Cdc6p-dependent loading of Mcm proteins onto pre-replicative chromatin in budding yeast.
The Cdc6 protein is essential for the assembly of pre-replicative complexes (pre-RCs) at origins of DNA replication in the budding yeast Saccharomyces cerevisiae. This reaction is blocked in vivo by the cyclin-dependent kinase Cdc28p, together with its regulatory subunits, the B type cyclins that are present throughout S, G2, and M phases. Because the destruction of B type cyclins and the consequent inactivation of the kinase are essential for exit from mitosis, pre-RC formation can only occur after passage through mitosis. Therefore, pre-RC formation has been proposed to be essential for coupling S phase and mitosis and for limiting DNA replication to once per cell cycle. The Mcm2-7 family of proteins has been implicated in limiting replication to once per cell cycle from experiments with Xenopus egg extracts. Here we show that the Mcm proteins of budding yeast are abundant and are quantitatively found in a chromatin-enriched fraction specifically during the G1 phase of the cell cycle. This chromatin binding depends on the de novo synthesis of Cdc6p, providing evidence that a conserved biochemical pathway plays a critical role in coordinating DNA replication with mitosis in both yeast and higher eukaryotes. Cdc6p and the origin recognition complex can be selectively removed from this chromatin-enriched fraction without removing the Mcm proteins. From these results, we propose that Cdc6p (and the origin recognition complex) nucleates the binding of Mcm proteins to chromatin, but once bound, the Mcm proteins appear to interact tightly with some other component of chromatin.
Donovan
S
S
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, United Kingdom.
Harwood
J
J
Drury
L S
LS
Diffley
J F
JF
D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002843:Chromatin / Q000302:isolation & purification / Q000378:metabolism*; D004261:DNA Replication; D005656:Fungal Proteins / Q000378:metabolism; D016193:G1 Phase; D046911:Macromolecular Substances; D008938:Mitosis; D008954:Models, Biological; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis; D018741:Replication Origin; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D002843:Chromatin; D005656:Fungal Proteins; D046911:Macromolecular Substances; D016271:Proto-Oncogene Proteins c-myc; D029701:Saccharomyces cerevisiae Proteins