Analysis of the Intrinsically Disordered N-Terminus of the DNA Junction-Resolving Enzyme T7 Endonuclease I: Identification of Structure Formed upon DNA Binding.
Analysis of the Intrinsically Disordered N-Terminus of the DNA Junction-Resolving Enzyme T7 Endonuclease I: Identification of Structure Formed upon DNA Binding.
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DOI：
10.1021/acs.biochem.6b00242
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发表时间：
2016-08-02
期刊：
影响因子：
2.9
通讯作者：
Norman DG
中科院分区：
文献类型：
--
作者：
Freeman AD;Stevens M;Declais AC;Leahy A;Mackay K;El Mkami H;Lilley DM;Norman DG
同源重组的四向（Holliday）DNA 连接是通过核酸酶对称切割两条链来处理的。这些连接解析酶以二聚体形式与四路连接结合，从而扭曲该过程中连接的结构。 T7 核酸内切酶 I 的晶体结构已确定为游离蛋白和具有 DNA 连接的复合物。在这两种晶体结构中，N 端 16 个氨基酸肽均不可见，但删除该肽对解析过程具有显着影响。在这里，我们通过在该区域内的独特位点包含自旋标记探针来研究 N 末端肽，并通过电子顺磁共振进行研究。连续波实验表明，这些标记在游离蛋白中是可移动的，但在结合 DNA 连接时受到限制，主要相互作用发生在残基 7-10 和 12 处。使用 PELDOR 测量二聚体两个肽内等效位置之间的距离表明，残基 2-12 的单体间距离很长且广泛分布在游离蛋白中，但显着缩短，并且在与 DNA 结合时变得更加明确。这些结果表明，N 端肽在与 DNA 连接点结合时变得更有组织性，并嵌入分支点的小沟中，这与表明在解析过程中发挥重要作用的生化数据一致。这项研究证明了蛋白质区域内存在晶体学无法观察到的结构。
The four-way (Holliday) DNA junction of homologous recombination is processed by the symmetrical cleavage of two strands by a nuclease. These junction-resolving enzymes bind to four-way junctions in dimeric form, distorting the structure of the junction in the process. Crystal structures of T7 endonuclease I have been determined as free protein, and the complex with a DNA junction. In neither crystal structure was the N-terminal 16-amino acid peptide visible, yet deletion of this peptide has a marked effect on the resolution process. Here we have investigated the N-terminal peptide by inclusion of spin-label probes at unique sites within this region, studied by electron paramagnetic resonance. Continuous wave experiments show that these labels are mobile in the free protein but become constrained on binding a DNA junction, with the main interaction occurring for residues 7–10 and 12. Distance measurements between equivalent positions within the two peptides of a dimer using PELDOR showed that the intermonomeric distances for residues 2–12 are long and broadly distributed in the free protein but are significantly shortened and become more defined on binding to DNA. These results suggest that the N-terminal peptides become more organized on binding to the DNA junction and nestle into the minor grooves at the branchpoint, consistent with the biochemical data indicating an important role in the resolution process. This study demonstrates the presence of structure within a protein region that cannot be viewed by crystallography.