AP4A IN CELL PROLIFERATION, DNA REPLICATION AND REPAIR

AP4A 在细胞增殖、DNA 复制和修复中的作用

• 批准号: 3305890
• 负责人: JAMBOOR K. VISHWANATHA
• 金额: $ 8.88万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-06 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3305890
• 关键词: DNA binding protein; DNA repair; DNA replication; HeLa cells; adenosine triphosphate; enzyme linked immunosorbent assay; gene expression; human genetic material tag; immunocytochemistry; laboratory rabbit; molecular cloning; molecular genetics; monoclonal antibody; nucleic acid probes; protein sequence; protein structure function; radioimmunoassay

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.

DNA复制和细胞增殖的调节控制是其中之一。 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 在不同点上的互动。 其中包括细胞的定向 进入细胞周期的S期，该期对数量敏感 环境影响，如生长刺激因子， 添加血清。 这样的刺激导致一种多型反应， 导致DNA复制的事件。 细胞的一种反应是 二核苷酸二腺苷水平显著升高 四磷酸，Ap 4A. Ap 4A参与多种细胞事件，如 如DNA复制和细胞增殖，DNA修复和血小板 聚集和血管紧张。 一种特异性结合 Ap 4A已在各种原核和真核细胞中检测到， 我们已经发现，在HeLa细胞中，这种结合蛋白是 能够复制SV 40的多蛋白DNA聚合酶复合物 体外DNA。 作为我们长期目标的一部分 在分子水平上调节真核生物中的DNA复制，我们 已经提出研究Ap 4A结合的生理作用， DNA复制过程中的蛋白质。 的持续时间 在本项目中，我们将结合蛋白纯化至均一， 表征结合蛋白的物理和生物化学性质， 性质，确定该蛋白在SV 40体外DNA中的作用 Ap 4A和结合蛋白在细胞中的复制能力 启动DNA复制。 我们将确定氨基酸 结合蛋白的组成和序列，并产生多克隆 和针对该蛋白质的单克隆抗体。 利用这些试剂，我们 将研究这种蛋白质在不同阶段的表达， 细胞周期和该蛋白的细胞内定位。 利用 的氨基酸序列数据，我们将生成寡核苷酸探针 并克隆结合蛋白的cDNA。 在延续这一 项目，我们将进行实验，以确定pbysiological作用， 这种结合蛋白参与Ap 4A调节的各种细胞事件。