Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712, United States.
Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas 78712, United States.
Bioconjug Chem. 2023 Jun 21;34(6):983-987. doi: 10.1021/acs.bioconjchem.3c00156. Epub 2023 May 15.
Histones catalyze the DNA strand incision at apurinic/apyrimidinic (AP) sites accompanied by formation of reversible but long-lived DNA-protein cross-links (DPCs) at 3'-DNA termini within single-strand breaks. These DPCs need to be removed because 3'-hydroxyl is required for gap-filling DNA repair synthesis but are challenging to study because of their reversible nature. Here we report a chemical approach to synthesize stable and site-specific 3'-histone-DPCs and their repair by three nucleases, human AP endonuclease 1, tyrosyl-DNA phosphodiesterase 1, and three-prime repair exonuclease 1. Our method employs oxime ligation to install an alkyne to 3'-DNA terminus, genetic incorporation of an azidohomoalanine to histone H4 at a defined position, and click reaction to conjugate DNA to H4 site-specifically. Using these model DPC substrates, we demonstrated that the DPC repair efficiency is highly affected by the local protein environment, and prior DPC proteolysis facilitates the repair.
组蛋白在无碱基/无嘧啶（AP）位点催化 DNA 链的切口，同时在单链断裂的 3'-DNA 末端形成可逆但长寿命的 DNA-蛋白质交联（DPC）。这些 DPC 需要被去除，因为 3'-羟基对于缺口填充 DNA 修复合成是必需的，但由于其可逆性质，很难进行研究。在这里，我们报告了一种化学方法来合成稳定的、定点的 3'-组蛋白-DPC，并通过三种核酸酶，即人 AP 内切核酸酶 1、酪氨酰-DNA 磷酸二酯酶 1 和 3'-末端修复外切核酸酶 1 对其进行修复。我们的方法采用肟连接将炔烃安装到 3'-DNA 末端，在特定位置将叠氮基同型丙氨酸遗传整合到组蛋白 H4 上，并通过点击反应将 DNA 特异性地连接到 H4 位点。使用这些模型 DPC 底物，我们证明了 DPC 修复效率受到局部蛋白质环境的高度影响，并且 DPC 蛋白水解有利于修复。
