Binding of cyclin-dependent kinases to ORC and Cdc6p regulates the chromosome replication cycle

Cdc6p and the origin recognition complex (ORC) are essential for assembly o f a pre-replicative complex (preRC) at origins of replication, before the i nitiation of DNA synthesis. In the absence of Cdc6p, cells fail to initiate DNA replication and undergo a "reductional" mitosis, in which the unreplic ated chromosomes are randomly segregated to the spindle poles. We show here that the cells harboring a mutation in the essential Cdc6p Walker A-box ar rest in late mitosis, probably at anaphase. This cell cycle block requires either the three Cdc28p phosphorylation sites within the N terminus of Cdc6 p or a short region (aa 8-17) that contains a Cy (Cyclin) interaction seque nce. These same two Cdc6p mutants that allow a reductional mitosis are defe ctive in binding Cdc28p kinase. In addition to Cdc6p, ORC also binds to cyc lin-dependent kinases (CDKs). Interestingly, Sic1p, a CDK inhibitor protein , blocked the S phase-specific Cdc28p-CIb5p kinase from interacting with OR C, but did not prevent the Gt-specific Cdc28p-CIn2p kinase-ORC interaction. We suggest that ORC, Cdc6p, and Sic1p bind to different CDKs in a cell cyc le-dependent manner to temporally regulate events that (i) allow preRC form ation after mitosis, (ii) prevent mitosis before DNA replication can occur, and (iii) promote initiation of DNA replication.