While the Mre11-Rad50-Nbs1 (MRN) complex has known roles in repair processes like homologous recombination and microhomology-mediated end-joining, its role in non-homologous 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. We therefore 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 non-ligatable ends. NHEJ repair was indicated by the more than 1000-fold decrease in excision in cells lacking Ku or DNA ligase 4. Most repaired excision sites had less than 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. cerevisiae. S. pombe NHEJ was reduced more than 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, an 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.
- Received February 8, 2017.
- Accepted February 24, 2017.
- Copyright © 2017, The Genetics Society of America