{"doi":"10.1101/2025.08.05.668694","title":"Thymine DNA Glycosylase Binds to R-Loops and Excises 5-Formyl and 5-Carboxyl Cytosine from DNA/RNA Hybrids","abstract":"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 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. GRAPHICAL ABSTRACT","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":558712,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9511,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":473966,"name":"L.S. Pidugu","orcid":"0000-0002-3426-9003","position":1,"is_corresponding":false},{"id":471208,"name":"Mary E. Cook","orcid":"0000-0002-6298-2586","position":2,"is_corresponding":false},{"id":1019606,"name":"Xinyu Y. Nie","orcid":"0009-0005-0756-0275","position":3,"is_corresponding":false},{"id":1459926,"name":"E. A. P. Tharaka Amarasekara","orcid":null,"position":4,"is_corresponding":false},{"id":559620,"name":"Jérôme S. Menet","orcid":"0000-0002-7682-0967","position":5,"is_corresponding":false},{"id":693866,"name":"Alexander C. Drohat","orcid":"0000-0002-7458-8770","position":6,"is_corresponding":false},{"id":479667,"name":"Jonathan T. Sczepanski","orcid":"0000-0002-9275-2597","position":7,"is_corresponding":false},{"id":1459925,"name":"Baiyu Zhu","orcid":null,"position":0,"is_corresponding":true}],"reference_count":87,"raw_metadata":null,"created_at":"2026-07-19T02:55:30.312295Z","pmid":"40799608","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}