DNA REPLICATION CHECKPOINT

真核细胞使用细胞周期检查点来确保有丝分裂被推迟，直到染色体完全复制和修复。酵母和哺乳动物细胞中的检查点突变体对抑制DNA合成或破坏DNA的药物高度敏感。哺乳动物细胞中的检查点突变也容易发生染色体重排、断裂或丢失，这些事件往往与癌症和其他疾病有关。这个项目的目的是在非常基础的水平上了解当药物羟基脲(HU)减缓或停止DNA复制时发生的检查点事件。这种复制或HU检查点可以防止有丝分裂的发生。这项研究将用裂殖酵母庞贝裂殖酵母进行。这种生物已经成为研究细胞周期和检查点控制的优秀模式系统，这些控制在大多数物种中通常是保守的，包括人类。研究将集中在CDS1上，这是一种似乎在复制检查点发挥核心作用的蛋白激酶。该项目有三个具体目标。第一个目标是了解CDS1如何帮助实施复制检查点。调控有丝分裂开始的蛋白质将作为CDS1的靶标进行评估。第二个目标是了解CDS1是如何被调控的。我们将研究激活Cds1并使其底物磷酸化的机制。第三个目的是揭示将CDS1的核定位限制在细胞周期的DNA合成(S)阶段的机制。这些研究将提高对CDS1作为裂解酵母检查点机制的重要元件的理解，并将为研究人类同源蛋白的功能提供一个框架。

Eukaryotic cells use cell cycle checkpoints to ensure that mitosis is delayed until chromosomes are completely replicated and repaired. Checkpoint mutants in yeast and mammalian cells are hypersensitive to agents that inhibit DNA synthesis or damage DNA. Checkpoint mutants in mammalian cells are also prone to rearrangement, fragmentation or loss of chromosomes, events that are often causally associated with cancer and other diseases. The aim of this project is to understand at a very basic level the checkpoint events that are engaged when DNA replication is slowed or halted by the drug hydroxyurea (HU). This replication or HU checkpoint prevents the onset of mitosis. The studies will be carried out with the fission yeast Schizosaccharomyces pombe. This organism has served as an outstanding model system for the investigation of cell cycle and checkpoint controls that are generally conserved amongst most species, including humans. The investigations will focus on Cds1, a protein kinase that appears to play a central role in the replication checkpoint. The project has three specific aims. The first aim is to understand how Cds1 helps to enforce the replication checkpoint. Proteins that regulate the onset of mitosis will be evaluated as targets of Cds1. The second aim is to understand how Cds1 is regulated. Mechanisms that activate Cds1 and cause it to phosphorylate its substrates will be investigated. The third aim is to uncover the mechanism that restricts nuclear localization of Cds1 to the DNA synthesis (S) phase of the cell cycle. These studies will improve the understanding of Cds1 as an important element of checkpoint mechanisms in fission yeast and will provide a framework for investigating the functions of homologous proteins in humans.