PHOSPHORYLATION OF DNA POLYMERASE ? PRIMASE

DNA复制的控制是基因工程的关键过程之一。 调节哺乳动物细胞周期。 细胞失调 循环可以出现在几个阶段，例如在“决定”， 开始DNA复制或在S期的适当完成，和 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 详细阐明控制DNA的分子机制 在哺乳动物细胞中复制。 乳多空病毒DNA在大肠杆菌中的复制 感染的细胞和无细胞反应已被证明是一种 这是一个非常有用的模型系统。 它促进了 许多细胞蛋白的鉴定和表征 复制所需的，并导致了对 SV 40 DNA复制机制 我们研究计划的一个方面 重点是蛋白磷酸化在调节 启动SV 40病毒所需的几种蛋白质的活性 无细胞系统中的DNA复制 例如磷酸化 细胞周期蛋白A依赖性激酶抑制DNA聚合酶α-引发酶 其SV 40来源依赖性复制活性。 我们用肽 作图和MALDI-MS以确定相关的磷酸化位点， 然后在这些位点上制造突变， 蛋白质在病毒DNA复制和最近设计的无细胞 人类DNA复制系统。 的可能性 DNA聚合酶α-引物酶的磷酸化状态影响细胞 体内的周期进展或基因组稳定性也受到限制。 调查 我们计划很快解决的另一个悬而未决的问题是， 控制细胞周期中PolA的去磷酸化。

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.