Thymine DNA glycosylase binds to R-loops and excises 5-formyl and 5-carboxyl cytosine from DNA/RNA hybrids

Once considered rare byproducts of transcription, R-loops are now recognized as important regulators of various nuclear processes. In particular, evidence indicates a role for R-loops in regulating DNA methylation dynamics. R-loops have been shown to promote active DNA demethylation-the enzymatic reversal of 5-methylcytosine back into cytosine-by recruiting associated proteins, providing an attractive targeting mechanism. Nevertheless, many aspects of this process, including whether the associated proteins bind to and function on DNA within R-loops, remain to be substantiated. Herein, we demonstrate that thymine DNA glycosylase (TDG), a key enzyme in the active DNA demethylation pathway, binds to synthetic R-loop substrates in vitro and can excise DNA demethylation intermediates 5-formylcytosine (5fC) and 5-carboxycytosine (5caC) from DNA in DNA/RNA hybrids. We also show that R-loops confer strand-specific TDG activity at CpGs, potentially explaining the asymmetric distribution of 5fC/5caC at gene promoters. Furthermore, we provide important mechanistic insights into base excision on DNA/RNA hybrid duplexes using ¹⁹F NMR. Finally, our findings raise the possibility that TDG-R-loop interactions could occur in mammalian cells. Collectively, our results establish a biochemical mechanism by which 5fC/5caC can be removed from DNA within DNA/RNA hybrids, which has important implication for the role of R-loops during DNA demethylation.