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

CDC6 is conserved during evolution and is essential and limiting for the in itiation of eukaryotic DNA replication. Human CDC6 activity is regulated by periodic transcription and CDK-regulated subcellular localization. Here, w e show that, in addition to being absent from nonproliferating cells, CDC6 is targeted for ubiquitin-mediated proteolysis by the anaphase promoting co mplex (APC)/cyclosome in G(1). A combination of point mutations in the dest ruction box and REN-box motifs in CDC6 stabilizes the protein in G(1) and i n quiescent cells. furthermore, APC, in association with CDH1, ubiquitinate s CDC6 in vitro, and both APC and CDH1 are required and limiting for CDC6 p roteolysis in vivo. Although a stable mutant of CDC6 is biologically active , overexpression of this mutant or wild-type CDC6 is not sufficient to indu ce multiple rounds of DNA replication in the same cell cycle. The APC-CDH1- dependent proteolysis of CDC6 in early G(1) and in quiescent cells suggests that this process is part of a mechanism that ensures the timely licensing of replication origins during G(1).