Mechanistically distinct BER processes are essential for the tolerance of exogenous 5hm-dC and enzymatic oxidative demethylation

Epigenetic information is transmitted through covalent DNA modifications that regulate chromatin structure and gene expression. 5-Hydroxymethylcytosine (5hmC) is a key epigenetic intermediate generated during TET-mediated oxidative demethylation of 5-methylcytosine. Whether 5hmC itself is intrinsically genotoxic remains unclear. Here, we combine genome-wide CRISPR loss-of-function screens, isogenic knockouts and mass spectrometry to systematically compare the cellular consequences of exogenous 5-hydroxymethyl-2'-cytidine (5hm-dC) exposure and endogenous oxidative demethylation in mammalian cells. We find that exogenous 5hm-dC causes toxicity, mediated by deamination to 5-hydroxymethyl-2'-deoxyuridine (5hm-dU), followed by excision by the glycosylase SMUG1, which generates base excision repair (BER) intermediates that compromise cell viability. In contrast, toxicity associated with enzymatic oxidative demethylation is primarily driven by TDG-dependent excision of oxidized methylcytosine derivatives. Despite these distinct initiating events, both genotoxins converge on a critical requirement for DNA polymerase {beta} (POL{beta}), indicating that efficient BER completion is essential to mitigate cytotoxic repair intermediates.