SPECIFIC REACTIVITY OF ABASIC SITES WITH POLYAMINES ENABLES THEIR GENOMIC MAPPING
Apurinic/apyrimidinic (AP) sites are among the most abundant DNA lesions and represent key intermediates in the base excision DNA repair pathway. Knowing their genomic distribution is important for understanding the mechanisms of genotoxic agents. Although several methods exist for genomic mapping of AP sites, some of them suffer from limited specificity and/or complex multi-step workflows. Here, we present APX-seq, a novel method based on the recently discovered reaction of AP sites with 1,2-diamines, for highly specific chemical labelling, isolation and enrichment of AP site-containing genomic fragments for subsequent DNA sequencing. Following in vitro optimization and validation, we applied APX-seq to the genome of cultured HepG2 cells. Our finding reveal that AP sites are broadly distributed in the genome and are enriched in euchromatin, consistent with the prior studies. Moreover, we show that treatment with genotoxic agents such as a DNA-alkylating chemical or X-ray radiation, barely alters the genomic landscape of AP sites. Overall design: APX-seq and AP-seq were performed in HepG2 cells subjected to four conditions: untreated (control), 5 mM MMS treatment, 10 mM MMS treatment, and 6 Gy X-ray radiation (XRD). Cells were harvested at defined time points following treatment for subsequent genomic DNA extraction and AP site profiling.