[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70nt的单链DNA结合时,则形成1～3个SSB2-DNA复合物.这些结果说明SSB2在与长度小于70 nt的单链DNA结合时,存在着多种构型.而这些构型的形成取决于单链DNA的长度、蛋白的浓度,并与SSB蛋白所受的预处理温度和反应温度有关.SSB3与59nt和70 nt结合时,最多形成3个或4个复合物.低温保存和高温下反应对SSB3蛋白的功能影响比SSB2更为显著,表现为构型明显减少或结合ssDNA能力下降.此外,在腾冲嗜热菌中,SSB2和SSB3的表达水平随温度变化而变化,SSB2在45℃～65℃间表达水平很高,在75℃时表达水平下降,而SSB3在45℃时表达水平较低,在55℃到75℃间表达水平较高,说明二者在腾冲嗜热菌中的表达可能受到培养温度的调控.[结论]腾冲嗜热菌中SSB2和SSB3具有全新的底物结合特征及其不同的体内表达模式.%[Objective] SSB2 and SSB3 are ssDNA-binding proteins of Thermoanaerobacter tengcongensis. This work aimed to disclose novel properties of both proteins. [ Methods ] We performed electrophorefic 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 consplexes. 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 - 70℃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 tempe~ture of treatment. To our knowledge, this is the fast disclosure of the characteristics of SSB2 and SSB3 on 35-70 nt oligonucleotides.
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