REGULATION OF INITIATION OF SV40 DNA REPLICATION

染色体DNA复制的启动很可能是至关重要的 控制点在维持细胞的正常增殖。 对涉及的机制和控制的重要见解 哺乳动物DNA合成来自一个模型系统的分析，该模型系统 复制乳头状病毒SV40的基因组。最近的研究表明 提示SV40DNA在体外复制的启动可能是 以由磷酸化状态调节的细胞周期的方式控制 病毒启动子蛋白中的SV40大T抗原。染色体 DNA复制可以类似地由细胞周期控制的启动 细胞启动蛋白的修饰。因此，蜂窝 T抗原磷酸化修饰可能是一种有用的模型 研究哺乳动物细胞中DNA复制的控制。 该项目旨在实现1)了解如何针对特定地点 T抗原去磷酸化刺激DNA复制的启动， 2)细胞周期调控因子的识别 修饰T抗原的磷酸化。细胞内的催化亚单位 磷酸蛋白磷酸酶2A(PP2Ac)对SV40早期的刺激作用 DNA复制并使有限数量的特定位点去磷酸化 在T抗原上。PP2Ac去除的关键磷酸盐将是 下定决心。去磷酸化的T抗原将被重新分离和检测 对于启动复制的活动，以确定是否 去磷酸化既是刺激的必要条件也是充分条件 活动。最近在T抗原上构建了点突变，缺乏 特定的磷酸化位点，也将被检测。去磷酸化- T抗原与碱基对相互作用的诱导变化 将评估SV40 DNA复制的最小来源。化验， 在复制条件下执行，将测量绑定中的更改 常数，T抗原自结合的变化，T抗原诱导 起源DNA结构的变化和T抗原解旋酶的变化 活动。T抗原与细胞蛋白相互作用的变化 参与细胞增殖的人将通过 免疫共沉淀实验。 PP2Ac刺激G1期细胞提取液中SV40 DNA复制 比S期细胞提取物的作用要大得多。这表明 存在一种细胞周期调节因子，它可以改变 T抗原的磷酸化状态。细胞因素(S)，最有可能 一种可逆地使T抗原失活的激酶将被鉴定出来，并 纯净的。T抗原磷酸酶和激酶的活性将是 通过细胞周期进行评估，以及调节这些因素的机制 将定义细胞周期中的活动。详细的分析 对SV40 DNA复制启动的控制可能会提供一些见解 对细胞DNA复制的控制，以及它在 癌细胞。

The initiation of chromosomal DNA replication is likely to be a vital control point in the maintenance of normal cell proliferation. Significant insights into the mechanisms and controls involved in mammalian DNA synthesis have derived from analysis of a model system, the replication of the genome of the papovavirus SV40. Recent studies have indicated that the initiation of SV40 DNA replication in vitro may be controlled in a cell cycle regulated manner by the phosphorylation state of the viral initiator protein, the SV40 large T antigen. Chromosomal DNA replication may similarly be initiated by the cell cycle controlled modification of cellular initiator proteins. Therefore, cellular modification of T antigen phosphorylation may be a useful model for studying the control of DNA replication in mammalian cells. This project aims to achieve 1) an understanding of how the site-specific dephosphorylation of T antigen stimulates initiation of DNA replication, and 2) the identification of the cell cycle regulated factors which modify T antigen phosphorylation. The catalytic subunit of the cellular phosphoprotein phosphatase 2A (PP2Ac) stimulates the early stages in SV40 DNA replication and dephosphorylates a limited number of specific sites on T antigen. The critical phosphates removed by PP2Ac will be determined. Dephosphorylated T antigen will be re-isolated and assayed for activity in initiation of replication, to determine if dephosphorylation is both necessary and sufficient for stimulation of activity. Recently constructed point mutants in T antigen, lacking the specific phosphorylation sites, will also be assayed. Dephosphorylation- induced changes in the interaction of T antigen with the 64 base pair minimal origin of SV40 DNA replication will be assessed. Assays, performed under replication conditions, will measure changes in binding constants, changes in T antigen self-association, T antigen-induced changes in origin DNA structure, and alterations in T antigen helicase activity. Alterations in T antigen interactions with cellular proteins involved in cellular proliferation will be assessed by co-immunoprecipitation experiments. PP2Ac stimulates SV40 DNA replication in extracts from Gl phase cells to a much greater extent than extracts from S phase cells. This suggests the existence of a cell cycle regulated factor which modifies the phosphorylation state of T antigen. The cellular factor(s), most likely a kinase, which reversibly inactivates T antigen will be identified and purified. Activities of the T antigen phosphatase and kinase will be assessed through the cell cycle, and the mechanism which modulates these activities through the cell cycle will be defined. A detailed analysis of the control of initiation of SV40 DNA replication may provide insights into the control of cellular DNA replication, and its abnormalities in cancerous cells.