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The active site of the junction-resolving enzyme T7 endonuclease I

机译：结连接酶T7核酸内切酶I的活性位点

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摘要

Endonuclease I is a junction-resolving enzyme encoded by bacteriophage T7, that selectively binds and cleaves four-way DNA junctions. We have recently solved the structure of this dimeric enzyme at atomic resolution, and identified the probable catalytic residues. The putative active site comprises the side-chains of three acidic amino acids (Glu20, Asp55 and Glu65) together with a lysine residue (Lys67), and shares strong similarities with a number of type II restriction enzymes. However, it differs from a typical restriction enzyme as the proposed catalytic residues in both active sites are contributed by both polypeptides of the dimer. Mutagenesis experiments confirm the importance of all the proposed active site residues. We have carried out in vitro complementation experiments using heterodimers formed from mutants in different active site residues, showing that Glu20 is located on a different monomer from the remaining amino acid residues comprising the active site. These experiments confirm that the helix-exchanged architecture of the enzyme creates a mixed active site in solution. Such a composite active site structure should result in unilateral cleavage by the complemented heterodimer; this has been confirmed by the use of a cruciform substrate. Based upon analogy with closely similar restriction enzyme active sites and our mutagenesis experiments, we propose a two-metal ion mechanism for the hydrolytic cleavage of DNA junctions.
机译：核酸内切酶I是由噬菌体T7编码的连接分辨酶，它选择性地结合并切割四向DNA连接。我们最近在原子分辨率上解决了该二聚酶的结构，并确定了可能的催化残基。推定的活性位点包括三个酸性氨基酸（Glu20，Asp55和Glu65）的侧链以及赖氨酸残基（Lys67），并且与许多II型限制酶具有相似性。但是，它与典型的限制性酶不同，因为两个活性位点中的拟议催化残基均由二聚体的两个多肽贡献。诱变实验证实了所有提议的活性位点残基的重要性。我们已经使用由不同活性位点残基中的突变体形成的异二聚体进行了体外互补实验，表明Glu20与构成活性位点的其余氨基酸残基位于不同的单体上。这些实验证实了该酶的螺旋交换结构在溶液中产生了混合的活性位点。这种复合的活性位点结构应导致互补的异二聚体单方面切割。这已经通过使用十字形基板得到了证实。基于相似的限制性内切酶活性位点的相似性以及我们的诱变实验，我们提出了一种水解DNA连接的双金属离子机理。

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