PHOSPHORYLATION OF DNA POLYMERASE ? PRIMASE

DNA复制的控制是人类进化过程中的关键过程之一 哺乳动物细胞周期的调节。细胞调节失调 循环可以在几个阶段出现，例如，在决定 开始脱氧核糖核酸复制或在适当完成S阶段，以及 是多种人类疾病的重要致病因素， 最常见的是癌症。我们研究的长期目标是 从分子细节上阐明控制DNA的机制 哺乳动物细胞中的复制。乳头状病毒DNA在宿主体内的复制 被感染的细胞和无细胞反应已被证明是一种 为此，模型系统非常有用。它促进了 多种细胞蛋白的鉴定与鉴定 是复制所必需的，并使我们对 SV40DNA复制机制的研究。我们研究计划的一个方面 重点介绍了蛋白质磷酸化在调节细胞周期中的作用 SV40病毒启动所需几种蛋白的活性 在无细胞系统中进行DNA复制。例如，磷酸化 细胞周期蛋白A依赖蛋白对DNA聚合酶α-启动酶的抑制作用 其SV40依赖于来源的复制活性。我们使用的是多肽 作图和MALDI-MS以定义相关的磷酸化位点， 然后在这些位置产生突变，并对突变体进行表征 病毒DNA复制中的蛋白质和最近设计的无细胞蛋白质 人类DNA复制系统。有没有可能 DNA聚合酶α-Primase的磷酸化状态对细胞的影响 体内的周期进程或基因组稳定性也处于 调查。我们计划很快解决的另一个未决问题是如何 细胞周期中Pola的去磷酸化受到控制。

Control of DNA replication is one of the key processes in the regulation of the mammalian cell cycle. Dysregulation of the cell cycle can arise at several stages, for example in the "decision" to begin DNA replication or in the proper completion of the S-phase, and is an important causative factor in a variety of human diseases, the most frequent being cancer. The long term goal of our research is to elucidate in molecular detail the mechanisms that control DNA replication in mammalian cells. Replication of papovaviral DNA in infected cells and in cell-free reactions has proven to be an extremely useful model system toward this end. It has facilitated the identification and characterization of many of the cellular proteins required for replication and has led to a basic understanding of the mechanism of SV40 DNA replication. One aspect of our research program focusses on the role of protein phosphorylation in regulating the activity of several proteins required for the initiation of SV40 viral DNA replication in a cell-free system. For example, phosphorylation of DNA polymerase alpha-primase by cyclin A-dependent kinases inhibits its SV40 origin-dependent replication activity. We are using peptide mapping and MALDI-MS to define the relevant phosphorylation sites, then creating mutations at these sites and characterizing the mutant proteins in viral DNA replication and in a recently devised cell-free human DNA replication system. The possibility that the phosphorylation state of DNA polymerase alpha-primase affects cell cycle progression or genomic stability in vivo is also under investigation. Another open question we plan to address soon is how dephosphorylation of pol a in the cell cycle is controlled.