REGULATION OF INITIATION OF SV40 DNA REPLICATION

染色体DNA复制的启动可能是一个至关重要的 控制点维持细胞正常增殖。 对参与的机制和控制的重要见解 哺乳动物的DNA合成来自于对模型系统的分析， 乳多空病毒SV 40基因组的复制。 最近的研究 表明SV 40 DNA在体外复制的起始可能是 以磷酸化状态调节的细胞周期方式控制 病毒起始蛋白SV 40大T抗原 染色体 类似地，DNA复制可以由细胞周期控制的 细胞起始蛋白的修饰。 因此，Cellular T抗原磷酸化的修饰可能是一个有用的模型， 研究哺乳动物细胞中DNA复制的控制。 该项目旨在实现1）了解如何网站的具体 T抗原的去磷酸化刺激DNA复制的起始， 以及2）鉴定细胞周期调节因子， 修饰T抗原磷酸化。 细胞的催化亚基 磷蛋白磷酸酶2A（PP 2Ac）刺激SV 40的早期阶段 DNA复制和去磷酸化有限数量的特定位点 T抗原 PP 2Ac去除的关键磷酸盐将是 测定将重新分离和测定脱磷酸化T抗原 复制启动时的活动，以确定是否 去磷酸化是刺激细胞增殖所必需的， 活动 最近构建的T抗原点突变体，缺乏 特异性磷酸化位点也将被分析。 去磷酸化- 诱导T抗原与64个碱基对相互作用的变化 将评估SV 40 DNA复制的最小起点。 分析， 在复制条件下执行，将测量绑定中的更改 常数，T抗原自身结合的变化，T抗原诱导的 原始DNA结构的变化和T抗原解旋酶的改变 活动 T抗原与细胞蛋白相互作用的改变 将通过以下方法评估参与细胞增殖的 免疫共沉淀实验。 PP 2Ac刺激来自G1期细胞的提取物中的SV 40 DNA复制， 比S期细胞的提取物更大的程度。 这表明 细胞周期调节因子的存在，其改变了 T抗原的磷酸化状态。 细胞因素，最有可能 将鉴定可逆地使T抗原失活的激酶， 提纯 T抗原磷酸酶和激酶的活性将被测定。 通过细胞周期评估，以及调节这些的机制 通过细胞周期的活动将被定义。 详细分析 对SV 40 DNA复制起始的控制可能提供一些见解， 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.