Genome-Wide Detection of DNA Lesions at Single-Base Resolution Using Glycosylase-Cleavage Ligation-Assisted Sequencing

Abstract Upon exposure to endogenous and exogenous damaging agents, tens of thousands of DNA lesions are generated in the mammalian genome, including uracil (U), 8-oxo-7,8-dihydroguanine (8OG), and apurinic/apyrimidinic (AP) sites. It has been documented that these lesions can impair genomic integrity by inducing single-strand breaks and affecting fundamental cellular processes such as gene transcription, DNA replication, and chromatin assembly. Comprehensive and accurate genome-wide mapping of DNA lesions is therefore essential for elucidating their biological and pathological roles. Here, we introduce glycosylase-cleavage ligation-assisted sequencing (GCLA-seq), a method for genome-wide mapping of glycosylase-targeted DNA lesions at single-base resolution. The combined use of DNA glycosylases and T4 DNA ligase in GCLA-seq achieves selective enrichment and amplification of lesion-containing DNA. Using this method, we achieved single-nucleotide-resolution mapping of U, 8OG, and AP sites. Compared with previous approaches for mapping DNA lesions, GCLA-seq does not require specific antibodies or complicated chemical labeling. It can be applied to the localization analysis of various types of DNA lesions, thereby overcoming the limitation that a specific mapping strategy should be developed for each lesion type. Together, these results establish GCLA-seq as an enzyme-based approach for single-base-resolution glycosylase-targeted lesion detection, with genome-wide uracil mapping demonstrated in mammalian genomic DNA.