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bioRxiv · 10.1101/2025.08.05.668694

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

Zhu, B.·Pidugu, L. S.·Cook, M. E.·Nie, X. Y.·Amarasekara, T.·Menet, J. S.·Drohat, A. C.·Sczepanski, J. T.
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Abstract

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 19F 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. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/668694v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@1ac2095org.highwire.dtl.DTLVardef@18f4952org.highwire.dtl.DTLVardef@16c403org.highwire.dtl.DTLVardef@9ed96c_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Zhu, B., Pidugu, L. S., Cook, M. E., Nie, X. Y., Amarasekara, T., Menet, J. S., Drohat, A. C., Sczepanski, J. T.. 2025-08-05. Thymine DNA Glycosylase Binds to R-Loops and Excises 5-Formyl and 5-Carboxyl Cytosine from DNA/RNA Hybrids. https://doi.org/10.1101/2025.08.05.668694

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