AP4A IN CELL PROLIFERATION, DNA REPLICATION AND REPAIR

对DNA复制和细胞增殖的调控是一种 生物体生命中最基本的过程。损失 细胞对正常调节机制的反应导致 癌细胞的癌性生长。真核生物DNA复制的调控 是一个涉及许多蛋白质-蛋白质和蛋白质-DNA的复杂过程 在不同的点上的互动。其中包括细胞的承诺 进入细胞周期的S阶段，这对一个数字很敏感 对环境的影响，如生长刺激因子和 添加血清。这样的刺激会导致多型性反应 导致DNA复制的事件。细胞的一种这样的反应是 二核苷酸，二腺苷的水平显著增加 四磷酸盐，Ap4A。Ap4A参与多种细胞事件，如 作为DNA复制和细胞增殖，DNA修复和血小板 聚集和血管紧张度。一种特定结合于 Ap4A已在各种原核和真核细胞中检测到，并 我们已经发现，在HeLa细胞中，这种结合蛋白是一个 能够复制SV40的多蛋白DNA聚合酶复合体 DNA在体外。作为我们理解的长期目标的一部分 在分子水平上调控真核生物的DNA复制 已经提出研究Ap4A结合的生理作用 在这项提案中，DNA复制过程中的蛋白质。在持续时间内 在这个项目中，我们将把结合蛋白提纯到同质性， 结合蛋白的物理和生化特征 性质，确定该蛋白在SV40体外DNA中的作用 细胞内Ap4A和结合蛋白的复制与能力 启动DNA复制。我们将测定氨基酸 结合蛋白的组成和序列分析及多克隆的产生 以及针对该蛋白质的克隆抗体。利用这些试剂，我们 将研究这种蛋白在不同阶段的表达情况 细胞周期和这种蛋白质在细胞内的位置。利用 在氨基酸序列数据中，我们将生成寡核苷酸探针 并克隆了结合蛋白的全长c DNA。在接下来的时间里 项目中，我们将进行实验，以确定 这种结合蛋白存在于Ap4A调节的各种细胞事件中。

The regulatory control of DNA replication and cell proliferation is one of the most fundamental processes in the life of an organism. Loss of cells's responsiveness to normal mechanisms of regulation results in cancerous growth of cells. The regulation of eukaryotic DNA replication is a complex process involving many protein-protein and protein-DNA interactions at various points. Among these are the commitment of cells to enter the S phase of the cell cycle, which is sensitive to a number of environmental influences such as the growth-stimulating factors and addition of serum. Such stimulation results in a pleiotypic response of events leading to DNA replication. One such response of cells is a marked increase in the level of the dinucleotide, diadenosine tetraphosphate, Ap4A. Ap4A is involved in multiple cellular events such as DNA replication and cell proliferation, DNA repair and platelet aggregation and vascular tonus. A protein that specifically binds to Ap4A has been detected in various prokaryotic and eukaryotic cells and we have found that in HeLa cells, this binding protein is part of a multiprotein DNA polymerase complex that is capable of replicating SV40 DNA in vitro. As part of our long-term goal of understanding the regulation of DNA replication in eukaryotes at the molecular level, we have proposed to study the physiological role of the Ap4A binding protein during DNA replication in this proposal. In the duration of this project, we will purify the binding protein to homogeneity, characterize the binding protein regarding its physical and biochemical properties, determine the role of this protein in SV40 in vitro DNA replication and the ability of Ap4A and the binding protein in initiating DNA replication. We will determine the amino acid composition and sequence of the binding protein and generate polyclonal and monoclonal antibodies to this protein. Utilizing these reagents, we will study the expression of this protein during various phases of the cell cycle and the intracellular location of this protein. Making use of the amino acid sequence data, we will generate oligonucleotide probes and clone the cDNA for the binding protein. In continuation this project, we will perform experiments to define the pbysiological role of this binding protein in the various cellular events that Ap4A modulates.