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Characterization of Brca2-deficient plants excludes the role of NHEJ and SSA in the meiotic chromosomal defect phenotype.

Dumont M, Massot S, Doutriaux MP, Gratias A - PLoS ONE (2011)

Bottom Line: The resulting nucleofilament can thus invade a homologous DNA sequence to copy and restore the original genetic information.Moreover, it is demonstrated that during meiosis, neither NHEJ nor SSA compensate for HR deficiency in BRCA2-inactivated plants.The possible mechanism(s) involved in the formation of these aberrant chromosomal bridges in the absence of HR during meiosis are discussed.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie des Plantes, CNRS UMR8618, Université Paris Sud-11, Orsay, France.

ABSTRACT
In somatic cells, three major pathways are involved in the repair of DNA double-strand breaks (DBS): Non-Homologous End Joining (NHEJ), Single-Strand Annealing (SSA) and Homologous Recombination (HR). In somatic and meiotic HR, DNA DSB are 5' to 3' resected, producing long 3' single-stranded DNA extensions. Brca2 is essential to load the Rad51 recombinase onto these 3' overhangs. The resulting nucleofilament can thus invade a homologous DNA sequence to copy and restore the original genetic information. In Arabidopsis, the inactivation of Brca2 specifically during meiosis by an RNAi approach results in aberrant chromosome aggregates, chromosomal fragmentation and missegregation leading to a sterility phenotype. We had previously suggested that such chromosomal behaviour could be due to NHEJ. In this study, we show that knock-out plants affected in both BRCA2 genes show the same meiotic phenotype as the RNAi-inactivated plants. Moreover, it is demonstrated that during meiosis, neither NHEJ nor SSA compensate for HR deficiency in BRCA2-inactivated plants. The role of the plant-specific DNA Ligase6 is also excluded. The possible mechanism(s) involved in the formation of these aberrant chromosomal bridges in the absence of HR during meiosis are discussed.

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Hypersentivity to MMS, gamma-rays and UV irradiation of nhej, ssa and nhej ssa plants.Before sowing, all seeds were surface-sterilized. (A) MMS hypersensitivity, 11 days post-germination. Seeds were sown on MS 0.5 agar 1% sucrose supplemented with MMS at various doses. (B) Gamma-irradiation hypersentivity, 7 days post-irradiation. After 48 h at 4°C in darkness, seeds were exposed to various doses of gamma-rays : 0, 100 and 200 grays before being sown on MS 0.5 agar. (C) UV hypersensitivity, 10 days post-irradiation. Seeds were sown in MS 0.5 agar. After 4 days of growth, the plantlets were exposed to UV-C, left in the dark for 3 days to avoid photoreactivation, and then exposed to light.
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pone-0026696-g005: Hypersentivity to MMS, gamma-rays and UV irradiation of nhej, ssa and nhej ssa plants.Before sowing, all seeds were surface-sterilized. (A) MMS hypersensitivity, 11 days post-germination. Seeds were sown on MS 0.5 agar 1% sucrose supplemented with MMS at various doses. (B) Gamma-irradiation hypersentivity, 7 days post-irradiation. After 48 h at 4°C in darkness, seeds were exposed to various doses of gamma-rays : 0, 100 and 200 grays before being sown on MS 0.5 agar. (C) UV hypersensitivity, 10 days post-irradiation. Seeds were sown in MS 0.5 agar. After 4 days of growth, the plantlets were exposed to UV-C, left in the dark for 3 days to avoid photoreactivation, and then exposed to light.

Mentions: Sensitivity to various DNA damaging agents is a classical assay to characterize DNA repair mutant plants as most of them show no obvious somatic phenotype. To control that our mutants were indeed affected in DNA repair, their sensitivity to MMS, gamma-ray and UV irradiation was assayed. In comparison to wild-type plants, root growth was affected in the nhej plants as well as in the ssa plants in the presence of MMS or after gamma exposure. Indeed, the MMS hypersensitivity was visible at 50 ppm and gamma–ray hypersensitivity was observed at 100 grays for each single mutant line. However, MMS-induced retarded growth was more pronounced in ercc1 than in ku80 and ligIV plants (Figure 5A). MMS is a methylating agent, and due to the occurrence and clustering of modified bases, it can generate both SSB and DSB, which is reflected in the fact that ercc1 mutants (deficient for both SSA and BER) appeared to be more sensitive to this genotoxic treatment. Reciprocally, ercc1 plants were less sensitive to gamma irradiation when compared to ku80 and ligIV (Figure 5B). Ionising radiations mainly give rise to clustered DNA damages (modified bases and abasic sites) that lead to DNA DSB. Such DNA strand breaks are mostly repaired by NHEJ as suggested by the higher hypersensitivity of ku80 and ligIV mutants to gamma-rays. Finally, as expected, only the ercc1 plants were hypersensitive to UV exposure (Figure 5C). All of these results confirmed that the different mutant Arabidopsis lines were affected in DNA DSB repair.


Characterization of Brca2-deficient plants excludes the role of NHEJ and SSA in the meiotic chromosomal defect phenotype.

Dumont M, Massot S, Doutriaux MP, Gratias A - PLoS ONE (2011)

Hypersentivity to MMS, gamma-rays and UV irradiation of nhej, ssa and nhej ssa plants.Before sowing, all seeds were surface-sterilized. (A) MMS hypersensitivity, 11 days post-germination. Seeds were sown on MS 0.5 agar 1% sucrose supplemented with MMS at various doses. (B) Gamma-irradiation hypersentivity, 7 days post-irradiation. After 48 h at 4°C in darkness, seeds were exposed to various doses of gamma-rays : 0, 100 and 200 grays before being sown on MS 0.5 agar. (C) UV hypersensitivity, 10 days post-irradiation. Seeds were sown in MS 0.5 agar. After 4 days of growth, the plantlets were exposed to UV-C, left in the dark for 3 days to avoid photoreactivation, and then exposed to light.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198793&req=5

pone-0026696-g005: Hypersentivity to MMS, gamma-rays and UV irradiation of nhej, ssa and nhej ssa plants.Before sowing, all seeds were surface-sterilized. (A) MMS hypersensitivity, 11 days post-germination. Seeds were sown on MS 0.5 agar 1% sucrose supplemented with MMS at various doses. (B) Gamma-irradiation hypersentivity, 7 days post-irradiation. After 48 h at 4°C in darkness, seeds were exposed to various doses of gamma-rays : 0, 100 and 200 grays before being sown on MS 0.5 agar. (C) UV hypersensitivity, 10 days post-irradiation. Seeds were sown in MS 0.5 agar. After 4 days of growth, the plantlets were exposed to UV-C, left in the dark for 3 days to avoid photoreactivation, and then exposed to light.
Mentions: Sensitivity to various DNA damaging agents is a classical assay to characterize DNA repair mutant plants as most of them show no obvious somatic phenotype. To control that our mutants were indeed affected in DNA repair, their sensitivity to MMS, gamma-ray and UV irradiation was assayed. In comparison to wild-type plants, root growth was affected in the nhej plants as well as in the ssa plants in the presence of MMS or after gamma exposure. Indeed, the MMS hypersensitivity was visible at 50 ppm and gamma–ray hypersensitivity was observed at 100 grays for each single mutant line. However, MMS-induced retarded growth was more pronounced in ercc1 than in ku80 and ligIV plants (Figure 5A). MMS is a methylating agent, and due to the occurrence and clustering of modified bases, it can generate both SSB and DSB, which is reflected in the fact that ercc1 mutants (deficient for both SSA and BER) appeared to be more sensitive to this genotoxic treatment. Reciprocally, ercc1 plants were less sensitive to gamma irradiation when compared to ku80 and ligIV (Figure 5B). Ionising radiations mainly give rise to clustered DNA damages (modified bases and abasic sites) that lead to DNA DSB. Such DNA strand breaks are mostly repaired by NHEJ as suggested by the higher hypersensitivity of ku80 and ligIV mutants to gamma-rays. Finally, as expected, only the ercc1 plants were hypersensitive to UV exposure (Figure 5C). All of these results confirmed that the different mutant Arabidopsis lines were affected in DNA DSB repair.

Bottom Line: The resulting nucleofilament can thus invade a homologous DNA sequence to copy and restore the original genetic information.Moreover, it is demonstrated that during meiosis, neither NHEJ nor SSA compensate for HR deficiency in BRCA2-inactivated plants.The possible mechanism(s) involved in the formation of these aberrant chromosomal bridges in the absence of HR during meiosis are discussed.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie des Plantes, CNRS UMR8618, Université Paris Sud-11, Orsay, France.

ABSTRACT
In somatic cells, three major pathways are involved in the repair of DNA double-strand breaks (DBS): Non-Homologous End Joining (NHEJ), Single-Strand Annealing (SSA) and Homologous Recombination (HR). In somatic and meiotic HR, DNA DSB are 5' to 3' resected, producing long 3' single-stranded DNA extensions. Brca2 is essential to load the Rad51 recombinase onto these 3' overhangs. The resulting nucleofilament can thus invade a homologous DNA sequence to copy and restore the original genetic information. In Arabidopsis, the inactivation of Brca2 specifically during meiosis by an RNAi approach results in aberrant chromosome aggregates, chromosomal fragmentation and missegregation leading to a sterility phenotype. We had previously suggested that such chromosomal behaviour could be due to NHEJ. In this study, we show that knock-out plants affected in both BRCA2 genes show the same meiotic phenotype as the RNAi-inactivated plants. Moreover, it is demonstrated that during meiosis, neither NHEJ nor SSA compensate for HR deficiency in BRCA2-inactivated plants. The role of the plant-specific DNA Ligase6 is also excluded. The possible mechanism(s) involved in the formation of these aberrant chromosomal bridges in the absence of HR during meiosis are discussed.

Show MeSH
Related in: MedlinePlus