Multiple sites of replication initiation in the human beta-globin gene locus

The cell cycle-dependent, ordered assembly of protein prereplicative comple xes suggests that eukaryotic replication origins determine when genomic rep lication initiates. By comparison, the factors that determine where replica tion initiates relative to the sites of prereplicative complex formation ar e not known. In the human globin gene locus previous work showed that repli cation initiates at a single site 5' to the beta -globin gene when protein synthesis is inhibited by emetine. The present study has examined the patte rn of initiation around the genetically defined P-globin replicator in loga rithmically 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 g lobin 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, th e abundance of short nascent strands at another initiation site similar to 20 kb upstream is similar to4-fold as great as that at the IR, The latter s ite abuts an early S phase replicating fragment previously defined at low r esolution in logarithmically dividing cells.