IDENTIFICATION OF PRIMARY INITIATION SITES FOR DNA-REPLICATION IN THEHAMSTER DIHYDROFOLATE-REDUCTASE GENE INITIATION ZONE

Mammalian replication origins appear paradoxical. While some studies c onclude that initiation occurs bidirectionally from specific loci, oth ers conclude that initiation occurs at many sites distributed througho ut large DNA regions, To clarify this issue, the relative number of ea rly replication bubbles was determined at 26 sites in a 110-kb locus c ontaining the dihydrofolate reductase (DHFR)-encoding gene in CHO cell s; 19 sites were located within an 11-kb sequence containing ori-beta. The ratio of similar to 0.8-kb nascent DNA strands to nonreplicated D NA at each site was quantified by competitive PCR. Nascent DNA was def ined either as DNA that was labeled by incorporation of bromodeoxyurid ine in vivo or as RNA-primed DNA that was resistant to lambda-exonucle ase. Two primary initiation sites were identified within the 12-kb reg ion, where two-dimensional gel electrophoresis previously detected a h igh frequency of replication bubbles. A sharp peak of nascent DNA occu rred at the ori-beta origin of bidirectional replication where initiat ion events were 12 times more frequent than at distal sequences. A sec ond peak occurred 5 kb downstream at a previously unrecognized origin (ori-beta'), Thus, the DHFR gene initiation zone contains at least thr ee primary initiation sites (ori-beta, ori-beta', and ori-gamma), sugg esting that initiation zones in mammals, like those in fission yeast, consist of multiple replication origins.