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Genetics. 2017 May;206(1):481-496. doi: 10.1534/genetics.117.200972. Epub 2017 Mar 14.

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

Author information

1
Department of Molecular Genetics.
2
Department of Genetics and Genomic Sciences, and.
3
Cellular and Molecular Medicine, and.
4
Department of Biology, John Carroll University, University Heights, OH 44118.
5
Biomedical Sciences Training Program, Case Western Reserve University School of Medicine, Cleveland, Ohio, 44106, and.
6
Department of Molecular Genetics, rungek@ccf.org.

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.

KEYWORDS:

Hermes; MRN; MRX; Mre11-Rad50-Nbs1; Mre11-Rad50-Xrs2; NHEJ; hAT; hairpin; nonhomologous end-joining; transposon

PMID:
28292918
PMCID:
PMC5419490
DOI:
10.1534/genetics.117.200972
[Indexed for MEDLINE]
Free PMC Article

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