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 inNature 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
LanguageEnglish
Published United States Nature Publishing Group 01.03.2023
Nature Publishing Group US
Subjects
Accumulation
Bacteria
Bioassays
Chromosomes - metabolism
Deoxyribonucleic acid
DNA
DNA - chemistry
DNA biosynthesis
DNA Replication
DNA-Binding Proteins - metabolism
Electron microscopy
Estrogens
Genomes
Genomic Instability
Humans
Hybrids
Okazaki fragments
R-loops
Replication
Replication forks
Ribonucleic acid
RNA
Transcription
Visualization
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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.
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ISSN:1545-9993
1545-9985
DOI:10.1038/s41594-023-00928-6