Investigation of Orc6- and ORCA-interacting Proteins

The Origin Recognition Complex (ORC) binds to the origin of replication and recruits the pre-replication complex to initiate DNA replication in all eukaryotes. The smallest subunit of ORC, Orc6, is essential in DNA replication and mitosis. Additionally, Origin Recognition Complex-Associated (ORCA) is a pre-replication complex protein which localizes to regions of heterochromatin and stabilizes ORC. The aim of this study was to further define the role of Orc6 and ORCA by identifying unknown proteins which interact with them. With this goal, we utilized the BioID approach, which involved the expression of fusion proteins containing the BirA biotin ligase. We therefore expressed the BirA-HA-Orc6, BirA-HA-ORCA, or BirA-HA-EYFP fusion protein in mammalian cells and potential Orc6- and ORCA-interacting proteins were labeled upon subsequent addition of biotin to the cells. Finally, we performed a pull-down of biotinylated proteins and mass spectrometry analysis is pending for the pull-down products.Ope