The fission yeast origin recognition complex is constitutively associated with chromatin and is differentially modified through the cell cycle

The origin recognition complex (ORC) binds to the well defined origins of D NA replication in budding yeast. Homologous proteins in other eukaryotes ha ve been identified but are less well characterised, We report here the char acterisation of a fission yeast ORC complex (SpORC). Database searches iden tified a fission yeast Orc5 homologue, SpOrc5 is essential for cell viabili ty and its deletion phenotype is identical to that of two previously identi fied ORC subunit homologues, SpOrc1 (Orp1/Cdc30) and SpOrc2 (Orp2), Co-immu noprecipitation experiments demonstrate that SpOrc1 forms a complex with Sp Orc2 and SpOrc5 and gel filtration chromatography shows that SpOrc1 and SpO rc5 fractionate as high molecular mass complexes. SpORC subunits localise t o the nucleus in a punctate distribution which persists throughout interpha se and mitosis, We developed a chromatin isolation protocol and show that S pOrc1, 2 and 5 are associated with chromatin at all phases of the cell cycl e. While the levels, nuclear localisation and chromatin association of SpOR C remain constant through the cell cycle, one of its subunits, SpOrc2, is d ifferentially modified. We show that SpOrc2 is a phosphoprotein which is hy permodified in mitosis and is rapidly converted to a faster migrating isofo rm as cells proceed into G(1) in preparation for S-phase.