The pre-replicative complex (pre-RC) is formed at all potential origins of 
replication through the action of the origin recognition complex (ORC), Cdc6, 
Cdt1, and the Mcm2-7 complex. The end result of pre-RC formation is the loading 
of the Mcm2-7 replicative helicase onto origin DNA. We examined pre-RC formation 
in vitro and found that it proceeds through separable binding events. 
Origin-bound ORC recruits Cdc6, and this ternary complex then promotes helicase 
loading in the presence of a pre-formed Mcm2-7-Cdt1 complex. Using a stepwise 
pre-RC assembly assay, we investigated the fate of pre-RC components during 
later stages of the reaction. We determined that helicase loading is accompanied 
by dissociation of ORC, Cdc6, and Cdt1 from origin DNA. This dissociation 
requires ATP hydrolysis at a late stage of pre-RC assembly. Our results indicate 
that pre-RC formation is a dynamic process.