Nonperiodic activity of the human anaphase-promoting complex-Cdh1 ubiquitin ligase results in continuous DNA synthesis uncoupled from mitosis

Ubiquitin-proteasome-mediated destruction of rate-limiting proteins is requ ired for timely progression through the main cell cycle transitions. The an aphase-promoting complex (APC), periodically activated by the Cdh1 subunit, represents one of the major cellular ubiquitin ligases which, in Saccharom yces cerevisiae and Drosophila spp., triggers exit from mitosis and during G(1) prevents unscheduled DNA replication. In this study we investigated th e importance of periodic oscillation of the APC-Cdh1 activity for the cell cycle progression in human cells. We show that conditional interference wit h the APC-Cdh1 dissociation at the G(1)/S transition resulted in an inabili ty to accumulate a surprisingly broad range of critical mitotic regulators including cyclin B1, cyclin A, Plk1, Pds1, mitosin (CENP-F), Aim1, and Cdc2 0. Unexpectedly, although constitutively assembled APC-Cdh1 also delayed G( 1)/S transition and lowered the rate of DNA synthesis during S phase, some of the activities essential for DNA replication became markedly amplified, mainly due to a progressive increase of E2F-dependent cyclin E transcriptio n and a rapid turnover of the p27(Kip1) cyclin-dependent kinase inhibitor. Consequently, failure to inactivate APC-Cdh1 beyond the G(1)/S transition n ot only inhibited productive cell division but also supported slow but unin terrupted DNA replication, precluding S-phase exit and causing massive over replication of the genome. Our data suggest that timely oscillation of the APC-Cdh1 ubiquitin ligase activity represents an essential step in coordina ting DNA replication with cell division and that failure of mechanisms regu lating association of APC with the Cdh1 activating subunit can undermine ge nomic stability in mammalian cells.