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 DNA replication. In metazoans, origin selection is poorly understood and it is unknown whether specific DNA binding by metazoan ORC controls replication. To address this problem, we used in vivo and in vitro approaches to demonstrate that Drosophila ORC (DmORC) binds to replication elements that direct repeated initiation of replication to amplify the Drosophila chorion gene loci in the follicle cells of egg chambers. Using immunolocalization, we observe that ACE3, a 440-bp chorion element that contains information sufficient 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 localization of DmORC is related to its DNA-binding properties, we find that purified DmORC binds to ACE3 and AER-d in vitro, and like its S. cerevisiae counterpart, this binding is dependent on ATP. Our findings suggest that sequence-specific DNA binding by ORC regulates initiation of metazoan DNA replication. Furthermore, adaptation of this experimental approach will allow for the identification of additional metazoan ORC DNA-binding sites and potentially origins of replication.
Austin
R J
RJ
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Orr-Weaver
T L
TL
Bell
S P
SP
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002823:Chorion / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004261:DNA Replication / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004330:Drosophila / Q000235:genetics* / Q000378:metabolism*; D005260:Female; D017344:Genes, Insect; D008856:Microscopy, Fluorescence; D051738:Origin Recognition Complex; D018741:Replication Origin
D017931:DNA Primers; D004268:DNA-Binding Proteins; D051738:Origin Recognition Complex; D004247:DNA