The CRL4Cdt2 ubiquitin ligase targets the degradation of p21Cip1 to control replication licensing

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Abstract
The faithful replication of genomic DNA is crucial for maintaining genome stability. In eukaryotes, DNA rereplication is prevented by the temporal regulation of replication licensing. Replication-licensing factors are required to form prereplicative complexes during G1 phase, but are inactivated in S phase to prevent rereplication. A vertebrate CUL4 CRL ubiquitin ligase (CRL4) complex containing Cdt2 as the substrate recognition subunit promotes proper DNA replication, in part, by degrading the replication-licensing factor Cdt1 during S phase. We show that the Caenorhabditis elegans CRL4Cdt2 complex has a conserved role in degrading Cdt1. Furthermore, we show that CRL4Cdt2 restrains replication licensing in both C. elegans and humans by targeting the degradation of the cyclin-dependent kinase (CDK) inhibitors CKI-1 and p21Cip1, respectively. Human CRL4Cdt2 targets the degradation of p21 in S phase, with the in vivo ubiquitylation of p21 by CRL4Cdt2 dependent on p21 binding to PCNA. Inactivation of Cdt2 induces rereplication, which requires the presence of the CDK inhibitor p21. Strikingly, coinactivation of CRL4Cdt2 and SCFSkp2 (which redundantly targets p21 degradation) prevents the nuclear export of the replication-licensing factor Cdc6 during S phase, and the block on nuclear export is dependent on p21. Our work defines the degradation of p21 as a critical aspect of replication licensing in human cells.