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PMID:28292918

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Citation

'Li, Y, Wang, J, Zhou, G, Lajeunesse, M, Le, N, Stawicki, BN, Corcino, YL, Berkner, KL and Runge, KW (2017) Non-Homologous End-Joining with Minimal Sequence Loss Is Promoted by the Mre11-Rad50-Nbs1-Ctp1 Complex in Schizosaccharomyces pombe. Genetics '

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 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. cerevisiaeS. 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.

Links

PubMed Online version:10.1534/genetics.117.200972

Keywords


Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

SCHPO:RAD32

GO:0006303: double-strand break repair via nonhomologous end joining

ECO:0000315:

P

Mre11, a protein required for double stranded break repair via non homologous end joining, forms a dimer to bridge the gap between the two broken ends of the double-stranded DNA. Figure 6 proves how dimerization of mre11 ( or rad32 in S. pombe) needs to occur for efficient non homologous end joining since the introduced mutations in the Mre11 dimerization domain, but not the nuclease domain, reduce NHEJ. This data is represented by the drastic decrease in PCR products since the bands for PCR products would indicate successful repair by non homologous end-joining. Therefore, mre11 dimerization is essential for NHEJ since there were no excision products from PCR.

complete
CACAO 12476

SCHPO:RAD50

GO:0006303: double-strand break repair via nonhomologous end joining

ECO:0000315:

P

Rad50 is part of the Mre11-Rad50-Nbs1 complex which plays a role in DNA repair processes including DNA damage checkpoint and recruitment of telomerase. Rad50 is responsible for stimulating ATP dependent DNA unwinding. Fig. 4. Efficient NHEJ requires Ku70, DNA ligase 4 and MRN. (Mre11-Rad50-Nbs1=MRN)

complete
CACAO 12784

Notes

See also

References

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