During cell division, initiator proteins target DNA replication origins to promote genome duplication. The Origin Recognition Complex (ORC) is the eukaryotic initiator protein that acts at a major regulatory point in the cell cycle to initiate DNA replication. In an attempt to learn more about the specific mechanism of eukaryotic initiation, I have used Transmission Electron Microscopy (TEM) and single particle image reconstruction methods to obtain intermediate resolution three-dimensional models of the Drosophila melanogaster ORC (DmORC) in various nucleotide and phosphorylation states. Additionally, I have investigated DmORC binding to various DNA templates by Atomic Force Microscopy (AFM). The archeal, prokaryotic and eukaryotic initiators are all members of the AAA+ switch/motor class of proteins and share initiator-specific features that reflect common mechanistic function. High-resolution structural data revealed that the bacterial initiator, ATP-DnaA, forms a right-handed helical filament that, for the first time, suggested a mechanism by which origin DNA is processed by an initiator complex. The computational and structural data presented in this thesis posit ORC and ATP-DnaA are structurally homologous and may engage origin DNA through a common mechanism.

Regulation of ORC by phosphorylation and nucleotide binding were also investigated in an attempted to visualize how modification by phosphorylation regulates the complex through conformational rearrangements. Single particle EM studies of ORC bound to nucleotide in both the hypo- and hyperphosphorylated state show nucleotide binding gives rise to an analogous conformation independent of phosphorylation state. At the intermediate resolution achieved in our studies, ATP promotes changes along the toroidal core of the complex with modest observable differences contributed by phosphorylation.

Despite the prediction that ORC and DnaA function through a common mechanism, to date there has been no observed DNA unwinding activity by a eukaryotic initiator. To explore ORC's interaction with DNA, DmORC was imaged with various linear and supercoiled DNA substrates using AFM. These data suggest ORC is wrapping an average of ∼130bp of linear DNA. This study represents the first structural characterization of a metazoan initiator and allows for a more detailed comparison of the initiators across the three domains of life.