Characterization of Cdc47p-minichromosome maintenance complexes in Saccharomyces cerevisiae: identification of Cdc45p as a subunit.

Cdc47p is a member of the minichromosome maintenance (MCM) family of polypeptides, which have a role in the early stages of chromosomal DNA replication. Here, we show that Cdc47p assembles into stable complexes with two other members of the MCM family, Cdc46p and Mcm3p. The assembly of Cdc47p into complexes with Cdc46p does not appear to be cell cycle regulated, making it unlikely that these interactions per se are a rate-limiting step in the control of S phase. Cdc45p is also shown to interact with Cdc47p in vivo and to be a component of high-molecular-weight MCM complexes in cell lysates. Like MCM polypeptides, Cdc45p is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae; however, Cdc45p remains in the nucleus throughout the cell cycle, whereas MCMs are nuclear only during G1. We characterize two mutations in CDC47 and CDC46 which arrest cells with unduplicated DNA as a result of single base substitutions. The corresponding amino acid substitutions in Cdc46p and Cdc47p severely reduce the ability of these polypeptides to assemble in a complex with each other in vivo and in vitro. This argues that assembly of Cdc47p into complexes with other MCM polypeptides is important for its role in the initiation of chromosomal DNA replication. Eukaryotic DNA replication is controlled through the activity of replication origins, which initiate new rounds of DNA synthesis once per S phase in each cell cycle (12). Although little is known about how the initiation of chromosomal DNA replication is controlled, it is clear that once DNA replication is initiated, negative controls block the reestablishment of the prereplicative state at replication origins. These negative controls persist throughout G2 and mitosis but are removed as cells enter G1 (34), thus ensuring that initiation events at origins are restricted to only once per cell cycle. An activity of Xenopus egg extracts known as replication licensing factor (RLF) has previously been implicated in the establishment of the prereplicative state of cells in G1 (1, 7). RLF activity is thought to function by establishing functionally competent preinitiation complexes at replication origins after breakdown of the nuclear envelope. After performing its licensing role and immediately after an initiation event, the model predicts that RLF is inactivated or destroyed, leaving replication origins in a postreplicative state (1). This provides a simple framework to explain how S phase and mitosis are maintained in the correct order and how replication origins function only once per cell cycle. The initiation of DNA replication in the budding yeast Saccharomyces cerevisiae requires the minichromosome maintenance (MCM) family of polypeptides, which include Mcm2p and Mcm3p (38), Cdc54p/Mcm4p (37), Cdc46p/Mcm5p (18), Mcm6p (5), and Cdc47p/Mcm7p (10). The six members of the MCM family carry a highly conserved motif (the MCM box) which resembles a conserved domain associated with transcription and replication factors with known or assumed DNA