Roles of Protein Structures on the DNA Recognition

为了分析DNA结合蛋白对DNA的特异性识别作用，我们用X射线和核磁共振的方法测定了许多DNA结合域的三维结构，例如原癌基因产物c-Myb，酵母磷酸代谢转录因子Ph4，大肠杆菌复制终止因子Pho4，胸腺嘧啶二聚体修复酶T4核酸内切酶V，干扰素反应转录因子IRF2。在识别蛋白质的序列中，α螺旋位于DNA的主槽中，螺旋中的几个氨基酸与含有Watson/Crick型碱基对的特定碱基相互作用。而另一方面，识别受损DNA的T4内切酶V破坏了Watson/Crick类型的碱基对，一个碱基被翻出来。在c-Myb DNA结合域的情况下，用核磁共振研究了其自由态和DNA结合态的动态结构；在自由态下，柔性连接子连接两个亚域，每个亚域是一个结构独立的单元；而在DNA结合态，两个亚域固定在DNA的主槽中，协同识别特定的碱基序列。此外，我们还测定了RuvC、核糖核酸聚合酶亚基α、嘌呤抑制因子PURR、OmpR、PHOB、端粒重复序列结合因子hTRF1、通用转录因子TFIIE等几种脱氧核糖核酸结合蛋白的三维结构，并讨论了它们与脱氧核糖核酸的结合模式。

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.