Cell Type-dependent Requirement for PIP Box-regulated Cdt1 Destruction During S Phase

Previous studies have shown that Cdt1 overexpression in cultured cells can trigger re-replication, but not whether CRL4Cdt2-triggered destruction of Cdt1 is required for normal mitotic cell cycle progression in vivo. We demonstrate that PIP box–mediated destruction of Cdt1Dup during S phase is necessary for the cell division cycle in Drosophila.DNA synthesis–coupled proteolysis of the prereplicative complex component Cdt1 by the CRL4Cdt2 E3 ubiquitin ligase is thought to help prevent rereplication of the genome during S phase. To directly test whether CRL4Cdt2-triggered destruction of Cdt1 is required for normal cell cycle progression in vivo, we expressed a mutant version of Drosophila Cdt1 (Dup), which lacks the PCNA-binding PIP box (DupΔPIP) and which cannot be regulated by CRL4Cdt2. DupΔPIP is inappropriately stabilized during S phase and causes developmental defects when ectopically expressed. DupΔPIP restores DNA synthesis to dup null mutant embryonic epidermal cells, but S phase is abnormal, and these cells do not progress into mitosis. In contrast, DupΔPIP accumulation during S phase did not adversely affect progression through follicle cell endocycles in the ovary. In this tissue the combination of DupΔPIP expression and a 50% reduction in Geminin gene dose resulted in egg chamber degeneration. We could not detect Dup hyperaccumulation using mutations in the CRL4Cdt2 components Cul4 and Ddb1, likely because these cause pleiotropic effects that block cell proliferation. These data indicate that PIP box–mediated destruction of Dup is necessary for the cell division cycle and suggest that Geminin inhibition can restrain DupΔPIP activity in some endocycling cell types