ROLE OF MAMMALIAN HELICASES IN DNA REPLICATION

哺乳动物解旋酶在 DNA 复制中的作用

基本信息

批准号：
3085841
负责人：
CHRISTOPHER B UMBRICHT
金额：
$ 7.42万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1989
资助国家：
美国
起止时间：
1989-07-12 至 1994-06-30
项目状态：
已结题

项目摘要

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.

这些研究的长期目标是描述分子水平、哺乳动物DNA复制及其调控细胞。能够复制的无细胞复制系统具有病毒复制起点的外源 DNA 模板该实验室开发的，允许哺乳动物蛋白质参与DNA复制，可通过生化方法鉴定例如分馏和重构实验。 DNA 解旋酶已知在复制中发挥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。我们将尝试纯化和表征参与的细胞解旋酶使用所获得的资源和技术专长进行复制由该实验室在我们的无细胞研究中复制系统。初始分馏步骤将使用经证明可用于分离的色谱柱迄今为止已鉴定的复制蛋白。后续步骤将包括亲和柱，例如 DNA-纤维素和 ATP-琼脂糖，以及将部分基于已发表的纯化经验来自小牛胸腺和鼠 FM3A 细胞的解旋酶。解旋酶在 SV40 复制中的作用定义为测定复制反应的重建纯化的蛋白质。他们的作用机制将被研究通过定义底物特异性和与其他物质的相互作用多种复制反应的纯化组分测定，例如起源依赖性展开、减少时间滞后的引发、对动力学和持续反应性的影响伸长、易位极性和调制拓扑异构酶诱导连接数的变化。单克隆将产生抗体，从而进行免疫亲和层析以利于纯化。他们将允许对细胞进行研究周期调节和亚细胞定位作为间接手段定义细胞解旋酶的作用。给予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进行尝试。阐明哺乳动物细胞的复制机制将有助于更好地了解其监管并提供洞察导致发电的基本变化和瘤形成的维持。这也可能提供工具和设计新的、更具体的抗肿瘤药物所需的目标代理。