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.