Cyclin-dependent kinases prevent DNA re-replication through multiple mechanisms

The stable propagation of genetic information requires that the entire geno me of an organism be faithfully replicated once and only once each cell cyc le. In eukaryotes, this replication is initiated at hundreds to thousands o f replication origins distributed over the genome, each of which must be pr ohibited from re-initiating DNA replication within every cell cycle. How ce lls prevent reinitiation has been a long-standing question in cell biology. In several eukaryotes, cyclin-dependent kinases (CDKs) have been implicate d in promoting the block to re-initiation(1), but exactly how they perform this function is unclear. Here we show that B-type CDKs in Saccharomyces ce revisiae prevent re-initiation through multiple overlapping mechanisms, inc luding phosphorylation of the origin recognition complex (ORC), downregulat ion of Cdc6 activity, and nuclear exclusion of the Mcm2-7 complex. Only whe n all three inhibitory pathways are disrupted do origins re-initiate DNA re plication in G2/M cells. These studies show that each of these three indepe ndent mechanisms of regulation is functionally important.