A vertebrate N-end rule degron reveals that Orc6 is required in mitosis for daughter cell abscission

Abstract

Orc6, an evolutionarily conserved component of the origin recognition complex, is essential for deoxyribonucleic acid (DNA) replication initiation from yeast to humans. Whether vertebrate Orc6 has a mitotic function remains unresolved. In vertebrates, but not yeast, its depletion causes centrosome amplification and multinucleate division, but replication stress indirectly causes similar abnormalities. In this paper, we exploit Varshavsky’s N-end rule to create a temperature-sensitive degron form of avian Orc6. Orc6 depletion during the S phase triggers centrosome amplification suppressed by G2 checkpoint inhibition, reflecting an indirect consequence of aberrant DNA replication. However, Orc6 depletion during mitosis suffices to cause asymmetric division and failure in cytokinesis, with a delay in daughter cell abscission revealed by a fluorescence-bleaching assay. A mutant lacking the C-terminal 25 residues cannot rescue these defects. Thus, vertebrate Orc6 is necessary during mitosis for the abscission stage of cytokinesis. Our findings exemplify N-end rule degrons as tools to unravel functions of a single protein during different phases of the vertebrate cell cycle.