The role of Orc6 in DNA replication and cytokinesis.

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This is an application for the Administrative Supplement to the currently active R01 GM121449 grant entitled “The Role of Orc6 in DNA replication and cytokinesis”. The major thrust of the parent grant is to understand the functions of replication initiators Orc6 and ORC in eukaryotic cells and tissues using Drosophila and human cells as model systems. ORC, heteromeric six-subunit protein complex, is essential for DNA replication. The smallest of ORC subunits, Orc6 protein, is important for DNA replication in all eukaryotes. We have shown that in Drosophila Orc6 has an additional role in cytokinesis through interaction with the septin complex, a highly conserved polymerizing protein assembly that is critical for cytokinesis in many species and is recognized as important component of the cytoskeleton. In preliminary studies we have identified structural features of Orc6 that contribute directly to the functions of the protein in replication and cytokinesis. The overall goal of this proposal is to define the distinct mechanisms of Orc6 function in DNA replication and in cytokinesis using Drosophila as a model system. All major objectives of the parent grant require constant purifications of the wild type and mutant protein complexes (such as ORC and septin complex and their subunits) to study protein functions and protein/DNA interactions. ChemiDoc Imagers offer best-in-class performance with ease of use for visible light (RGB) and far red/near infrared (FR/NIR) fluorescence and chemiluminescence detection and all general gel documentation applications. Stain-free imaging enables immediate visualization of proteins without gel staining and instant verification of protein transfer to blots. The Administrative Supplement will allow us to purchase a new ChemiDoc MP that will greatly facilitate and advance our studies and workflow. The purchase of new equipment required for the project and associated research expenses will provide the necessary investment into the research-oriented economy. We believe that the discoveries made with the help of new ChemiDoc MP system funded by the Administrative Supplement will significantly strengthen our chances for obtaining future NIH support through competitive renewal application. This in turn will ensure preservation of the newly created and current research positions supported by the parent grant.