Origin Recognition Complex
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, our 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).Hyperphosphorylation of the DmORC by cyclin dependent kinases allows nucleotide binding but inhibits the ATPase activity of Orc1, and ablates the ATP-dependent interaction of ORC with DNA. We used single particle EM of ORC bound to nucleotide in both the dephosphorylated and hyper-phosphorylated states to show that nucleotide binding gives rise to an analogous conformation, independent of phosphorylation state. ATP promotes changes along the toroidal core of the complex with negligible differences contributed by phosphorylation. Thus, hyperphosphorylation does not induce meso-scale rearrangement of the ORC structure. We also performed atomic force microscopy (AFM) studies that showed that the contour length of a 688 bp linear DNA fragment shortens by the equivalent of ∼130 bp upon ORC binding, suggesting that ORC may wrap the DNA in a manner akin to what has been shown for DnaA (Clarey et al., JSB 2008). Based on existing data and our structures, we propose a model for subunit arrangement for the AAA+ and winged helix domains. Our initial subunit mapping experiments positioned the Orc5 subuni,t which is mostly constituted of the conserved AAA ATPase domain, is located in the toroidal region. In collaboration with the Berger lab, we are now mapping each of the other components to generated a full architectural map of ORC.