Elevated Levels of the Polo Kinase Cdc5 Override the Mec1/ATR Checkpoint in Budding Yeast by Acting at Different Steps of the Signaling Pathway

Checkpoints are surveillance mechanisms that constitute a barrier to oncogenesis by preserving genome integrity. Loss of checkpoint function is an early event in tumorigenesis. Polo kinases (Plks) are fundamental regulators of cell cycle progression in all eukaryotes and are frequently overexpressed in tumors. Through their polo box domain, Plks target multiple substrates previously phosphorylated by CDKs and MAPKs. In response to DNA damage, Plks are temporally inhibited in order to maintain the checkpoint-dependent cell cycle block while their activity is required to silence the checkpoint response and resume cell cycle progression. Here, we report that, in budding yeast, overproduction of the Cdc5 polo kinase overrides the checkpoint signaling induced by double strand DNA breaks (DSBs), preventing the phosphorylation of several Mec1/ATR targets, including Ddc2/ATRIP, the checkpoint mediator Rad9, and the transducer kinase Rad53/CHK2. We also show that high levels of Cdc5 slow down DSB processing in a Rad9-dependent manner, but do not prevent the binding of checkpoint factors to a single DSB. Finally, we provide evidence that Sae2, the functional ortholog of human CtIP, which regulates DSB processing and inhibits checkpoint signaling, is regulated by Cdc5. We propose that Cdc5 interferes with the checkpoint response to DSBs acting at multiple levels in the signal transduction pathway and at an early step required to resect DSB ends.

检验点（checkpoint）是一类监视机制，通过维持基因组完整性构成抵御肿瘤发生的屏障。检验点功能缺失是致瘤过程中的早期事件。Polo样激酶（Polo kinases, Plks）是所有真核生物细胞周期进程的核心调控因子，且在肿瘤中常出现过表达。通过其Polo盒结构域（polo box domain），Polo样激酶可靶向多种此前已被细胞周期蛋白依赖性激酶（CDKs）和丝裂原活化蛋白激酶（MAPKs）磷酸化的底物。当遭遇DNA损伤时，Polo样激酶会被暂时性抑制，以维持依赖于检验点的细胞周期阻滞；而当需要终止检验点应答、恢复细胞周期进程时，则需要Polo样激酶的活性。本研究显示，在酿酒酵母（budding yeast）中，过量表达Polo样激酶Cdc5可绕过双链DNA断裂（double strand DNA breaks, DSBs）诱导的检验点信号通路，阻断多个Mec1/ATR靶标的磷酸化，其中包括Ddc2/ATRIP、检验点中介蛋白Rad9以及转导激酶Rad53/CHK2。本研究同时发现，高表达Cdc5会以Rad9依赖的方式延缓双链DNA断裂的加工过程，但不会阻碍检验点因子与单个双链DNA断裂位点的结合。此外，本研究提供证据表明，调控双链DNA断裂加工并抑制检验点应答的人源CtIP功能同源物Sae2，其活性受Cdc5的调控。本研究提出，Cdc5可通过作用于信号转导通路的多个层级以及双链DNA断裂末端切除所需的早期步骤，来干扰针对双链DNA断裂的检验点应答。