Cell cycle– and cell growth–regulated proteolysis of mammalian CDC6 is dependent on APC–CDH1

<jats:p>CDC6 is conserved during evolution and is essential and limiting for the initiation of eukaryotic DNA replication. Human CDC6 activity is regulated by periodic transcription and CDK-regulated subcellular localization. Here, we show that, in addition to being absent from nonproliferating cells, CDC6 is targeted for ubiquitin-mediated proteolysis by the anaphase promoting complex (APC)/cyclosome in G<jats:sub>1</jats:sub>. A combination of point mutations in the destruction box and KEN-box motifs in CDC6 stabilizes the protein in G<jats:sub>1</jats:sub> and in quiescent cells. Furthermore, APC, in association with CDH1, ubiquitinates CDC6 in vitro, and both APC and CDH1 are required and limiting for CDC6 proteolysis in vivo. Although a stable mutant of CDC6 is biologically active, overexpression of this mutant or wild-type CDC6 is not sufficient to induce multiple rounds of DNA replication in the same cell cycle. The APC–CDH1-dependent proteolysis of CDC6 in early G<jats:sub>1</jats:sub> and in quiescent cells suggests that this process is part of a mechanism that ensures the timely licensing of replication origins during G<jats:sub>1</jats:sub>.</jats:p>