EUCARYOTIC DNA-DEPENDENT ATPASES

这项提案描述了识别参与蛋白质的实验。 真核核酸代谢，纯化它们，并阐明它们 功能。最初的实验旨在确定DNA的作用- 核酸代谢中的依赖ATPase。单抗是一种 识别至少两种不同形式的小牛胸腺，依赖于DNA ATPase将被用来定义该酶的完整性。这两种形式的 该酶识别引物-模板连接(DNA聚合酶底物) 作为ATP水解酶的效应器。因此，这种酶有望 在DNA复制中发挥作用。建议的实验目的是：1) 利用单抗建立一种纯化人胸腺病毒的方法 完整的依赖DNA的ATPase。2)定义该ATPase的关系 到其他层析上不同的DNA依赖的ATPase。3)定义 通过使用单抗这种酶在细胞过程中的作用 渗透性细胞系统中的抗体及其在细胞周期中的应用 同步。4)识别额外的蛋白质，与 酶(S)相互作用。 虽然在研究真核DNA方面存在相当大的困难 结合蛋白及其在核酸新陈代谢中的作用， 这个项目将使用最新的技术来完成它的 目标。这些技术包括克隆的生产和使用。 抗体、免疫亲和层析和特殊亲和层析，以及 有效“冻结”的紫外光激光交联技术 蛋白质-核酸与五纳秒的光脉冲相互作用。 了解特定的蛋白质-核酸相互作用将有助于我们 在更好地理解DNA复制等过程中，可能会给我们 了解正常系统在哪里出错，导致各种 遗传性疾病，包括不能怀孕，出生缺陷，以及 各种癌症。从识别特定对象中收集的信息 蛋白质-核酸相互作用可能是药物设计的关键 来预防或控制某些疾病状态。

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.