Chromatin-bound Cdc6 persists in S and G(2) phases in human cells, while soluble Cdc6 is destroyed in a cyclin A-cdk2 dependent process

Cdc6 is essential for the initiation of DNA replication in all organisms in which it has been studied, In addition, recombinant Cdc6 can stimulate ini tiation in G(1) nuclei in vitro. We have analysed the behaviour of recombin ant Cdc6 in mammalian cell extracts under in vitro replication conditions. We find that Cdc6 is imported into the nucleus in G1 phase, where it binds to chromatin and remains relatively stable. In S phase, exogenous Cdc6 is d estroyed in a process that requires import into the nucleus and phosphoryla tion by a chromatin-bound protein kinase, Recombinant cyclin A-cdk2 can com pletely substitute for the nucleus in promoting destruction of soluble Xeno pus and human Cdc6, Despite this regulated destruction, endogenous Cdc6 persists in the nucleus after initiation, although the amount falls, Cdc6 levels remain constant i n G(2) then fall again before mitosis, We propose that cyclin A-cdk2 phosph orylation results in destruction of any Cdc6 not assembled into replication complexes, but that assembled proteins remain, in the phosphorylated state , in the nucleus. This process could contribute to the prevention of reinit iation in human cells by making free Cdc6 unavailable for re-assembly into replication complexes after G(1) phase.