Direct visualization of transcription-replication conflicts reveals post-replicative DNA:RNA hybrids

Henriette Stoy, Katharina Zwicky, Danina Kuster, Kevin S. Lang, Jana Krietsch, Magdalena P. Crossley, Jonas A. Schmid, Karlene A. Cimprich, Houra Merrikh, Massimo Lopes

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

Abstract

Transcription-replication collisions (TRCs) are crucial determinants of genome instability. R-loops were linked to head-on TRCs and proposed to obstruct replication fork progression. The underlying mechanisms, however, remained elusive due to the lack of direct visualization and of non-ambiguous research tools. Here, we ascertained the stability of estrogen-induced R-loops on the human genome, visualized them directly by electron microscopy (EM), and measured R-loop frequency and size at the single-molecule level. Combining EM and immuno-labeling on locus-specific head-on TRCs in bacteria, we observed the frequent accumulation of DNA:RNA hybrids behind replication forks. These post-replicative structures are linked to fork slowing and reversal across conflict regions and are distinct from physiological DNA:RNA hybrids at Okazaki fragments. Comet assays on nascent DNA revealed a marked delay in nascent DNA maturation in multiple conditions previously linked to R-loop accumulation. Altogether, our findings suggest that TRC-associated replication interference entails transactions that follow initial R-loop bypass by the replication fork.

Original languageEnglish (US)
Pages (from-to)348-359
Number of pages12
JournalNature Structural and Molecular Biology
Volume30
Issue number3
DOIs
StatePublished - Mar 2023

Bibliographical note

Publisher Copyright:
© 2023, The Author(s).

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

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