posted on 2022-01-03, 14:35authored byFeng Tang, Jun Yuan, Bi-Feng Yuan, Yinsheng Wang
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.