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Published on: September 11, 2022
Nonhomologous End-Joining with Minimal Sequence Loss Is Promoted by the Mre11-Rad50-Nbs1-Ctp1 Complex in
Yanhui Li1,2, Jinyu Wang1,2, Gang Zhou3
1Department of Molecular Genetics.
Abstract:
While the Mre11-Rad50-Nbs1 (MRN) complex has known roles in repair processes like homologous recombination and microhomology-mediated end-joining, its role in nonhomologous end-joining (NHEJ) is unclear as Saccharomyces cerevisiae, Schizosaccharomyces pombe, and mammals have different requirements for repairing cut DNA ends. Most double-strand breaks (DSBs) require nucleolytic processing prior to DNA ligation. Therefore, we studied repair using the Hermes transposon, whose excision leaves a DSB capped by hairpin ends similar to structures generated by palindromes and trinucleotide repeats. We generated single Hermes insertions using a novel S. pombe transient transfection system, and used Hermes excision to show a requirement for MRN in the NHEJ of nonligatable ends. NHEJ repair was indicated by the >1000-fold decrease in excision in cells lacking Ku or DNA ligase 4. Most repaired excision sites had <5 bp of sequence loss or mutation, characteristic for NHEJ and similar excision events in metazoans, and in contrast to the more extensive loss seen in S. cerevisiaeS. pombe NHEJ was reduced >1000-fold in cells lacking each MRN subunit, and loss of MRN-associated Ctp1 caused a 30-fold reduction. An Mre11 dimer is thought to hold DNA ends together for repair, and Mre11 dimerization domain mutations reduced repair 300-fold. In contrast, a mre11 mutant defective in endonucleolytic activity, the same mutant lacking Ctp1, or the triple mutant also lacking the putative hairpin nuclease Pso2 showed wild-type levels of repair. Thus, MRN may act to recruit the hairpin opening activity that allows subsequent repair.
Insights
The Mre11-Rad50-Nbs1 (MRN) complex is crucial for repairing DNA double-strand breaks (DSBs) with hairpin ends via nonhomologous end-joining (NHEJ). MRN likely recruits nucleases to process these complex DNA ends for repair.
Area of Science:
- Molecular Biology
- Genetics
- DNA Repair
Background:
- The Mre11-Rad50-Nbs1 (MRN) complex is essential for DNA double-strand break (DSB) repair, but its specific role in nonhomologous end-joining (NHEJ) remains unclear across different species.
- NHEJ typically requires processing of DNA ends, particularly those with complex structures like hairpins, before ligation.
Purpose of the Study:
- To investigate the role of the MRN complex in the NHEJ pathway, specifically for repairing DSBs with hairpin ends.
- To elucidate the mechanism by which MRN facilitates the repair of nonligatable DNA ends.
Main Methods:
- Utilized a novel transient transfection system in *Schizosaccharomyces pombe* (S. pombe).
- Employed the *Hermes* transposon system to generate DSBs with hairpin ends.
- Assessed NHEJ efficiency by measuring the frequency of transposon excision in wild-type and mutant strains lacking key DNA repair proteins (Ku, DNA ligase 4, MRN subunits, Ctp1).
Main Results:
- *S. pombe* NHEJ of hairpin ends requires the MRN complex, with mutations in MRN subunits or Ctp1 significantly reducing repair efficiency (>1000-fold and 30-fold, respectively).
- Mutations affecting Mre11 dimerization impaired repair (300-fold reduction), suggesting a role in holding DNA ends together.
- Mutants defective in Mre11's endonucleolytic activity or lacking the hairpin nuclease Pso2 showed wild-type repair levels, indicating these activities are not essential for MRN-mediated hairpin end repair.
Conclusions:
- The MRN complex plays a critical role in the NHEJ pathway for repairing DNA DSBs with hairpin structures.
- MRN likely functions by facilitating the processing of hairpin ends, possibly by recruiting nucleolytic activities, rather than directly ligating the ends.
- This study clarifies the distinct roles of MRN in NHEJ compared to other DNA repair pathways and species.
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