Physical Chemistry of Recombinational intermediates

进化是所有物种的一个普遍过程，它为适应不断变化的环境提供了必要的灵活性。 我们试图构建和表征模拟重组结构的合成核酸模型系统，特别是霍利迪连接，这是一个四链分支的中间体。 在Mg 2+存在下，霍利迪连接形成堆叠的X结构，在其不存在下，其结构是开放的;这两种结构似乎与细胞过程有关。 我们建议检查两个单独的霍利迪路口和复杂的图案组成的几个霍利迪路口，以确定功能的结构，其转换和分辨率。 我们还将使用霍利迪衍生的构建体表征蛋白质-核酸相互作用。具体目标是：[1]使用由四个霍利迪结制成的平行四边形作为表征霍利迪结的工具：我们的目标是检查堆叠X结构中螺旋之间的旋转角度。 我们将使用一个基于平行四边形的系统，使不失真的对称固定路口，固定其分支点，这个系统将使我们能够表征热力学和分辨率的路口，否则移动的同源序列。 投影图还提供了一个关于旋转角度的机械潜力的窗口。[2]我们将通过同步加速器产生的羟基自由基跟踪开放和堆叠结构之间的转变。[3]我们计划使用连接封闭实验来定量表征霍利迪路口和双交叉分子，在其功能的关键因素的灵活性。[4]一个新的模体，paranemic交叉DNA的性质，表明它可能作为一个中间体在同源重组的同源识别。 我们将确定它的形成是否可以由超螺旋驱动，我们将使用分子作为一个系统来测量平行交叉结构和两个平行并置螺旋之间的自由能差。[5]我们将使用Holliday衍生的系统来测试SoxR调节蛋白与DNA结合时的解旋。 这些研究旨在测试关键假设，并对涉及遗传重组的DNA结构和过程进行中心观察。

Recombination is a universal process in all species that affords the flexibility necessary for adaptation to a changing environment. We seek to construct and characterize synthetic nucleic acid model systems that mimic structures involved in recombination, particularly the Holliday junction, which is a four-stranded branched intermediate. In the presence of Mg2+, the Holliday junction forms a stacked-X structure, and in its absence its structure is open; both structures appear relevant to cellular processes. We propose to examine both the individual Holliday junction and complex motifs composed of several Holliday junctions to determine features of the structure, its transformations and its resolution. We will also characterize a protein-nucleic acid interaction using a Holliday-derived construct. The specific aims are: [1] To use parallelograms made from four Holliday junctions as tools to characterize Holliday junctions: We aim to examine the swivel angles between the helices in the stacked-X structure. We will use a parallelogram-based system to make undistorted symmetric immobile junctions, that fix their branch points; this system will enable us to characterize both the thermodynamics and resolution of junctions with otherwise mobile homologous sequences. Parallelograms also offer a window on the mechanical potential of the swivel angles. [2] We will follow the transition between the open and stacked structures by means of synchrotron-generated hydroxyl radicals. [3] We plan to use ligation-closure experiments to characterize quantitatively the flexibility of Holliday junctions and double crossover molecules, a key factor in their functioning. [4] A new motif, paranemic crossover DNA has properties that suggest it may act as an intermediate in homology recognition in homologous recombination. We will establish whether its formation can be driven by supercoiling, and we will use the molecule as a system to measure the free energy difference between a parallel crossover structure and two parallel juxtaposed helices. [5] We will use a Holliday-derived system to test the unwinding of SoxR regulatory protein when it binds to DNA. These studies are designed to test key hypotheses and make central observations on DNA structures and processes that are involved in genetic recombination.