Nucleic Acids Research, 2001, Vol. 29, No. 3 809-817
© 2001 Oxford University Press
Multiple sites of replication initiation in the human ß-globin gene locus
Department of Biochemistry and Molecular Biology, Wright State University, Dayton, OH 45435, USA
The cell cycle-dependent, ordered assembly of protein prereplicative complexes suggests that eukaryotic replication origins determine when genomic replication initiates. By comparison, the factors that determine where replication initiates relative to the sites of prereplicative complex formation are not known. In the human globin gene locus previous work showed that replication initiates at a single site 5' to the ß-globin gene when protein synthesis is inhibited by emetine. The present study has examined the pattern of initiation around the genetically defined ß-globin replicator in logarithmically growing HeLa cells, using two PCR-based nascent strand assays. In contrast to the pattern of initiation detected in emetine-treated cells, analysis of the short nascent strands at five positions spanning a 40 kb globin gene region shows that replication initiates at more than one site in non-drug-treated cells. Quantitation of nascent DNA chains confirmed that replication begins at several locations in this domain, including one near the initiation region (IR) identified in emetine-treated cells. However, the abundance of short nascent strands at another initiation site 
20 kb upstream is 4-fold as great as that at the IR. The latter site abuts an early S phase replicating fragment previously defined at low resolution in logarithmically dividing cells.
* To whom correspondence should be addressed. Tel: +1 937 775 3125; Fax: +1 937 775 3730; Email: michael.leffak{at}wright.edu
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