GENETIC AND MOLECULAR ANALYSIS OF YEAST DNA REPLICATION

因为癌细胞显示出异常的染色体数目和高 突变率，对染色体DNA机制的了解 复制和修复可用于临床诊断、预后和 在治疗方案的设计上。这项建议继续进行研究。 真核细胞DNA复制启动的调控 以发芽酵母细胞为模型系统的蛋白质磷酸化。 CDC7/Dbf4和CDC28/Clb蛋白激酶的重要作用 DNA复制的调控是这项研究的主要焦点。两者都有 蛋白激酶被假设为在MCM-复合体中磷酸化 DNA复制的起源。通过这种方式，这些激酶可能会重塑。 染色质结构起始于细胞周期的G1期 通过DNA复制机制获取起源。因为所有这些 蛋白质在从酵母到人类的进化过程中是保守的，这 监管机制可能是通用的。第一个中的主要假设 具体目的是这两个激酶参与移除 通过MCM-复合体的磷酸化抑制DNA复制。 将使用遗传和生化方法相结合的方法来测试 每个特定目标中的假设。突变将被分离出来，移除 该抑制从而允许绕过该磷酸化步骤。 MCM底物中的磷酸化部位将通过 电喷雾质谱仪。另一个目标将集中在监管上 在细胞周期中被其他激酶磷酸化 以及Dbf4蛋白水平的调节。其他的激活会是 通过蛋白质纯化鉴定。DBF4蛋白合成和 降解将使用标记的Dbf4蛋白进行分析。RAD53 检查点蛋白激酶作为CDC7/Dbf4激酶的可能调节因子 也将进行研究。接下来的两个目标集中在 CDC7/Dbf4激酶在减数分裂和错误探针DNA修复中(诱导- 突变)，这是两个尚未得到很好研究的过程。 假设是上游的调控事件，如绑定 在这两个细胞过程中也被使用，但在下游 底物磷酸化等事件是不同的。因为 CDC7/Dbf4激酶不用于减数分裂复制，减数分裂 复制可能受到不同的监管，即使它使用类似 复制蛋白和起源。最终目标将检查角色 蛋白激酶在DNA复制中的作用。相似的基因和 生化方法也将被用于这些后一种目标。

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.