The Chinese Hamster Dihydrofolate Reductase Replication Origin Beta Is Active at Multiple Ectopic Chromosomal Locations and Requires Specific DNA Sequence Elements for Activity

Abstract

To identify cis-acting genetic elements essential for mammalian chromosomal DNA replication, a 5.8-kb fragment from the Chinese hamster dihydrofolate reductase (DHFR) locus containing the origin beta (ori-β) initiation region was stably transfected into random ectopic chromosomal locations in a hamster cell line lacking the endogenous DHFR locus. Initiation at ectopic ori-β in uncloned pools of transfected cells was measured using a competitive PCR-based nascent strand abundance assay and shown to mimic that at the endogenous ori-β region in Chinese hamster ovary K1 cells. Initiation activity of three ectopic ori-β deletion mutants was reduced, while the activity of another deletion mutant was enhanced. The results suggest that a 5.8-kb fragment of the DHFR ori-β region is sufficient to direct initiation and that specific DNA sequences in the ori-β region are required for efficient initiation activity.

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The Chinese Hamster Dihydrofolate Reductase Replication Origin Beta Is Active at Multiple Ectopic Chromosomal Locations and Requires Specific DNA Sequence Elements for Activity

ACKNOWLEDGMENTS

We thank A. Schmidt and S. Dehde for their important contributions to the early phase of this project, M. Giacca for valuable technical advice, A. Carothers for DR12 cells, N. Heintz for DHFR DNA fragments, and A. Schmidt, M. Giacca, J. Hamlin, N. Heintz, and M. L. DePamphilis for sharing DHFR DNA sequence data. We appreciate the help of A. K. Patten with homology searches, R. Stein with statistical analysis, J. Francis with mutant construction, and U. Herbig and V. Podust with the manuscript.

The financial support of the NIH (GM 52948 and training grant CA 09385), the Army Breast Cancer Program (BC980907), and Vanderbilt University is gratefully acknowledged.