Genome-scale mapping of pre-replication complex proteins has not been reported 
in mammalian cells. Poor enrichment of these proteins at specific sites may be 
due to dispersed binding, poor epitope availability or cell cycle stage-specific 
binding. Here, we have mapped sites of biotin-tagged ORC and MCM protein binding 
in G1-synchronized populations of Chinese hamster cells harboring amplified 
copies of the dihydrofolate reductase (DHFR) locus, using avidin-affinity 
purification of biotinylated chromatin followed by high-density microarray 
analysis across the DHFR locus. We have identified several sites of significant 
enrichment for both complexes distributed throughout the previously identified 
initiation zone. Analysis of the frequency of initiations across stretched DNA 
fibers from the DHFR locus confirmed a broad zone of de-localized initiation 
activity surrounding the sites of ORC and MCM enrichment. Mapping positions of 
mononucleosomal DNA empirically and computing nucleosome-positioning information 
in silico revealed that ORC and MCM map to regions of low measured and predicted 
nucleosome occupancy. Our results demonstrate that specific sites of ORC and MCM 
enrichment can be detected within a mammalian initiation zone, and suggest that 
initiation zones may be regions of generally low nucleosome occupancy where 
flexible nucleosome positioning permits flexible pre-RC assembly sites.