Department of Chemistry, University of Utah, 315 S 1400 East, Salt Lake City, Utah84112-0850, United States.
Anal Chem. 2022 Nov 1;94(43):15027-15032. doi: 10.1021/acs.analchem.2c02951. Epub 2022 Oct 21.
The binding interaction between the DNA repair enzyme apurinic/apyrimidinic endonuclease-1 (APE1) with promoter G-quadruplex (G4) folds bearing an abasic site (AP) can serve as a gene regulatory switch during oxidative stress. Prior fluorescence-based analysis in solution suggested APE1 binds the promoter G4 but whether this interaction was specific or not remained an open question. Second harmonic generation (SHG) was used in this work to measure the noncanonical DNA-protein binding interaction in a label-free assay with high sensitivity to demonstrate the interaction is ordered and specific. The binding of APE1 to the promoter G4 with AP sites modeled by a tetrahydrofuran analogue produced dissociation constants of ∼100 nM that differed from duplex and single-stranded DNA control studies. The SHG measurements confirmed APE1 binds the G4 folds in a specific manner resolving a remaining question regarding how this endonuclease with gene regulatory features engages G4 folds. The studies demonstrate the power of SHG to interrogate noncanonical DNA-protein interactions providing a foundational example for the use of this analytical method in future biochemical analyses.
DNA 修复酶脱嘌呤/脱嘧啶核酸内切酶 1（APE1）与携带碱基缺失（AP）的启动子 G-四链体（G4）折叠之间的结合相互作用可以作为氧化应激过程中的基因调控开关。之前在溶液中的荧光分析表明 APE1 结合启动子 G4，但这种相互作用是否具有特异性仍是一个悬而未决的问题。这项工作中使用二次谐波产生（SHG）在无标记测定中以高灵敏度测量非规范的 DNA-蛋白质结合相互作用，以证明该相互作用是有序和特异的。APE1 与 AP 位点模拟的四氢呋喃类似物的 启动子 G4 的结合产生的离解常数约为 100 nM，与双链和单链 DNA 对照研究不同。SHG 测量结果证实 APE1 以特异的方式结合 G4 折叠，解决了关于具有基因调控特征的这种内切酶如何与 G4 折叠结合的剩余问题。这些研究证明了 SHG 检测非规范 DNA-蛋白质相互作用的能力，为该分析方法在未来生化分析中的应用提供了基础范例。
