| Ubiquitin-mediated proteolysis is a rapid and irreversible mechanism utilized by multiple cellular processes, including gene transcription, signal transduction, and differentiation. Ubiquitination constitutes three enzymatic reactions: ubiquitin activation by E1 ubiquitin activating enzyme, transfer of activated ubiquitin to E2 ubiquitin conjugating enzyme, and attachment of ubiquitin to substrate with an E3 ubiquitin ligase. Assembly of an ubiquitin chain on a substrate targets it to the 26S proteasome. Since they bind substrate directly, E3 activity and substrate selection is tightly regulated.; Cullin-RING E3 ubiquitin ligases drive cell cycle progression via the timely and ordered elimination of positive and negative regulators of cyclin dependent kinases. The founding member of the CRL family is SCF (Skp1-Cdc53/Cullin 1-F box protein). Mammals encode six different cullins (Cul1, Cul2, Cul3, Cul4A, Cul4B, Cul5), and all function as E3s. SCF is best characterized for control of the G1/S transition, and misregulation of SCF substrates is associated with unchecked proliferation and tumor progression. In the cell cycle, disruption of G1 progression, S phase entry, or the completion of one full and accurate round of DNA replication can produce genomic instability. However, the contribution of other CRLs to the maintenance of genomic integrity is uncharacterized.; In this study, we sought to identify novel targets of ubiquitin-mediated proteolysis in the cell cycle. We identified three substrates of Cul4: the replication initiation factor Cdt1, the cyclin-dependent kinase inhibitor p27Kip1, and cyclin E. Cdt1 proteolysis following DNA damage is an evolutionarily conserved response, and constitutes a checkpoint for DNA replication in G1. To characterize the mechanism of Cdt1 degradation, we identified a novel WD40 repeat protein, l(2)dtl, as a Cul4 E3 subunit. Finally, Cul4 targets p27 and cyclin E at the G1/S transition and this pathway is evolutionarily conserved. Overall, we propose roles for Cul4 in the maintenance of genomic stabi1ity. |
