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PCNA functions as a molecular platform to trigger Cdt1 destruction and prevent re-replication

Nature Cell Biology volume 8, pages 84–90 (2006)Cite this article

Abstract

Ubiquitin-mediated proteolysis of the replication licensing factor Cdt1 (Cdc10-dependent transcript 1) in S phase is a key mechanism that limits DNA replication to a single round per cell cycle in metazoans1,2,3,4,5,6. In Xenopus egg extracts, Cdt1 is destroyed on chromatin during DNA replication1. Here, we report that replication-dependent proteolysis of Cdt1 requires its interaction with proliferating cell nuclear antigen (PCNA), a homotrimeric processivity factor for DNA polymerases7. Cdt1 binds to PCNA through a consensus PCNA-interaction motif that is conserved in Cdt1 of all metazoans, and removal of PCNA from egg extracts inhibits replication-dependent Cdt1 destruction. Mutation of the PCNA-interaction motif yields a stabilized Cdt1 protein that induces re-replication. DDB1, a component of the Cul4 E3 ubiquitin ligase that mediates human Cdt1 proteolysis in response to DNA damage8, is also required for replication-dependent Cdt1 destruction. Cdt1 and DDB1 interact in extracts, and DDB1 chromatin loading is dependent on the binding of Cdt1 to PCNA, which indicates that PCNA docking activates the pre-formed Cdt1–Cul4DDB1 ligase complex. Thus, PCNA functions as a platform for Cdt1 destruction, ensuring efficient and temporally restricted inactivation of a key cell-cycle regulator.

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Figure 1: Cdt1 binds to PCNA via a conserved PIP box.
Figure 2: Replication of M13 ssDNA induces PCNA-dependent Cdt1 destruction.
Figure 3: PCNA-dependent Cdt1 destruction in LSS limits DNA replication to a single round.
Figure 4: DDB1 is required for Cdt1 destruction.
Figure 5: DDB1 is recruited to chromatin by Cdt1.

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Acknowledgements

We thank T. Takahashi for numerous helpful suggestions, and W. Harper and D. Finley for insightful comments on the manuscript. We also thank C. Cvetic and T. Prokhorova for preparation of the Orc2 and Cdt1 antisera, and J. Pascal and T. Ellenberger for providing recombinant PCNA. E.E.A is an Howard Hughes Medical Institute pre-doctoral fellow. This work was supported by an American Cancer Society grant (106201) to J.C.W.

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  1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, 02115, MA, USA

    Emily E. Arias & Johannes C. Walter

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Correspondence to Johannes C. Walter.

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Arias, E., Walter, J. PCNA functions as a molecular platform to trigger Cdt1 destruction and prevent re-replication. Nat Cell Biol 8, 84–90 (2006). https://doi.org/10.1038/ncb1346

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