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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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Meiotic defects in brca2a brca2b mutant plants and in wild-type Brca2-inactivated plants.(A) Wild-type and brca2 double mutant plants exhibt no growth defect except for sterility. Chloralhydrate discolored siliques are full of seeds in wild-type plants in comparison with the discolored siliques of the brca2 double mutant plants. (B) Observation of meiocytes by DAPI staining in Brca2-deficient plants, transformed or not with the full length cDNA of AtBRCA2a, and in brca2a brca2b homozygous double mutant plants. (A–E) Different stages of meiosis in the wild-type plants. Meiosis is normal. (A) Prophase I stage, (B) diakinesis, the five bivalents are attached by a chiasma, (C) metaphase I with five aligned bivalents, (D) anaphase I, bivalents segregate into two sets of five univalents, (E) anaphase II, with four groups that contain five chromosomes each after sister chromatid separation. (F–J) Different stages of meiosis in wild-type plants transformed with the pDMC1::RNAi/BRCA2 construct. (F) Prophase I, (G) no normal diakinesis phase (H) metaphase I with condensed and entangled chromosomes, (I) anaphase I, with entangled and stretched chromosomes. (J) Anaphase II, with bridges extending between chromosomes. (K–O) Different stages of meiosis in brca2 double mutant plants. (K) Prophase I, (L) anaphase I, entangled and stretched chromosomes. (M) Metaphase II with entangled chromosomes. (N) anaphase II, fragmentated chromosomes. (O) telophase II with chromosome missegregation. (P–T) Different stages of meiosis in brca2 double mutant plants, transformed with the pDMC1::cDNA AtBRCA2a. Meiosis is restored to normal. (P) Prophase I stage, (Q) diakinesis, (R) metaphase I, (S) anaphase I, (T) anaphase II. Bar 10 µm.
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pone-0026696-g002: Meiotic defects in brca2a brca2b mutant plants and in wild-type Brca2-inactivated plants.(A) Wild-type and brca2 double mutant plants exhibt no growth defect except for sterility. Chloralhydrate discolored siliques are full of seeds in wild-type plants in comparison with the discolored siliques of the brca2 double mutant plants. (B) Observation of meiocytes by DAPI staining in Brca2-deficient plants, transformed or not with the full length cDNA of AtBRCA2a, and in brca2a brca2b homozygous double mutant plants. (A–E) Different stages of meiosis in the wild-type plants. Meiosis is normal. (A) Prophase I stage, (B) diakinesis, the five bivalents are attached by a chiasma, (C) metaphase I with five aligned bivalents, (D) anaphase I, bivalents segregate into two sets of five univalents, (E) anaphase II, with four groups that contain five chromosomes each after sister chromatid separation. (F–J) Different stages of meiosis in wild-type plants transformed with the pDMC1::RNAi/BRCA2 construct. (F) Prophase I, (G) no normal diakinesis phase (H) metaphase I with condensed and entangled chromosomes, (I) anaphase I, with entangled and stretched chromosomes. (J) Anaphase II, with bridges extending between chromosomes. (K–O) Different stages of meiosis in brca2 double mutant plants. (K) Prophase I, (L) anaphase I, entangled and stretched chromosomes. (M) Metaphase II with entangled chromosomes. (N) anaphase II, fragmentated chromosomes. (O) telophase II with chromosome missegregation. (P–T) Different stages of meiosis in brca2 double mutant plants, transformed with the pDMC1::cDNA AtBRCA2a. Meiosis is restored to normal. (P) Prophase I stage, (Q) diakinesis, (R) metaphase I, (S) anaphase I, (T) anaphase II. Bar 10 µm.

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)

Meiotic defects in brca2a brca2b mutant plants and in wild-type Brca2-inactivated plants.(A) Wild-type and brca2 double mutant plants exhibt no growth defect except for sterility. Chloralhydrate discolored siliques are full of seeds in wild-type plants in comparison with the discolored siliques of the brca2 double mutant plants. (B) Observation of meiocytes by DAPI staining in Brca2-deficient plants, transformed or not with the full length cDNA of AtBRCA2a, and in brca2a brca2b homozygous double mutant plants. (A–E) Different stages of meiosis in the wild-type plants. Meiosis is normal. (A) Prophase I stage, (B) diakinesis, the five bivalents are attached by a chiasma, (C) metaphase I with five aligned bivalents, (D) anaphase I, bivalents segregate into two sets of five univalents, (E) anaphase II, with four groups that contain five chromosomes each after sister chromatid separation. (F–J) Different stages of meiosis in wild-type plants transformed with the pDMC1::RNAi/BRCA2 construct. (F) Prophase I, (G) no normal diakinesis phase (H) metaphase I with condensed and entangled chromosomes, (I) anaphase I, with entangled and stretched chromosomes. (J) Anaphase II, with bridges extending between chromosomes. (K–O) Different stages of meiosis in brca2 double mutant plants. (K) Prophase I, (L) anaphase I, entangled and stretched chromosomes. (M) Metaphase II with entangled chromosomes. (N) anaphase II, fragmentated chromosomes. (O) telophase II with chromosome missegregation. (P–T) Different stages of meiosis in brca2 double mutant plants, transformed with the pDMC1::cDNA AtBRCA2a. Meiosis is restored to normal. (P) Prophase I stage, (Q) diakinesis, (R) metaphase I, (S) anaphase I, (T) anaphase II. Bar 10 µm.
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pone-0026696-g002: Meiotic defects in brca2a brca2b mutant plants and in wild-type Brca2-inactivated plants.(A) Wild-type and brca2 double mutant plants exhibt no growth defect except for sterility. Chloralhydrate discolored siliques are full of seeds in wild-type plants in comparison with the discolored siliques of the brca2 double mutant plants. (B) Observation of meiocytes by DAPI staining in Brca2-deficient plants, transformed or not with the full length cDNA of AtBRCA2a, and in brca2a brca2b homozygous double mutant plants. (A–E) Different stages of meiosis in the wild-type plants. Meiosis is normal. (A) Prophase I stage, (B) diakinesis, the five bivalents are attached by a chiasma, (C) metaphase I with five aligned bivalents, (D) anaphase I, bivalents segregate into two sets of five univalents, (E) anaphase II, with four groups that contain five chromosomes each after sister chromatid separation. (F–J) Different stages of meiosis in wild-type plants transformed with the pDMC1::RNAi/BRCA2 construct. (F) Prophase I, (G) no normal diakinesis phase (H) metaphase I with condensed and entangled chromosomes, (I) anaphase I, with entangled and stretched chromosomes. (J) Anaphase II, with bridges extending between chromosomes. (K–O) Different stages of meiosis in brca2 double mutant plants. (K) Prophase I, (L) anaphase I, entangled and stretched chromosomes. (M) Metaphase II with entangled chromosomes. (N) anaphase II, fragmentated chromosomes. (O) telophase II with chromosome missegregation. (P–T) Different stages of meiosis in brca2 double mutant plants, transformed with the pDMC1::cDNA AtBRCA2a. Meiosis is restored to normal. (P) Prophase I stage, (Q) diakinesis, (R) metaphase I, (S) anaphase I, (T) anaphase II. Bar 10 µm.
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