ROLE OF MAMMALIAN HELICASES IN DNA REPLICATION

这些研究的长期目标是描述， 哺乳动物DNA复制及其调控 细胞 一种无细胞复制系统， 具有病毒复制起点的外源DNA模板已经被 在这个实验室里开发的， 参与DNA复制的生物化学方法 例如分馏和重构实验。 已知DNA解旋酶在复制中起核心作用 原核和病毒基因组，因此可以预期， 在哺乳动物的复制中同样重要。 我们将尝试 为了纯化和表征涉及以下的细胞解旋酶： 利用获得的资源和技术专长进行复制 这个实验室在研究我们的无细胞 复制系统 最初的分馏步骤将使用 色谱柱，证明是有用的分离， 迄今为止发现的复制蛋白。 后续步骤将 包括亲和柱如DNA-纤维素和ATP-琼脂糖， 将部分基于已发表的净化经验， 小牛胸腺和鼠FM 3A细胞的解旋酶。 解旋酶在SV 40复制中的作用将由以下定义： 测定复制反应的重建， 纯化的蛋白质。 将研究其作用机制 通过定义底物特异性和与其它底物的相互作用， 在各种各样的复制反应的纯化组分中， 分析，如来源依赖性解旋，减少时滞 的引发，对动力学和持续合成能力的影响， 延伸、易位的极性和 拓扑异构酶诱导的连接数的变化。 单克隆 抗体将被提高，允许免疫亲和层析 以便于纯化。 他们将允许调查细胞 周期调节和亚细胞定位作为间接手段， 定义了细胞解旋酶的作用。 如果有足够的量 纯蛋白质的制备、蛋白质测序和解旋酶基因的克隆 将尝试。 哺乳动物细胞复制机制的阐明 将导致更好地了解其监管，并提供 深入了解负责生成的根本变化 和维持瘤形成。 这也可以提供工具， 制定新的和更具体的战略所需的目标 剂.

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.