DNA STRUCTURE IN RECOMBINATION BY THE YEAST ENZYME FLP

DNA的定点重组是许多生物学中的基本事件 涉及基因组重排的过程。目前，有很大的 对特性良好的特定部位重组系统感兴趣，这些重组系统可以 用于将基因靶向于真核生物染色体上的特定位置 活着。因此，重要的是要了解物理和化学 控制效率、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 利用链球菌FLP重组酶进行定点重组。 酿酒。FLP是唯一一种具有 在任何可察觉的程度和许多方面都被描述为 FLP与其靶位的相互作用尚不清楚。 这项建议侧重于DNA二级结构和三级结构的作用 在重组反应中和重组位序列方面 它们对于功能组件的组装和组织非常重要 重组酶-DNA复合体。重组酶-DNA突触的结构 复合体(参与位点的中间核蛋白中间体 配对和链交换)将通过结合物理 以及生化方法。旋转扩散技术，它测量 溶液中大分子整体自转运动的速率，将 用于确定突触复合体中DNA的几何形状。这 信息I将与拓扑学和螺旋学的结果相结合 为获得二级和三级结构模型而进行的阶段性研究 突触复合体中的DNA。从这些研究中得到的几何学 将与预测的四链霍利迪进行比较 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 将被用来解释某些重组位点突变的影响 它们对速度和效率有明显和令人费解的影响 重组。

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.