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DNA-protein Crosslinking Sequencing for Genome-wide Mapping of Thymidine Glycol

ABSTRACT: Thymidine glycol (Tg) is the most prevalent form of oxidatively induced pyrimidine lesion 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, its 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 is capitalized on the specificity of a 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 HEK293T cells and the isogenic NTHL1 and NEIL1 double knock out cell lines. We found that Tg is depleted in genomic regions associated with active transcription, but enriched at the nucleosome-binding sites, especially at heterochromatin sites. Collectively, our approach allows for comprehensive analysis of Tg in the human genome and provides a robust tool for future functional studies of Tg in DNA. It can be envisaged that the method can be adapted for mapping other modified nucleosides in genomic DNA in the future.

ORGANISM(S): Homo sapiens

PROVIDER: GSE184204 | GEO | 2021/09/18

REPOSITORIES: GEO

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