PIP-box-mediated degradation prohibits re-accumulation of Cdc6 during S phase.

Cdc6 and Cdt1 initiate DNA replication licensing when cells exit mitosis. In cycling cells, Cdc6 is efficiently degraded from anaphase onwards as a result of APC/C-Cdh1 activity. When APC/C-Cdh1 is switched off again, at the end of G1 phase, Cdc6 could thus re-accumulate, risking the re-licensing of DNA as long as Cdt1 is present. Here, we carefully investigated the dynamics of Cdt1 and Cdc6 in cycling cells. We reveal a novel APC/C-Cdh1-independent degradation pathway that prevents nuclear Cdc6 re-accumulation at the G1-S transition and during S phase. Similar to Cdt1, nuclear clearance of Cdc6 depends on an N-terminal PIP-box and the Cdt2-containing CRL4 complex. When cells reach G2 phase, Cdc6 rapidly re-accumulates but, at this time, Cdt1 is mostly absent and expression of Cdc6 is limited to the cytoplasm. We propose that Cdk1 contributes to the nuclear export of Cdc6 at the S-to-G2 transition. In summary, our results show that different control mechanisms of Cdc6 restrain erroneous licensing of DNA replication during G1, S and G2 phase.