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Journal of Biological Chemistry



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During DNA replication or repair, the DNA polymerase cofactor, proliferating cell nuclear antigen (PCNA), homotrimerizes and encircles the replicating DNA, thereby acting as a DNA clamp that promotes DNA polymerase processivity. The formation of the PCNA trimer is also essential for targeting the replication-licensing protein, chromatin-licensing, and DNA replication factor 1 (CDT1), for ubiquitin-dependent proteolysis to prevent chromosomal DNA re-replication. CDT1 uses its PCNA-interacting peptide box (PIP box) to interact with PCNA, and the CRL4 E3 ubiquitin ligase subunit CDT2 is recruited through the formation of PCNA–CDT1 complexes. However, it remains unclear how CDT1 and many other PIP box–containing proteins are marked for degradation by the CRL4CDT2 ubiquitin ligase during DNA replication or damage. Here, using recombinant protein expression coupled with site-directed mutagenesis, we report that CDT2 and PCNA directly interact and this interaction depends on the presence of a highly conserved, C-terminal PIP box–like region in CDT2. Deletion or mutation of this region abolished the CDT2–PCNA interaction between CDT2 and PCNA both in vitro and in vivo. Moreover, PCNA-dependent CDT1 degradation in response to DNA damage and replication during the cell cycle requires an intact PIP box in CDT2. The requirement of the PIP boxes in both CDT2 and its substrate CDT1 suggests that the formation of the PCNA trimeric clamp around DNA during DNA replication and repair may bring together CDT1 and CRL4CDT2 ubiquitin E3 ligase to target CDT1 for proteolysis in a DNA synthesis–dependent manner.



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This research was originally published in the Journal of Biological Chemistry. Leng, F., Saxena, L., Hoang, N., Zhang, C., Lee, L., Li, W., ... & Zhang, H. Proliferating cell nuclear antigen interacts with the CRL4 ubiquitin ligase subunit CDT2 in DNA synthesis–induced degradation of CDT1. J. Biol. Chem. Year; 293(49):18879-18889. © the American Society for Biochemistry and Molecular Biology or © the Author(s).

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