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.
Dalton
S
S
Department of Biochemistry, University of Adelaide, South Australia. sdalton@biochem.adelaide.edu.au
Hopwood
B
B
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D015825:Chromosomes, Fungal / Q000378:metabolism*; D004261:DNA Replication / Q000502:physiology; D004271:DNA, Fungal / Q000096:biosynthesis; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000378:metabolism*; D064168:Minichromosome Maintenance Complex Component 7; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D017354:Point Mutation; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins
C102209:CDC45 protein, S cerevisiae; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C069200:MCM5 protein, S cerevisiae; C489423:MCM5 protein, human; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; C491088:MCM7 protein, S cerevisiae; D064168:Minichromosome Maintenance Complex Component 7