A Genome-wide Screen Identifies p97 as an Essential Regulator of DNA Damage-Dependent CDT1 Destruction

Several proteins, including the replication licensing factor CDT1 and the histone methyltransferase SET8, are targeted for proteolysis during DNA replication and repair by the E3 ubiquitin ligase CRL4CDT2. CRL4CDT2 function is coupled to replication and repair because it only ubiquitinates substrates that associate with chromatin-bound PCNA. Here, we report a genome-wide siRNA screen that identifies multiple factors necessary for CDT1 destruction after UV irradiation. Among these, nucleotide excision repair factors promote CDT1 destruction due to a role in recruiting PCNA to damaged DNA. The COP9/Signalosome regulates CDT2 stability through CUL4 deneddylation. Finally, the p97 AAA+-ATPase and its cofactor UFD1 are required for proteasome-dependent removal of ubiquitinated CDT1 and SET8 from chromatin and their subsequent degradation both in vivo and in a Xenopus egg extract system in vitro. This study provides insight into and a resource for the further exploration of pathways that promote timely degradation of chromatin-associated CRL4CDT2 substrates. [Display omitted] ► A siRNA screen uncovers new modules in the degradation of the licensing factor CDT1 ► NER factors and the CSN function upstream of CDT1 ubiquitination ► p97 and UFD1 are required for CDT1 destruction downstream of ubiquitination ► p97 catalytic activity is required for removal of ubiquitinated CDT1 from chromatin