DNA STRUCTURE IN RECOMBINATION BY THE YEAST ENZYME FLP

酵母酶 FLP 重组的 DNA 结构

• 批准号: 2185311
• 负责人: STEPHEN D. LEVENE
• 金额: $ 10.49万
• 依托单位: UNIVERSITY OF TEXAS DALLAS
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2185311
• 关键词: DNA binding protein; Saccharomyces cerevisiae; chemical cleavage; dichroism; electron microscopy; fungal genetics; gene rearrangement; molecular cloning; nucleic acid structure; recombinase; synapses

Site-specific recombination of DNA is an essential event in many biological processes that involve genomic rearrangements. Currently, there is great interest in well-characterized site-specific recombination systems that can be used to target genes to specific locations on eukaryotic chromosomes in vivo. It is therefore important to understand the physical and chemical factors that govern the efficiency, product distribution, and target specificity of recombination. The overall goal of the proposed program is to understand the role of DNA structure in site-specific recombination by the FLP recombinase of S. cerevisiae. FLP is the only eukaryotic site-specific recombinase that has been characterized to any appreciable extent and many aspects of the interaction of FLP with its target site are unknown. This proposal focuses on the role of DNA secondary and tertiary structure in the recombination reaction and on aspects of recombination site sequence that are important for the assembly and organization of functional recombinase-DNA complexes. The structure of the recombinase-DNA synaptic complex (the intermediate nucleo-protein intermediate involved in site pairing and strand exchange) will be studied by a combination of physical and biochemical approaches. Rotational diffusion techniques, which measure the rate of overall rotational motion of macromolecules in solution, will be applied to determine the geometry of DNA in the synaptic complex. This information I will be combined with results from topological and helical phasing studies to obtain a model for the secondary and tertiary structure of DNA in the synaptic complex. The geometry obtained from these studies will be compared to that predicted for four-stranded Holliday intermediates, proposed to be a key intermediate in FLP recombination. Knowledge of the detailed structure of DNA in the synaptic intermediate will be used to explain the effects of certain recombination site mutations that have pronounced and puzzling effects on the rate and efficiency of recombination.

DNA的位点特异性重组是许多生物学过程中必不可少的事件。 涉及基因组重排的过程。 目前， 对充分表征位点特异性重组系统感兴趣，所述系统可以 用于将基因靶向真核生物染色体上的特定位置， vivo. 因此，重要的是要了解物理和化学 决定效率、产品分布和目标的因素 重组的特异性。 该计划的总体目标是了解DNA的作用 S.的FLP重组酶位点特异性重组中的结构。 啤酒。 FLP是唯一的真核生物位点特异性重组酶， 在任何明显的程度和许多方面的特点， FLP与其靶位点相互作用是未知的。 该建议侧重于DNA二级和三级结构的作用 在重组反应和重组位点序列方面 这对功能的组装和组织很重要， 重组酶-DNA复合物。 重组酶-DNA突触的结构 复合物（参与位点的中间核蛋白中间体） 配对和链交换）将通过物理 和生物化学方法。 旋转扩散技术， 溶液中大分子整体旋转运动速率将 可以应用于确定突触复合体中DNA的几何形状。 这 信息I将与拓扑和螺旋的结果相结合， 分阶段研究，以获得二级和三级结构的模型 突触复合体中的DNA。 从这些研究中获得的几何形状 将与四股霍利迪的预测进行比较 中间体，被认为是FLP重组的关键中间体。 了解突触中间体中DNA的详细结构 将用于解释某些重组位点突变的影响 这些因素对生物多样性的速率和效率产生了显著而令人困惑的影响， 重组