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Published on: March 31, 2022
A CRISPR-Cas9-based system for the dose-dependent study of DNA double-strand break sensing and repair
Morgane Auboiron1, Jocelyn Coiffard1, Sylvain Kumanski1
1Centre de Recherche en Biologie cellulaire de Montpellier (CRBM), Université de Montpellier - Centre National de la Recherche Scientifique, Montpellier, France.
Abstract:
The integrity of DNA is put at risk by different lesions, among which double-strand breaks (DSBs) occur at a lower frequency but have the most life-threatening consequences. The study of DSB repair requires tools that can induce the accumulation of these breaks and includes the use of chemical genotoxins, ionizing radiation, or the expression of sequence-specific nucleases. While genotoxins and irradiation allow for dose-dependent studies, nuclease expression permits assessments at precise locations. In this work, we have leveraged the repetitive nature of the Ty transposon elements in the genome of Saccharomyces cerevisiae and the cutting activity of the RNA-guided Cas9 nuclease to create a tool that combines sequence specificity and dose-dependency. In particular, we can achieve the controlled induction of 0, 1, 15, or 59 DSBs in cells with an otherwise identical genetic background. We make the first application of this tool to better understand the behavior of the apical kinase of the DNA damage response Tel1 in the nuclear space. We found that Tel1 is capable of forming nuclear foci, which are clustered by condensin when DSBs occur in Ty elements. In striking contrast with other DSB-related protein foci, Tel1 foci are in tight contact with the nuclear periphery, therefore suggesting a role for the nuclear membrane in their congregation.
Insights
Researchers developed a novel tool to precisely control DNA double-strand breaks (DSBs) in yeast. This method revealed that Tel1 kinase forms nuclear foci clustered by condensin and localized to the nuclear periphery.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- DNA double-strand breaks (DSBs) are critical DNA lesions requiring efficient repair.
- Existing methods for inducing DSBs (genotoxins, radiation, nucleases) have limitations in precision or dose control.
- Studying DSB repair dynamics necessitates tools for controlled induction of breaks.
Purpose of the Study:
- To develop a novel tool for sequence-specific and dose-dependent induction of DSBs in Saccharomyces cerevisiae.
- To investigate the behavior and localization of the Tel1 kinase in response to precisely controlled DSBs.
Main Methods:
- Leveraged repetitive Ty transposon elements and CRISPR-Cas9 nuclease activity in yeast.
- Engineered a system for controlled induction of 0, 1, 15, or 59 DSBs.
- Applied this tool to study Tel1 kinase localization and foci formation.
Main Results:
- Successfully created a tool for controlled DSB induction with varying numbers of breaks.
- Demonstrated that Tel1 kinase forms nuclear foci in response to DSBs within Ty elements.
- Observed that Tel1 foci are clustered by condensin and exhibit tight association with the nuclear periphery.
Conclusions:
- The developed tool enables precise, dose-dependent DSB induction for studying DNA repair.
- Tel1 kinase foci formation is influenced by DSB location and condensin.
- The nuclear periphery may play a role in Tel1 congregation during DNA damage response.
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