SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Ave., Novosibirsk 630090, Russia.
Department of Natural Sciences, Novosibirsk State University, 2 Pirogova Street, Novosibirsk 630090, Russia.
Int J Mol Sci. 2022 Nov 1;23(21):13353. doi: 10.3390/ijms232113353.
Azide-alkyne cycloaddition ("click chemistry") has found wide use in the analysis of molecular interactions in living cells. 5-ethynyl-2-(hydroxymethyl)tetrahydrofuran-3-ol (EAP) is a recently developed apurinic/apyrimidinic (AP) site analog functionalized with an ethynyl moiety, which can be introduced into cells in DNA constructs to perform labeling or cross-linking in situ. However, as a non-natural nucleoside, EAP could be subject to removal by DNA repair and misreading by DNA polymerases. Here, we investigate the interaction of this clickable AP site analog with DNA polymerases and base excision repair enzymes. Similarly to the natural AP site, EAP was non-instructive and followed the "A-rule", directing residual but easily detectable incorporation of dAMP by DNA polymerase I Klenow fragment, bacteriophage RB69 DNA polymerase and human DNA polymerase β. On the contrary, EAP was blocking for DNA polymerases κ and λ. EAP was an excellent substrate for the major human AP endonuclease APEX1 and AP exonucleases Xth and Nfo but was resistant to the AP lyase activity of DNA glycosylases. Overall, our data indicate that EAP, once within a cell, would represent a replication block and would be removed through an AP endonuclease-initiated long-patch base excision repair pathway.
叠氮-炔环加成反应（“点击化学”）在分析活细胞中的分子相互作用方面得到了广泛应用。5-乙炔基-2-（羟甲基）四氢呋喃-3-醇（EAP）是一种最近开发的无嘌呤/无嘧啶（AP）位点类似物，其功能化有一个乙炔部分，可以在 DNA 构建体中引入细胞中，以进行原位标记或交联。然而，作为一种非天然核苷，EAP 可能会被 DNA 修复去除，并被 DNA 聚合酶误读。在这里，我们研究了这种可点击的 AP 位点类似物与 DNA 聚合酶和碱基切除修复酶的相互作用。与天然 AP 位点类似，EAP 是无指导的，并遵循“A 规则”，指导 DNA 聚合酶 I Klenow 片段、噬菌体 RB69 DNA 聚合酶和人 DNA 聚合酶 β 残留但容易检测到的 dAMP 掺入。相反，EAP 对 DNA 聚合酶 κ 和 λ 具有阻断作用。EAP 是人类主要的 AP 内切酶 APEX1 和 AP 外切酶 Xth 和 Nfo 的极好底物，但对 DNA 糖苷酶的 AP 裂合酶活性具有抗性。总的来说，我们的数据表明，EAP 一旦进入细胞，将代表一个复制阻断，并将通过 AP 内切酶起始的长补丁碱基切除修复途径被去除。
