EUCARYOTIC DNA-DEPENDENT ATPASES

真核DNA依赖的ATP酶

• 批准号: 3468012
• 负责人: Joel W Hockensmith
• 金额: $ 11.42万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-12-01 至 1994-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3468012
• 关键词: DNA; DNA binding protein; DNA directed DNA polymerase; DNA replication; adenosinetriphosphatase; affinity chromatography; chemical binding; conformation; cow; crosslink; enzyme mechanism; lasers; monoclonal antibody; nucleic acid metabolism; protein purification; protein sequence; synchronous cell division; thymus; tissue /cell culture; ultraviolet radiation; western blottings

This proposal describes experiments to identify proteins involved in eucaryotic nucleic acid metabolism, to purify them, and to elucidate their function. Initial experiments are aimed at identifying the role of DNA- dependent ATPases in nucleic acid metabolism. Monoclonal antibodies that recognize at least two distinct forms of a calf thymus, DNA-dependent ATPase will be used to define the integrity of that enzyme. Both forms of the enzyme recognize primer-template junctions (a DNA polymerase substrate) as effectors for ATP hydrolysis. Consequently, this enzyme is expected to play a role in DNA replication. The proposed experiments are aimed at: 1) using the monoclonal antibodies to develop a method of purification for the intact DNA-dependent ATPase. 2) Defining the relationship of this ATPase to other chromatographically distinct DNA-dependent ATPases. 3) Defining the role of this enzyme in cellular processes through the use of monoclonal antibodies in permeabilized cell systems and using cell cycle synchronization. 4) Identifying additional proteins with which the enzyme(s) interacts. While there has been considerable difficulty in the study of eucaryotic DNA binding proteins and their role in nucleic acid metabolism in the past, this project will use the latest technologies in order to accomplish its goals. These technologies include production and use of monoclonal antibodies, immunoaffinity and specialized affinity chromatography, and an ultraviolet laser cross-linking technique which effectively "freezes" protein-nucleic acid interactions with a five nanosecond pulse of light. An understanding of specific protein-nucleic acid interactions will aid us in better understanding processes such as DNA replication and may give us an understanding of where normal systems go awry resulting in various genetic disorders, including inability to conceive, birth defects, and various cancers. The information gleaned from identification of specific protein-nucleic acid interactions may be critical for the design of drugs that prevent or control certain disease states.

该提案描述了鉴定参与蛋白质的实验， 真核核酸代谢，纯化它们，并阐明它们的 功能 最初的实验旨在确定DNA的作用- 依赖ATP酶的核酸代谢。 的单克隆抗体 识别至少两种不同形式的小牛胸腺，DNA依赖性 ATP酶将用于确定该酶的完整性。 两种形式的 该酶识别引物-模板连接（DNA聚合酶底物） 作为ATP水解的效应物。 因此，这种酶预计将 在DNA复制中发挥作用。 实验的目的是：1） 利用单克隆抗体开发了纯化方法 完整的DNA依赖性ATP酶。 2)定义这种ATP酶的关系 与其他色谱上不同的DNA依赖性ATP酶。 3)限定 这种酶在细胞过程中的作用，通过使用单克隆 透化细胞系统中的抗体和使用细胞周期 同步 4)鉴定与细胞凋亡相关的其他蛋白质 酶相互作用。 虽然在真核DNA的研究中存在相当大的困难， 结合蛋白及其在核酸代谢中的作用， 该项目将使用最新的技术，以实现其 目标. 这些技术包括单克隆抗体的生产和使用。 抗体、免疫亲和和专门的亲和层析，以及 紫外激光交联技术，有效地“冻结” 蛋白质-核酸相互作用与5纳秒的光脉冲。 对特定蛋白质-核酸相互作用的理解将有助于我们 更好地理解DNA复制等过程， 了解正常系统在哪里出错，导致各种 遗传性疾病，包括无法怀孕，出生缺陷， 各种癌症。 从特定的识别中收集的信息 蛋白质-核酸相互作用对于药物的设计可能是至关重要的 来预防或控制某些疾病