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Updated: Oct 29, 2025
Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells
Published on: September 8, 2010
DNA-RNA hybrids at DSBs interfere with repair by homologous recombination
Pedro Ortega1, José Antonio Mérida-Cerro1, Ana G Rondón1
1Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Universidad de Sevilla-CSIC-Universidad Pablo de Olavide, Seville, Spain.
Abstract:
DNA double-strand breaks (DSBs) are the most harmful DNA lesions and their repair is crucial for cell viability and genome integrity. The readout of DSB repair may depend on whether DSBs occur at transcribed versus non-transcribed regions. Some studies have postulated that DNA-RNA hybrids form at DSBs to promote recombinational repair, but others have challenged this notion. To directly assess whether hybrids formed at DSBs promote or interfere with the recombinational repair, we have used plasmid and chromosomal-based systems for the analysis of DSB-induced recombination in Saccharomyces cerevisiae. We show that, as expected, DNA-RNA hybrid formation is stimulated at DSBs. In addition, mutations that promote DNA-RNA hybrid accumulation, such as hpr1∆ and rnh1∆ rnh201∆, cause high levels of plasmid loss when DNA breaks are induced at sites that are transcribed. Importantly, we show that high levels or unresolved DNA-RNA hybrids at the breaks interfere with their repair by homologous recombination. This interference is observed for both plasmid and chromosomal recombination and is independent of whether the DSB is generated by endonucleolytic cleavage or by DNA replication. These data support a model in which DNA-RNA hybrids form fortuitously at DNA breaks during transcription and need to be removed to allow recombinational repair, rather than playing a positive role.
Insights
DNA-RNA hybrids form at DNA double-strand breaks (DSBs) but do not promote repair. Instead, high levels of these hybrids interfere with the homologous recombination repair of DSBs, hindering cell viability.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- DNA double-strand breaks (DSBs) are critical DNA lesions impacting cell viability and genome stability.
- The role of DNA-RNA hybrids at DSBs in repair pathways remains controversial.
- DSB repair outcomes may differ based on their location within transcribed or non-transcribed genomic regions.
Purpose of the Study:
- To investigate whether DNA-RNA hybrids at DSBs promote or impede recombinational repair.
- To elucidate the functional significance of DNA-RNA hybrids in the context of DSB repair.
Main Methods:
- Utilized plasmid and chromosomal systems in *Saccharomyces cerevisiae* to analyze DSB-induced recombination.
- Assessed the impact of mutations (e.g., *hpr1∆*, *rnh1∆ rnh201∆*) that enhance DNA-RNA hybrid accumulation.
- Investigated DSB repair mechanisms following both endonucleolytic cleavage and replication-dependent breaks.
Main Results:
- DNA-RNA hybrid formation is demonstrably stimulated at DSBs.
- Mutations leading to increased DNA-RNA hybrids resulted in significant plasmid loss at transcribed DSB sites.
- Elevated or persistent DNA-RNA hybrids at DSBs were found to interfere with homologous recombination repair.
Conclusions:
- DNA-RNA hybrids form incidentally at DSBs during transcription.
- Removal of DNA-RNA hybrids is necessary for efficient homologous recombination repair of DSBs.
- These findings challenge the notion that DNA-RNA hybrids actively promote DSB repair, suggesting an inhibitory role.
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