title: Nonhomologous End-Joining with Minimal Sequence Loss Is Promoted by the Mre11-Rad50-Nbs1-Ctp1 Complex inSchizosaccharomyces pombe.
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 asSaccharomyces 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 theHermestransposon, whose excision leaves a DSB capped by hairpin ends similar to structures generated by palindromes and trinucleotide repeats. We generated singleHermesinsertions using a novelS. pombetransient transfection system, and usedHermesexcision 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 inS. cerevisiaeS. pombeNHEJ 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, amre11mutant 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.
authors.affiliation: Department of Molecular Genetics.|Department of Genetics and Genomic Sciences, and.
authors.forename: Yanhui
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Molecular Genetics.|Department of Genetics and Genomic Sciences, and.
authors.forename: Jinyu
authors.initials: J
authors.lastname: Wang
authors.affiliation: Cellular and Molecular Medicine, and.
authors.forename: Gang
authors.initials: G
authors.lastname: Zhou
authors.affiliation: Department of Biology, John Carroll University, University Heights, OH 44118.
authors.forename: Michael
authors.initials: M
authors.lastname: Lajeunesse
authors.affiliation: Biomedical Sciences Training Program, Case Western Reserve University School of Medicine, Cleveland, Ohio, 44106, and.
authors.forename: Nga
authors.initials: N
authors.lastname: Le
authors.affiliation: Biomedical Sciences Training Program, Case Western Reserve University School of Medicine, Cleveland, Ohio, 44106, and.
authors.forename: Brittany N
authors.initials: BN
authors.lastname: Stawicki
authors.affiliation: Biomedical Sciences Training Program, Case Western Reserve University School of Medicine, Cleveland, Ohio, 44106, and.
authors.forename: Yalitza Lopez
authors.initials: YL
authors.lastname: Corcino
authors.affiliation: Department of Genetics and Genomic Sciences, and.
authors.forename: Kathleen L
authors.initials: KL
authors.lastname: Berkner
authors.affiliation: Department of Molecular Genetics, rungek@ccf.org.|Department of Genetics and Genomic Sciences, and.
authors.forename: Kurt W
authors.identifier: 0000-0001-8492-0530
authors.initials: KW
authors.lastname: Runge
mesh_terms: D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics*; D004247:DNA / Q000235:genetics; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D004706:Endodeoxyribonucleases / Q000235:genetics*; D005090:Exodeoxyribonucleases / Q000235:genetics*; D046912:Multiprotein Complexes / Q000235:genetics; D012568:Schizosaccharomyces / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics*
chemical_list: D002868:Chromosomal Proteins, Non-Histone; C525888:Ctp1 protein, S pombe; D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; C478035:Nbs1 protein, S pombe; C480032:Rad50 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; C092453:Mre11 protein, S pombe; C000621194:Pso2 protein, S pombe
keywords: Hermes; MRN; MRX; Mre11-Rad50-Nbs1; Mre11-Rad50-Xrs2; NHEJ; hAT; hairpin; nonhomologous end-joining; transposon