Single-nucleotide-resolution genomic maps of O6-methylguanine from the glioblastoma drug temozolomide

Temozolomide kills cancer cells by forming O⁶-methylguanine (O⁶-MeG), which leads to cell cycle arrest and apoptosis. However, O⁶-MeG repair by O⁶-methylguanine-DNA methyltransferase (MGMT) contributes to drug resistance. Characterizing genomic profiles of O⁶-MeG could elucidate how O⁶-MeG accumulation is influenced by repair, but there are no methods to map genomic locations of O⁶-MeG. Here, we developed an immunoprecipitation- and polymerase-stalling-based method, termed O⁶-MeG-seq, to locate O⁶-MeG across the whole genome at single-nucleotide resolution. We analyzed O⁶-MeG formation and repair with regards to sequence contexts and functional genomic regions in a glioblastoma-derived cell line and evaluated the impact of MGMT expression. O⁶-MeG signatures were highly similar to mutational signatures from patients previously treated with temozolomide. Furthermore, MGMT did not preferentially repair O⁶-MeG with respect to sequence context, chromatin state or gene expression level, however, may protect oncogenes from mutations. Finally, we found an MGMT-independent strand bias in O⁶-MeG accumulation in highly expressed genes. These data provide high resolution insight on how O⁶-MeG formation and repair are impacted by genome structure and nucleotide sequence. Further, O⁶-MeG-seq is expected to enable future studies of DNA modification signatures as diagnostic markers for addressing drug resistance and preventing secondary cancers.