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Structural maintenance of chromosomes (SMC) proteins promote homolog-independent recombination repair in meiosis crucial for germ cell genomic stability.

Bickel JS, Chen L, Hayward J, Yeap SL, Alkers AE, Chan RC - PLoS Genet. (2010)

Bottom Line: Chromosome fragments associated with HR defects have only been reported in mutants, which have disrupted inter-homolog crossover.Surprisingly, the smc-5 and smc-6 mutations did not disrupt the formation of chiasmata, the cytologically visible linkages between homologous chromosomes formed from meiotic inter-homolog crossovers.Together, these results demonstrate that the successful completion of homolog-independent recombination is crucial for germ cell genomic stability.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.

ABSTRACT
In meiosis, programmed DNA breaks repaired by homologous recombination (HR) can be processed into inter-homolog crossovers that promote the accurate segregation of chromosomes. In general, more programmed DNA double-strand breaks (DSBs) are formed than the number of inter-homolog crossovers, and the excess DSBs must be repaired to maintain genomic stability. Sister-chromatid (inter-sister) recombination is postulated to be important for the completion of meiotic DSB repair. However, this hypothesis is difficult to test because of limited experimental means to disrupt inter-sister and not inter-homolog HR in meiosis. We find that the conserved Structural Maintenance of Chromosomes (SMC) 5 and 6 proteins in Caenorhabditis elegans are required for the successful completion of meiotic homologous recombination repair, yet they appeared to be dispensable for accurate chromosome segregation in meiosis. Mutations in the smc-5 and smc-6 genes induced chromosome fragments and dismorphology. Chromosome fragments associated with HR defects have only been reported in mutants, which have disrupted inter-homolog crossover. Surprisingly, the smc-5 and smc-6 mutations did not disrupt the formation of chiasmata, the cytologically visible linkages between homologous chromosomes formed from meiotic inter-homolog crossovers. The mutant fragmentation defect appeared to be preferentially enhanced by the disruptions of inter-homolog recombination but not by the disruptions of inter-sister recombination. Based on these findings, we propose that the C. elegans SMC-5/6 proteins are required in meiosis for the processing of homolog-independent, presumably sister-chromatid-mediated, recombination repair. Together, these results demonstrate that the successful completion of homolog-independent recombination is crucial for germ cell genomic stability.

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Loss of function mutations in smc-5 and smc-6 conferred hypersensitivity to ionizing radiation (IR) and increased DNA-damage responses in germ cells.(A) Graph of the viability of the eggs produced from mock and radiation-exposed germ cells in mutant F1 and wild-type L4-stage larvae as described [31]. 297 eggs or more were counted for each genotype and at each dose of IR. (B) Relative mRNA abundance for egl-1 after normalization to gamma tubulin tbg-1 as measured by quantitative RT-PCR [32]. Error bars represent standard errors. The asterisks (*) indicate significant changes from the wild-type control (p<0.001, t-Test). Gel analysis confirmed the size of each RT-PCR product and is shown underneath the graph. (C) Micrographs of germ cell corpses (white arrows) detected by CED-1::GFP fluorescence showed a greater number of corpses in the smc-5(tm2868) mutant germline. The average number of corpses per gonad and the standard error are indicated. Scale bars = 10 µm.
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pgen-1001028-g003: Loss of function mutations in smc-5 and smc-6 conferred hypersensitivity to ionizing radiation (IR) and increased DNA-damage responses in germ cells.(A) Graph of the viability of the eggs produced from mock and radiation-exposed germ cells in mutant F1 and wild-type L4-stage larvae as described [31]. 297 eggs or more were counted for each genotype and at each dose of IR. (B) Relative mRNA abundance for egl-1 after normalization to gamma tubulin tbg-1 as measured by quantitative RT-PCR [32]. Error bars represent standard errors. The asterisks (*) indicate significant changes from the wild-type control (p<0.001, t-Test). Gel analysis confirmed the size of each RT-PCR product and is shown underneath the graph. (C) Micrographs of germ cell corpses (white arrows) detected by CED-1::GFP fluorescence showed a greater number of corpses in the smc-5(tm2868) mutant germline. The average number of corpses per gonad and the standard error are indicated. Scale bars = 10 µm.

Mentions: In order to determine if the smc-5 and smc-6 mutants have defects in the maintenance of germ cell genomic stability, we tested whether germ cells lacking SMC-5/6 functions are hypersensitive to ionizing radiation (IR). We followed a published protocol that examines the viability of eggs produced from radiation-damaged germ cells [31]. For each dose of gamma radiation exposure examined, the smc-5 and smc-6 mutants exhibited drastically reduced viability in comparison to wild type, consistent with the mutant germ cells being hypersensitive to DNA damage (Figure 3A).


Structural maintenance of chromosomes (SMC) proteins promote homolog-independent recombination repair in meiosis crucial for germ cell genomic stability.

Bickel JS, Chen L, Hayward J, Yeap SL, Alkers AE, Chan RC - PLoS Genet. (2010)

Loss of function mutations in smc-5 and smc-6 conferred hypersensitivity to ionizing radiation (IR) and increased DNA-damage responses in germ cells.(A) Graph of the viability of the eggs produced from mock and radiation-exposed germ cells in mutant F1 and wild-type L4-stage larvae as described [31]. 297 eggs or more were counted for each genotype and at each dose of IR. (B) Relative mRNA abundance for egl-1 after normalization to gamma tubulin tbg-1 as measured by quantitative RT-PCR [32]. Error bars represent standard errors. The asterisks (*) indicate significant changes from the wild-type control (p<0.001, t-Test). Gel analysis confirmed the size of each RT-PCR product and is shown underneath the graph. (C) Micrographs of germ cell corpses (white arrows) detected by CED-1::GFP fluorescence showed a greater number of corpses in the smc-5(tm2868) mutant germline. The average number of corpses per gonad and the standard error are indicated. Scale bars = 10 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2908675&req=5

pgen-1001028-g003: Loss of function mutations in smc-5 and smc-6 conferred hypersensitivity to ionizing radiation (IR) and increased DNA-damage responses in germ cells.(A) Graph of the viability of the eggs produced from mock and radiation-exposed germ cells in mutant F1 and wild-type L4-stage larvae as described [31]. 297 eggs or more were counted for each genotype and at each dose of IR. (B) Relative mRNA abundance for egl-1 after normalization to gamma tubulin tbg-1 as measured by quantitative RT-PCR [32]. Error bars represent standard errors. The asterisks (*) indicate significant changes from the wild-type control (p<0.001, t-Test). Gel analysis confirmed the size of each RT-PCR product and is shown underneath the graph. (C) Micrographs of germ cell corpses (white arrows) detected by CED-1::GFP fluorescence showed a greater number of corpses in the smc-5(tm2868) mutant germline. The average number of corpses per gonad and the standard error are indicated. Scale bars = 10 µm.
Mentions: In order to determine if the smc-5 and smc-6 mutants have defects in the maintenance of germ cell genomic stability, we tested whether germ cells lacking SMC-5/6 functions are hypersensitive to ionizing radiation (IR). We followed a published protocol that examines the viability of eggs produced from radiation-damaged germ cells [31]. For each dose of gamma radiation exposure examined, the smc-5 and smc-6 mutants exhibited drastically reduced viability in comparison to wild type, consistent with the mutant germ cells being hypersensitive to DNA damage (Figure 3A).

Bottom Line: Chromosome fragments associated with HR defects have only been reported in mutants, which have disrupted inter-homolog crossover.Surprisingly, the smc-5 and smc-6 mutations did not disrupt the formation of chiasmata, the cytologically visible linkages between homologous chromosomes formed from meiotic inter-homolog crossovers.Together, these results demonstrate that the successful completion of homolog-independent recombination is crucial for germ cell genomic stability.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.

ABSTRACT
In meiosis, programmed DNA breaks repaired by homologous recombination (HR) can be processed into inter-homolog crossovers that promote the accurate segregation of chromosomes. In general, more programmed DNA double-strand breaks (DSBs) are formed than the number of inter-homolog crossovers, and the excess DSBs must be repaired to maintain genomic stability. Sister-chromatid (inter-sister) recombination is postulated to be important for the completion of meiotic DSB repair. However, this hypothesis is difficult to test because of limited experimental means to disrupt inter-sister and not inter-homolog HR in meiosis. We find that the conserved Structural Maintenance of Chromosomes (SMC) 5 and 6 proteins in Caenorhabditis elegans are required for the successful completion of meiotic homologous recombination repair, yet they appeared to be dispensable for accurate chromosome segregation in meiosis. Mutations in the smc-5 and smc-6 genes induced chromosome fragments and dismorphology. Chromosome fragments associated with HR defects have only been reported in mutants, which have disrupted inter-homolog crossover. Surprisingly, the smc-5 and smc-6 mutations did not disrupt the formation of chiasmata, the cytologically visible linkages between homologous chromosomes formed from meiotic inter-homolog crossovers. The mutant fragmentation defect appeared to be preferentially enhanced by the disruptions of inter-homolog recombination but not by the disruptions of inter-sister recombination. Based on these findings, we propose that the C. elegans SMC-5/6 proteins are required in meiosis for the processing of homolog-independent, presumably sister-chromatid-mediated, recombination repair. Together, these results demonstrate that the successful completion of homolog-independent recombination is crucial for germ cell genomic stability.

Show MeSH
Related in: MedlinePlus