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Nucleic Acids Research 2005 33(14):4412-4424; doi:10.1093/nar/gki747
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Published online 5 August 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

Article

Initiation of DNA replication at the human ß-globin 3' enhancer

Alla Buzina1, Mirit I. Aladjem2, John L. Kolman3, Geoffrey M. Wahl3 and James Ellis1,4,*

1Developmental Biology Program, Hospital for Sick Children Toronto, Ontario, Canada 2National Cancer Institute Bethesda, MD 3Gene Expression Laboratory, The Salk Institute San Diego, CA 4Department of Molecular and Medical Genetics, University of Toronto Toronto, Ontario, Canada

*To whom correspondence should be addressed. Tel: 416 813 7295; Fax: 416 813 8883; Email: jellis{at}sickkids.ca

Received March 24, 2005. Revised May 30, 2005. Accepted July 15, 2005.

The origin of DNA replication in the human ß-globin gene contains an initiation region (IR) and two flanking auxiliary elements. Two replicator modules are located within the upstream auxiliary sequence and the IR core, but the functional sequences in the downstream auxiliary element are unknown. Here, we use a combination of benzoylated-naphthoylated DEAE (BND) cellulose purification and nascent strand abundance assays to show that replication initiation occurs at the ß-globin 3' enhancer on human chromosome 11 in the Hu11 hybrid murine erythroleukemia (MEL) cell line. To examine replicator function, 3' enhancer fragments were inserted into an ectopic site in MEL cells via an optimized FRT/EGFP-FLP integration system. These experiments demonstrate that the 1.6 kb downstream auxiliary element is a third replicator module called bGRep-E in erythroid cells. The minimal 260 bp 3' enhancer is required but not sufficient to initiate efficient replication, suggesting cooperation with adjacent sequences. The minimal 3' enhancer also cooperates with elements in an expressing HS3ß/{gamma}-globin construct to initiate replication. These data indicate that the ß-globin replicator has multiple initiation sites in three closely spaced replicator modules. We conclude that a mammalian enhancer can cooperate with adjacent sequences to create an efficient replicator module.


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