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

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 res...

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Published in:Nature structural & molecular biology Vol. 30; no. 3; pp. 348 - 359
Main Authors: Stoy, Henriette, Zwicky, Katharina, Kuster, Danina, Lang, Kevin S, Krietsch, Jana, Crossley, Magdalena P., Schmid, Jonas A., Cimprich, Karlene A., Merrikh, Houra, Lopes, Massimo
Format: Journal Article
Language:English
Published: New York Nature Publishing Group US 01.03.2023
Nature Publishing Group
Subjects:
101/28
631/1647/328/1259
631/337/1427
631/337/151/1431
631/337/151/2356
631/337/572
Accumulation
Bacteria
Bioassays
Biochemistry
Biological Microscopy
Biomedical and Life Sciences
Chromosomes - metabolism
Deoxyribonucleic acid
DNA
DNA - chemistry
DNA biosynthesis
DNA Replication
DNA-Binding Proteins - metabolism
Electron microscopy
Estrogens
Genomes
Genomic Instability
Humans
Hybrids
Life Sciences
Membrane Biology
Okazaki fragments
Protein Structure
R-loops
Replication
Replication forks
Ribonucleic acid
RNA
Transcription
Visualization
ISSN:1545-9993, 1545-9985, 1545-9985
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Summary: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. The authors develop an EM-based method to directly visualize R-loops. Applying this method to transcription-replication conflicts in human and bacterial cells, they show that DNA:RNA hybrids accumulate primarily behind replication forks, and are linked to fork slowing and fork reversal.
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ISSN:1545-9993
1545-9985
1545-9985
DOI:10.1038/s41594-023-00928-6