Author SummaryIn eukaryotic organisms, genome replication starts simultaneously from many sites on the DNA, called origins of replication. In budding yeast, these origins are activated by a kinase, Clb5/6-Cdk1. Until the start of S-phase, when the replication origins are activated, this kinase is kept inactive by an inhibitor, Sic1, which has multiple phosphorylation sites. Sic1 phosphorylation at the onset of S-phase leads to its rapid destruction, unleashing a stockpile of Clb5/6-Cdk1. Here, we show using live-cell fluorescent microscopy that Clb5/6-Cdk1 phosphorylation of Sic1 creates a feedback loop that functions as a switch. Our experiments reveal that the feedback loop shields Sic1 destruction from molecular fluctuations and environmental variability, ensuring that the switch flips decisively. We also demonstrate that a multisite phosphorylation scheme is not required for rapid Sic1 destruction. Sic1 can also be phosphorylated by another kinase, called Cln1/2-Cdk1. We demonstrate that this seemingly redundant interaction is responsible for robust timing of Sic1 destruction. Our experiments and mathematical model identify the contribution of each component to the function of this biochemical circuit.