Spontaneous deamination of DNA cytosine and adenine into uracil and hypoxanthine, respectively, causes C to T and A to G transition mutations if left unrepaired. Endonuclease Q (EndoQ) initiates the repair of these premutagenic DNA lesions in prokaryotes by cleaving the phosphodiester backbone 5′ of either uracil or hypoxanthine bases or an apurinic/apyrimidinic (AP) lesion generated by the excision of these damaged bases. To understand how EndoQ achieves selectivity toward these structurally diverse substrates without cleaving undamaged DNA, we determined the crystal structures of Pyrococcus furiosus EndoQ bound to DNA substrates containing uracil, hypoxanthine, or an AP lesion. The structures show that substrate engagement by EndoQ depends both on a highly distorted conformation of the DNA backbone, in which the target nucleotide is extruded out of the helix, and direct hydrogen bonds with the deaminated bases. A concerted swing motion of the zinc-binding and C-terminal helical domains of EndoQ toward its catalytic domain allows the enzyme to clamp down on a sharply bent DNA substrate, shaping a deep active-site pocket that accommodates the extruded deaminated base. Within this pocket, uracil and hypoxanthine bases interact with distinct sets of amino acid residues, with positioning mediated by an essential magnesium ion. The EndoQ–DNA complex structures reveal a unique mode of damaged DNA recognition and provide mechanistic insights into the initial step of DNA damage repair by the alternative excision repair pathway. Furthermore, we demonstrate that the unique activity of EndoQ is useful for studying DNA deamination and repair in mammalian systems.

如果不加以修复，DNA 胞嘧啶和腺嘌呤分别自发脱氨成尿嘧啶和次黄嘌呤，会导致 C 到 T 和 A 到 G 的转换突变。核酸内切酶 Q (EndoQ) 通过切割尿嘧啶或次黄嘌呤碱基的磷酸二酯骨架 5' 或通过切除这些受损碱基产生的无嘌呤/无嘧啶 (AP) 损伤来启动原核生物中这些诱变前 DNA 损伤的修复。为了了解 EndoQ 如何在不切割未受损 DNA 的情况下实现对这些结构多样的底物的选择性，我们确定了Pyrococcus furiosus的晶体结构EndoQ 与含有尿嘧啶、次黄嘌呤或 AP 病变的 DNA 底物结合。这些结构表明，EndoQ 与底物的结合取决于 DNA 骨架的高度扭曲构象，其中靶核苷酸被挤出螺旋，以及与脱氨基碱基的直接氢键。EndoQ 的锌结合结构域和 C 端螺旋结构域向其催化结构域的协同摆动运动允许酶夹住急剧弯曲的 DNA 底物，形成一个深的活性位点口袋，容纳挤出的脱氨基碱基。在这个口袋中，尿嘧啶和次黄嘌呤碱基与不同组的氨基酸残基相互作用，定位由必需的镁离子介导。EndoQ-DNA 复合结构揭示了一种独特的受损 DNA 识别模式，并为通过替代切除修复途径进行 DNA 损伤修复的初始步骤提供了机制见解。此外，我们证明 EndoQ 的独特活性可用于研究哺乳动物系统中的 DNA 脱氨基和修复。