DNA-RNA hybrids at DSBs interfere with repair by homologous recombination

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 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.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for all figures.

Article and author information

Author details

  1. Pedro Ortega

    Genetics, CABIMER, Universidad de Sevilla, Seville, Spain
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4216-3695
  2. Jose Antonio Mérida-Cerro

    Genetics, CABIMER, Universidad de Sevilla, Seville, Spain
    Competing interests
    No competing interests declared.
  3. Ana G Rondón

    Universidad de Sevilla, CABIMER, Universidad de Sevilla, Seville, Spain
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9481-1255
  4. Belén Gómez-González

    Department of Genetics, CABIMER, Universidad de Sevilla, Sevilla, Spain
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1655-8407
  5. Andrés Aguilera

    Department of Molecular Biology, CABIMER, Universidad de Sevilla, Seville, Spain
    For correspondence
    andres.aguilera@cabimer.es
    Competing interests
    Andrés Aguilera, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4782-1714

Funding

H2020 European Research Council (ERC2014 AdG669898 TARLOOP)

  • Andrés Aguilera

Ministerio de Economía y Competitividad (BFU2016-75058-P)

  • Andrés Aguilera

Ministerio de Ciencia, Innovación y Universidades (PDI2019-104270GB-I00)

  • Andrés Aguilera

Junta de Andalucía (P12-BIO-1238)

  • Andrés Aguilera

European Union, FEDER

  • Andrés Aguilera

Ministerio de Educación, Cultura y Deporte (PhD FPU fellowship)

  • Pedro Ortega

Junta de Andalucía (PhD fellowship)

  • Jose Antonio Mérida-Cerro

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Wolf-Dietrich Heyer, University of California, Davis, United States

Publication history

  1. Received: April 28, 2021
  2. Accepted: July 7, 2021
  3. Accepted Manuscript published: July 8, 2021 (version 1)
  4. Version of Record published: July 19, 2021 (version 2)

Copyright

© 2021, Ortega et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Pedro Ortega
  2. Jose Antonio Mérida-Cerro
  3. Ana G Rondón
  4. Belén Gómez-González
  5. Andrés Aguilera
(2021)
DNA-RNA hybrids at DSBs interfere with repair by homologous recombination
eLife 10:e69881.
https://doi.org/10.7554/eLife.69881

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