The role of Orc6 in DNA replication and cytokinesis.

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Abstract. The Origin Recognition Complex (ORC), a heteromeric six-subunit protein complex, is essential for DNA replication. ORC's functions also extend beyond 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 solved the structure of human Orc6 and identified its structural features that contribute directly to the functions of the protein in replication and cytokinesis. We characterized the mutations in different domains of Orc6 leading to Meier-Gorlin syndrome (MGS) in humans, an autosomal recessive disorder characterized by primordial dwarfism and developmental abnormalities. We showed that despite different underlying molecular mechanisms these mutations resulted in similar phenotypes, defects in pre-RC formation and impaired DNA replication. Thus, the diverse molecular functions of Orc6 can be experimentally dissected to obtain a complete understanding of its discrete roles in the cell. 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. We established a humanized Orc6-based Drosophila model system which will be used to analyze the effects of naturally occurring and structure-guided targeted mutations of human Orc6 in protein functions. Regulatory insights into replicative and non-replicative functions of ORC in metazoans are limited, despite decades of research. Our completion of this project will help to fill this gap in our understanding of diverse ORC's functions in metazoans and provide clues as to how a specific mutation or mis-regulation would lead to growth and developmental defects.