Protein- mediated stabilization and nicking of the nontemplate DNA strand dramatically affect R- loop formation in vitro

• 成果类型: Article
• 署名作者: Holleman, Ethan; Catley, Thomas E.; Sereiva, Tadas; Hartono, Stella R.; Pyne, Alice L. B.; Chedin, Frederic
• 署名单位: University of California System; University of California Davis; University of Sheffield
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-13533
• DOI: 10.1073/pnas.2509309122
• 发表日期: 2025-09-18
• 关键词: genome; transcription; replication; hybrids; cas9

摘要

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 frequencywithout 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 hot-spots 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.