STRUCTURE AND REPLICATION OF DNA

描述：拟议的研究旨在广泛地促进 应用电子显微镜认识DNA的功能 重组、复制和病毒领域的方法学(EM) 结构。 RecA促进的链交换(SE)的细节和情况 防止或刺激RecA行动将被特别考虑 参考：(I)recf、recr、reco、Ruva、RuvB和SSB如何调制 电子显微镜观察RecA在同源重组和重组修复中的作用 为测试特定模型而设计的体外实验分析 过程；(2)过程中存在三链结构 RecA介导的SE；(Iii)可以观察和分析的实际变化 通过EM与两个连续的ATP非依赖性和依赖性相关 SE的阶段；(Iv)沿着RecA细丝的位置(随机的，在末端， 在一波中)RecA的缔合-解离； 存在于活跃的RecA细丝中；以及(Vi) RuvB和受其他因素影响的方式。 在另一个项目中，对伽马R6K质粒源的起始进行控制 将会被检查。Pr蛋白和Six在AT富集区的解离 超活性突变体及其对超螺旋张力和DNAA的影响 应用EM、S1核酸酶等方法研究整合宿主因子 敏感度和放射性掺入。这将需要 体外R6K的几个特性的标准化和简化 系统。用金标抗体和EM，个别特异性蛋白 将在伽马起源的起爆复合体中确定。 第三个项目涉及HIV-1整合酶。一个尚未确定身份的人 整合酶(IN)活性产生的供体-靶向重组结构 将研究用非离子洗涤剂从HIV-1病毒粒子中分离出来。这个 结构已经通过凝胶的迁移率确定，并可能是一种潜在的 协同反应的中间体。将使用金色标记的HIV-1 IN 以帮助确定在低和低的情况下，复合体是否具有不同的结构 可能有助于解释特定缺失的高盐检测条件 在低盐条件下生产的一体化产品中发现的。 呼肠孤病毒结构是最终目标的主题。工作将继续从 证明了10个RNA片段中至少有7个的3‘末端 呼肠孤病毒RNA与病毒蛋白密切接触。这些蛋白质 参与这一绑定或联系的人将被确定。结构细节 将对与病毒发生的转录事件进行检查， 以及非常古老的观察结果，即呼肠孤病毒RNA基因组不需要被分段 在某些情况下将被重新审查。帽子装订的使用 将探索最近分离出来的蛋白质。

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.