Design principles of the yeast G1/S switch.
Design principles of the yeast G1/S switch.
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DOI：
10.1371/journal.pbio.1001673
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发表时间：
2013-10
期刊：
影响因子：
9.8
通讯作者：
Tang C
中科院分区：
文献类型：
--
作者：
Yang X;Lau KY;Sevim V;Tang C
单细胞显微镜和计算模型为出芽酵母启动DNA复制的G1/S开关提供了新的机制见解，揭示了Clb5/6-Cdk1和Sic1反馈回路以及生化电路设计的新规则。出芽酵母细胞周期G1/S转变的一个标志是b型细胞周期蛋白- cdk化学计量抑制剂Sic1的蛋白水解降解。删除SIC1或改变SIC1降解动力学增加了基因组的不稳定性。我们确定了G1/S回路部分的一些关键事实：Sic1在多个位点上的磷酸化对于其破坏是必要的，并且在体外，上游激酶Cln1/2-Cdk1和下游激酶Clb5/6-Cdk1都可以磷酸化Sic1，具有不同的特异性、协同性和进程性。然而，由于体外，体内和理论研究之间的差异，该系统如何作为一个整体工作仍然存在争议。在这里，通过监测在不同系统扰动下单个细胞中Sic1的实时破坏，我们提供了一个清晰的画面，说明该电路如何在体内作为一个开关发挥作用。我们发现，Cln1/2-Cdk1设定了Sic1破坏的适当时间，但对其破坏速度没有贡献；因此，它只是一个触发器。Sic1的抑制靶点Clb5/6-Cdk1通过双负反馈回路控制Sic1的破坏速度，确保了Clb5/6-Cdk1活性的全或无转变。此外，我们证明了Sic1的单磷突变体的降解是快速的和开关式的，就像野生型一样。我们的数学模型证实了我们对电路的理解，并证明了两种激酶之间的底物共享不是冗余，而是设计的一部分，以克服Sic1降解的时间和清晰度之间的权衡。我们的研究为酵母G1/S开关的设计特征提供了直接的机制见解。在真核生物中，基因组复制同时从DNA上的许多位点开始，称为复制起点。在出芽酵母中，这些起源被一种名为Clb5/6-Cdk1的激酶激活。在s期开始之前，当复制起点被激活时，该激酶被具有多个磷酸化位点的抑制剂Sic1保持非活性。在s期开始时Sic1磷酸化导致其快速破坏，释放Clb5/6-Cdk1储备。在这里，我们使用活细胞荧光显微镜显示，Clb5/6-Cdk1的Sic1磷酸化产生了一个反馈回路，其功能类似于开关。我们的实验表明，反馈回路保护Sic1免受分子波动和环境变化的破坏，确保开关果断翻转。我们还证明了快速破坏Sic1不需要多位点磷酸化方案。Sic1也可以被另一种称为Cln1/2-Cdk1的激酶磷酸化。我们证明了这种看似冗余的相互作用是Sic1破坏的健壮定时的原因。我们的实验和数学模型确定了每个组成部分对这个生化回路功能的贡献。
Single-cell microscopy and computational modeling offer novel mechanistic insight into the G1/S switch that initiates DNA replication in budding yeast, revealing a Clb5/6-Cdk1 and Sic1 feedback loop and new rules of biochemical circuit design. A hallmark of the G1/S transition in budding yeast cell cycle is the proteolytic degradation of the B-type cyclin-Cdk stoichiometric inhibitor Sic1. Deleting SIC1 or altering Sic1 degradation dynamics increases genomic instability. Certain key facts about the parts of the G1/S circuitry are established: phosphorylation of Sic1 on multiple sites is necessary for its destruction, and both the upstream kinase Cln1/2-Cdk1 and the downstream kinase Clb5/6-Cdk1 can phosphorylate Sic1 in vitro with varied specificity, cooperativity, and processivity. However, how the system works as a whole is still controversial due to discrepancies between in vitro, in vivo, and theoretical studies. Here, by monitoring Sic1 destruction in real time in individual cells under various perturbations to the system, we provide a clear picture of how the circuitry functions as a switch in vivo. We show that Cln1/2-Cdk1 sets the proper timing of Sic1 destruction, but does not contribute to its destruction speed; thus, it acts only as a trigger. Sic1's inhibition target Clb5/6-Cdk1 controls the speed of Sic1 destruction through a double-negative feedback loop, ensuring a robust all-or-none transition for Clb5/6-Cdk1 activity. Furthermore, we demonstrate that the degradation of a single-phosphosite mutant of Sic1 is rapid and switch-like, just as the wild-type form. Our mathematical model confirms our understanding of the circuit and demonstrates that the substrate sharing between the two kinases is not a redundancy but a part of the design to overcome the trade-off between the timing and sharpness of Sic1 degradation. Our study provides direct mechanistic insight into the design features underlying the yeast G1/S switch. In 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.
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影响因子：
64.5
作者：
Feldman, RMR;Correll, CC;Deshaies, RJ
通讯作者：
Deshaies, RJ
影响因子：
10.5
作者：
EPSTEIN, CB;CROSS, FR
通讯作者：
CROSS, FR
影响因子：
4.4
作者：
Gillespie, DT
通讯作者：
Gillespie, DT
DOI：
10.1098/rstb.1993.0078
发表时间：
1993-06-29
期刊：
PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY OF LONDON SERIES B-BIOLOGICAL SCIENCES
影响因子：
--
作者：
MOLL, T;SCHWOB, E;NASMYTH, K
通讯作者：
NASMYTH, K
影响因子：
64.8
作者：
Nash, P;Tang, XJ;Tyers, M
通讯作者：
Tyers, M