Nucleic Acid Structure Research Group, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
Biochemistry. 2011 Nov 22;50(46):9963-72. doi: 10.1021/bi2011898. Epub 2011 Oct 26.
The four-way (Holliday) DNA junction is the central intermediate in homologous recombination. It is ultimately resolved into two nicked-duplex species by the action of a junction-resolving enzyme. These enzymes are highly selective for the structure of branched DNA, yet as a class these proteins impose significant distortion on their target junctions. Bacteriophage T7 endonuclease I selectively binds and cleaves DNA four-way junctions. The protein is an extremely stable dimer, comprising two globular domains joined by a β-strand bridge with each active site including amino acids from both polypeptides. The crystal structure of endonuclease I has been solved both as free protein and in complex with a DNA junction, showing that the protein, as well as the junction, becomes distorted on binding. We have therefore used site-specific spin-labeling in conjunction with EPR distance measurements to analyze induced fit in the binding of endonuclease I to a DNA four-way junction. The results support the change in protein structure as it binds to the junction. In addition, we have examined the structure of wild type and catalytically inactive mutants alone and in complex with DNA. We demonstrate the presence of hitherto undefined metastable conformational states within endonuclease I, showing how these states can be influenced by DNA-junction binding or mutations within the active sites. In addition, we demonstrate a previously unobserved instability in the N-terminal α1-helix upon active site mutation. These studies reveal that structural changes in both DNA and protein occur in the action of this junction-resolving enzyme.
四链 DNA 连接点是同源重组的中心中间体。它最终通过连接点解析酶的作用分解为两个带有缺口的双链体。这些酶对分支 DNA 的结构具有高度选择性，但作为一类，这些蛋白质会对其靶连接点造成显著的扭曲。噬菌体 T7 内切酶 I 选择性地结合并切割 DNA 四链连接点。该蛋白是一种极其稳定的二聚体，由两个球状结构域通过β-链桥连接而成，每个活性位点都包含来自两条多肽的氨基酸。内切酶 I 的晶体结构已经被解决，无论是在游离蛋白还是与 DNA 连接点的复合物中，都表明在结合时，蛋白和连接点都会发生扭曲。因此，我们使用了定点自旋标记与 EPR 距离测量相结合的方法来分析内切酶 I 与 DNA 四链连接点结合的诱导适应。结果支持了在结合连接点时蛋白结构的变化。此外，我们还单独研究了野生型和催化失活突变体的结构，以及它们与 DNA 的复合物。我们证明了内切酶 I 中存在迄今为止尚未定义的亚稳定构象状态，展示了这些状态如何受到 DNA 连接点结合或活性位点突变的影响。此外，我们还证明了在活性位点突变时，N 端α1-螺旋存在以前未观察到的不稳定性。这些研究揭示了在这种连接点解析酶的作用下，DNA 和蛋白质都会发生结构变化。
