CDC45, A NOVEL YEAST GENE THAT FUNCTIONS WITH THE ORIGIN RECOGNITION COMPLEX AND MCM PROTEINS IN INITIATION OF DNA-REPLICATION

The CDC45 gene of Saccharomyces cerevisiae was isolated by complementa tion of the cold-sensitive cdc45-1 mutant and shown to be essential fo r cell viability. Although CDC45 genetically interacts with a group of MCM genes (CDC46, CDC47, and CDC54), the predicted sequence of its pr otein product reveals no significant sequence similarity to any known Mcm family member. Further genetic characterization of the cdc45-1 mut ant demonstrated that it is synthetically lethal with orc2-1, mcm2-1, and mcm3-1. These results not only reveal a functional connection betw een the origin recognition complex (ORC) and Cdc45p but also extend th e CDC45-MCM genetic interaction to all known MCM family members that w ere shown to be involved in replication initiation. Initiation of DNA replication in cdc45-1 cells was defective, causing a delayed entry in to S phase at the nonpermissive temperature, as well as a high plasmid loss rate which could be suppressed by tandem copies of replication o rigins. Furthermore, two-dimensional gels directly showed that chromos omal origins fired less frequently in cdc45-1 cells at the nonpermissi ve temperature. These findings suggest that Cdc45p, ORC, and Mcm prote ins act in concert for replication initiation throughout the genome.