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Updated: Dec 12, 2025
Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase
Published on: April 29, 2010
Topoisomerase 1 prevents replication stress at R-loop-enriched transcription termination sites
Alexy Promonet1, Ismaël Padioleau1,2, Yaqun Liu3
1Institut de Génétique Humaine, CNRS et Université de Montpellier, Equipe labélisée Ligue contre le Cancer, Montpellier, France.
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.
Insights
Topoisomerase I (Top1) depletion causes toxic R-loops at gene terminators, leading to DNA double-strand breaks and replication stress. Fork pausing at terminators maintains genome integrity by preventing replication-transcription conflicts.
Area of Science:
- Genetics
- Molecular Biology
- Genomics
Background:
- R-loops, structures of RNA:DNA hybrids, play dual roles in chromosome function.
- Topoisomerase I (Top1) is crucial for managing DNA supercoiling and preventing aberrant R-loop formation.
Purpose of the Study:
- To identify toxic R-loops in the human genome.
- To investigate the consequences of Topoisomerase I depletion on R-loop formation and genome stability.
Main Methods:
- Mapping of RNA:DNA hybrids, replication stress markers (phosphorylated RPA), and DNA double-strand breaks (DSBs).
- Utilizing cells depleted for Topoisomerase I (Top1).
Main Results:
- RNA:DNA hybrids were detected at both transcription start sites (TSS) and terminators (TTS) of highly expressed genes.
- Replication stress markers were specifically found at TTS, which are replicated head-on to transcription.
- Top1 depletion led to DSBs accumulation at TTS, persistent checkpoint activation, γ-H2AX spreading, and replication fork slowdown.
Conclusions:
- Fork pausing at TTS in highly expressed genes with R-loops prevents head-on replication-transcription conflicts.
- Topoisomerase I (Top1) plays a critical role in maintaining genome integrity by regulating R-loops and replication dynamics.
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