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

描述(申请人提供)：真核细胞复制它们的染色体。DNA复制控制中的错误可能会导致染色体不稳定。我们的长期目标是了解真核生物中DNA复制控制的分子机制。CDC6P是从酵母到人类的关键DNA复制组件。我们的初步结果表明，酵母GSK-3激酶(Mck1p)靶向DNA复制因子CDC6p的降解，这反过来又抑制DNA的重新复制。已知CDC6的N-末端区域被Cyclin/CDK复合体磷酸化，使其在来源许可后被降解。我们将研究Mck1p激酶控制的新的CDC6P降解机制，以抑制DNA的重新复制。在这个提议中，有三个目标将检验我的假设，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，而Mck1抑制的CDC6降解是通过SCFCDC4复合体介导的。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激酶从酵母到人类都是保守的。本研究结果可用于理解高等真核生物中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性的关键步骤，了解染色体不稳定性是肿瘤发生的一个标志，因此了解染色体不稳定性是非常重要的。

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.