Abstract
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.
Highlights
In the cell cycle of the budding yeast Saccharomyces cerevisiae, DNA replication initiation is driven by a sharp rise of Clb5/6-Cdk1 activity [1,2,3]
Sic1 phosphorylation at the onset of S-phase leads to its rapid destruction, unleashing a stockpile of Clb5/6Cdk1
Our experiments reveal that the feedback loop shields Sic1 destruction from molecular fluctuations and environmental variability, ensuring that the switch flips decisively
Summary
In the cell cycle of the budding yeast Saccharomyces cerevisiae, DNA replication initiation is driven by a sharp rise of Clb5/6-Cdk activity [1,2,3]. As the cell passes Start [4,5], the commitment to the round of cell division, the transcriptional inhibitor Whi, is phosphorylated and excluded from the nucleus [6,7], and two transcription factor complexes Swi4/Swi (SBF) [8] and Mbp1/ Swi (MBF) [9] are activated. Unlike Cln1/2-Cdk, which is the major driver of cell cycle progression in the late G1 phase by phosphorylating many G1/S targets [12], Clb5/6-Cdk is rendered inactive throughout G1 phase by the inhibitor Sic until Sic is phosphorylated and degraded (Figure 1) [13,14]. Strains with either SIC1 deleted or altered Sic degradation dynamics show a significant increase in genomic instability (Figure S4), underlining the importance of Sic for a proper S-phase entry [15,16]
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