STRUCTURE AND REPLICATION OF DNA

DNA 的结构和复制

• 批准号: 2020657
• 负责人: ROSS B INMAN
• 金额: $ 20.05万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-01-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2020657
• 关键词: DNA binding protein; DNA replication; DNA replication origin; Reoviridae; electron microscopy; genetic recombination; human immunodeficiency virus 1; integrase; nuclease; nucleic acid structure; tissue /cell culture; transcription factor; virus protein

DESCRIPTION: The proposed research is aimed broadly to contribute to an understanding of DNA function by application of electron microscopic methodology (EM) in the areas of recombination, replication and virus structure. Details of RecA promoted strand exchange (SE) and the circumstances preventing or stimulating RecA action will be considered with particular reference to: (i) how RecF, RecR, RecO, RuvA, RuvB and SSB modulate the action of RecA in homologous recombination and recombinational repair by EM analysis of in vitro experiments designed to test specific models for this process; (ii) the existence of triple stranded structures during RecA-mediated SE; (iii) the actual changes that can be observed and analyzed by EM correlating with the two successive ATP-independent and ATP-dependent phases of SE; (iv) the positions along the RecA filament (random, at ends, in a wave) of the association-dissociations of RecA; (v) whether SSB is present within active RecA filaments; and (vi) the translocation ability of RuvB and mode of influence by other factors. In another project, control of initiation at the gamma R6K plasmid origin will be examined. Unwinding at the AT-rich region by pr protein and six hyperactive mutants and the effect of super helical tension and DnaA or Integration Host Factor will be studied applying methods of EM, S1 nuclease sensitivity and radioactive incorporation. This will require standardization and simplification of several features of the in vitro R6K system. With gold labeled antibodies and EM, individual specific proteins will be identified in initiation complexes at the gamma origin. A third project involves HIV-1 Integrase. An as yet unidentified donor-target recombinant structure produced by integrase (IN) activity isolated by non-ionic detergent from HIV-1 virions will be studied. The structure has been identified by gel mobility and may be a potential intermediate in the concerted reaction. Gold labeled HIV-1 IN will be used to help determine if the complex has a different structure under low and high salt assay conditions which might help explain the specific deletions that are found in integration products produced under low salt conditions. Reovirus structure is the subject of the last aim. Work will continue from the demonstration that the 3' ends of a least 7 of the 10 RNA segments of reovirus RNA are in intimate contact with viral protein. The proteins involved in this binding or contact will be identified. Structural details of the transcription event that takes place with the virus will be examined, and very old observations that the reovirus RNA genome need not be segmented under certain conditions will be reexamined. The use of a cap-binding protein that has been recently isolated will be explored.

描述：拟议的研究旨在广泛地促进 应用电子显微镜了解DNA的功能 重组、复制和病毒领域的EM方法 结构 RecA促进链交换（SE）的详细信息和情况 预防或刺激RecA作用将被特别考虑， （i）RecF、RecR、RecO、RuvA、RuvB和SSB如何调节 RecA在EM同源重组和重组修复中的作用 设计用于测试特定模型的体外实验分析 （二）三股结构的存在， RecA介导的SE;（iii）可以观察和分析的实际变化 通过与两个连续的ATP非依赖性和ATP依赖性相关的EM SE的相位;（iv）沿着RecA细丝的沿着位置（随机，在末端， （v）是否SSB是 存在于活性RecA细丝内;和（vi） RuvB和其他因素的影响模式。 在另一个项目中，在γ R6 K质粒起点控制起始 将被审查。 pr蛋白在AT富集区解旋， 超活性突变体和超螺旋张力和DnaA或 将应用EM、S1核酸酶方法研究整合宿主因子 灵敏度和放射性掺入。 这将需要 体外R6 K的几个特征的标准化和简化 系统 用金标抗体和EM， 将在γ起源处的起始复合物中被识别。 第三个项目涉及HIV-1整合酶。 一个身份不明的 整合酶（IN）活性产生的供体-靶重组结构 将研究通过非离子去污剂从HIV-1病毒体中分离的病毒。 的 结构已确定的凝胶流动性，并可能是一个潜在的 协调反应的中间体。 将使用金标HIV-1 IN 以帮助确定复合物是否在低和低温度下具有不同的结构， 可能有助于解释特定缺失的高盐测定条件 在低盐条件下生产的集成产品中发现。 呼肠孤病毒的结构是本课题的最后一个目标。 工作将继续从 证明了10个RNA片段中至少7个的3'端是由RNA片段的3'端连接的。 呼肠孤病毒RNA与病毒蛋白紧密接触。 的蛋白质 参与这种绑定或接触将被识别。 结构细节 将检查病毒发生的转录事件， 以及呼肠孤病毒RNA基因组不需要分段的古老观察 在一定的条件下，将被重新审查。 一个瓶盖的使用 将探索最近分离的蛋白质。