Department of Chemistry, University of California, Riverside, Riverside, California 92521-0403, United States.
Sauvage Center for Molecular Sciences, Department of Chemistry, Wuhan University, Wuhan 430072, China.
J Am Chem Soc. 2022 Jan 12;144(1):454-462. doi: 10.1021/jacs.1c10490. Epub 2022 Jan 3.
Thymidine glycol (Tg) is the most prevalent form of oxidatively induced pyrimidine lesions in DNA. Tg can arise from direct oxidation of thymidine in DNA. In addition, 5-methyl-2'-deoxycytidine (5-mdC) can be oxidized to 5-mdC glycol, and its subsequent deamination also yields Tg. However, Tg's distribution in the human genome remains unknown. Here, we presented a DNA-protein cross-linking sequencing (DPC-Seq) method for genome-wide mapping of Tg in human cells. Our approach capitalizes on the specificity of a bifunctional DNA glycosylase, i.e., NTHL1, for the covalent labeling, as well as DPC pulldown, SDS-PAGE fractionation, and membrane transfer for highly efficient and selective enrichment of Tg-bearing DNA. By employing DPC-Seq, we detected thousands of Tg sites in the human genome, where dual ablation of NTHL1 and NEIL1, the major DNA glycosylases responsible for Tg repair, led to pronounced increases in the number of Tg peaks. In addition, Tg is depleted in genomic regions associated with active transcription but enriched at nucleosome-binding sites, especially at heterochromatin sites marked with H3K9me2. Collectively, we developed a DPC-Seq method for highly efficient enrichment of Tg-containing DNA and for genome-wide mapping of Tg in human cells. Our work offers a robust tool for future functional studies of Tg in DNA, and we envision that the method can also be adapted for mapping other modified nucleosides in genomic DNA in the future.
胸腺嘧啶乙二醇（Tg）是 DNA 中最常见的氧化诱导嘧啶损伤形式。Tg 可以直接由 DNA 中的胸腺嘧啶氧化产生。此外，5-甲基-2'-脱氧胞苷（5-mdC）可以氧化为 5-mdC 二醇，其随后的脱氨也会产生 Tg。然而，Tg 在人类基因组中的分布仍然未知。在这里，我们提出了一种用于在人类细胞中进行全基因组 Tg 作图的 DNA-蛋白质交联测序（DPC-Seq）方法。我们的方法利用了双功能 DNA 糖苷酶（即 NTHL1）的特异性，用于共价标记以及 DPC 下拉、SDS-PAGE 分馏和膜转移，以高效且选择性地富集携带 Tg 的 DNA。通过使用 DPC-Seq，我们在人类基因组中检测到数千个 Tg 位点，其中 NTHL1 和 NEIL1 的双重缺失，这两种主要的负责 Tg 修复的 DNA 糖苷酶，导致 Tg 峰数量显著增加。此外，Tg 在与活跃转录相关的基因组区域中被耗尽，但在核小体结合位点富集，尤其是在标记有 H3K9me2 的异染色质位点。总之，我们开发了一种用于高效富集含 Tg 的 DNA 和在人类细胞中进行全基因组 Tg 作图的 DPC-Seq 方法。我们的工作为 Tg 在 DNA 中的未来功能研究提供了一种强大的工具，我们设想该方法将来也可以适应于在基因组 DNA 中对其他修饰核苷进行作图。
