IV型限制酶ScoMcrA中SRA结构域介导的二聚体化对硫结合结构域功能的影响机制

SET and RING-associated domain-mediated dimerization in type IV restriction enzyme ScoMcrA promotes the affinity of sulfur binding domain to sulfur modified DNA

摘要:

[背景] 部分细菌的DNA骨架会发生磷硫酰化修饰，硫结合结构域（Sulfur Binding Domain，SBD）可以特异性识别这种生理修饰。与绝大多数SBD-HNH双结构域核酸酶不同，ScoMcrA的SBD和HNH结构域中间插入了一个特异性识别5-甲基胞嘧啶（5mC）修饰DNA的SET and RING-Associated （SRA）结构域。晶体结构显示，单独的SBD是单体，而SBD-SRA是双体。[目的] 探究ScoMcrA中SRA结构域的存在对SBD识别硫修饰DNA的影响及影响方式。[方法] 凝胶迁移实验（Electrophoresis Mobility Shift Assay，EMSA）比较SBD、SBD-SRA对硫修饰DNA结合力的差异；对参与SBD-SRA二聚体化的关键氨基酸残基突变，并检测点突变对SBD-SRA蛋白二聚体化及结合硫修饰DNA的影响。[结果] 相较于SBD结构域，SBD-SRA双结构域对磷硫酰化修饰DNA的结合能力明显增强。对SBD-SRA双体互作界面进行单点突变基本不影响其对硫修饰DNA的结合，当二聚体化界面连续的L₂₆₁LGET₂₆₅突变成A₂₆₁AAAA₂₆₅时，突变体对硫修饰DNA的结合力下降到与SBD相似的水平。[结论] 根据EMSA实验结果可以初步判断，SRA结构域介导的SBD-SRA双体化能增强SBD对硫修饰DNA的结合力；L₂₆₁LGET₂₆₅是SRA结构域上影响SBD对硫修饰DNA结合力的关键氨基酸位点。

Abstract:

[Background] Some bacterial DNA skeleton undergo phosphorothioate modification, sulfur binding domain (SBD) can specifically recognize this physiological modification. Unlike most SBD-HNH di-domain nucleases, SET and RING-associated (SRA) domain, specifically recognize DNA 5-methylcytosine (5mC), is inserted between SBD and HNH domains of ScoMcrA. The crystal structures show that single SBD is a monomer and SBD-SRA is a dimer.[Objective] The effects of the presence of SRA domain on the binding of sulfur modified DNA by SBD, and the way SRA domain affect the phosphorothioated DNA recognition. [Methods] Electrophoresis mobility shift assay (EMSA) was applied to compare binding affinity of SBD and SBD-SRA to sulfur modified DNA respectively. The key amino acid residues involved in dimerization of SBD-SRA were mutated to examine the binding affinity of mutant proteins to phosphorothioated DNA. [Results] Compared with SBD domain alone, the di-domain protein SBD-SRA showed enhanced affinity to sulfur modified DNA. The single point mutation of ten amino acid residues at the dimer-forming interface of SBD and SRA domain seldom affect its binding affinity to sulfur modified DNA. By comparison, L₂₆₁LGET₂₆₅ are simultaneously mutated to A₂₆₁AAAA₂₆₅ on SBD-SRA, the binding affinity of the mutant to sulfur modified DNA decreased to a level similar to that of SBD. [Conclusion] According to EMSA results, we primarily came to the conclusion that SRA domain can improve the binding ability of SBD to sulfur modified DNA in SBD-SRA di-domain protein; L₂₆₁LGET₂₆₅ is the key amino acid sites in the SRA domain that affects the binding ability of SBD to sulfur modified DNA.