Roles of Protein Structures on the DNA Recognition

为了分析DNA结合蛋白的特异性DNA识别，我们利用x射线和核磁共振的方法确定了许多DNA结合域在其DNA结合状态下的三维结构，如原癌基因产物c-Myb，酵母磷酸盐代谢转录因子Pho4，大肠杆菌复制终止因子Tus，胸腺嘧啶二聚体修复酶T4内切酶V，干扰素应答转录因子IRF2。在识别蛋白质的序列中，α螺旋位于DNA的主要凹槽中，螺旋上的几个氨基酸与持有沃森/克里克型碱基对的特定碱基相互作用。而另一方面，T4内切酶V，识别受损DNA，破坏沃森/克里克型碱基对，碱基被翻转。在c-Myb dna结合域的情况下，利用核磁共振研究了其游离态和dna结合态的动态结构；在自由状态下，两个子结构域由一个柔性连接子连接，每个子结构域是一个结构独立的单元，而在DNA结合状态下，两个子结构域固定在DNA的主槽中，并协同识别特定的碱基序列。此外，我们还确定了几种dna结合蛋白的三维结构，包括RuvC、RNA聚合酶亚基α、嘌呤抑制因子PurR、OmpR、PhoB、端粒重复结合因子hTRF1、一般转录因子TFIIE等，并讨论了它们的dna结合模式。

In order to analyze the specific DNA-recognition of DNA-binding proteins, we have determined three dimensional structures of many DNA-binding domains in their DNA bound states, for example, a protooncogene product, c-Myb, a yeast phosphate metabolic transcription factor, Pho4, a E. coli replication termination factor, Tus, a thymine dimer repair enzyme, T4 endonuclease V, and an interferon responsive transcription factor, IRF2 by using X-ray and NMR methods. In the sequence recognizing proteins, an α helix is located in the major groove of DNA and several amino acids from the helix are interacting with specific bases holding the Watson/Crick type base pairs. While, on the other hand, T4 endonuclease V, which recognizes a damaged DNA, disrupts a Watson/Crick type base pair and a base is flipped out. In the case of c-Myb DNA-binding domain, the dynamic structures of its free and DNA-bound states have been investigated by using NMR ; in the free state a flexible linker connects two subdomains and each subdomain is a structurally independent unit, while, in the DNA-bound state the two subdomains are fixed in the major groove of DNA and recognize a specific base sequence cooperatively. In addition, we have determined three dimensional structures of several DNA-binding proteins including RuvC, RNA polymerase subunit α , purine repressor PurR, OmpR, PhoB, telomere repeat binding factor hTRF1, a general transcription factor TFIIE in their DNA-free states and their DNA-binding modes have been discussed.