Physical Chemistry of Recombinational intermediates

重组是所有物种的普遍过程，它提供了适应不断变化的环境所必需的灵活性。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Holliday结，这是一个四链分支中间体。在Mg2+存在的情况下，Holliday结形成一个堆叠的x型结构，而在Mg2+不存在的情况下，Holliday结的结构是开放的；这两种结构似乎都与细胞过程有关。我们建议研究单个的Holliday结和由几个Holliday结组成的复杂母题，以确定结构的特征，其转换和分辨率。我们还将使用holliday衍生结构表征蛋白质-核酸相互作用。使用由四个Holliday结点组成的平行四边形作为表征Holliday结点的工具：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来制作未扭曲的对称不动结点，固定它们的分支点；该系统将使我们能够表征热力学和分辨率的连接，否则移动同源序列。平行四边形也为旋转角度的机械潜力提供了一个窗口。我们将通过同步加速器产生的羟基自由基来跟踪开放和堆叠结构之间的转变。我们计划使用结扎闭合实验来定量表征Holliday结和双交叉分子的灵活性，这是它们功能的关键因素。作为一种新的基序，偏位交叉DNA具有可能在同源重组中作为同源识别的中间体的特性。我们将确定它的形成是否可以由超卷曲驱动，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。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，并对涉及基因重组的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.