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


Stoy, Henriette; Zwicky, Katharina; Kuster, Danina; Lang, Kevin S; Krietsch, Jana; Crossley, Magdalena P; Schmid, Jonas A; Cimprich, Karlene A; Merrikh, Houra; Lopes, Massimo (2023). Direct visualization of transcription-replication conflicts reveals post-replicative DNA:RNA hybrids. Nature Structural & Molecular Biology, 30(3):348-359.

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.

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.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Molecular Cancer Research
07 Faculty of Science > Institute of Molecular Cancer Research
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Scopus Subject Areas:Life Sciences > Structural Biology
Life Sciences > Molecular Biology
Language:English
Date:2 March 2023
Deposited On:16 Mar 2023 10:15
Last Modified:29 Apr 2024 01:36
Publisher:Nature Publishing Group
ISSN:1545-9985
OA Status:Hybrid
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.1038/s41594-023-00928-6
PubMed ID:36864174
  • Content: Published Version
  • Language: English
  • Licence: Creative Commons: Attribution 4.0 International (CC BY 4.0)