Binding of AP endonuclease-1 to G-quadruplex DNA depends on the N-terminal domain, Mg(2+) and ionic strength.
Binding of AP endonuclease-1 to G-quadruplex DNA depends on the N-terminal domain, Mg(2+) and ionic strength.
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DOI：
10.1021/acsbiomedchemau.1c00031
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发表时间：
2021-12-15
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影响因子：
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通讯作者：
Burrows CJ
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文献类型：
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作者：
Fleming AM;Manage SAH;Burrows CJ
碱基切除修复酶无嘌呤/脱嘧啶核酸内切酶-1(APE1)也参与转录调控。当蛋白与启动子G-四链(G4)结合时，APE1可以在两条途径中发挥作用，该启动子带有一个基本位点(用四氢呋喃，F模拟)，导致非正则折叠上的酶停滞，以招募激活的转录因子。目前还缺乏研究APE1‘S与血管内皮生长因子启动子G4结合和催化活性的生化和生物物理研究，目前的工作提供了对这一主题的见解。在这里，天然的APE1被用于切割试验，而催化不活跃的突变体D210A被用于与双链DNA(DsDNA)与天然G4或G4与F在不同位置的结合试验，揭示了相互作用对阳离子浓度K+和Mg2+以及蛋白质的N-末端结构域的依赖关系。在0、1或10 mM镁离子中的测定发现，dsDNA和G4底物需要阳离子来结合和催化，其中G4结合随着[Mg2+]的增加而增加。用50 mM K+离子和140 mM生理K+离子进行的研究表明，含F的dsDNA被APE1结合和切割；而含有F的G4S在低盐中切割很差，在高盐中根本不切割，但结合仍然很强。使用Δ33或Δ61 N端截短的APE1蛋白，对双链DNA与F的结合和切割影响最小；相反，G4S需要N端才能结合，没有N端的催化几乎消失。有了这些知识，我们发现APE1可以在溶液中重塑含F的血管内皮生长因子启动子dsDNA→G4折叠。最后，添加G4配体吡哆醇他汀抑制含F的G4S的APE1结合和切割，但不抑制dsDNA。讨论了这一结果的生物和药物化学意义。
The base excision repair enzyme apurinic/apyrimidinic endonuclease-1 (APE1) is also engaged in transcriptional regulation. APE1 can function in both pathways when the protein binds to a promoter G-quadruplex (G4) bearing an abasic site (modeled with tetrahydrofuran, F) that leads to enzymatic stalling on the non-canonical fold to recruit activating transcription factors. Biochemical and biophysical studies to address APE1’s binding and catalytic activity with the vascular endothelial growth factor (VEGF) promoter G4 are lacking, and the present work provides insight on this topic. Herein, the native APE1 was used for cleavage assays, and the catalytically inactive mutant D210A was used for binding assays with double-stranded DNA (dsDNA) versus the native G4 or the G4 with F at various positions, revealing dependencies of the interaction on the cation concentrations K+ and Mg2+ and the N-terminal domain of the protein. Assays in 0, 1, or 10 mM Mg2+ found that dsDNA and G4 substrates required the cation for both binding and catalysis, in which G4 binding increased with [Mg2+]. Studies with 50 versus physiological 140 mM K+ ions showed that F-containing dsDNA was bound and cleaved by APE1; whereas, the G4s with F were poorly cleaved in low salt and not cleaved at all at higher salt while the binding remained robust. Using Δ33 or Δ61 N-terminal truncated APE1 proteins, the binding and cleavage of dsDNA with F was minimally impacted; in contrast, the G4s required the N-terminus for binding and catalysis is nearly abolished without the N-terminus. With this knowledge, we found APE1 could remodel the F-containing VEGF promoter dsDNA→G4 folds in solution. Lastly, the addition of the G4 ligand pyridostatin inhibited APE1 binding and cleavage of F-containing G4s but not dsDNA. The biological and medicinal chemistry implications of the results are discussed.