腾冲嗜热菌单链 DNA 结合蛋白 SSB2 和 SSB3 新的单链 DNA 结合特性

Novel ssDNA-binding properties of SSB2 and SSB3 from Thermoanaerobacter tengcongensis

摘要:

摘要：【目的】揭示腾冲嗜热菌中两个单链DNA结合蛋白SSB2和SSB3的全新的底物结合功能及其不同的体内表达模式。【方法】利用腾冲嗜热菌复制起始位点附近的长度较短的单链DNA为底物，采用非变性聚丙烯酰胺凝胶电泳及western blot方法，研究SSB2和SSB3体外单链DNA结合特征和体内表达模式。【结果】SSB2 与35nt的复制起始区单链DNA（ssDNA）结合, 形成单个SSB2-DNA复合物；当与59 nt ssDNA结合时，可以随着蛋白浓度的递增形成一个或两个SSB2-DNA复合物；而与70n

Abstract:

Abstract: [Objective] SSB2 and SSB3 are ssDNA-binding proteins of Thermoanaerobacter tengcongensis. This work aimed to disclose novel properties of both proteins. [Methods] We performed electrophoretic mobility shift assays (EMSAs) using oligonucleotides spanning the replication origin of T. tengcongensis and non-denaturing polyacrylamide gels. Western blotting assays were used to study the expression patterns of both proteins. [Results] SSB2 bound to 35-nt, 59-nt and 70-nt ssDNA spanning the replication origin and formed one, two or three DNA-protein complexes. The number of the SSB2-DNA complexes was determined by both the length of the ssDNA and the concentration of SSB2. SSB3 formed one more DNA-protein complex with 59-nt or 70-nt ssDNA in comparison with SSB2. Storage of the proteins at -70oC led to the disappearance of one SSB2-(70-nt) complex, or two SSB3-(59-nt) complexes or three SSB3-(70-nt) complexes in the EMSA, indicating the distinct loss of the SSBs’s conformations. Moreover, SSB2 and SSB3 displayed different expression patterns at variable incubation temperatures in vivo. [Conclusion] SSB2 and SSB3 could bind ssDNA with various conformations that were determined by the length of ssDNA, the concentration of the proteins, as well as the temperature of treatment. To our knowledge, this is the first disclosure of the characteristics of SSB2 and SSB3 on 35-70 nt oligonucleotides.