BRCA1/BRC-1 and SMC-5/6 regulate DNA repair pathway engagement during Caenorhabditis elegans meiosis
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Evaluation Summary:
DNA double-strand breaks are a major threat to genome stability. In this study, the roles of two DNA repair proteins, Brc-1 and Smc-5, are investigated in C. elegans meiotic cells, to investigate the DSB repair pathways using the homolog or the sister chromatid as template . The results highlight a regulatory role of Brc-1 and Smc-5 as repressors of repair with the sister chromatid. The experiments are generally well executed, and the findings will be of interest to the DNA repair and C. elegans meiosis communities.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)
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Abstract
The preservation of genome integrity during sperm and egg development is vital for reproductive success. During meiosis, the tumor suppressor BRCA1/BRC-1 and structural maintenance of chromosomes 5/6 (SMC-5/6) complex genetically interact to promote high fidelity DNA double strand break (DSB) repair, but the specific DSB repair outcomes these proteins regulate remain unknown. Using genetic and cytological methods to monitor resolution of DSBs with different repair partners in Caenorhabditis elegans , we demonstrate that both BRC-1 and SMC-5 repress intersister crossover recombination events. Sequencing analysis of conversion tracts from homolog-independent DSB repair events further indicates that BRC-1 regulates intersister/intrachromatid noncrossover conversion tract length. Moreover, we find that BRC-1 specifically inhibits error prone repair of DSBs induced at mid-pachytene. Finally, we reveal functional interactions of BRC-1 and SMC-5/6 in regulating repair pathway engagement: BRC-1 is required for localization of recombinase proteins to DSBs in smc-5 mutants and enhances DSB repair defects in smc-5 mutants by repressing theta-mediated end joining (TMEJ). These results are consistent with a model in which some functions of BRC-1 act upstream of SMC-5/6 to promote recombination and inhibit error-prone DSB repair, while SMC-5/6 acts downstream of BRC-1 to regulate the formation or resolution of recombination intermediates. Taken together, our study illuminates the coordinated interplay of BRC-1 and SMC-5/6 to regulate DSB repair outcomes in the germline.
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Evaluation Summary:
DNA double-strand breaks are a major threat to genome stability. In this study, the roles of two DNA repair proteins, Brc-1 and Smc-5, are investigated in C. elegans meiotic cells, to investigate the DSB repair pathways using the homolog or the sister chromatid as template . The results highlight a regulatory role of Brc-1 and Smc-5 as repressors of repair with the sister chromatid. The experiments are generally well executed, and the findings will be of interest to the DNA repair and C. elegans meiosis communities.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The authors analyze the roles of BRC-1 and SMC-5 in C. elegans meiosis taking advantage of specific assays to distinguish DSB repair pathways: an inter-sister assay (ICR) (Mos1 induced DSB), an inter-homolog assay (IH)(Mos1 induced DSB), a SCE assay based on Edu labelling of sister chromatids, and other assays such as radiation sensitivity. In addition, due to the controlled timing of DSB induction, by recovering progeny at specific time points, the authors evaluate the properties of cells at leptotene-mid pachytene or at late pachytene-diplotene. The authors also take advantage of SNP in the ICR assay to measure conversion tract length.
The main findings are:
- Intersister crossovers are increased in brc-1 and smc-5.
- Intersister non-crossovers are increased in smc-5.
- Interhomolog recombination is …Reviewer #1 (Public Review):
The authors analyze the roles of BRC-1 and SMC-5 in C. elegans meiosis taking advantage of specific assays to distinguish DSB repair pathways: an inter-sister assay (ICR) (Mos1 induced DSB), an inter-homolog assay (IH)(Mos1 induced DSB), a SCE assay based on Edu labelling of sister chromatids, and other assays such as radiation sensitivity. In addition, due to the controlled timing of DSB induction, by recovering progeny at specific time points, the authors evaluate the properties of cells at leptotene-mid pachytene or at late pachytene-diplotene. The authors also take advantage of SNP in the ICR assay to measure conversion tract length.
The main findings are:
- Intersister crossovers are increased in brc-1 and smc-5.
- Intersister non-crossovers are increased in smc-5.
- Interhomolog recombination is increased in both brc-1 and smc-5 for late prophase cells.
- Increased mutation rate in brc-1.
- Shorter non crossover conversion tracts (ICR assay) in brc-1.
- TMEJ involved in DSB repair in brc-1 smc-5 double mutant.
- Independent localization of Brc-1 and smc-5.Having assays for specific events allows gaining more direct information on the DSB repair phenotypes of such mutants. The conversion tract assay is the most convincing and clear data which fits well with the role of Brc-1 in end resection. However the results of the ICR and IH assays are interesting but do not fit with previous observations on the role of Brc-1 and Smc-5 based on analysis of meiotic phenotypes, Rad-51 foci and diakinesis, these discrepancies should be addressed.
The experimental approach has some issues that should be addressed: i) the two main windows (inter-homolog and non-inter homolog) are defined based on meiotic progression in wild type. The timing in the mutants and upon Mos1 induction (which could also affect the timing of meiotic progression) should be determined. In particular, the increase of interhomolog events in brc-1 is left without a validated interpretation. ii) Potentially the phenotypes observed in the ICR and IH assays (but not EdU) may be specific to Mos1-induced DSB and may not apply to Spo11-induced breaks. iii) The use of the Edu assay could be clarified, it seems that the interpretation of configurations is challenging, thus potentially leading to selection bias among diakinesis.
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Reviewer #2 (Public Review):
The manuscript by Toraason et al focuses on the role of the conserved BRC-1/BRCA1 and SMC-5/6 proteins and their role in homologous recombination (HR) during C. elegans meiosis, and in response to ionizing irradiations. 1) The manuscript provides convincing evidence (using previously established reporter systems where GFP expression is restored, and the labeling of individual sister chromatids) that BRC-1 and SMC-5/6 restrict repair between siter chromatids in meiotic prophase, but do not overtly affect CO/non CO outcome between homologs, except for relatively late stage prophase cells (22-34 hour window). The authors also provide evidence that BRC-1 but not SMC-5/6 is required for long gene conversion tracks in HR. 2) Careful radiation sensitivity assays are conducted to assess the dependence of repair of …
Reviewer #2 (Public Review):
The manuscript by Toraason et al focuses on the role of the conserved BRC-1/BRCA1 and SMC-5/6 proteins and their role in homologous recombination (HR) during C. elegans meiosis, and in response to ionizing irradiations. 1) The manuscript provides convincing evidence (using previously established reporter systems where GFP expression is restored, and the labeling of individual sister chromatids) that BRC-1 and SMC-5/6 restrict repair between siter chromatids in meiotic prophase, but do not overtly affect CO/non CO outcome between homologs, except for relatively late stage prophase cells (22-34 hour window). The authors also provide evidence that BRC-1 but not SMC-5/6 is required for long gene conversion tracks in HR. 2) Careful radiation sensitivity assays are conducted to assess the dependence of repair of various cells meiotic prophase cells engaged or not engaged with homolog pairing on BRC-1, SMC-5, microhomology mediated end joining and end joining pathways. 3) Finally, locating BRC-1 and SMC-5 protein it is shown that there is no interdependence of protein localization.
I am overall supportive of the manuscript, which think provides important information of BRC-1 and SMC5/6 function in meiotic germ cells. It appears likely that the roles of these proteins are conserved, C. elegans being a most useful model.
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Reviewer #3 (Public Review):
The manuscript by Toraason et al investigates the role of BRC-1/BRCA1 and the SMC-5/6 complex in repair pathway choice during C. elegans meiosis. The authors use a recently developed system to detect crossover and non-crossover repair events that use the sister chromatid or the same chromosome for repair of a site-specific induced DSB, a related system to look at repair outcomes using the homolog as a repair template, and a cytological approach to detect inter-sister exchanges. The authors show that BRC-1 and SMC-5 both function during meiosis to limit the formation of inter-sister crossovers but are not essential for interhomolog recombination. BRC-1 also suppresses error-prone DNA repair processes during mid-pachytene and promotes the formation of long non-crossover conversion tracts, functions that may …
Reviewer #3 (Public Review):
The manuscript by Toraason et al investigates the role of BRC-1/BRCA1 and the SMC-5/6 complex in repair pathway choice during C. elegans meiosis. The authors use a recently developed system to detect crossover and non-crossover repair events that use the sister chromatid or the same chromosome for repair of a site-specific induced DSB, a related system to look at repair outcomes using the homolog as a repair template, and a cytological approach to detect inter-sister exchanges. The authors show that BRC-1 and SMC-5 both function during meiosis to limit the formation of inter-sister crossovers but are not essential for interhomolog recombination. BRC-1 also suppresses error-prone DNA repair processes during mid-pachytene and promotes the formation of long non-crossover conversion tracts, functions that may not be reliant on SMC-5/6. Finally, the authors show genetic interactions consistent with a role of BRC-1 regulating theta-mediated end joining in smc-5 mutants; however, BRC-1 and SMC-5 do not appear to regulate one-another's localization.
The manuscript is focused on examining the consequences of brc-1 and smc-5 mutations on repair pathway choice in C. elegans meiosis. It achieves that goal. The experiments are generally well done, and the results will be of interest to investigators studying DNA repair and meiotic recombination in C. elegans.
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