{"doi":"10.1073/pnas.2509309122","title":"Protein-mediated stabilization and nicking of the nontemplate DNA strand dramatically affect R-loop formation in vitro","abstract":"R-loops are an important class of non-B DNA structures that form co-transcriptionally. Using in vitro transcription and unbiased quantitative sequencing readouts, we show that the addition of single-strand DNA binding proteins co-transcriptionally can drive a 3- to 5-fold increase of R-loop frequency without significant changes to R-loop distribution. We propose that this is caused by stabilizing and preventing the collapse of short nascent R-loops. This suggests that R-loop formation is highly dynamic and highlights single strand binding proteins as players in cellular R-loop regulation. We further show that nontemplate strand DNA nicks are powerful initiators of R-loop formation, increasing R-loop frequencies by up to two orders of magnitude. Atomic force microscopy revealed that the nontemplate strand in nick-initiated structures is often flayed away from the RNA:DNA hybrid and engaged in self-pairing, creating unique forked R-loop features. DNA nicks, one of the most frequent DNA lesions in cells, are therefore potential hotspots for opportunistic R-loop initiation and may cause the formation of a distinct class of R-loops. Overall, this work highlights the importance of the displaced single-strand on R-loop initiation and dynamics.","journal":"Proceedings of the National Academy of Sciences","year":2025,"id":532933,"datarank":0.21612714171626074,"base_score":1.3862943611198906,"endowment":1.3862943611198906,"self_citation_contribution":0.20794415416798362,"citation_network_contribution":0.008182987548277131,"self_endowment_contribution":0.20794415416798362,"citer_contribution":0.008182987548277131,"corpus_percentile":null,"corpus_rank":null,"citation_count":3,"citer_count":2,"citers_with_citation_signal":1,"citers_with_endowment":1,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9573,"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":1414446,"name":"Thomas E. Catley","orcid":"0000-0002-8919-2701","position":1,"is_corresponding":false},{"id":555041,"name":"Tadas Sereiva","orcid":null,"position":2,"is_corresponding":false},{"id":290916,"name":"Stella R. Hartono","orcid":"0000-0001-6234-3445","position":3,"is_corresponding":false},{"id":1414447,"name":"Alice L. B. Pyne","orcid":"0000-0002-2658-8987","position":4,"is_corresponding":false},{"id":247517,"name":"Frédéric Chédin","orcid":"0000-0002-1306-5335","position":5,"is_corresponding":false},{"id":1414911,"name":"Ethan T Holleman","orcid":null,"position":0,"is_corresponding":true}],"reference_count":45,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:51:27.893975Z","pmid":"40966285","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":[]}