The Human β-Globin Replication Initiation Region Consists of Two Modular Independent Replicators

Abstract

Previous studies have shown that mammalian cells contain replicator sequences, which can determine where DNA replication initiates. However, the specific sequences that confer replicator activity were not identified. Here we report a detailed analysis of replicator sequences that dictate initiation of DNA replication from the human β-globin locus. This analysis suggests that the β-globin replication initiation region contains two adjacent, redundant replicators. Each replicator was capable of initiating DNA replication independently at ectopic sites. Within each of these two replicators, we identified short, discrete, nonredundant sequences, which cooperatively determine replicator activity. Experiments with somatic cell hybrids further demonstrated that the requirements for initiation at ectopic sites were similar to the requirements for initiation within native human chromosomes. The replicator clustering and redundancy exemplified in the human β-globin locus may account for the extreme difficulty in identifying replicator sequences in mammalian cells and suggest that mammalian replication initiation sites may be determined by cooperative sequence modules.

We thankfully acknowledge Mel DePamphilis for critical reading of the manuscript and for numerous helpful suggestions. We are grateful to Geoffrey M. Wahl, Yves G. Pommier, Kurt W. Kohn, Tsutomo Shimura, Haiqing Fu, Elsa Bronze Da Rocha, and Fred E. Indig for helpful suggestions. We thank Luo-Wei Rodewald for assistance in making the deletion mutants shown in Fig. 1 and Agnes Telling for assistance in making the DT-40 cells containing human chromosome 11 and deletions in the human β-globin locus in these cells.

Work in M.I.A.'s laboratory is supported by the NIH's intramural program. M.G. is supported by NIH grants DK44746 and HL57620.