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GEMC1 is a TopBP1-interacting protein required for chromosomal DNA replication.

Balestrini A, Cosentino C, Errico A, Garner E, Costanzo V - Nat. Cell Biol. (2010)

Bottom Line: Phosphorylated xGEMC1 stimulates initiation of DNA replication, whereas depletion of xGEMC1 prevents the onset of DNA replication owing to the impairment of Cdc45 loading onto chromatin.Similarly, inhibition of GEMC1 expression with morpholino and siRNA oligos prevents DNA replication in embryonic and somatic vertebrate cells.These data suggest that GEMC1 promotes initiation of chromosomal DNA replication in multicellular organisms by mediating TopBP1- and Cdk2-dependent recruitment of Cdc45 onto replication origins.

View Article: PubMed Central - PubMed

Affiliation: Genome Stability, London Research Institute, Hertfordshire, UK.

ABSTRACT
Many of the factors required for chromosomal DNA replication have been identified in unicellular eukaryotes. However, DNA replication is poorly understood in multicellular organisms. Here, we report the identification of GEMC1 (geminin coiled-coil containing protein 1), a novel vertebrate protein required for chromosomal DNA replication. GEMC1 is highly conserved in vertebrates and is preferentially expressed in proliferating cells. Using Xenopus laevis egg extract we show that Xenopus GEMC1 (xGEMC1) binds to the checkpoint and replication factor TopBP1, which promotes binding of xGEMC1 to chromatin during pre-replication complex (pre-RC) formation. We demonstrate that xGEMC1 interacts directly with replication factors such as Cdc45 and the kinase Cdk2-CyclinE, through which it is heavily phosphorylated. Phosphorylated xGEMC1 stimulates initiation of DNA replication, whereas depletion of xGEMC1 prevents the onset of DNA replication owing to the impairment of Cdc45 loading onto chromatin. Similarly, inhibition of GEMC1 expression with morpholino and siRNA oligos prevents DNA replication in embryonic and somatic vertebrate cells. These data suggest that GEMC1 promotes initiation of chromosomal DNA replication in multicellular organisms by mediating TopBP1- and Cdk2-dependent recruitment of Cdc45 onto replication origins.

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Effects resulting from inhibition of GEMC1 expression in Xenopus embryosa) WB of xGEMC1 extracted from Xenopus embryos that were untreated (Un) or injected with control (C-MO) and morpholino oligos complementary to xGEMC1 DNA sequence (xGEMC1-MO) taken at the indicated stages. Lower panel shows coumassie staining of histone proteins from total embryo lysates. b) Morphology of Xenopus embryos that were untreated (Un), injected with control (C-MO) or anti xGEMC1 morpholino oligos (xGEMC1-MO) taken at the indicated stages. c) Quantification of experiment shown in (b). 100 embryos were counted for each treatment. Graph shows a typical result. d) Sections of fixed Xenopus embryos injected with control (C-MO) or with anti xGEMC1 morpholino oligos (xGEMC1-MO) and stained with ematossilin-eosin. Area in the rectangle shows 5x magnification. e) DNA content reduction in embryos that were uninjected (Not injected), injected with control morpholino oligos (Control MO) or with anti xGEMC1 morpholino oligos (xGEMC1-MO). Data are mean ± SD of 3 independent experiments; * and ** P <0.001 compared with not injected control, t-test) f) Cleavage of 35S-labeled PARP induced by lysates of embryos that were uninjected (Not injected), injected with control morpholino oligos (Control MO) or anti xGEMC1 morpholino oligos (xGEMC1-MO). See Supplementary Fig 6 for un-cropped gels shown in this figure.
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Figure 4: Effects resulting from inhibition of GEMC1 expression in Xenopus embryosa) WB of xGEMC1 extracted from Xenopus embryos that were untreated (Un) or injected with control (C-MO) and morpholino oligos complementary to xGEMC1 DNA sequence (xGEMC1-MO) taken at the indicated stages. Lower panel shows coumassie staining of histone proteins from total embryo lysates. b) Morphology of Xenopus embryos that were untreated (Un), injected with control (C-MO) or anti xGEMC1 morpholino oligos (xGEMC1-MO) taken at the indicated stages. c) Quantification of experiment shown in (b). 100 embryos were counted for each treatment. Graph shows a typical result. d) Sections of fixed Xenopus embryos injected with control (C-MO) or with anti xGEMC1 morpholino oligos (xGEMC1-MO) and stained with ematossilin-eosin. Area in the rectangle shows 5x magnification. e) DNA content reduction in embryos that were uninjected (Not injected), injected with control morpholino oligos (Control MO) or with anti xGEMC1 morpholino oligos (xGEMC1-MO). Data are mean ± SD of 3 independent experiments; * and ** P <0.001 compared with not injected control, t-test) f) Cleavage of 35S-labeled PARP induced by lysates of embryos that were uninjected (Not injected), injected with control morpholino oligos (Control MO) or anti xGEMC1 morpholino oligos (xGEMC1-MO). See Supplementary Fig 6 for un-cropped gels shown in this figure.

Mentions: To validate these results we depleted xGEMC1 protein from Xenopus embryos by selectively inactivating xGEMC1 expression with morpholino antisense oligos (MO)16. We selected MO oligos that were able to inhibit xGEMC1 translation in the reticulocyte system (xGEMC1-MO) (Supplementary Fig 5e). When injected into fertilized eggs, xGEMC1-MO oligos inhibited xGEMC1 expression (Fig 4a), whereas control oligos (C-MO) did not affect xGEMC1 (Fig 4a). Depletion of xGEMC1 induced severe delay in embryo development and gross anatomical defects with poor yolk resorbption and no distinct structures such as the gut, the optic vesicle or the tail. These defects were detected in most of the embryos injected with xGEMC1-MO oligos (Fig 4b and 4c). The most significant effects were detected after complete down-regulation of xGEMC1 protein levels around neurula stage (Fig 4b). The delayed effects of morpholino oligos were likely due to persistence of high levels of xGEMC1 protein up to late developmental stages. Histological analysis revealed a dramatic decrease in cell density and DNA content when xGEMC1 was inhibited (Fig 4d and 4e). This was accompanied by apoptosis induction (Fig 4f). To confirm that xGEMC1 has a role in DNA replication in mammalian organisms we isolated the mouse homologue of GEMC1 (mGEMC1) whose mRNA levels were assed by RT-PCR (Fig 5a). A decrease in mGEMC1 mRNA obtained with siRNA oligos (Fig 5a and 5b) led to severe inhibition of cell proliferation (Fig 5c and 5d). The extent of inhibition was similar to the one obtained with inhibition of Cdc45 expression (Fig 5c, 5d and Supplementary Fig 5f). Ectopic expression of RNAi resistant mGEMC1 protein, obtained by introducing silent mutations in mGEMC1 encoding cDNA, which was expressed under the control of doxycycline regulated promoter, restored proliferation in mGEMC1 silenced cells (Fig 5e and 5f).


GEMC1 is a TopBP1-interacting protein required for chromosomal DNA replication.

Balestrini A, Cosentino C, Errico A, Garner E, Costanzo V - Nat. Cell Biol. (2010)

Effects resulting from inhibition of GEMC1 expression in Xenopus embryosa) WB of xGEMC1 extracted from Xenopus embryos that were untreated (Un) or injected with control (C-MO) and morpholino oligos complementary to xGEMC1 DNA sequence (xGEMC1-MO) taken at the indicated stages. Lower panel shows coumassie staining of histone proteins from total embryo lysates. b) Morphology of Xenopus embryos that were untreated (Un), injected with control (C-MO) or anti xGEMC1 morpholino oligos (xGEMC1-MO) taken at the indicated stages. c) Quantification of experiment shown in (b). 100 embryos were counted for each treatment. Graph shows a typical result. d) Sections of fixed Xenopus embryos injected with control (C-MO) or with anti xGEMC1 morpholino oligos (xGEMC1-MO) and stained with ematossilin-eosin. Area in the rectangle shows 5x magnification. e) DNA content reduction in embryos that were uninjected (Not injected), injected with control morpholino oligos (Control MO) or with anti xGEMC1 morpholino oligos (xGEMC1-MO). Data are mean ± SD of 3 independent experiments; * and ** P <0.001 compared with not injected control, t-test) f) Cleavage of 35S-labeled PARP induced by lysates of embryos that were uninjected (Not injected), injected with control morpholino oligos (Control MO) or anti xGEMC1 morpholino oligos (xGEMC1-MO). See Supplementary Fig 6 for un-cropped gels shown in this figure.
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Figure 4: Effects resulting from inhibition of GEMC1 expression in Xenopus embryosa) WB of xGEMC1 extracted from Xenopus embryos that were untreated (Un) or injected with control (C-MO) and morpholino oligos complementary to xGEMC1 DNA sequence (xGEMC1-MO) taken at the indicated stages. Lower panel shows coumassie staining of histone proteins from total embryo lysates. b) Morphology of Xenopus embryos that were untreated (Un), injected with control (C-MO) or anti xGEMC1 morpholino oligos (xGEMC1-MO) taken at the indicated stages. c) Quantification of experiment shown in (b). 100 embryos were counted for each treatment. Graph shows a typical result. d) Sections of fixed Xenopus embryos injected with control (C-MO) or with anti xGEMC1 morpholino oligos (xGEMC1-MO) and stained with ematossilin-eosin. Area in the rectangle shows 5x magnification. e) DNA content reduction in embryos that were uninjected (Not injected), injected with control morpholino oligos (Control MO) or with anti xGEMC1 morpholino oligos (xGEMC1-MO). Data are mean ± SD of 3 independent experiments; * and ** P <0.001 compared with not injected control, t-test) f) Cleavage of 35S-labeled PARP induced by lysates of embryos that were uninjected (Not injected), injected with control morpholino oligos (Control MO) or anti xGEMC1 morpholino oligos (xGEMC1-MO). See Supplementary Fig 6 for un-cropped gels shown in this figure.
Mentions: To validate these results we depleted xGEMC1 protein from Xenopus embryos by selectively inactivating xGEMC1 expression with morpholino antisense oligos (MO)16. We selected MO oligos that were able to inhibit xGEMC1 translation in the reticulocyte system (xGEMC1-MO) (Supplementary Fig 5e). When injected into fertilized eggs, xGEMC1-MO oligos inhibited xGEMC1 expression (Fig 4a), whereas control oligos (C-MO) did not affect xGEMC1 (Fig 4a). Depletion of xGEMC1 induced severe delay in embryo development and gross anatomical defects with poor yolk resorbption and no distinct structures such as the gut, the optic vesicle or the tail. These defects were detected in most of the embryos injected with xGEMC1-MO oligos (Fig 4b and 4c). The most significant effects were detected after complete down-regulation of xGEMC1 protein levels around neurula stage (Fig 4b). The delayed effects of morpholino oligos were likely due to persistence of high levels of xGEMC1 protein up to late developmental stages. Histological analysis revealed a dramatic decrease in cell density and DNA content when xGEMC1 was inhibited (Fig 4d and 4e). This was accompanied by apoptosis induction (Fig 4f). To confirm that xGEMC1 has a role in DNA replication in mammalian organisms we isolated the mouse homologue of GEMC1 (mGEMC1) whose mRNA levels were assed by RT-PCR (Fig 5a). A decrease in mGEMC1 mRNA obtained with siRNA oligos (Fig 5a and 5b) led to severe inhibition of cell proliferation (Fig 5c and 5d). The extent of inhibition was similar to the one obtained with inhibition of Cdc45 expression (Fig 5c, 5d and Supplementary Fig 5f). Ectopic expression of RNAi resistant mGEMC1 protein, obtained by introducing silent mutations in mGEMC1 encoding cDNA, which was expressed under the control of doxycycline regulated promoter, restored proliferation in mGEMC1 silenced cells (Fig 5e and 5f).

Bottom Line: Phosphorylated xGEMC1 stimulates initiation of DNA replication, whereas depletion of xGEMC1 prevents the onset of DNA replication owing to the impairment of Cdc45 loading onto chromatin.Similarly, inhibition of GEMC1 expression with morpholino and siRNA oligos prevents DNA replication in embryonic and somatic vertebrate cells.These data suggest that GEMC1 promotes initiation of chromosomal DNA replication in multicellular organisms by mediating TopBP1- and Cdk2-dependent recruitment of Cdc45 onto replication origins.

View Article: PubMed Central - PubMed

Affiliation: Genome Stability, London Research Institute, Hertfordshire, UK.

ABSTRACT
Many of the factors required for chromosomal DNA replication have been identified in unicellular eukaryotes. However, DNA replication is poorly understood in multicellular organisms. Here, we report the identification of GEMC1 (geminin coiled-coil containing protein 1), a novel vertebrate protein required for chromosomal DNA replication. GEMC1 is highly conserved in vertebrates and is preferentially expressed in proliferating cells. Using Xenopus laevis egg extract we show that Xenopus GEMC1 (xGEMC1) binds to the checkpoint and replication factor TopBP1, which promotes binding of xGEMC1 to chromatin during pre-replication complex (pre-RC) formation. We demonstrate that xGEMC1 interacts directly with replication factors such as Cdc45 and the kinase Cdk2-CyclinE, through which it is heavily phosphorylated. Phosphorylated xGEMC1 stimulates initiation of DNA replication, whereas depletion of xGEMC1 prevents the onset of DNA replication owing to the impairment of Cdc45 loading onto chromatin. Similarly, inhibition of GEMC1 expression with morpholino and siRNA oligos prevents DNA replication in embryonic and somatic vertebrate cells. These data suggest that GEMC1 promotes initiation of chromosomal DNA replication in multicellular organisms by mediating TopBP1- and Cdk2-dependent recruitment of Cdc45 onto replication origins.

Show MeSH
Related in: MedlinePlus