期刊
GENETICS
卷 206, 期 1, 页码 481-496出版社
GENETICS SOCIETY AMERICA
DOI: 10.1534/genetics.117.200972
关键词
Mre11-Rad50-Nbs1; MRN; Mre11-Rad50-Xrs2; MRX; nonhomologous end-joining; NHEJ; hairpin; hAT; transposon; Hermes
资金
- National Institutes of Health (NIH) [RO1 GM-050752, AG-019960]
- National Science Foundation [1516220]
- Case Western Reserve University-Cleveland Clinic Comprehensive Cancer Center
- NIH R01 grants [HL-55566, HL-81093]
- Div Of Molecular and Cellular Bioscience
- Direct For Biological Sciences [1516220] Funding Source: National Science Foundation
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. cerevisiae. S. 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.
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