Multiple functions of the Drosophila Orc6 protein

Abstract

The Origin recognition complex (ORC) is important for the DNA replication in eukaryotic cells. ORC is also involved in other cell functions. The smallest Drosophila ORC subunit, Orc6, consists of two functional domains. Larger N‐terminal domain directly involved in DNA binding and is important for ORC‐dependent DNA replication. Smaller C‐terminal domain is important for cytokinesis.Deletion of Orc6 gene was created in Drosophila. Mutant lethal alleles of Orc6 are defective in both DNA replication and cell proliferation during larval stages of development. Homozygous Orc6 mutants display disc‐less phenotype and show abnormal chromosome condensation and segregation. Mutant cells arrest in both G1 and mitosis stages of the cell cycle. Orc6 mutation can be rescued to viability by full‐length Orc6 transgene. The expression of a transgene carrying the core replicative N‐terminal domain of the Orc6 protein results a restoration of DNA replication in mutant larval tissues. The mutant cells carrying this transgene do not exhibit G1 arrest but nevertheless accumulate at mitosis.The cytokinetic function of DmOrc6 is associated with the C‐terminal domain of the protein. Orc6 interacts with the septin protein Pnut, which together with the septins Sep1 and Sep2 forms a septin complex important for cytokinesis. Septin complexes exhibit GTPase activity and can assemble into large filaments. The interaction of Orc6 with the septin complex is dependent on the coiled‐coil domain of Pnut. Furthermore, the binding of Orc6 to Pnut increases the intrinsic GTPase activity of the Drosophila septin complex, while in the absence of GTP it enhances septin complex filament formation.Together our results suggest an active role for Orc6 in DNA replication and septin complex function. Orc6 might be a part of a control mechanism directing the cytokinesis machinery during the final steps of mitosis.