Thymine DNA Glycosylase Binds to R-Loops and Excises 5-Formyl and 5-Carboxyl Cytosine from DNA/RNA Hybrids

R-loops are three-stranded nucleic acid structures consisting of a DNA/RNA hybrid and a displaced single-stranded DNA. 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 (5mC) back into cytosine—by recruiting associated proteins, providing an attractive targeting mechanism. Nevertheless, many important aspects of this process, including whether the associated proteins bind to and function on R-loops, remain to be substantiated. In this study, we demonstrate for the first time that thymine DNA glycosylase (TDG), a key enzyme in the active DNA demethylation pathway, binds tightly to R-loops in vitro and can excise DNA demethylation intermediates 5-formylcytosine (5fC) and 5-carboxycytosine (5caC) from DNA in DNA/RNA hybrid duplexes. We also show that R-loops guide the strand-specific activity of TDG at CpG sites, 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 ¹⁹ F NMR. Finally, our findings suggest that TDG–R-loop interactions may be widespread in human cells. Collectively, our results provide strong evidence that R-loops play a critical role in DNA demethylation and support a mechanism in which 5fC/5caC are directly removed from DNA/RNA hybrids in cells.