Inhibition of DNA re-replication by cyclin/CDK and GSK-3 kinase in S. cerevisiae

描述（由申请人提供）：真核细胞复制其染色体。DNA复制控制中的错误可能导致染色体不稳定。我们的长期目标是了解真核生物DNA复制控制的分子机制。Cdc6p是从酵母到人类的关键DNA复制成分。我们的初步研究结果表明，酵母GSK-3激酶（Mck1p）可靶向DNA复制因子Cdc6p降解，从而抑制DNA的再复制。已知Cdc6的n端区域在获得来源许可后被Cyclin/CDK复合物磷酸化降解。我们将研究由Mck1p激酶控制的Cdc6p降解抑制DNA再复制的新机制。在这个提议中，三个目标将验证我的假设，即Cdk1和GSK3对Cdc6p的顺序磷酸化是否控制了Cdc6p降解的时间。Aim1将检测Cdc6p是否被Mck1激酶直接磷酸化，Mck1依赖性Cdc6的降解是通过SCFCDC4复合物介导的。Aim2将测试Mck1p以不同于CDK的机制靶向Cdc6p的可能性。Aim3将测试CDK对Cdc6的磷酸化是否是Mck1p磷酸化的先决条件。Cdc6p和GSK-3激酶从酵母到人类是保守的。本研究结果可用于了解高等真核生物GSK-3激酶降解Cdc6的分子机制。准确的DNA复制控制是避免染色体不稳定的关键步骤，因为染色体不稳定是肿瘤发生的一个标志，因此了解染色体不稳定是很重要的。

DESCRIPTION (provided by applicant): Eukaryotic cells duplicate their chromosomes. Errors in the DNA replication control could lead to chromosome instability. Our long-term goal is to understand the molecular mechanism of DNA replication control in eukaryotes. Cdc6p is a key DNA replication component from yeast to humans. Our preliminary results showed that yeast GSK-3 kinase (Mck1p) targets DNA replication factor Cdc6p for degradation, which in turn inhibits DNA re-replication. It is known that N-terminal region of Cdc6 is phosphorylated by Cyclin/CDK complex for its degradation after the origin is licensed. We will study the novel Cdc6p degradation mechanism controlled by Mck1p kinase to inhibit DNA re- replication. In this proposal, three aims will test my hypothesis if a sequential phosphorylation of Cdc6p by Cdk1 and GSK3 controls the timing of Cdc6p degradation. Aim1 will test if Cdc6p is directly phosphorylated by Mck1 kinase, and Mck1-depedent Cdc6 degradation is mediated through SCFCDC4 complex. Aim2 will test the possibility that Mck1p targets Cdc6p in a distinct mechanism from CDK. Aim3 will test if the phosphorylation of Cdc6 by CDK is a pre-requisite for phosphorylation by Mck1p. Cdc6p and GSK-3 kinase are conserved from yeast to humans. The results obtained from this study can be applied to understand the molecular mechanism of Cdc6 degradation by GSK-3 kinase in higher eukaryotes. The accurate DNA replication control is a critical step to avoid chromosome instability, and it is important to understand since the chromosome instability is a hallmark of tumorigenesis.