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

Nature Cell Biology volume 12, pages 484–491 (2010)Cite this article

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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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Figure 1: Identification of GEMC1.
Figure 2: Analysis of GEMC1 function in DNA replication.
Figure 3: Identification of proteins that interact with GEMC1.
Figure 4: Effects of inhibiting GEMC1 expression in Xenopus embryos.
Figure 5: Effects of inhibiting GEMC1 expression in mammalian cells.

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Acknowledgements

We thank T. Hunt, members of Clare Hall Laboratories and members of the Genome Stability Unit for their comments, H. Mahbubani and J. Kirk for technical support with X. laevis, J. Gannon for technical assistance in antibody production and M. Wu for embryo injection. This work was supported by grants from Cancer Research UK. V. C. is also supported by the Lister Institute of Preventive Medicine, the European Research Council (ERC) start-up grant (N. 206281) and the EMBO Young Investigator Program (YIP).

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Authors and Affiliations

  1. Genome Stability,

    Alessia Balestrini, Claudia Cosentino, Elizabeth Garner & Vincenzo Costanzo

  2. Cell cycle Laboratories, London Research Institute, Hertfordshire, EN6 3LD, UK

    Alessia Errico

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  1. Alessia Balestrini
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  2. Claudia Cosentino
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A.B, C.C, A.E and E.G performed the experiments. V.C conceived the experiments and wrote the manuscript.

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Correspondence to Vincenzo Costanzo.

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Balestrini, A., Cosentino, C., Errico, A. et al. GEMC1 is a TopBP1-interacting protein required for chromosomal DNA replication. Nat Cell Biol 12, 484–491 (2010). https://doi.org/10.1038/ncb2050

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