STRUCTURE AND SYNTHESIS OF DNA

DNA 的结构和合成

• 批准号: 2174945
• 负责人: PAUL T ENGLUND
• 金额: $ 43.83万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-01-01 至 1994-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2174945
• 关键词: Crithidia; DNA directed DNA polymerase; DNA replication; DNA replication origin; Mastigophora; Trypanosoma; affinity chromatography; carbon; cell components; circular DNA; density gradient ultracentrifugation; electron microscopy; gel electrophoresis; laboratory rat; microorganism genetics; mitochondrial DNA; nucleic acid sequence; nucleic acid structure; phosphorus; radionuclides; radiotracer

The long term objectives of this project are to investigate the structure and mechanism of replication of kinetoplast DNA (kDNA) in trypanosomatids. Kinetoplast DNA is a network containing thousands of topologically interlocked DNA minicircles. Minicircle replication occurs after release of the circles from the network. The following projects will be initiated. First, minicircle replication will be established in vitro. The substrate will be a plasmid containing a minicircle replication origin, and proteins will be provided by a mitochondrial extract. Second, proteins involved in kDNA replication will be purified and studied. Purification will rely heavily on affinity techniques using monoclonal antibodies, and these antibodies will also be used to immunolocalize the proteins and to study their mechanism of action. Third, using the in vitro system and purified proteins, minicircle sequences important to replication will be studied. These experiments will involve in vitro replication or binding of proteins with normal or mutated minicircle sequences. Fourth, topology of kDNA networks will be investigated, in hopes of determining their pattern of organization. These studies will involve characterization of minicircle oligomers released from networks by random minicircle cleavage. Finally, the structure of networks in vivo will be investigated using high resolution fluorescence microscopic techniques that allow three dimensional image reconstruction. These studies should allow determination of changes in network structure which occur during replication, and, using in situ hybridization, the localization of the minicircle bent helix. Overall, these studies should provide basic knowledge of DNA structure and replication; in addition, since humans contain no DNA resembling a kDNA network, they may suggest novel modes of anti-parasite chemotherapy.

该项目的长期目标是调查结构 和锥虫中动力学DNA（kDNA）复制的机理。 Kinetoplast DNA是一个包含数千个拓扑的网络 互锁的DNA小环。 释放后发生微圆复制 网络中的圆圈。 将启动以下项目。 首先，将在体外建立微圆复制。 基板 将是包含微圆复制起源的质粒，蛋白质 线粒体提取物将提供。 第二，蛋白质参与 KDNA复制将被纯化和研究。 纯化将依靠 使用单克隆抗体对亲和力技术进行严重的亲和力技术，这些技术 抗体也将用于免疫蛋白质并研究 他们的作用机理。 第三，使用体外系统并纯化 将研究蛋白质，小圆序列对复制很重要的序列。 这些实验将涉及体外复制或蛋白质结合 带有正常或突变的微角序列。 第四，kDNA的拓扑 将研究网络，以期确定其模式 组织。 这些研究将涉及微圆的表征 通过随机微孔裂解从网络释放的低聚物。 最后， 体内网络的结构将使用高 分辨率荧光显微镜技术允许三维 图像重建。 这些研究应允许确定变化 在复制过程中发生的网络结构中，并使用原位 杂交，小圆弯螺旋的定位。 全面的， 这些研究应提供DNA结构的基本知识和 复制；另外，由于人类没有类似于kDNA的DNA 网络，他们可能建议抗寄生虫化学疗法的新型模式。