N 2-Alkyl-dG lesions elicit R-loop accumulation in the genome

Humans are exposed to DNA alkylating agents through endogenous metabolism, environmental exposure and cancer chemotherapy. The resulting alkylated DNA adducts may elicit genome instability by perturbing DNA replication and transcription. R-loops regulate various cellular processes, including transcription, DNA repair, and telomere maintenance. However, unscheduled R-loops are also recognized as potential sources of DNA damage and genome instability. In this study, by employing fluorescence microscopy and R-loop sequencing approaches, we uncovered, for the first time, that minor-groove N2-alkyl-dG lesions elicit elevated R-loop accumulation in chromatin and in plasmid DNA in cells. We also demonstrated that the N2-alkyl-dG-induced R-loops impede transcription elongation and compromise genome integrity. Moreover, genetic depletion of DDX23, a R-loop helicase, renders cells more sensitive toward benzo[a]pyrene diolepoxide, a carcinogen that induces mainly the minor-groove N2-dG adduct. Together, our work unveiled that unrepaired minor-groove N2-alkyl-dG lesions may perturb genome integrity through augmenting R-loop levels in chromatin. Our findings suggest a potential therapeutic strategy involving the combination of R-loop helicase inhibitors with DNA alkylating drugs.