The fission yeast origin recognition complex is constitutively associated with chromatin and is differentially...

The origin recognition complex (ORC) binds to the well defined origins of DNA replication in budding yeast. Homologous proteins in other eukaryotes have been identified but are less well characterised. We report here the characterisation of a fission yeast ORC complex (SpORC). Database searches identified a fission yeast Orc5 homologue. SpOrc5 is essential for cell viability and its deletion phenotype is identical to that of two previously identified ORC subunit homologues, SpOrc1 (Orp1/Cdc30) and SpOrc2 (Orp2). Co-immunoprecipitation experiments demonstrate that SpOrc1 forms a complex with SpOrc2 and SpOrc5 and gel filtration chromatography shows that SpOrc1 and SpOrc5 fractionate as high molecular mass complexes. SpORC subunits localise to the nucleus in a punctate distribution which persists throughout interphase and mitosis. We developed a chromatin isolation protocol and show that SpOrc1, 2 and 5 are associated with chromatin at all phases of the cell cycle. While the levels, nuclear localisation and chromatin association of SpORC remain constant through the cell cycle, one of its subunits, SpOrc2, is differentially modified. We show that SpOrc2 is a phosphoprotein which is hypermodified in mitosis and is rapidly converted to a faster migrating isoform as cells proceed into G(1) in preparation for S-phase.