AN ESSENTIAL ROLE FOR THE CDC6 PROTEIN IN FORMING THE PRE-REPLICATIVECOMPLEXES OF BUDDING YEAST

ORIGINS Of DNA replication in Saccharomyces cerevisiae are bound by tw o protein complexes during the cell cycle(1,2). Post-replicative compl exes closely resemble those generated in vitro by purified origin reco gnition complex (ORC)(1,3-5), which is essential for DNA replication i n vivo(6-11). Pre-replicative complexes (pre-RCs) are characterized by an extended region of nuclease protection overlapping the ORC footpri nt(1). We show here that the Cdc6 protein (Cdc6p), which is necessary for origin firing in vivo(12-16), is essential for the establishment a nd maintenance of pre-RCs, suggesting that it is a component of these complexes. Without Cdc6p, G1 origins closely resemble post-replicative origins, providing evidence that ORC is also a component of pre-RCs, These results suggest that pre-RCs play an essential role in initiatin g DNA replication and support a two-step mechanism for the assembly of functional initiation complexes.