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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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Observation of meiocytes by DAPI staining in nhej, ssa, nhej ssa and lig6 mutant plants transformed with the pDMC1::RNAi/BRCA2 construct.Different stages of meiosis were observed in plants transformed with pDMC1::RNAi/BRCA2 in nhej mutant plants, ku80 (A–D) or lig4 (E–H), and in ssa mutant plants, ercc1 (I–L), in nhej ssa double mutant plants, ercc1 ku80 (M–P) or ercc1 lig4 (Q–T) and in lig6 mutant plants (U–X). (A, E, I, M, Q, U) prophase I. (B, F, J, N, R, V) metaphase I. (C, G, K, O, S, W) anaphase I. (D, H, L, P, T, X) anaphase II. Bar 10 µm.
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pone-0026696-g006: Observation of meiocytes by DAPI staining in nhej, ssa, nhej ssa and lig6 mutant plants transformed with the pDMC1::RNAi/BRCA2 construct.Different stages of meiosis were observed in plants transformed with pDMC1::RNAi/BRCA2 in nhej mutant plants, ku80 (A–D) or lig4 (E–H), and in ssa mutant plants, ercc1 (I–L), in nhej ssa double mutant plants, ercc1 ku80 (M–P) or ercc1 lig4 (Q–T) and in lig6 mutant plants (U–X). (A, E, I, M, Q, U) prophase I. (B, F, J, N, R, V) metaphase I. (C, G, K, O, S, W) anaphase I. (D, H, L, P, T, X) anaphase II. Bar 10 µm.

Mentions: The meiotic behaviour was examined after DAPI staining of the chromosomes in the meiocytes of several independent transformed plants that were inactivated for the Brca2 function: 175 meiotic figures from two ku80, 170 meioses from two ligIV and 34 meioses from two ercc1 lines independently transformed with the pDMC1::RNAi/BRCA2 construct were observed. As a control, they were compared to the meioses of one ku80, one ligIV and one ercc1 plant containing the RNAi/0 construct. All of the observed control plant meiotic figures were normal in the single mutants affected for either NHEJ (ku80, ligIV) or SSA (ercc1), as well as in the nhej ssa double mutant (Supplementary Figure S1). On the other hand, meiosis was profoundly disturbed in meiocytes of these same mutant lines transformed with the pDMC1::RNAi/BRCA2 construct: chromosomal entangling without bivalent formation, fragmentation, and missegregation of chromosomes (Figure 6). Such observations have been previously reported in wild-type pDMC1::RNAi/BRCA2 plants [39]. These observations suggested that, contrary to our hypothesis, in the absence of Brca2 during meiosis, neither NHEJ nor SSA were responsible for an alternative meiotic DSB repair that would have been revealed because of the absence of HR [39]. The impact of the inactivation of both pathways in the absence of Brca2 during meiosis was also examined. Meiotic figures from one pDMC1::RNAi/BRCA2 transformant for ercc1 ku80 (257 meiotic events, among them 80 were post-prophase) and two pDMC1::RNAi/BRCA2 transformants for ercc1 lig4 (115 meiosis, including 63 post-prophase stages) were observed. In all double mutant plants transformed with pDMC1::RNAi/BRCA2, the brca2 meiotic phenotype remained unaltered (Figure 6).


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)

Observation of meiocytes by DAPI staining in nhej, ssa, nhej ssa and lig6 mutant plants transformed with the pDMC1::RNAi/BRCA2 construct.Different stages of meiosis were observed in plants transformed with pDMC1::RNAi/BRCA2 in nhej mutant plants, ku80 (A–D) or lig4 (E–H), and in ssa mutant plants, ercc1 (I–L), in nhej ssa double mutant plants, ercc1 ku80 (M–P) or ercc1 lig4 (Q–T) and in lig6 mutant plants (U–X). (A, E, I, M, Q, U) prophase I. (B, F, J, N, R, V) metaphase I. (C, G, K, O, S, W) anaphase I. (D, H, L, P, T, X) anaphase II. Bar 10 µm.
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Related In: Results  -  Collection

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pone-0026696-g006: Observation of meiocytes by DAPI staining in nhej, ssa, nhej ssa and lig6 mutant plants transformed with the pDMC1::RNAi/BRCA2 construct.Different stages of meiosis were observed in plants transformed with pDMC1::RNAi/BRCA2 in nhej mutant plants, ku80 (A–D) or lig4 (E–H), and in ssa mutant plants, ercc1 (I–L), in nhej ssa double mutant plants, ercc1 ku80 (M–P) or ercc1 lig4 (Q–T) and in lig6 mutant plants (U–X). (A, E, I, M, Q, U) prophase I. (B, F, J, N, R, V) metaphase I. (C, G, K, O, S, W) anaphase I. (D, H, L, P, T, X) anaphase II. Bar 10 µm.
Mentions: The meiotic behaviour was examined after DAPI staining of the chromosomes in the meiocytes of several independent transformed plants that were inactivated for the Brca2 function: 175 meiotic figures from two ku80, 170 meioses from two ligIV and 34 meioses from two ercc1 lines independently transformed with the pDMC1::RNAi/BRCA2 construct were observed. As a control, they were compared to the meioses of one ku80, one ligIV and one ercc1 plant containing the RNAi/0 construct. All of the observed control plant meiotic figures were normal in the single mutants affected for either NHEJ (ku80, ligIV) or SSA (ercc1), as well as in the nhej ssa double mutant (Supplementary Figure S1). On the other hand, meiosis was profoundly disturbed in meiocytes of these same mutant lines transformed with the pDMC1::RNAi/BRCA2 construct: chromosomal entangling without bivalent formation, fragmentation, and missegregation of chromosomes (Figure 6). Such observations have been previously reported in wild-type pDMC1::RNAi/BRCA2 plants [39]. These observations suggested that, contrary to our hypothesis, in the absence of Brca2 during meiosis, neither NHEJ nor SSA were responsible for an alternative meiotic DSB repair that would have been revealed because of the absence of HR [39]. The impact of the inactivation of both pathways in the absence of Brca2 during meiosis was also examined. Meiotic figures from one pDMC1::RNAi/BRCA2 transformant for ercc1 ku80 (257 meiotic events, among them 80 were post-prophase) and two pDMC1::RNAi/BRCA2 transformants for ercc1 lig4 (115 meiosis, including 63 post-prophase stages) were observed. In all double mutant plants transformed with pDMC1::RNAi/BRCA2, the brca2 meiotic phenotype remained unaltered (Figure 6).

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