GENETIC AND MOLECULAR ANALYSIS OF YEAST DNA REPLICATION

因为癌细胞显示异常的染色体数目和高 突变率，染色体DNA的机制知识 复制和修复可用于临床诊断、预后和 in the design设计of therapies治疗. 这一建议是一项正在进行的研究的继续 真核生物DNA复制起始的调控 蛋白质磷酸化使用芽殖酵母细胞作为模型系统。 Cdc 7/Dbf 4和Cdc 28/Clb蛋白激酶的作用同样重要， DNA复制的调节因子是这项研究的主要焦点。 两 蛋白激酶被假设为磷酸化Mcm-复合物， DNA复制的起源 通过这种方式，激酶可以重塑 在细胞周期的G1期起源的染色质结构，以允许 通过DNA复制机制到达起点。 因为所有这些 蛋白质在从酵母到人类的进化中是保守的， 监管机制可能是普遍的。第一个主要假设是 具体的目的是这两种激酶参与去除 通过Mcm复合物的磷酸化抑制DNA复制。 将使用遗传和生物化学相结合的方法来测试 每个具体目标的假设。 突变将被分离， 该抑制从而允许绕过磷酸化步骤。 Mcm底物中的磷酸化位点将通过 电喷雾质谱另一个目标将集中在监管上， 在细胞周期中通过其他激酶的磷酸化 和Dbf 4蛋白水平的调节。 其他激酶将 通过蛋白质纯化鉴定。 Dbf 4蛋白质合成和 将使用标记的Dbf 4蛋白分析降解。 Rad53 检查点蛋白激酶作为Cdc 7/Dbf 4激酶的可能调节剂 也将被研究。 接下来的两个目标集中在 Cdc 7/Dbf 4激酶在减数分裂和错误探针DNA修复中的作用（诱导- 诱变），这是两个尚未充分研究的过程。 假设是上游调控事件，如结合 的Dbf 4也用于这两个细胞过程，但下游 诸如底物磷酸化的事件是不同的。 因为 Cdc 7/Dbf 4激酶不用于减数分裂复制，减数分裂 复制可能受到不同的调节，即使它使用类似的 复制蛋白和起源。 最后的目标将审查的作用 Pakl蛋白激酶在DNA复制中的作用。 相似的基因和 生物化学方法也将用于这些后一目的。

Because cancer cells display abnormal chromosome numbers and high mutation rates, knowledge of the mechanism of chromosomal DNA replication and repair can be used in clinical diagnosis, prognosis and in the design of therapies. This proposal continues an ongoing study of the regulation of the initiation of eukaryotic DNA replication by protein phosphorylation using budding yeast cells as a model system. The roles of both Cdc7/Dbf4 and Cdc28/Clb protein kinases as important regulators of DNA replication are the main focus of this study. Both protein kinases are hypothesized to phosphorylate the Mcm-complex at origins of DNA replication. In this way, the kinases may remodel chromatin structure at origins in G1 phase of the cell cycle to permit access to origins by the DNA replication machinery. Because all these proteins are conserved in evolution from yeast to humans, this regulatory mechanism may be universal. The major hypothesis in the first specific aim is that these two kinases are involved in removing the inhibition to DNA replication by phosphorylation of the Mcm-complex. A combined genetic and biochemical approaches will be used to test the hypotheses in each Specific Aim. Mutations will be isolated that remove the inhibition thereby allowing for bypass of the phosphorylation step. Sites of phosphorylation in the Mcm substrates will be identified by electrospray mass spectrometry. Another aim will focus on the regulation of this kinase during the cell cycle by phosphorylation by other kinases and the modulation of Dbf4 protein levels. Other kinases will be identified by protein purification. Dbf4 protein synthesis and degradation will be analyzed using a tagged Dbf4 protein. The Rad53 checkpoint protein kinase as a possible regulatory of Cdc7/Dbf4 kinase will also be studied. The next two aims are focused on the role of Cdc7/Dbf4 kinase in meiosis and in error-probe DNA repair (induced- mutagenesis), which are two processes that have not been well-studied. The hypothesis is that upstream regulatory events such as the binding of Dbf4 are also used in these two cellular processes, but downstream events such as substrate phosphorylation are different. Because Cdc7/Dbf4 kinase is not used for meiotic replication, meiotic replication may be regulated differently even though it uses similar replication proteins and origins. The final aim will examine the role of the Pakl protein kinase in DNA replication. Similar genetic and biochemical method will be employed in these latter aims as well.