旋酶;利用荧光偏振和快速停留技术得到SsDHX36的最佳酶学反应条件,发现SsDHX36对G4 DNA具有平行构型的亲和选择性,对富含鸟嘌呤的ssDNA具有结合和解旋的底物偏好性;结合单分子荧光共振能量转移技术对SsDHX36和DmDHX36进行活性比较,发现两者与G4 DNA结合的构型选择性和对富含鸟嘌呤DNA的底物偏好性是保守的。但两者对不同序列长度ssDNA的亲和性差异显著,SsDHX36受序列长度影响较大且亲和力随序列长度的增加而增强,而DmDHX36则完全相反。本文克隆纯化了SsDHX36解旋酶并研究了其酶学活性,着重分析了SsDHX36和DmDHX36的功能保守性,为理解DHX36的保守功能机制,探究基于DHX36与富含鸟嘌呤序列互作的免疫反应酶学活性基础和靶向药物设计提供了理论与实验基础。

The DEAH-box helicase 36(DHX36) widely exists in organisms and can be used as an exogenous nucleic acid sensor involved in the immune response. It has attracted considerable attention due to its high affinity and unwinding activity for G-quadruplexes. The three-dimensional structures of DHX36 helicases from three species have been reported, but the study of their conserved enzymatic activity is rarely reported. Taking the wild boar(Sus scrofa) DHX36 helicase(SsDHX36) as the research object, this paper systematically studied its enzymatic properties using biochemical and biophysical research techniques and compared its activity with that of the lower invertebrate Drosophila melanogaster DHX36 helicase(DmDHX36) to explore the functional conservation of DHX36 among species. In this study, the SsDHX36 helicase with a purity level above 95% was isolated and purified in a prokaryotic expression system. The optimal enzymatic reaction conditions of SsDHX36 were obtained by the fluorescence polarization method and the stopped-flow technique. It was discovered that SsDHX36 has an affinity with parallel topological selectivity for G4 DNA and a substrate preference for guanine-rich ssDNA in binding and unwinding. Combined with the single-molecular Forster Resonance Energy Transfer(FRET) technique, the activities of SsDHX36 and DmDHX36 were compared. It was found that they were conserved in topological selectivity for G4 DNA binding and the preference for guanine-rich DNA substrates. However, the results revealed that the two helicases differed significantly in the affinity for ssDNAs of different sequence lengths, with SsDHX36 being more influenced by sequence lengths and the affinity increasing with sequence length, while DmDHX36 was the opposite. In sum, SsDHX36 was cloned and purified, and then the enzymatic activity of SsDHX36 was systematically studied. The functional conservation of SsDHX36 and DmDHX36 was analyzed, which provided a theoretical and experimental basis for understanding the mechanism of DHX36. Our work will provide a basis for exploring the enzymatic activity of immune reactions based on the interaction between DHX36 and guanine-rich sequences, and the design of targeted drugs.