A cytokinetic function of Drosophila ORC6 protein resides in a domain distinct from its replication activity.
Coordination between separate pathways may be facilitated by the requirements for common protein factors, a finding congruent with the link between proteins regulating DNA replication with other important cellular processes. We report that the smallest of Drosophila origin recognition complex subunits, Orc6, was found in embryos and cell culture localized to the cell membrane and cleavage furrow during cell division as well as in the nucleus. A two-hybrid screen revealed that Orc6 interacts with the Drosophila peanut (pnut), a member of the septin family of proteins important for cell division. This interaction, mediated by a distinct C-terminal domain of Orc6, was substantiated in Drosophila cells by coimmunoprecipitation from extracts and cytological methods. Silencing of Orc6 expression with double-stranded RNA resulted in a formation of multinucleated cells and also reduced DNA replication. Deletion of the C-terminal Orc6-peanut interaction domain and subsequent overexpression of the Orc6 mutant protein resulted in the formation of multinucleated cells that had replicated DNA. This mutant protein does not localize to the membrane or cleavage furrows. These results suggest that Orc6 has evolved a domain critical mainly for cytokinesis.
Chesnokov
Igor N
IN
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham School of Medicine, Birmingham, AL 35294, USA. ichesnokov@uab.edu
Chesnokova
Olga N
ON
Botchan
Michael
M
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000502:physiology*; D004330:Drosophila; D029721:Drosophila Proteins / Q000737:chemistry* / Q000502:physiology*; D016193:G1 Phase; D015723:Gene Library; D020868:Gene Silencing; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008840:Microfilament Proteins / Q000737:chemistry; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D051738:Origin Recognition Complex; D010455:Peptides / Q000737:chemistry; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000378:metabolism; D013997:Time Factors; D020798:Two-Hybrid System Techniques
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D008164:Luminescent Proteins; D008840:Microfilament Proteins; C477598:ORC6 protein, Drosophila; D051738:Origin Recognition Complex; D010455:Peptides; D012330:RNA, Double-Stranded; C087195:pnut protein, Drosophila; D049452:Green Fluorescent Proteins; D004247:DNA