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

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

作　　者：张树利[1,2] 杨海花[1] 李磊[1] 田宇清[1] 谭华荣[1] 

机构地区：[1]中国科学院微生物研究所,北京100101 [2]中国科学院研究生院,北京100039

出　　处：《微生物学报》2009年第4期453-459,共7页Acta Microbiologica Sinica

基　　金：Supported by the National Natural Science Foundation of China (30621005);the National Programs for High Technology Research and Development of China (2009CB118905)~~

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59ntssDNA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70nt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70nt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 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 - 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 warious 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 East disclosure of the characteristics of SSB2 and SSB3 on 35-70 nt oligonucleotides.

关 键 词：单链DNA结合蛋白 蛋白构型 蛋白表达模式 

分 类 号：Q93[生物学—微生物学]