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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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Induction of cohesion defects by downregulation of human MCM-BP.(A) MCM-BP transcript levels in siRNA transfected HEK-293T cells versus untransfected and transfected control cells. Samples were harvested 48 h after transfection. All values were normalized against the expression level of the TBP and UBC genes. (B) MCM-BP, MCM4, MCM6, and MCM7 protein levels in samples as described in (A). (C) Quantification of nuclei showing totally separated sister chromatids (n>100 per condition, obtained from two independent transfection experiments). The asterisk marks a significant difference by Student's t-test (P-value <0.05). (C–E) Representative images of mitotic spread of untransformed (D), control transfected (E), and MCM-BP siRNA-transfected HEK-293T cells (F). Insets show higher magnification images of single sister chromatid pairs.
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pgen-1000817-g007: Induction of cohesion defects by downregulation of human MCM-BP.(A) MCM-BP transcript levels in siRNA transfected HEK-293T cells versus untransfected and transfected control cells. Samples were harvested 48 h after transfection. All values were normalized against the expression level of the TBP and UBC genes. (B) MCM-BP, MCM4, MCM6, and MCM7 protein levels in samples as described in (A). (C) Quantification of nuclei showing totally separated sister chromatids (n>100 per condition, obtained from two independent transfection experiments). The asterisk marks a significant difference by Student's t-test (P-value <0.05). (C–E) Representative images of mitotic spread of untransformed (D), control transfected (E), and MCM-BP siRNA-transfected HEK-293T cells (F). Insets show higher magnification images of single sister chromatid pairs.

Mentions: A role for MCM-BP, the human ETG1 homolog, in sister chromatid cohesion was assessed with the RNA interference technique by means of pooled short interference RNAs (siRNAs). In human embryonic kidney 293T (HEK-293T) cells transfected with siRNAs that targeted MCM-BP, the MCM-BP expression was much lower than that of untransfected or transfected control cells (Figure 7A). Protein gel blotting with a specific antibody confirmed that the decrease in MCM-BP transcription was accompanied by a reduced MCM-BP protein abundance (Figure 7B). No change in protein level was seen for MCM4, MCM6, and MCM7, which are the most conserved and core subunits of the MCM complex, demonstrating that the siRNAs targeted specifically MCM-BP without affecting the abundance of other proteins of the MCM complex. As seen in mitotic spreads (Figure 7C–7F), depletion of MCM-BP strongly affected sister chromatid cohesion, resulting in a higher proportion of nuclei with completely separated sister chromatids (38% versus 2% or 3% in untransfected or transfected control cells, respectively). Thus, as observed for its plant counterpart, MCM-BP seems to be important for sister chromatid cohesion.


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)

Induction of cohesion defects by downregulation of human MCM-BP.(A) MCM-BP transcript levels in siRNA transfected HEK-293T cells versus untransfected and transfected control cells. Samples were harvested 48 h after transfection. All values were normalized against the expression level of the TBP and UBC genes. (B) MCM-BP, MCM4, MCM6, and MCM7 protein levels in samples as described in (A). (C) Quantification of nuclei showing totally separated sister chromatids (n>100 per condition, obtained from two independent transfection experiments). The asterisk marks a significant difference by Student's t-test (P-value <0.05). (C–E) Representative images of mitotic spread of untransformed (D), control transfected (E), and MCM-BP siRNA-transfected HEK-293T cells (F). Insets show higher magnification images of single sister chromatid pairs.
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Related In: Results  -  Collection

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

pgen-1000817-g007: Induction of cohesion defects by downregulation of human MCM-BP.(A) MCM-BP transcript levels in siRNA transfected HEK-293T cells versus untransfected and transfected control cells. Samples were harvested 48 h after transfection. All values were normalized against the expression level of the TBP and UBC genes. (B) MCM-BP, MCM4, MCM6, and MCM7 protein levels in samples as described in (A). (C) Quantification of nuclei showing totally separated sister chromatids (n>100 per condition, obtained from two independent transfection experiments). The asterisk marks a significant difference by Student's t-test (P-value <0.05). (C–E) Representative images of mitotic spread of untransformed (D), control transfected (E), and MCM-BP siRNA-transfected HEK-293T cells (F). Insets show higher magnification images of single sister chromatid pairs.
Mentions: A role for MCM-BP, the human ETG1 homolog, in sister chromatid cohesion was assessed with the RNA interference technique by means of pooled short interference RNAs (siRNAs). In human embryonic kidney 293T (HEK-293T) cells transfected with siRNAs that targeted MCM-BP, the MCM-BP expression was much lower than that of untransfected or transfected control cells (Figure 7A). Protein gel blotting with a specific antibody confirmed that the decrease in MCM-BP transcription was accompanied by a reduced MCM-BP protein abundance (Figure 7B). No change in protein level was seen for MCM4, MCM6, and MCM7, which are the most conserved and core subunits of the MCM complex, demonstrating that the siRNAs targeted specifically MCM-BP without affecting the abundance of other proteins of the MCM complex. As seen in mitotic spreads (Figure 7C–7F), depletion of MCM-BP strongly affected sister chromatid cohesion, resulting in a higher proportion of nuclei with completely separated sister chromatids (38% versus 2% or 3% in untransfected or transfected control cells, respectively). Thus, as observed for its plant counterpart, MCM-BP seems to be important for sister chromatid cohesion.

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