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XRCC4 and MRE11 Roles and Transcriptional Response to Repair of TALEN-Induced Double-Strand DNA Breaks
Ronald Benjamin1,2, Atoshi Banerjee1,2, Xiaogang Wu1
1Nevada Institute of Personalized Medicine, University of Nevada Las Vegas, Las Vegas, NV 89154, USA.
Abstract:
Double-strand breaks (DSB) are one of the most lethal forms of DNA damage that, if left unrepaired, can lead to genomic instability, cellular transformation, and cell death. In this work, we examined how repair of transcription activator-like effector nuclease (TALEN)-induced DNA damage was altered when knocking out, or inhibiting a function of, two DNA repair proteins, XRCC4 and MRE11, respectively. We developed a fluorescent reporter assay that uses TALENs to introduce DSB and detected repair by the presence of GFP fluorescence. We observed repair of TALEN-induced breaks in the XRCC4 knockout cells treated with mirin (a pharmacological inhibitor of MRE11 exonuclease activity), albeit with ~40% reduced efficiency compared to normal cells. Editing in the absence of XRCC4 or MRE11 exonuclease was robust, with little difference between the indel profiles amongst any of the groups. Reviewing the transcriptional profiles of the mirin-treated XRCC4 knockout cells showed 307 uniquely differentially expressed genes, a number far greater than for either of the other cell lines (the HeLa XRCC4 knockout sample had 83 genes, and the mirin-treated HeLa cells had 30 genes uniquely differentially expressed). Pathways unique to the XRCC4 knockout+mirin group included differential expression of p53 downstream pathways, and metabolic pathways indicating cell adaptation for energy regulation and stress response. In conclusion, our study showed that TALEN-induced DSBs are repaired, even when a key DSB repair protein or protein function is not operational, without a change in indel profiles. However, transcriptional profiles indicate the induction of unique cellular responses dependent upon the DNA repair protein(s) hampered.
Insights
Double-strand breaks (DSB) are repaired even without key DNA repair proteins XRCC4 or MRE11. Transcriptional analysis reveals unique cellular responses to DNA damage when these repair pathways are hindered.
Area of Science:
- Molecular Biology
- Genetics
- Cellular Biology
Background:
- Double-strand breaks (DSB) represent a critical form of DNA damage with severe consequences if unrepaired, including genomic instability and cell death.
- Understanding the intricate DNA repair mechanisms is crucial for comprehending cellular responses to genotoxic stress.
Purpose of the Study:
- To investigate the impact of disrupting XRCC4 and MRE11 function on the repair of transcription activator-like effector nuclease (TALEN)-induced DNA double-strand breaks.
- To analyze the resulting indel profiles and transcriptional changes in cells with compromised DNA repair pathways.
Main Methods:
- Development of a fluorescent reporter assay utilizing TALENs to induce DSBs and detect repair via GFP fluorescence.
- Employing CRISPR/Cas9 technology to create XRCC4 knockout cells and mirin to inhibit MRE11 exonuclease activity.
- Comparative analysis of indel profiles and whole-genome transcriptional profiling.
Main Results:
- TALEN-induced DSBs were repaired in XRCC4 knockout cells treated with mirin, albeit with approximately 40% reduced efficiency compared to control cells.
- No significant differences in indel profiles were observed across groups with compromised XRCC4 or MRE11 function.
- Transcriptional analysis revealed 307 uniquely differentially expressed genes in XRCC4 knockout+mirin treated cells, significantly more than in other experimental groups.
Conclusions:
- TALEN-induced DSBs can be repaired even when essential DNA repair proteins like XRCC4 or MRE11 are functionally impaired, without altering indel profiles.
- Disruption of specific DNA repair proteins induces unique transcriptional responses, including alterations in p53 downstream pathways and metabolic pathways related to cell adaptation and stress response.
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