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Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy
Published on: June 25, 2013
Ctp1 and Exonuclease 1, alternative nucleases regulated by the MRN complex, are required for efficient meiotic
Joseph A Farah1, Gareth A Cromie, Gerald R Smith
1Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA.
Abstract:
Double-strand breaks (DSBs) in DNA are lethal unless repaired. Faithful repair requires processing of the DSB ends and interaction with intact homologous DNA, which can produce genetic recombinants. To determine the role of nucleases in DSB end-processing and joint molecule resolution, we studied recombination at the site of a single DSB, generated by induction of the I-SceI endonuclease, during meiosis of fission yeast lacking Rec12 (Spo11 homolog) and, hence, other DSBs. We find that in the presence of the MRN (Rad32-Rad50-Nbs1) complex efficient recombination requires Ctp1, the ortholog of the nuclease Sae2, but not the nuclease activity of MRN. In the absence of MRN, exonuclease 1 (Exo1) becomes the major nuclease required for efficient recombination. Our data indicate that MRN enables access of Ctp1 to the DSB but blocks access of Exo1. In our assay, the Rad16-Swi10 nuclease, required for nucleotide excision-repair, is required for efficient recombination, presumably to remove heterologous DNA at the end of the I-SceI cut site. Another nuclease, the Mus81-Eme1 Holliday junction resolvase, is required to generate crossovers accompanying gene conversion at the I-SceI cut site. Additional, previously published evidence indicates that these 5 nucleases play similar roles in wild-type fission yeast meiotic recombination and in the repair of spontaneous and damage-induced mitotic DSBs. We propose that in wild-type meiosis MRN, in conjunction with Ctp1, removes the covalently attached Rec12 protein from the DNA end, which is then resected by Ctp1 and other activities to produce the single-stranded DNA necessary for further steps of DSB repair.
Insights
DNA double-strand breaks (DSBs) are repaired by homologous recombination. This study reveals nucleases Ctp1 and Exo1 play key roles in DSB repair, with MRN complex regulating their access to DNA breaks during meiosis.
Area of Science:
- Molecular Biology
- Genetics
- DNA Repair Mechanisms
Background:
- DNA double-strand breaks (DSBs) are critical DNA lesions that must be repaired to maintain genomic stability.
- Homologous recombination is a major pathway for DSB repair, involving end processing and strand invasion.
- Nucleases play essential roles in processing DSB ends and resolving recombination intermediates.
Purpose of the Study:
- To elucidate the roles of specific nucleases in DNA double-strand break (DSB) end-processing during meiotic recombination.
- To determine how the MRN complex influences the activity of other nucleases at DSB sites.
- To understand the mechanisms underlying joint molecule resolution and crossover formation in DSB repair.
Main Methods:
- Studied meiotic recombination at a single DSB site induced by the I-SceI endonuclease in fission yeast.
- Utilized strains deficient in key nucleases, including Rec12 (Spo11 homolog), MRN complex, Ctp1, Exo1, Rad16-Swi10, and Mus81-Eme1.
- Analyzed genetic recombination outcomes to infer the functions of individual nucleases in DSB repair pathways.
Main Results:
- Efficient recombination at DSB sites requires Ctp1 in the presence of the MRN complex, but not MRN's nuclease activity.
- Exonuclease 1 (Exo1) becomes the primary nuclease for recombination when MRN is absent, indicating functional redundancy.
- MRN complex facilitates Ctp1 access to DSBs while restricting Exo1 access; Rad16-Swi10 and Mus81-Eme1 nucleases are also crucial for specific repair steps.
Conclusions:
- The MRN complex regulates the access of Ctp1 and Exo1 nucleases to DSB ends, directing repair pathway choice.
- Ctp1 and Exo1 act sequentially or in parallel, with Ctp1 likely involved in removing the covalently attached Rec12 protein.
- These nucleases (MRN, Ctp1, Exo1, Rad16-Swi10, Mus81-Eme1) have conserved roles in meiotic and mitotic DSB repair across eukaryotes.
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