Cell cycle regulation by PP1 and Cdc7

我的长期研究目标是解开人类细胞如何在DNA复制和细胞周期控制的背景下保持遗传稳定性的机制。为了保持遗传稳定性，整个基因组必须忠实地复制，并且每个细胞周期只复制一次。后者是通过严格调节复制前复合物（pre-RC;“许可”）的逐步形成和复制起始的有序激活来实现的。现有数据表明，Cdc 7在许可和复制启动过程中发挥着关键作用。Cdc 7本身在被Dbf 4调节亚基结合时被激活。然而，Dbf 4与Cdc 7结合激活Cdc 7的确切机制尚不清楚。一个流行的模型是Cdc 7在高水平Dbf 4的情况下被Cdk介导的磷酸化激活。然而，与此模型不同的是，我们最近发现非磷酸化Cdc 7对DNA复制起点具有高亲和力并激活DNA复制。此外，Cdk 1/细胞周期蛋白B介导的Cdc 7磷酸化导致其从染色质解离，阻止DNA（再）复制，从而确保每个细胞周期一次的DNA复制。我们还发现，Cdc 7作为复制激活剂是“恢复”的PP 1介导的去磷酸化细胞退出有丝分裂。还发现PP 1在pre-RC已经形成之后激活复制检查点。因此，PP 1可以作为复制促进剂和抑制剂发挥作用：前者通过恢复Cdc 7功能，后者通过激活复制检查点（即，抑制复制起始）。因此，人类细胞需要在Cdc 7、Cdk 1和PP 1之间具有密切的串扰以维持遗传稳定性。基于这些观察，我们假设PP 1介导的Cdc 7去磷酸化是一种主动调节机制，以促进复制在两个不同的步骤：许可和启动激活步骤。为了验证这一假设，我们建议：（一）系统地研究PP 1介导的Cdc 7去磷酸化的作用，在复制许可和激活过程中，在正常的，永生化的，和癌症的人类细胞;和（ii）确定Cdc 7去磷酸化的潜在作用，在胞质分裂的调节。这项研究的数据将揭示人类细胞在DNA复制和细胞周期控制的背景下如何保持或失去遗传稳定性的机制。

My long-term research goal is unravelling the mechanism of how human cells maintain genetic stability in the context of DNA replication and cell cycle control. To maintain genetic stability, the entire genome must replicate faithfully and only once per cell cycle. The latter is achieved by strictly regulating the stepwise formation of the pre-replication complex (pre-RC; “licensing”) and the orderly activation of replication initiation. Available data indicate that Cdc7 plays critical roles for both licensing and replication initiation processes. Cdc7 itself is activated when it is bound by the Dbf4 regulatory subunit. However, the exact mechanism when Dbf4 binds to Cdc7 to activate it is poorly understood. A prevalent model has been that Cdc7 is activated by Cdk-mediated phosphorylation in the context of high levels of Dbf4. Contrarily to this model, however, we have recently found that non-phosphorylated Cdc7 has high affinity for the origin of DNA replication and activates DNA replication. Furthermore, Cdk1/cyclin B-mediated Cdc7 phosphorylation leads to its dissociation from chromatin, preventing DNA (re)replication and, thus, ensuring once-per-cell cycle DNA replication. We also found that Cdc7 as the replication activator is “restored” by PP1-mediated dephosphorylation as cells exit mitosis. PP1 has also been found to activate a replication checkpoint after pre-RC is already formed. Thus, PP1 may function as a replication promoter as well as an inhibitor: for the former by restoring Cdc7 function and the latter by activating replication checkpoint (i.e., inhibiting replication initiation). Thus, human cells need to have intimate crosstalk between Cdc7, Cdk1 and PP1 to maintain genetic stability. Based on these observations, we postulate that the PP1-mediated Cdc7 dephosphorylation is an active regulation mechanism to promote replication at two different steps: the licensing and initiation activation steps. To test this hypothesis, we propose to: (i) systematically examine the role of PP1-mediated Cdc7 dephosphorylation in the replication licensing and activation process in normal, immortalized, and cancerous human cells; and (ii) determine the potential role of Cdc7 dephosphorylation in the regulation of cytokinesis. Data from this study will shed new light on the mechanism of how human cells maintain or lose genetic stability in the context of DNA replication and cell cycle control.