DNA replication, as with all macromolecular synthesis steps, is controlled in 
part at the level of initiation. Although the origin recognition complex (ORC) 
binds to origins of DNA replication, it does not solely determine their 
location. To initiate DNA replication ORC requires Cdc6 to target initiation to 
specific DNA sequences in chromosomes and with Cdt1 loads the ring-shaped 
mini-chromosome maintenance (MCM) 2-7 DNA helicase component onto DNA. ORC and 
Cdc6 combine to form a ring-shaped complex that contains six AAA+ subunits. ORC 
and Cdc6 ATPase mutants are defective in MCM loading, and ORC ATPase mutants 
have reduced activity in ORC x Cdc6 x DNA complex formation. Here we analyzed 
the role of the Cdc6 ATPase on ORC x Cdc6 complex stability in the presence or 
absence of specific DNA sequences. Cdc6 ATPase is activated by ORC, regulates 
ORC x Cdc6 complex stability, and is suppressed by origin DNA. Mutations in the 
conserved origin A element, and to a lesser extent mutations in the B1 and B2 
elements, induce Cdc6 ATPase activity and prevent stable ORC x Cdc6 formation. 
By analyzing ORC x Cdc6 complex stability on various DNAs, we demonstrated that 
specific DNA sequences control the rate of Cdc6 ATPase, which in turn controls 
the rate of Cdc6 dissociation from the ORC x Cdc6 x DNA complex. We propose a 
mechanism explaining how Cdc6 ATPase activity promotes origin DNA sequence 
specificity; on DNA that lacks origin activity, Cdc6 ATPase promotes 
dissociation of Cdc6, whereas origin DNA down-regulates Cdc6 ATPase resulting in 
a stable ORC x Cdc6 x DNA complex, which can then promote MCM loading. This 
model has relevance for origin specificity in higher eukaryotes.