Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712, United States; Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, United States.
Center for Biomedical Research Support, Biological Mass Spectrometry Facility, The University of Texas at Austin, Austin, TX 78712, United States.
DNA Repair (Amst). 2022 Nov;119:103391. doi: 10.1016/j.dnarep.2022.103391. Epub 2022 Aug 27.
The apurinic/apyrimidinic (abasic, AP) site is one of the most abundant DNA lesions. Previous studies by others demonstrated that human AlkB homologue 1 (ALKBH1) catalyzes the DNA strand incision at an AP site, resulting in suicidal cross-linking of the enzyme to the 3'-DNA end. Prior site-directed mutagenesis experiments had reported that Cys129 of ALKBH1 is the predominant nucleophile that conjugates to the C3' position of the incised AP site, 3'-phospho-α,β-unsaturated aldehyde (3'-PUA), to form a 3'-PUA-ALKBH1 cross-link. However, direct evidence to support this mechanism was lacking. The 3'-PUA-ALKBH1 cross-link is so far the only adduct that has been found to form via a Michael addition reaction between a protein and 3'-PUA. It is unclear whether and how this type of cross-link is repaired. In this study, we first demonstrated that the 3'-PUA-ALKBH1 cross-link is fairly stable under physiological temperature and pH as only ~10% of the adduct decomposed after a 3-day incubation. Using a gel-based assay with an aldehyde-reacting probe, we demonstrated that the 3'-PUA-ALKBH1 cross-link has a free aldehyde group that is in line with the Michael addition mechanism. Moreover, we found that the 3'-PUA-ALKBH1 cross-link can be excised by human tyrosyl-DNA phosphodiesterase 1 (TDP1) and the removal efficiency is significantly enhanced if the adduct is pre-digested by trypsin. Notably, we employed TDP1 as a molecular tool to homogeneously release the cross-linked peptides from DNA to facilitate liquid chromatography tandem mass spectrometry analysis, and demonstrated that Cys129 and Cys371 of ALKBH1 cross-link to 3'-PUA.
无碱基（AP）位点是最丰富的 DNA 损伤之一。其他人的先前研究表明，人类 AlkB 同源物 1（ALKBH1）催化 AP 位点处的 DNA 链断裂，导致酶与 3'-DNA 末端发生自杀交联。先前的定点突变实验报告称，ALKBH1 的半胱氨酸 129 是主要的亲核试剂，与切口 AP 位点的 C3'位置结合，形成 3'-磷酸-α,β-不饱和醛（3'-PUA），形成 3'-PUA-ALKBH1 交联。然而，缺乏支持该机制的直接证据。到目前为止，3'-PUA-ALKBH1 交联是唯一通过蛋白质与 3'-PUA 之间的迈克尔加成反应形成的加合物。目前尚不清楚这种类型的交联是否以及如何被修复。在这项研究中，我们首先证明 3'-PUA-ALKBH1 交联在生理温度和 pH 下相当稳定，只有 ~10%的加合物在 3 天孵育后分解。使用基于凝胶的醛反应探针测定法，我们证明 3'-PUA-ALKBH1 交联具有游离的醛基，符合迈克尔加成机制。此外，我们发现 3'-PUA-ALKBH1 交联可以被人酪氨酸-DNA 磷酸二酯酶 1（TDP1）切除，如果加合物先用胰蛋白酶预先消化，则去除效率显著提高。值得注意的是，我们采用 TDP1 作为分子工具将交联肽从 DNA 上均匀释放出来，以促进液相色谱串联质谱分析，并证明 ALKBH1 的半胱氨酸 129 和 371 与 3'-PUA 交联。
