Forced binding of the origin of replication complex to chromosomal sites inDrosophilaS2 cells creates an origin of replication

Abstract

Origins of replication in higher eukaryotes appear to lack specific sequence characteristics and those mapped often appear to be spread over several kilobases. This has complicated the study of site-specific events at origins of replication in vivo. Here we show that fusion of a Gal4-binding domain to proteins of the origin of replication complex (Orc) is sufficient to direct initiation to Gal4-binding sites inserted in the Drosophila S2 cell chromosome. The activation appears to go via an authentic route, taking place only in the S phase of the cell cycle and involving the formation of a prereplication complex. We have also shown that the origin-associated acetylation of histone H4 at K12 can be directed to the region of Orc binding by the presence of Orc. We expect that this system can provide a useful tool for the study of site-specific events at origins of replication in higher eukaryotes and a means to dissect Orc-dependent and Orc-independent events at origins.