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XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis

Nature volume 404, pages 622–625 (2000)Cite this article

Abstract

In eukaryotic cells, chromosomal DNA replication begins with the formation of pre-replication complexes at replication origins. Formation and maintenance of pre-replication complexes is dependent upon CDC6 (ref. 1), a protein which allows assembly of MCM2–7 proteins2,3,4, which are putative replicative helicases5. The functional assembly of MCM proteins into chromatin corresponds to replication licensing. Removal of these proteins from chromatin in S phase is crucial in origins firing regulation6. We have identified a protein that is required for the assembly of pre-replication complexes, in a screen for maternally expressed genes in Xenopus. This factor (XCDT1) is a relative of fission yeast cdt1, a protein proposed to function in DNA replication7, and is the first to be identified in vertebrates. Here we show, using Xenopus in vitro systems, that XCDT1 is required for chromosomal DNA replication. XCDT1 associates with pre-replicative chromatin in a manner dependent on ORC protein and is removed from chromatin at the time of initiation of DNA synthesis. Immunodepletion and reconstitution experiments show that XCDT1 is required to load MCM2–7 proteins onto pre-replicative chromatin. These findings indicate that XCDT1 is an essential component of the system that regulates origins firing during S phase.

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Figure 1: Xenopus CDT1 is homologous to fission yeast cdt1 protein.
Figure 2: Xenopus CDT1 is required for chromosomal DNA replication.
Figure 3: XCDT1 is a chromatin-associated protein.
Figure 4: XCDT1 is required for the assembly of MCM proteins onto pre-replicative chromatin.

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Acknowledgements

We thank S. Bocquet for technical assistance in the raising of antibodies; S. Cotterill for the cdc6 antibody; J. Blow and A. Rowles for the ORC1 antibody; and C. Jaulin, D. Fisher and J.-M. Lemaître for helpful technical tips. This work has been supported by grants from ARC and Ligue Nationale contre le Cancer. D. M. was supported by an EC fellowship (Biomedicine and Health) and a grant from Fondation pour la Recherche Médicale.

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Authors and Affiliations

  1. Institute of Human Genetics, CNRS, Genome Dynamics and Development, 141, rue de la Cardonille, Montpellier, 34396 Cedex 5, France

    Domenico Maiorano & Marcel Méchali

  2. Institut Jacques Monod, Université Paris VII, 2 Place Jussieu, Paris, Cedex 05, France

    Jacques Moreau

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  1. Domenico Maiorano
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Correspondence to Marcel Méchali.

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Maiorano, D., Moreau, J. & Méchali, M. XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis. Nature 404, 622–625 (2000). https://doi.org/10.1038/35007104

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