Topoisomerase 1 prevents replication stress at R-loop-enriched transcription termination sites

  • Nat Commun. 2020 Aug 7;11(1):3940. doi: 10.1038/s41467-020-17858-2.
Alexy Promonet  #  1 ,  Ismaël Padioleau  #  1  2 ,  Yaqun Liu  #  3 ,  Lionel Sanz  4 ,  Anna Biernacka  5 ,  Anne-Lyne Schmitz  1 ,  Magdalena Skrzypczak  5 ,  Amélie Sarrazin  6 ,  Clément Mettling  7 ,  Maga Rowicka  8 ,  Krzysztof Ginalski  5 ,  Frédéric Chedin  4 ,  Chun-Long Chen  9 ,  Yea-Lih Lin  10 ,  Philippe Pasero  11
Affiliations
  • 1. Institut de Génétique Humaine, CNRS et Université de Montpellier, Equipe labélisée Ligue contre le Cancer, Montpellier, France.
  • 2. Institut Gustave Roussy, Villejuif, France.
  • 3. Institut Curie, PSL Research University, CNRS, UMR3244, Sorbonne Université, Paris, France.
  • 4. Department of Molecular and Cellular Biology, University of California, Davis, CA, 95616, USA.
  • 5. Laboratory of Bioinformatics and Systems Biology, Centre of New Technologies, University of Warsaw, Warsaw, Poland.
  • 6. BioCampus Montpellier, CNRS et Université de Montpellier, Montpellier, France.
  • 7. Institut de Génétique Humaine, CNRS et Université de Montpellier, Montpellier, France.
  • 8. Department of Biochemistry and Molecular Biology, University of Texas Medical Branch at Galveston, Galveston, TX, USA.
  • 9. Institut Curie, PSL Research University, CNRS, UMR3244, Sorbonne Université, Paris, France. [email protected].
  • 10. Institut de Génétique Humaine, CNRS et Université de Montpellier, Equipe labélisée Ligue contre le Cancer, Montpellier, France. [email protected].
  • 11. Institut de Génétique Humaine, CNRS et Université de Montpellier, Equipe labélisée Ligue contre le Cancer, Montpellier, France. [email protected].
  • # Contributed equally.
PMID: 32769985 DOI: 10.1038/s41467-020-17858-2
Abstract

R-loops have both positive and negative impacts on chromosome functions. To identify toxic R-loops in the human genome, here, we map RNA:DNA hybrids, replication stress markers and DNA double-strand breaks (DSBs) in cells depleted for Topoisomerase I (Top1), an enzyme that relaxes DNA supercoiling and prevents R-loop formation. RNA:DNA hybrids are found at both promoters (TSS) and terminators (TTS) of highly expressed genes. In contrast, the phosphorylation of RPA by ATR is only detected at TTS, which are preferentially replicated in a head-on orientation relative to the direction of transcription. In Top1-depleted cells, DSBs also accumulate at TTS, leading to persistent checkpoint activation, spreading of γ-H2AX on chromatin and global replication fork slowdown. These data indicate that fork pausing at the TTS of highly expressed genes containing R-loops prevents head-on conflicts between replication and transcription and maintains genome integrity in a Top1-dependent manner.