Distal Sequences, but Not ori-β/OBR-1, Are Essential for Initiation of DNA Replication in the Chinese Hamster DHFR Origin

In the Chinese hamster dihydrofolate reductase replication initiation zone, the ori-β locus is preferred over other start sites. To test the hypothesis that ori-β contains a genetic replicator, we restored a deletion in the 3′ end of the DHFR gene with a cosmid that provides the missing sequence and simultaneously knocks out the downstream ori-β locus. Replication initiates normally in ori-β knockout cell lines, and the DHFR domain is still synthesized in early S phase. However, initiation is completely suppressed in the starting deletion variant lacking the 3′ end of the gene. We conclude that ori-β does not contain an essential replicator, but that distant sequence elements have profound effects on origin activity in this locus.