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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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Upregulation of G2/M-specific genes in ETG1-deficient plants.(A) Plot of average signal log ratios (SLRs; logarithms to the base 2) between wild-type and etg1 mutant plants for 9,910 genes during the 22-h cell cycle of Arabidopsis [26]. Genes were sorted into 10 bins representing the 10 time points of measurements, based on their maximal expression during the cell cycle. Cells were synchronized by a release from an aphidicolin arrest. Synchronized cells completed S phase in 5 h and went through mitosis from 9 to 14 h. The solid line represents the best-fitted curve. (B) GO analysis of the 196 upregulated genes in the first leaves of etg1 mutant plants. The yellow-to-orange color of the circles correspond to the level of significance of the overrepresented GO category of ≤0.01 according to a multiple t test with false discovery rate–corrected P value. The size of the circle is proportional to the number of genes in the category. (C) Comparison of the distribution of cell cycle phase-dependent upregulated genes in etg1 plants and plants treated with UV-B- or bleomycin. Microarray data sets of UV-B, and bleomycin treatment were imported from [28] and [29], respectively. S (red), G2 (blue), M (yellow), and G1 (green) phase-specific gene expression patterns were defined by Menges et al. [26].
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pgen-1000817-g001: Upregulation of G2/M-specific genes in ETG1-deficient plants.(A) Plot of average signal log ratios (SLRs; logarithms to the base 2) between wild-type and etg1 mutant plants for 9,910 genes during the 22-h cell cycle of Arabidopsis [26]. Genes were sorted into 10 bins representing the 10 time points of measurements, based on their maximal expression during the cell cycle. Cells were synchronized by a release from an aphidicolin arrest. Synchronized cells completed S phase in 5 h and went through mitosis from 9 to 14 h. The solid line represents the best-fitted curve. (B) GO analysis of the 196 upregulated genes in the first leaves of etg1 mutant plants. The yellow-to-orange color of the circles correspond to the level of significance of the overrepresented GO category of ≤0.01 according to a multiple t test with false discovery rate–corrected P value. The size of the circle is proportional to the number of genes in the category. (C) Comparison of the distribution of cell cycle phase-dependent upregulated genes in etg1 plants and plants treated with UV-B- or bleomycin. Microarray data sets of UV-B, and bleomycin treatment were imported from [28] and [29], respectively. S (red), G2 (blue), M (yellow), and G1 (green) phase-specific gene expression patterns were defined by Menges et al. [26].

Mentions: ETG1-deficient plants suffer from endogenous DNA stress and display a transient cell cycle arrest [24]. To gain more insight into this defective cell cycle, we examined transcript levels of 22,750 genes by using Affymetrix ATH1 GeneChip arrays. Triplicate batches of proliferating first leaves of 9-day-old wild-type and two independent etg1-1 and etg1-2 mutant plants were harvested for total RNA preparation. Statistical analysis identified a total of 219 genes differentially expressed between wild-type and etg1 plants at a P-value <0.01, among which 89% upregulated and 11% downregulated and displaying a 1.3- to 14.8-fold change in expression. Strikingly, of the 195 upregulated genes, 103 (53%) showed an expression peak during mitosis (Table S1 and Table S2; Figure S1). Transcription of genes expressed specifically during mitosis is regulated by a common upstream cis-acting element (ynCAACGG), designated mitosis-specific activator (MSA). In total, 82 upregulated genes in etg1 plants possessed an MSA element within the first 1 kb region upstream of the translation start, which is significantly more than expected by chance (P-value <0.001) and indicative for an arrest in late G2 or mitosis. This hypothesis was corroborated by an overall transcriptional induction of G2 and M-phase expressed genes in etg1 knockout plants, as demonstrated by plotting the average signal log ratios (SLRs) between the expression levels in wild-type and etg1 mutant plants of 9,910 genes previously defined as cell cycle regulated [26] (Figure 1A).


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)

Upregulation of G2/M-specific genes in ETG1-deficient plants.(A) Plot of average signal log ratios (SLRs; logarithms to the base 2) between wild-type and etg1 mutant plants for 9,910 genes during the 22-h cell cycle of Arabidopsis [26]. Genes were sorted into 10 bins representing the 10 time points of measurements, based on their maximal expression during the cell cycle. Cells were synchronized by a release from an aphidicolin arrest. Synchronized cells completed S phase in 5 h and went through mitosis from 9 to 14 h. The solid line represents the best-fitted curve. (B) GO analysis of the 196 upregulated genes in the first leaves of etg1 mutant plants. The yellow-to-orange color of the circles correspond to the level of significance of the overrepresented GO category of ≤0.01 according to a multiple t test with false discovery rate–corrected P value. The size of the circle is proportional to the number of genes in the category. (C) Comparison of the distribution of cell cycle phase-dependent upregulated genes in etg1 plants and plants treated with UV-B- or bleomycin. Microarray data sets of UV-B, and bleomycin treatment were imported from [28] and [29], respectively. S (red), G2 (blue), M (yellow), and G1 (green) phase-specific gene expression patterns were defined by Menges et al. [26].
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2806904&req=5

pgen-1000817-g001: Upregulation of G2/M-specific genes in ETG1-deficient plants.(A) Plot of average signal log ratios (SLRs; logarithms to the base 2) between wild-type and etg1 mutant plants for 9,910 genes during the 22-h cell cycle of Arabidopsis [26]. Genes were sorted into 10 bins representing the 10 time points of measurements, based on their maximal expression during the cell cycle. Cells were synchronized by a release from an aphidicolin arrest. Synchronized cells completed S phase in 5 h and went through mitosis from 9 to 14 h. The solid line represents the best-fitted curve. (B) GO analysis of the 196 upregulated genes in the first leaves of etg1 mutant plants. The yellow-to-orange color of the circles correspond to the level of significance of the overrepresented GO category of ≤0.01 according to a multiple t test with false discovery rate–corrected P value. The size of the circle is proportional to the number of genes in the category. (C) Comparison of the distribution of cell cycle phase-dependent upregulated genes in etg1 plants and plants treated with UV-B- or bleomycin. Microarray data sets of UV-B, and bleomycin treatment were imported from [28] and [29], respectively. S (red), G2 (blue), M (yellow), and G1 (green) phase-specific gene expression patterns were defined by Menges et al. [26].
Mentions: ETG1-deficient plants suffer from endogenous DNA stress and display a transient cell cycle arrest [24]. To gain more insight into this defective cell cycle, we examined transcript levels of 22,750 genes by using Affymetrix ATH1 GeneChip arrays. Triplicate batches of proliferating first leaves of 9-day-old wild-type and two independent etg1-1 and etg1-2 mutant plants were harvested for total RNA preparation. Statistical analysis identified a total of 219 genes differentially expressed between wild-type and etg1 plants at a P-value <0.01, among which 89% upregulated and 11% downregulated and displaying a 1.3- to 14.8-fold change in expression. Strikingly, of the 195 upregulated genes, 103 (53%) showed an expression peak during mitosis (Table S1 and Table S2; Figure S1). Transcription of genes expressed specifically during mitosis is regulated by a common upstream cis-acting element (ynCAACGG), designated mitosis-specific activator (MSA). In total, 82 upregulated genes in etg1 plants possessed an MSA element within the first 1 kb region upstream of the translation start, which is significantly more than expected by chance (P-value <0.001) and indicative for an arrest in late G2 or mitosis. This hypothesis was corroborated by an overall transcriptional induction of G2 and M-phase expressed genes in etg1 knockout plants, as demonstrated by plotting the average signal log ratios (SLRs) between the expression levels in wild-type and etg1 mutant plants of 9,910 genes previously defined as cell cycle regulated [26] (Figure 1A).

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