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EMBO J. 2006 Mar 8;25(5):1126-36. Epub 2006 Feb 16.

Two E3 ubiquitin ligases, SCF-Skp2 and DDB1-Cul4, target human Cdt1 for proteolysis.

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  • 1Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Higashi-ku, Fukuoka, Japan. hideon@molbiol.med.kyushu-u.ac.jp

Abstract

Replication licensing is carefully regulated to restrict replication to once in a cell cycle. In higher eukaryotes, regulation of the licensing factor Cdt1 by proteolysis and Geminin is essential to prevent re-replication. We show here that the N-terminal 100 amino acids of human Cdt1 are recognized for proteolysis by two distinct E3 ubiquitin ligases during S-G2 phases. Six highly conserved amino acids within the 10 first amino acids of Cdt1 are essential for DDB1-Cul4-mediated proteolysis. This region is also involved in proteolysis following DNA damage. The second E3 is SCF-Skp2, which recognizes the Cy-motif-mediated Cyclin E/A-cyclin-dependent kinase-phosphorylated region. Consistently, in HeLa cells cosilenced of Skp2 and Cul4, Cdt1 remained stable in S-G2 phases. The Cul4-containing E3 is active during ongoing replication, while SCF-Skp2 operates both in S and G2 phases. PCNA binds to Cdt1 through the six conserved N-terminal amino acids. PCNA is essential for Cul4- but not Skp2-directed degradation during DNA replication and following ultraviolet-irradiation. Our data unravel multiple distinct pathways regulating Cdt1 to block re-replication.

PMID:
16482215
[PubMed - indexed for MEDLINE]
PMCID:
PMC1409712
Free PMC Article

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