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Coordination of Cell Cycle Events

  • Author(s): Seoane, Agustin Ignacio
  • Advisor(s): Morgan, David O
  • et al.
Abstract

Establishment of sister chromatid cohesion is essential for cell division. In budding yeast, establishment depends on acetylation of cohesin by Eco1, an acetyltransferase whose levels are high during S phase and then decline when DNA replication is complete. Interestingly, three different kinases act sequentially to generate a phosphodegron motif that triggers Eco1 destruction after S phase. Phosphorylation by Cdk1 in early S phase primes an adjacent serine for phosphorylation by the Dbf4-Cdc7 kinase. This second phosphorylation primes a third site for phosphorylation by the GSK-3 homolog, Mck1. Together, these phosphorylation sites create a degron with the proper spacing to be recognized by the ubiquitin ligase SCFCdc4. Although all three kinases are active during S phase, Eco1 is not degraded until S phase is complete. Here, we show that the activity of Dbf4-Cdc7 toward Eco1 is delayed until after S phase. Overproduction of Dbf4 results in early Eco1 degradation, arguing that Dbf4-Cdc7 activity is normally limited to provide enough kinase activity to fire replication origins but not enough to target Eco1. The Mcm replicative helicase complex is known to be a major target of Dbf4-Cdc7, and we tested the hypothesis that the Mcm complex at licensed origins sequesters Dbf4-Cdc7, thereby preventing Eco1 phosphorylation until all origins have fired. Consistent with this hypothesis, we found that Eco1 was degraded early when we blocked Mcm loading at origins by repressing CDC6 expression in G1. Conversely, Eco1 degradation was delayed when we repressed CDC45 expression to maintain Mcm complexes at origins. Forcing Mcm to remain in the nucleus did not affect Eco1 degradation timing, suggesting that soluble Mcm complexes do not sequester Dbf4-Cdc7. Together, these results suggest that the Mcm complex at unfired replication origins sequesters Dbf4-Cdc7 during S phase, and only after Mcm activation is the kinase freed to phosphorylate Eco1. This mechanism allows Eco1 to establish cohesion during S phase before it is degraded.

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