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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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The brca2 single and double mutants.(A) Position of the T-DNA insertions in AtBRCA2a and AtBRCA2b. The structure of the AtBRCA2a and AtBRCA2b genes is represented by shaded boxes (exons) and thin lines (introns). The T-DNA insertion position is indicated. Each primer pair used to identify the mutants by PCR are compiled on the diagram in black and primer pairs used for RT-PCR analyses are given in red; their localization is correct but not to scale. (B) Schematically represented Brca2 protein with the position of the BRC repeats and the NLS relative to the T-DNA insertions, as indicated by a star. For convenience, and because they share 94.5% of identity, a single Brca2 protein is represented. (C) RT-PCR analysis of AtBRCA2 transcripts in the single and double brca2 mutants. RNA was extracted from young floral buds of wild-type plants (2 different plants, a and b) as well as of brca2a, brca2b and brca2a brca2b (2 different plants, a and b) mutant plants and was then reverse-transcribed. Double-stranded cDNAs were then PCR-amplified using the primer pairs represented in red in Figure 1A. The constitutive ACTIN gene transcript was used as a control.
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pone-0026696-g001: The brca2 single and double mutants.(A) Position of the T-DNA insertions in AtBRCA2a and AtBRCA2b. The structure of the AtBRCA2a and AtBRCA2b genes is represented by shaded boxes (exons) and thin lines (introns). The T-DNA insertion position is indicated. Each primer pair used to identify the mutants by PCR are compiled on the diagram in black and primer pairs used for RT-PCR analyses are given in red; their localization is correct but not to scale. (B) Schematically represented Brca2 protein with the position of the BRC repeats and the NLS relative to the T-DNA insertions, as indicated by a star. For convenience, and because they share 94.5% of identity, a single Brca2 protein is represented. (C) RT-PCR analysis of AtBRCA2 transcripts in the single and double brca2 mutants. RNA was extracted from young floral buds of wild-type plants (2 different plants, a and b) as well as of brca2a, brca2b and brca2a brca2b (2 different plants, a and b) mutant plants and was then reverse-transcribed. Double-stranded cDNAs were then PCR-amplified using the primer pairs represented in red in Figure 1A. The constitutive ACTIN gene transcript was used as a control.

Mentions: In a previous study, AtBRCA2a and AtBRCA2b expression was inactivated during meiosis by RNAi using an inverted 510 pb-fragment of the BRCA2 cDNA under the control of the meiotic-specific promoter of DMC1 (pDMC1) [39]. In this work, single and double T-DNA insertion mutants for AtBRCA2 were isolated and their phenotype compared to the RNAi-inactivated plants (named pDMC1::RNAi/BRCA2 ). First, brca2 plants mutated in the AtBRCA2 genes via either a T-DNA insertion located in the 10th intron of AtBRCA2a (in the Cter DNA binding domain) or an insertion in the 4th exon of AtBRCA2b (in the Nter domain of the protein, containing the BRC motifs) were isolated (Figure 1A and Figure 1B). AtBRCA2 transcripts were analysed by RT-PCR, using primers flanking the insertion sites in wild-type and in brca2 single mutant plants. Transcripts of the disrupted genes were not detected in the corresponding mutant lines, whereas transcripts of each AtBRCA2 gene were amplified in wild-type plants. This strongly suggested that the two single brca2 lines were mutants (Figure 1C). Each single mutant showed normal development and fertility. By crossing the single mutants, the double brca2a brca2b mutant was obtained. These latter plants showed no growth defect and behaved as the wild-type under normal greenhouse conditions. However, they were partially sterile producing very short and mostly empty siliques (Figure 2A). Moreover, the presence of meiotic defects was observed after DAPI staining of the chromosomes in the meiocytes. Indeed, all meiotic figures showed chromosomal entangling without bivalent formation, bridges and fragmentation, leading to chromosomal missegregation (Figure 2B) as previously described for pDMC1::RNAi/BRCA2 plants. A transgene containing a full length AtBRCA2a cDNA under the control of the promoter of the meiotic recombinase Dmc1 (pDMC1::cDNA AtBRCA2a) was introduced in 13 brac2a brca2b double mutant plants. 11 transformant plants presented a restored phenotype: 9 were completely fertile as demonstrated by the observation of wild-type siliques content and normal meiosis (Figure 2) and 2 were partially fertile (as they presented some siliques that developed as sterile). Only 1 transformant was sterile with developmental defects. As a control, 11 brca2a brca2b double mutant plants were transformed with a transgene containing the pDMC1::RNAi/0 construct, corresponding to the “empty vector” [39]: all of them were sterile (data not shown). These results reinforce the evidence for the role of AtBRCA2 at meiosis, previously uncovered by our RNAi strategy.


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)

The brca2 single and double mutants.(A) Position of the T-DNA insertions in AtBRCA2a and AtBRCA2b. The structure of the AtBRCA2a and AtBRCA2b genes is represented by shaded boxes (exons) and thin lines (introns). The T-DNA insertion position is indicated. Each primer pair used to identify the mutants by PCR are compiled on the diagram in black and primer pairs used for RT-PCR analyses are given in red; their localization is correct but not to scale. (B) Schematically represented Brca2 protein with the position of the BRC repeats and the NLS relative to the T-DNA insertions, as indicated by a star. For convenience, and because they share 94.5% of identity, a single Brca2 protein is represented. (C) RT-PCR analysis of AtBRCA2 transcripts in the single and double brca2 mutants. RNA was extracted from young floral buds of wild-type plants (2 different plants, a and b) as well as of brca2a, brca2b and brca2a brca2b (2 different plants, a and b) mutant plants and was then reverse-transcribed. Double-stranded cDNAs were then PCR-amplified using the primer pairs represented in red in Figure 1A. The constitutive ACTIN gene transcript was used as a control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026696-g001: The brca2 single and double mutants.(A) Position of the T-DNA insertions in AtBRCA2a and AtBRCA2b. The structure of the AtBRCA2a and AtBRCA2b genes is represented by shaded boxes (exons) and thin lines (introns). The T-DNA insertion position is indicated. Each primer pair used to identify the mutants by PCR are compiled on the diagram in black and primer pairs used for RT-PCR analyses are given in red; their localization is correct but not to scale. (B) Schematically represented Brca2 protein with the position of the BRC repeats and the NLS relative to the T-DNA insertions, as indicated by a star. For convenience, and because they share 94.5% of identity, a single Brca2 protein is represented. (C) RT-PCR analysis of AtBRCA2 transcripts in the single and double brca2 mutants. RNA was extracted from young floral buds of wild-type plants (2 different plants, a and b) as well as of brca2a, brca2b and brca2a brca2b (2 different plants, a and b) mutant plants and was then reverse-transcribed. Double-stranded cDNAs were then PCR-amplified using the primer pairs represented in red in Figure 1A. The constitutive ACTIN gene transcript was used as a control.
Mentions: In a previous study, AtBRCA2a and AtBRCA2b expression was inactivated during meiosis by RNAi using an inverted 510 pb-fragment of the BRCA2 cDNA under the control of the meiotic-specific promoter of DMC1 (pDMC1) [39]. In this work, single and double T-DNA insertion mutants for AtBRCA2 were isolated and their phenotype compared to the RNAi-inactivated plants (named pDMC1::RNAi/BRCA2 ). First, brca2 plants mutated in the AtBRCA2 genes via either a T-DNA insertion located in the 10th intron of AtBRCA2a (in the Cter DNA binding domain) or an insertion in the 4th exon of AtBRCA2b (in the Nter domain of the protein, containing the BRC motifs) were isolated (Figure 1A and Figure 1B). AtBRCA2 transcripts were analysed by RT-PCR, using primers flanking the insertion sites in wild-type and in brca2 single mutant plants. Transcripts of the disrupted genes were not detected in the corresponding mutant lines, whereas transcripts of each AtBRCA2 gene were amplified in wild-type plants. This strongly suggested that the two single brca2 lines were mutants (Figure 1C). Each single mutant showed normal development and fertility. By crossing the single mutants, the double brca2a brca2b mutant was obtained. These latter plants showed no growth defect and behaved as the wild-type under normal greenhouse conditions. However, they were partially sterile producing very short and mostly empty siliques (Figure 2A). Moreover, the presence of meiotic defects was observed after DAPI staining of the chromosomes in the meiocytes. Indeed, all meiotic figures showed chromosomal entangling without bivalent formation, bridges and fragmentation, leading to chromosomal missegregation (Figure 2B) as previously described for pDMC1::RNAi/BRCA2 plants. A transgene containing a full length AtBRCA2a cDNA under the control of the promoter of the meiotic recombinase Dmc1 (pDMC1::cDNA AtBRCA2a) was introduced in 13 brac2a brca2b double mutant plants. 11 transformant plants presented a restored phenotype: 9 were completely fertile as demonstrated by the observation of wild-type siliques content and normal meiosis (Figure 2) and 2 were partially fertile (as they presented some siliques that developed as sterile). Only 1 transformant was sterile with developmental defects. As a control, 11 brca2a brca2b double mutant plants were transformed with a transgene containing the pDMC1::RNAi/0 construct, corresponding to the “empty vector” [39]: all of them were sterile (data not shown). These results reinforce the evidence for the role of AtBRCA2 at meiosis, previously uncovered by our RNAi strategy.

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