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

doi:10.1038/s41594-023-00928-6

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

Veterinary and Biomedical Sciences

Research output: Contribution to journal › Article › peer-review

16 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 language	English (US)
Pages (from-to)	348-359
Number of pages	12
Journal	Nature Structural and Molecular Biology
Volume	30
Issue number	3
DOIs	https://doi.org/10.1038/s41594-023-00928-6
State	Published - Mar 2023

Bibliographical note

Funding Information:
We are grateful to J. Lüthi, F. Roessler, and the Center for Microscopy and Image Analysis of the University of Zurich for assistance with microscopy and imaging analysis. We thank S. Hamperl for technical assistance and critical reading of the manuscript, and members of the Lopes lab for useful discussions and suggestions on the manuscript. Work in the Lopes lab was supported by the SNF grants 31003A_169959 and 310030_189206, and by support of the Forschungskredit UZH to H. S.. K. A. C. is an ACS Research Professor and is supported by the NIH grant R01GM119334, while work in the Merrikh lab was supported by the NIH grant R01GM128191.

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

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

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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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AU - Schmid, Jonas A.

AU - Cimprich, Karlene A.

AU - Merrikh, Houra

AU - Lopes, Massimo

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N2 - 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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Direct visualization of transcription-replication conflicts reveals post-replicative DNA:RNA hybrids

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