ROLE OF MAMMALIAN HELICASES IN DNA REPLICATION

这些研究的长期目标是在 哺乳动物的分子水平、DNA复制及其调控 细胞。一种可复制的无细胞复制系统 具有病毒复制起点的外源DNA模板已被 在这个实验室里开发的，让哺乳动物的蛋白质 参与DNA复制将通过生化方法进行鉴定 例如分馏和重组实验。 众所周知，DNA解旋酶在复制过程中起着核心作用 原核生物和病毒基因组，因此可以预期 在哺乳动物复制中也同样重要。我们将尝试 细胞解旋酶(S)的纯化与鉴定 使用所获得的资源和技术专业知识进行复制 由这个实验室在我们的细胞自由调查中 复制系统。初始分馏步骤将使用 事实证明，色谱柱在分离 到目前为止已经确定了复制蛋白。后续步骤将 包括亲和柱，例如DNA-纤维素和ATP-琼脂糖，并 将在一定程度上基于已发表的净化经验 来自小牛胸腺和小鼠FM3A细胞的解旋酶。 解旋酶在SV40复制中的作用将由 用来检测复制反应的重组 纯化的蛋白质。他们的作用机制将被调查。 通过定义底物专一性和与其他 S品种复制反应的纯化组分 分析，如依赖原产地的解卷，减少时间延迟 引发，对动力学和过程的影响 易位的伸长、极性和调节 拓扑异构酶引起连接数的变化。单克隆 抗体将被提高，从而允许免疫亲和层析 以便于提纯。他们将允许对细胞进行调查 作为间接手段的周期调节和亚细胞定位 定义了细胞解旋酶的作用。如果有足够的数量 纯蛋白、蛋白质测序及解旋酶基因的克隆 将会被尝试。 哺乳动物细胞内复制机制的阐明 将导致对其监管的更好理解，并提供 洞察导致新一代人的根本变化 以及肿瘤的维持性。这也可以提供工具和 设计新的、更特异的抗肿瘤药物所需的靶点 探员们。

The long range goal of these studies is to described, at the molecular level, DNA replication and its regulation in mammalian cells. A cell free replication system capable of replicating exogenous DNA templates with a viral origin of replication has been developed in this laboratory, allowing the mammalian proteins involved in DNA replication to be identified by biochemical methods such as fractionation and reconstitution experiments. DNA helicases are known to play a central role in the replication of prokaryotic and viral genomes, and can therefor be expected to be similarly important in mammalian replication. We will attempt to purify and characterize the cellular helicase(s) involved in replication using the resources and technical expertise acquired by this laboratory in the investigation of our cell free replication system. The initial fractionation steps will use the chromatographic columns that proved useful in isolating the replication proteins identified so far. Subsequent steps will include affinity columns such as DNA-cellulose and ATP-agarose and will be based in part on published experience with the purification of helicases from calf thymus and murine FM3A cells. The role of helicases in SV40 replication will be defined by assaying for reconstitution of the replication reaction with purified proteins. Their mechanism of action will be investigated by defining substrate specificity and interactions with other purified components of the replication reaction in s variety of assays, such as origin dependent unwinding, reduction of time lag of initiation, influence on kinetics and processivity of elongation, polarity of translocation, and modulation of topoisomeraseinduced changes in linking number. Monoclonal antibodies will be raised, allowing immunoaffinity-chromatography to facilitate purification. They will allow investigation of cell cycle regulation and subcellular localization as indirect means of defining the role of cellular helicases. Given a sufficient amount of pure protein, protein sequencing and cloning of a helicase gene will be attempted. The elucidation of the mechanisms of replication in mammalian cells will lead to a better understanding of its regulation and provide insight into the fundamental alterations responsible for generation and maintenance of neoplasia. This may also provide the tools and targets necessary to design new and more specific antineoplastic agents.