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The MCM-binding protein ETG1 aids sister chromatid cohesion required for postreplicative homologous recombination repair.

Takahashi N, Quimbaya M, Schubert V, Lammens T, Vandepoele K, Schubert I, Matsui M, Inzé D, Berx G, De Veylder L - PLoS Genet. (2010)

Bottom Line: Here, we demonstrate that depletion of the evolutionarily conserved minichromosome maintenance helicase-binding protein ETG1 of Arabidopsis thaliana resulted in a stringent late G2 cell cycle arrest.We conclude that the ETG1 replication factor is required for efficient cohesion and that cohesion establishment is essential for proper development of plants suffering from endogenous DNA stress.Cohesion defects observed upon knockdown of its human counterpart suggest an equally important developmental role for the orthologous mammalian ETG1 protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Systems Biology, Flanders Institute for Biotechnology (VIB), Gent, Belgium.

ABSTRACT
The DNA replication process represents a source of DNA stress that causes potentially spontaneous genome damage. This effect might be strengthened by mutations in crucial replication factors, requiring the activation of DNA damage checkpoints to enable DNA repair before anaphase onset. Here, we demonstrate that depletion of the evolutionarily conserved minichromosome maintenance helicase-binding protein ETG1 of Arabidopsis thaliana resulted in a stringent late G2 cell cycle arrest. This arrest correlated with a partial loss of sister chromatid cohesion. The lack-of-cohesion phenotype was intensified in plants without functional CTF18, a replication fork factor needed for cohesion establishment. The synergistic effect of the etg1 and ctf18 mutants on sister chromatid cohesion strengthened the impact on plant growth of the replication stress caused by ETG1 deficiency because of inefficient DNA repair. We conclude that the ETG1 replication factor is required for efficient cohesion and that cohesion establishment is essential for proper development of plants suffering from endogenous DNA stress. Cohesion defects observed upon knockdown of its human counterpart suggest an equally important developmental role for the orthologous mammalian ETG1 protein.

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Inefficient DNA repair upon loss of sister chromatid cohesion.(A) Real-time RT-PCR analysis of DNA stress-inducible genes PARP2, BRCA, and CYCB1;1 in wild-type (Col-0), etg1-1, wild-type (Nos-0), ctf18-1, and etg1-1 ctf18-1 plants. Total RNA prepared from 8-day-old seedlings was amplified by RT-PCR. All values were normalized against the expression level of the ACTIN2 gene. (B) Statistical analysis of a comet assay. The average %-values of DNA in tails of nuclei of 7-day-old wild-type (Col-0), etg1-1, wild-type (Nos-0), ctf18-1, and etg1-1 ctf18-1 seedlings. Error bars indicate SD. (C) Examples of comets from plant nuclei with undamaged (top) or damaged (bottom) DNA. (D) Kinetics of DSB repair in wild-type versus etg1-1 mutant plants. Fractions of remaining DSB were calculated for 0, 5, 10, 20, and 60 min recovery time after treatment with 50 µg/ml bleomycin for 1 h. Maximum damage was normalized as 100% at t = 0. (E–G) Wild-type (Col-0, left) and etg1-1 (right) plants were grown on medium holding 50 ppm methyl methane sulfonate (E), 3 µg/ml mitomycin C (F), or no supplement (G). Plants were photographed 3 weeks after sowing.
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pgen-1000817-g006: Inefficient DNA repair upon loss of sister chromatid cohesion.(A) Real-time RT-PCR analysis of DNA stress-inducible genes PARP2, BRCA, and CYCB1;1 in wild-type (Col-0), etg1-1, wild-type (Nos-0), ctf18-1, and etg1-1 ctf18-1 plants. Total RNA prepared from 8-day-old seedlings was amplified by RT-PCR. All values were normalized against the expression level of the ACTIN2 gene. (B) Statistical analysis of a comet assay. The average %-values of DNA in tails of nuclei of 7-day-old wild-type (Col-0), etg1-1, wild-type (Nos-0), ctf18-1, and etg1-1 ctf18-1 seedlings. Error bars indicate SD. (C) Examples of comets from plant nuclei with undamaged (top) or damaged (bottom) DNA. (D) Kinetics of DSB repair in wild-type versus etg1-1 mutant plants. Fractions of remaining DSB were calculated for 0, 5, 10, 20, and 60 min recovery time after treatment with 50 µg/ml bleomycin for 1 h. Maximum damage was normalized as 100% at t = 0. (E–G) Wild-type (Col-0, left) and etg1-1 (right) plants were grown on medium holding 50 ppm methyl methane sulfonate (E), 3 µg/ml mitomycin C (F), or no supplement (G). Plants were photographed 3 weeks after sowing.

Mentions: Previously, we reported that ETG1-deficient plants suffer from replication stress, resulting in transcriptional induction of DNA repair genes and activation of the DNA replication checkpoint [24]. To assess the level of DNA damage in cohesion mutants, we compared the expression levels of the marker genes coding for poly(ADP-ribose) polymerase 2 (PARP2), breast cancer (BRCA), and B-type cyclin 1 (CYCB1;1) by real-time reverse-transcription (RT)-PCR in wild-type versus etg1-1, ctf18-1, and etg1-1 ctf18-1 mutant plants. γ-irradiation, UV light, and radiomimetic agents (such as bleomycin) are known to induce PARP2, BRCA, and CYCB1;1 expression [29],[34]. The expression level of these DNA stress genes was significantly upregulated in the etg1-1 mutant, confirming previous data [24]. By contrast, the cohesion mutant ctf18-1 showed no signs of DNA stress (Figure 6A). Interestingly, expression of the PARP2, BRCA, and CYCB1;1 genes was hyper-induced in etg1-1 ctf18-1 double mutant plants (Figure 6A), indicating severe DNA stress. To examine the level of DNA damage, 8-day-old seedlings were assayed by comet assay. In agreement with previous analyses, etg1-1, but not ctf18-1, mutants, exhibited significant DNA damage in comparison with control plants, whereas the DNA damage level of the etg1-1 ctf18-1 double mutant seedlings was much higher than that observed in the single etg1-1 mutants (Figure 6B and 6C).


The MCM-binding protein ETG1 aids sister chromatid cohesion required for postreplicative homologous recombination repair.

Takahashi N, Quimbaya M, Schubert V, Lammens T, Vandepoele K, Schubert I, Matsui M, Inzé D, Berx G, De Veylder L - PLoS Genet. (2010)

Inefficient DNA repair upon loss of sister chromatid cohesion.(A) Real-time RT-PCR analysis of DNA stress-inducible genes PARP2, BRCA, and CYCB1;1 in wild-type (Col-0), etg1-1, wild-type (Nos-0), ctf18-1, and etg1-1 ctf18-1 plants. Total RNA prepared from 8-day-old seedlings was amplified by RT-PCR. All values were normalized against the expression level of the ACTIN2 gene. (B) Statistical analysis of a comet assay. The average %-values of DNA in tails of nuclei of 7-day-old wild-type (Col-0), etg1-1, wild-type (Nos-0), ctf18-1, and etg1-1 ctf18-1 seedlings. Error bars indicate SD. (C) Examples of comets from plant nuclei with undamaged (top) or damaged (bottom) DNA. (D) Kinetics of DSB repair in wild-type versus etg1-1 mutant plants. Fractions of remaining DSB were calculated for 0, 5, 10, 20, and 60 min recovery time after treatment with 50 µg/ml bleomycin for 1 h. Maximum damage was normalized as 100% at t = 0. (E–G) Wild-type (Col-0, left) and etg1-1 (right) plants were grown on medium holding 50 ppm methyl methane sulfonate (E), 3 µg/ml mitomycin C (F), or no supplement (G). Plants were photographed 3 weeks after sowing.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2806904&req=5

pgen-1000817-g006: Inefficient DNA repair upon loss of sister chromatid cohesion.(A) Real-time RT-PCR analysis of DNA stress-inducible genes PARP2, BRCA, and CYCB1;1 in wild-type (Col-0), etg1-1, wild-type (Nos-0), ctf18-1, and etg1-1 ctf18-1 plants. Total RNA prepared from 8-day-old seedlings was amplified by RT-PCR. All values were normalized against the expression level of the ACTIN2 gene. (B) Statistical analysis of a comet assay. The average %-values of DNA in tails of nuclei of 7-day-old wild-type (Col-0), etg1-1, wild-type (Nos-0), ctf18-1, and etg1-1 ctf18-1 seedlings. Error bars indicate SD. (C) Examples of comets from plant nuclei with undamaged (top) or damaged (bottom) DNA. (D) Kinetics of DSB repair in wild-type versus etg1-1 mutant plants. Fractions of remaining DSB were calculated for 0, 5, 10, 20, and 60 min recovery time after treatment with 50 µg/ml bleomycin for 1 h. Maximum damage was normalized as 100% at t = 0. (E–G) Wild-type (Col-0, left) and etg1-1 (right) plants were grown on medium holding 50 ppm methyl methane sulfonate (E), 3 µg/ml mitomycin C (F), or no supplement (G). Plants were photographed 3 weeks after sowing.
Mentions: Previously, we reported that ETG1-deficient plants suffer from replication stress, resulting in transcriptional induction of DNA repair genes and activation of the DNA replication checkpoint [24]. To assess the level of DNA damage in cohesion mutants, we compared the expression levels of the marker genes coding for poly(ADP-ribose) polymerase 2 (PARP2), breast cancer (BRCA), and B-type cyclin 1 (CYCB1;1) by real-time reverse-transcription (RT)-PCR in wild-type versus etg1-1, ctf18-1, and etg1-1 ctf18-1 mutant plants. γ-irradiation, UV light, and radiomimetic agents (such as bleomycin) are known to induce PARP2, BRCA, and CYCB1;1 expression [29],[34]. The expression level of these DNA stress genes was significantly upregulated in the etg1-1 mutant, confirming previous data [24]. By contrast, the cohesion mutant ctf18-1 showed no signs of DNA stress (Figure 6A). Interestingly, expression of the PARP2, BRCA, and CYCB1;1 genes was hyper-induced in etg1-1 ctf18-1 double mutant plants (Figure 6A), indicating severe DNA stress. To examine the level of DNA damage, 8-day-old seedlings were assayed by comet assay. In agreement with previous analyses, etg1-1, but not ctf18-1, mutants, exhibited significant DNA damage in comparison with control plants, whereas the DNA damage level of the etg1-1 ctf18-1 double mutant seedlings was much higher than that observed in the single etg1-1 mutants (Figure 6B and 6C).

Bottom Line: Here, we demonstrate that depletion of the evolutionarily conserved minichromosome maintenance helicase-binding protein ETG1 of Arabidopsis thaliana resulted in a stringent late G2 cell cycle arrest.We conclude that the ETG1 replication factor is required for efficient cohesion and that cohesion establishment is essential for proper development of plants suffering from endogenous DNA stress.Cohesion defects observed upon knockdown of its human counterpart suggest an equally important developmental role for the orthologous mammalian ETG1 protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Systems Biology, Flanders Institute for Biotechnology (VIB), Gent, Belgium.

ABSTRACT
The DNA replication process represents a source of DNA stress that causes potentially spontaneous genome damage. This effect might be strengthened by mutations in crucial replication factors, requiring the activation of DNA damage checkpoints to enable DNA repair before anaphase onset. Here, we demonstrate that depletion of the evolutionarily conserved minichromosome maintenance helicase-binding protein ETG1 of Arabidopsis thaliana resulted in a stringent late G2 cell cycle arrest. This arrest correlated with a partial loss of sister chromatid cohesion. The lack-of-cohesion phenotype was intensified in plants without functional CTF18, a replication fork factor needed for cohesion establishment. The synergistic effect of the etg1 and ctf18 mutants on sister chromatid cohesion strengthened the impact on plant growth of the replication stress caused by ETG1 deficiency because of inefficient DNA repair. We conclude that the ETG1 replication factor is required for efficient cohesion and that cohesion establishment is essential for proper development of plants suffering from endogenous DNA stress. Cohesion defects observed upon knockdown of its human counterpart suggest an equally important developmental role for the orthologous mammalian ETG1 protein.

Show MeSH
Related in: MedlinePlus