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Mol Cell Biol, June 1998, p. 3266-3277, Vol. 18, No. 6
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Identification of Primary Initiation Sites for DNA Replication in the Hamster Dihydrofolate Reductase Gene Initiation Zone

Takehiko Kobayashi,dagger Theo Rein, and Melvin L. DePamphilis*

National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-2753

Received 11 November 1997/Returned for modification 18 December 1997/Accepted 27 February 1998

Mammalian replication origins appear paradoxical. While some studies conclude that initiation occurs bidirectionally from specific loci, others conclude that initiation occurs at many sites distributed throughout large DNA regions. To clarify this issue, the relative number of early replication bubbles was determined at 26 sites in a 110-kb locus containing the dihydrofolate reductase (DHFR)-encoding gene in CHO cells; 19 sites were located within an 11-kb sequence containing ori-beta . The ratio of ~0.8-kb nascent DNA strands to nonreplicated DNA at each site was quantified by competitive PCR. Nascent DNA was defined either as DNA that was labeled by incorporation of bromodeoxyuridine in vivo or as RNA-primed DNA that was resistant to lambda -exonuclease. Two primary initiation sites were identified within the 12-kb region, where two-dimensional gel electrophoresis previously detected a high frequency of replication bubbles. A sharp peak of nascent DNA occurred at the ori-beta origin of bidirectional replication where initiation events were 12 times more frequent than at distal sequences. A second peak occurred 5 kb downstream at a previously unrecognized origin (ori-beta '). Thus, the DHFR gene initiation zone contains at least three primary initiation sites (ori-beta , ori-beta ', and ori-gamma ), suggesting that initiation zones in mammals, like those in fission yeast, consist of multiple replication origins.

* Corresponding author. Mailing address: National Institute of Child Health and Human Development, NIH, Bldg. 6, Rm. 416, Bethesda, MD 20892-2753. Phone: (301) 570-1977. Fax: (301) 570-8797. E-mail: depamphm{at}box-d.nih.gov.

dagger Present address: National Institute for Basic Biology, 38 Nishigonaka, Myodaijicho, Okazaki, 444, Japan.

Mol Cell Biol, June 1998, p. 3266-3277, Vol. 18, No. 6
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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