Nonhomologous End-Joining with Minimal Sequence Loss Is Promoted by the Mre11-Rad50-Nbs1-Ctp1 Complex in .

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 , , 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 transposon, whose excision leaves a DSB capped by hairpin ends similar to structures generated by palindromes and trinucleotide repeats. We generated single insertions using a novel transient transfection system, and used 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 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 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.