Origins of replication are expected to recruit initiation proteins like origin 
recognition complex (ORC) and Cdc6 in eukaryotes and provide a platform for 
unwinding DNA. Here we test whether localization of initiation proteins onto DNA 
is sufficient for origin function. Different components of the ORC complex and 
Cdc6 stimulated prereplicative complex (pre-RC) formation and replication 
initiation when fused to the GAL4 DNA-binding domain and recruited to plasmid 
DNA containing a tandem array of GAL4-binding sites. Replication occurred once 
per cell cycle and was inhibited by Geminin, indicating that the plasmid was 
properly licensed during the cell cycle. The GAL4 fusion protein recruits other 
polypeptides of the ORC-Cdc6 complex, and nascent strand abundance was highest 
near the GAL4-binding sites. Therefore, the artificial origin recapitulates many 
of the regulatory features of physiological origins and is valuable for studies 
on replication initiation in mammalian cells. We demonstrated the utility of 
this system by showing the functional importance of the ATPase domains of human 
Cdc6 and Orc1 and the dispensability of the N-terminal segments of Orc1 and Orc2 
in this assay. Artificial recruitment of a eukaryotic cellular replication 
initiation factor to a DNA sequence can create a functional origin of 
replication, providing a robust genetic assay for these factors and a novel 
approach to generating episomal vectors for gene therapy.