In eukaryotic cells, firing of DNA replication origins normally does not recur until after M phase. This characteristic is thought to be due to the properties of "initiation" proteins like Orc, Cdc6, and Mcms. Using formaldehyde cross-linking, we show that Cdc6p and Mcm7p associate specifically with replication origins during G1 but not during G2 in S. cerevisiae. Mcm7p's association with origins depends on Cdc6p. Ectopic expression of Cdc6p enables it to associate with origins during G2, but this fails to recruit Mcm7p. Our data suggest that the loading of Mcm proteins onto origins is regulated by two mechanisms: first, by Cdc6p occupancy, and second, by S- and M-CDKs, whose activity during S, G2, and M phases prevents Mcm loading.
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