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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Cell Cycle Proteins / antagonists & inhibitors
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism*
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Cell Cycle Proteins / pharmacology
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Cells, Cultured
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Cullin Proteins / antagonists & inhibitors
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Cullin Proteins / genetics
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Cullin Proteins / metabolism*
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Cyclin A / metabolism
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Cyclin E / metabolism
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DNA Damage
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DNA Replication / radiation effects
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DNA-Binding Proteins / antagonists & inhibitors
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / metabolism*
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Fibroblasts / metabolism
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Geminin
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Gene Silencing
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HeLa Cells
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Humans
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Kidney / embryology
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Kidney / metabolism
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Mice
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Peptide Hydrolases / chemistry*
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Phosphorylation
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Proliferating Cell Nuclear Antigen / metabolism
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RNA, Small Interfering / pharmacology
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S-Phase Kinase-Associated Proteins / antagonists & inhibitors
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S-Phase Kinase-Associated Proteins / genetics
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S-Phase Kinase-Associated Proteins / metabolism*
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SKP Cullin F-Box Protein Ligases / antagonists & inhibitors
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SKP Cullin F-Box Protein Ligases / genetics
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SKP Cullin F-Box Protein Ligases / metabolism*
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Ubiquitin / metabolism
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Ultraviolet Rays
Substances
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CDT1 protein, human
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CUL4A protein, human
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Cell Cycle Proteins
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Cullin Proteins
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Cyclin A
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Cyclin E
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DDB1 protein, human
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DNA-Binding Proteins
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GMNN protein, human
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Geminin
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Proliferating Cell Nuclear Antigen
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RNA, Small Interfering
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S-Phase Kinase-Associated Proteins
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Ubiquitin
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SKP Cullin F-Box Protein Ligases
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Peptide Hydrolases