Drosophila ORC specifically binds to ACE3, an origin of DNA replication control element

In the yeast Saccharomyces cerevisiae, sequence-specific DNA binding by the origin recognition complex (ORC) is responsible for selecting origins of D NA replication. In metazoans, origin selection is poorly understood and it is unknown whether specific DNA binding by metazoan ORC controls replicatio n. To address this problem, we used in vivo and in vitro approaches to demo nstrate that Drosophila ORC (DmORC) binds to replication elements that dire ct repeated initiation of replication to amplify the Drosophila chorion gen e loci in the follicle cells of egg chambers. Using immunolocalization, we observe that ACE3, a 440-bp chorion element that contains information suffi cient to drive amplification, directs DmORC localization in follicle cells. Similarly, in vivo cross-linking and chromatin immunoprecipitation assays demonstrate association of DmORC with both ACE3 and two other amplification control elements, AER-d and ACE1. To demonstrate that the in vivo localiza tion of DmORC is related to its DNA-binding properties, we find that purifi ed DmORC binds to ACE3 and AER-d in vitro, and like its S. cerevisiae count erpart, this binding is dependent on ATP. Our findings suggest that sequenc e-specific DNA binding by ORC regulates initiation of metazoan DNA replicat ion. furthermore, adaptation of this experimental approach will allow for t he identification of additional metazoan ORC DNA-binding sites and potentia lly origins of replication.