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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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T-DNA insertion and expression in lig6 mutant.(A) Position of the T-DNA insertion in AtLIG6. The structure of the AtLIG6 gene 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 indicated in black while primer pairs used for RT-PCR analyses are given in red; their localization is correct but not to of scale. (B) RT-PCR analysis of AtLIG6 transcripts in lig6-/- mutant plants. RNA, extracted from floral buds of wild-type or lig6 mutant plants was reverse-transcribed. Double-stranded cDNAs were amplified by RT-PCR, performed with three different primer pairs: 5′ or 3′ to the T-DNAand flanking the T-DNA. The position of each primer is given above (Figure 7A). The constitutive ACTIN gene was used as a control.
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pone-0026696-g007: T-DNA insertion and expression in lig6 mutant.(A) Position of the T-DNA insertion in AtLIG6. The structure of the AtLIG6 gene 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 indicated in black while primer pairs used for RT-PCR analyses are given in red; their localization is correct but not to of scale. (B) RT-PCR analysis of AtLIG6 transcripts in lig6-/- mutant plants. RNA, extracted from floral buds of wild-type or lig6 mutant plants was reverse-transcribed. Double-stranded cDNAs were amplified by RT-PCR, performed with three different primer pairs: 5′ or 3′ to the T-DNAand flanking the T-DNA. The position of each primer is given above (Figure 7A). The constitutive ACTIN gene was used as a control.

Mentions: Homozygous lig6 plants containing a T-DNA insertion in exon 11 of the gene were obtained from the SALK collection (SALK_065307) (Figure 7A). All plants grew normally, they were fertile and undertook normal meioses (data not shown). These observations are in agreement with what was previously observed in a different lig6 insertional line (Waterworth et al, 2010) [44]. RT-PCR analyses detected transcripts on both sides of the T-DNA insertion but no transcripts could be found when primers flanking the T-DNA insertion were used (Figure 7B). As the T-DNA insertion is positioned in an exon, 42 bp from the codon of the catalytic lysine just upstream from the conserved motif II [49] lying in the core domain, and more specifically around the nucleotide binding pocket responsible for the nucleotidyl transfer, the catalytic activity of a putatively expressed protein in this mutant is probably non-functional.


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)

T-DNA insertion and expression in lig6 mutant.(A) Position of the T-DNA insertion in AtLIG6. The structure of the AtLIG6 gene 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 indicated in black while primer pairs used for RT-PCR analyses are given in red; their localization is correct but not to of scale. (B) RT-PCR analysis of AtLIG6 transcripts in lig6-/- mutant plants. RNA, extracted from floral buds of wild-type or lig6 mutant plants was reverse-transcribed. Double-stranded cDNAs were amplified by RT-PCR, performed with three different primer pairs: 5′ or 3′ to the T-DNAand flanking the T-DNA. The position of each primer is given above (Figure 7A). The constitutive ACTIN gene was used as a control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026696-g007: T-DNA insertion and expression in lig6 mutant.(A) Position of the T-DNA insertion in AtLIG6. The structure of the AtLIG6 gene 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 indicated in black while primer pairs used for RT-PCR analyses are given in red; their localization is correct but not to of scale. (B) RT-PCR analysis of AtLIG6 transcripts in lig6-/- mutant plants. RNA, extracted from floral buds of wild-type or lig6 mutant plants was reverse-transcribed. Double-stranded cDNAs were amplified by RT-PCR, performed with three different primer pairs: 5′ or 3′ to the T-DNAand flanking the T-DNA. The position of each primer is given above (Figure 7A). The constitutive ACTIN gene was used as a control.
Mentions: Homozygous lig6 plants containing a T-DNA insertion in exon 11 of the gene were obtained from the SALK collection (SALK_065307) (Figure 7A). All plants grew normally, they were fertile and undertook normal meioses (data not shown). These observations are in agreement with what was previously observed in a different lig6 insertional line (Waterworth et al, 2010) [44]. RT-PCR analyses detected transcripts on both sides of the T-DNA insertion but no transcripts could be found when primers flanking the T-DNA insertion were used (Figure 7B). As the T-DNA insertion is positioned in an exon, 42 bp from the codon of the catalytic lysine just upstream from the conserved motif II [49] lying in the core domain, and more specifically around the nucleotide binding pocket responsible for the nucleotidyl transfer, the catalytic activity of a putatively expressed protein in this mutant is probably non-functional.

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