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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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RT-PCR analysis of genes involved in NHEJ and SSA in the single and double brca2 mutants.RNA was extracted from young floral buds and reverse-transcribed, as described in Figure 1C. Double-stranded cDNAs were PCR-amplified using primer pair 454/455 for AtKU80 (see primer positions in Figure 4 and sequences in Table1), 336/445 for AtLIGIV and 452/453 for AtERCC1 (see Table 1 for sequences). The constitutive ACTIN gene transcript used as a control is presented in Figure 1C.
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pone-0026696-g003: RT-PCR analysis of genes involved in NHEJ and SSA in the single and double brca2 mutants.RNA was extracted from young floral buds and reverse-transcribed, as described in Figure 1C. Double-stranded cDNAs were PCR-amplified using primer pair 454/455 for AtKU80 (see primer positions in Figure 4 and sequences in Table1), 336/445 for AtLIGIV and 452/453 for AtERCC1 (see Table 1 for sequences). The constitutive ACTIN gene transcript used as a control is presented in Figure 1C.

Mentions: In order to identify the molecular pathways involved in the aberrant cytological phenotype observed in the Brca2-deficient plants during meiosis, mutant plants deficient in either the NHEJ (ku80-/- and ligIV-/-) or the SSA (ercc1-/-) pathways were characterized. Examining amplification of these transcripts specifically in meiocytes was not possible, as meiocytes would have to be specifically dissected which is technically difficult. However, as shown in Figure 3, all these three genes, and thus the pathways they are involved in, were found expressed in young flower buds, where meiosis takes place, in single as well as in double brca2 mutant plants. Two mutant lines have been previously described: SALK_044027, where the T-DNA insertion is in exon 6 of the AtLIGIV gene [42], [43] and SALK_033397 which contains a T-DNA insertion in exon 3 of AtERCC1 [16]. The absence of transcripts corresponding to the affected gene was confirmed for each mutant line by RT-PCR using primers flanking each T-DNA insertion (data not shown). The ku mutant line used in this study (SALK_112921) had not been characterized to date. It contains a T-DNA in the 6th intron of the AtKU80 gene (Figure 4A). RT-PCR analysis of the 5′ and 3′ regions flanking the T-DNA insertion revealed the presence of AtKU80 transcripts in both wild-type and ku80 mutant plants (Figure 4B). However, no transcripts could be detected in ku80 mutant plants when primers flanking the T-DNA insertion were used, suggesting that splicing of the 6th intron did not occur in the ku80 mutant. As the insertion site is positioned in the region encoding the domain involved in hetero-dimerization with Ku70, it is most likely that a putative protein, lacking this domain, would be non-functional. Thus, these ku80 plants were considered as functional mutants. The mutant plants, whatever the affected DNA repair pathway, exhibited no obvious developmental defects under normal growth conditions and were fertile, as previously described for ercc1, ku80 and ligIV Arabidopsis mutants [9], [13], [16].


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)

RT-PCR analysis of genes involved in NHEJ and SSA in the single and double brca2 mutants.RNA was extracted from young floral buds and reverse-transcribed, as described in Figure 1C. Double-stranded cDNAs were PCR-amplified using primer pair 454/455 for AtKU80 (see primer positions in Figure 4 and sequences in Table1), 336/445 for AtLIGIV and 452/453 for AtERCC1 (see Table 1 for sequences). The constitutive ACTIN gene transcript used as a control is presented in Figure 1C.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026696-g003: RT-PCR analysis of genes involved in NHEJ and SSA in the single and double brca2 mutants.RNA was extracted from young floral buds and reverse-transcribed, as described in Figure 1C. Double-stranded cDNAs were PCR-amplified using primer pair 454/455 for AtKU80 (see primer positions in Figure 4 and sequences in Table1), 336/445 for AtLIGIV and 452/453 for AtERCC1 (see Table 1 for sequences). The constitutive ACTIN gene transcript used as a control is presented in Figure 1C.
Mentions: In order to identify the molecular pathways involved in the aberrant cytological phenotype observed in the Brca2-deficient plants during meiosis, mutant plants deficient in either the NHEJ (ku80-/- and ligIV-/-) or the SSA (ercc1-/-) pathways were characterized. Examining amplification of these transcripts specifically in meiocytes was not possible, as meiocytes would have to be specifically dissected which is technically difficult. However, as shown in Figure 3, all these three genes, and thus the pathways they are involved in, were found expressed in young flower buds, where meiosis takes place, in single as well as in double brca2 mutant plants. Two mutant lines have been previously described: SALK_044027, where the T-DNA insertion is in exon 6 of the AtLIGIV gene [42], [43] and SALK_033397 which contains a T-DNA insertion in exon 3 of AtERCC1 [16]. The absence of transcripts corresponding to the affected gene was confirmed for each mutant line by RT-PCR using primers flanking each T-DNA insertion (data not shown). The ku mutant line used in this study (SALK_112921) had not been characterized to date. It contains a T-DNA in the 6th intron of the AtKU80 gene (Figure 4A). RT-PCR analysis of the 5′ and 3′ regions flanking the T-DNA insertion revealed the presence of AtKU80 transcripts in both wild-type and ku80 mutant plants (Figure 4B). However, no transcripts could be detected in ku80 mutant plants when primers flanking the T-DNA insertion were used, suggesting that splicing of the 6th intron did not occur in the ku80 mutant. As the insertion site is positioned in the region encoding the domain involved in hetero-dimerization with Ku70, it is most likely that a putative protein, lacking this domain, would be non-functional. Thus, these ku80 plants were considered as functional mutants. The mutant plants, whatever the affected DNA repair pathway, exhibited no obvious developmental defects under normal growth conditions and were fertile, as previously described for ercc1, ku80 and ligIV Arabidopsis mutants [9], [13], [16].

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