During cell division, initiator proteins target and bind to DNA replication origins to trigger genome duplication. Despite extensive research into the mechanism of DNA replication initiation, the precise molecular events of this process have not been well defined. Several similarities exist between prokaryotic and eukaryotic replication initiation allowing the prokaryotic system to be used as a general guideline when studying the eukaryotic system. These similarities include the role of ATP, the requirement for a negatively supercoiled DNA template, and the fact that both initiators function as AAA+ oligomers. In collaboration with the Botchan lab, electron microscopy studies of the metazoan Drosophila melanogaster Origin Recognition Complex (DmORC) in both the apo- and ATP-γS-bound forms reveal a semicircular core domain flanked by ancillary structural elements. Addition of ATP-γS promotes a conformational change in the core that compacts the complex and rearranges the secondary structural features (Clarey et al., NSMB 2006). Phylogenetic and structural analyses indicate that each of the five AAA+ domains in ORC contain a conserved structural element that, in the crystal structure of the bacterial initiator DnaA, promotes the formation of a right-handed AAA+ helix indicating that a helical AAA+ substructures may be a feature of all initiator complexes (Erzberger et al., NSMB 2006). Consistent with this model, a helical DnaA pentamer can be docked into the core of ORC with excellent pitch and twist complementarities. Furthermore, subunit mapping indicates the Orc5 subunit is located in this spiral region. Orc5 is an ideal candidate to test this hypothesis since it is almost entirely composed of the AAA+ domain.

The position of the helical pentamer suggests that the observed ATP-dependent conformational changes observed in ORC derive from a reorientation of AAA+ domains within the core of the complex. By analogy to the DNA wrapping activity of DnaA, these structural alterations may modulate the superstructure of eukaryotic replication origins. Further subunit mapping studies as well as mapping of the AAA+ domains will directly test this hypothesis. Though differences in prokaryotic and eukaryotic initiation exist, such as origin structure and initiator regulation by phosphorylation, the similarity in structure and function is striking. Therefore, from the structural homologies to DnaA and known biochemical results, we posit that ORC together with Cdc6 prepares the DNA template for helicase loading through mechanisms related to the pathway established for DnaA in prokaryotes.