DNA synthesis at individual replication forks requires the essential initiation factor Cdc45p

Cdc45p assembles at replication origins before initiation and is required f or origin firing in Saccharomyces cerevisiae. A heat-inducible cdc45 degron mutant was constructed that promotes rapid degradation of Cdc45p at the re strictive temperature, Consistent with a role in initiation, loss of Cdc45p in G(I) prevents all detectable DNA replication without preventing subsequ ent entry into mitosis, Loss of Cdc45p activity during S-phase blocks S-pha se completion but not activation of replication checkpoints. Using density substitution, me show that after allowing replication fork establishment, C dc45p inactivation prevents the subsequent progression of individual replic ation forks, This provides the first direct functional evidence that Cdc45p plays an essential role during elongation. Thus, like the large T antigen in SV40 replication, Cdc45p plays a central role in both initiation and elo ngation phases of chromosomal DNA replication.