Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, Rhode Island 02881, United States.
Department of Chemistry and Biochemistry, University of Lethbridge, 4401 University Drive West, Lethbridge, Alberta T1K 3M4, Canada.
J Am Chem Soc. 2024 Apr 17;146(15):10381-10392. doi: 10.1021/jacs.3c12890. Epub 2024 Apr 4.
DNA cross-links severely challenge replication and transcription in cells, promoting senescence and cell death. In this paper, we report a novel type of DNA interstrand cross-link (ICL) produced as a side product during the attempted repair of 1,-ethenoadenine (εA) by human α-ketoglutarate/Fe(II)-dependent enzyme ALKBH2. This stable/nonreversible ICL was characterized by denaturing polyacrylamide gel electrophoresis analysis and quantified by high-resolution LC-MS in well-matched and mismatched DNA duplexes, yielding 5.7% as the highest level for cross-link formation. The binary lesion is proposed to be generated through covalent bond formation between the epoxide intermediate of εA repair and the exocyclic -amino group of adenine or the -amino group of cytosine residues in the complementary strand under physiological conditions. The cross-links occur in diverse sequence contexts, and molecular dynamics simulations rationalize the context specificity of cross-link formation. In addition, the cross-link generated from attempted εA repair was detected in cells by highly sensitive LC-MS techniques, giving biological relevance to the cross-link adducts. Overall, a combination of biochemical, computational, and mass spectrometric methods was used to discover and characterize this new type of stable cross-link both in vitro and in human cells, thereby uniquely demonstrating the existence of a potentially harmful ICL during DNA repair by human ALKBH2.
DNA 交联严重挑战细胞中的复制和转录，促进衰老和细胞死亡。在本文中，我们报告了一种新型的 DNA 链间交联（ICL），它是人类 α-酮戊二酸/Fe(II)依赖性酶 ALKBH2 试图修复 1,-亚乙基腺嘌呤（εA）时作为副产物产生的。这种稳定/不可逆的 ICL 通过变性聚丙烯酰胺凝胶电泳分析进行表征，并通过高分辨率 LC-MS 在匹配和不匹配的 DNA 双链体中进行定量，交联形成的最高水平为 5.7%。该二元损伤是通过εA 修复的环氧化物中间体与生理条件下互补链中腺嘌呤的环外 -氨基或胞嘧啶残基的 -氨基之间形成共价键而产生的。交联发生在不同的序列背景下，分子动力学模拟解释了交联形成的上下文特异性。此外，通过高度敏感的 LC-MS 技术在细胞中检测到来自试图修复的εA 的交联，从而为交联加合物赋予了生物学相关性。总的来说，采用了生化、计算和质谱方法的组合，在体外和人类细胞中发现和表征了这种新型稳定交联，从而独特地证明了人类 ALKBH2 在 DNA 修复过程中存在潜在有害的 ICL。
