LOCAL AND GLOBAL STRUCTURES OF SUPERCOILED DNA

描述：（申请人摘要中的逐字记录）DNA超螺旋作用于 在几乎所有的遗传过程中起着关键作用，但我们对遗传过程的理解 超螺旋DNA的结构与其在 生物学仍然相当有限。超螺旋的一个重要特征是， 它极大地增加了遥远的分离的相互作用的可能性， DNA片段。许多遗传事件需要蛋白质之间的联系， 到DNA上遥远的位点。例子包括DNA复制，基因控制 表达、位点特异性重组和其它基因组重排。 超螺旋DNA的另一个基本性质是负超螺旋 促进DNA的局部结构转变。某些短的DNA区域可以 采用不同的构象，在某些情况下，采用特定的酶或 调节蛋白靶向这些区域以发挥其调节作用。 这项赠款将测试的想法，地方和全球结构的 超螺旋DNA是连接的，使得局部结构转变可以具有 对超螺旋DNA的总体几何形状或形貌有显著影响。 有待检验的一个假设是，局部替代构象可以定义 DNA分子的形状可能会影响两个 沿着一个DNA分子。我们将测试形成的影响， 十字形，分子内三链体DNA（H-DNA）和左手Z-DNA， 质粒拓扑学我们还将确定是否有一个强大的蛋白质， 对替代结构的亲和力，包括来自人类的Z-α结构域 RNA编辑酶，参与改变的整体几何形状的 分子。此外，我们将测试一个新的生物学想法-也就是说，我们将 测试DNA的地形或位置定位 超螺旋内的序列可以调节DNA结构转变。这是 一个重要而新颖的概念，因为它提供了另一个层次的监管 DNA的二级结构转换，这显然涉及生物学 流程.这将通过将反向重复序列定位在顶端区域来确定。 或质粒中的非顶端位置。上述研究将利用 原子力显微镜（AFM），二维琼脂糖凝胶电泳，和 DNA和核蛋白结构研究的化学探针分析 配合物

Description:(Verbatim from the applicant's abstract) DNA supercoiling plays a critical role in virtually all genetic processes, yet our understanding of the relationship between the structure of supercoiled DNA and its specific roles in biology is still rather limited. One important feature of supercoiling is that it dramatically increases the probability of interaction of distantly separated DNA segments. Many genetic events require communication between proteins bound to distant sites on DNA. Examples include DNA replication, control of gene expression, site-specific recombination and other genome rearrangements. Another fundamental property of supercoiled DNA is that negative supercoiling facilitates local structural transitions in DNA. Certain short DNA regions can adopt alternative conformations and, in some cases specific enzymes or regulatory proteins target these regions to exert their regulatory effects. This grant will test the idea that the local and global structures of supercoiled DNA are linked, such that local structural transitions can have a significant impact on the overall geometry or topography of supercoiled DNA. One hypothesis to be tested is that local alternative conformations can define the shape of DNA molecule in a way that may effect the juxtaposition of two sites along a DNA molecule. We will test the influence of the formation of cruciforms, intramolecular triplex DNA (H-DNA), and left-handed Z-DNA on plasmid topography. We will also determine if proteins that have a strong affinity for alternative structures, including the Z-alpha domain from a human RNA editing enzyme, participate in changing of the overall geometry of the molecule. In addition, we will test a new biological idea - that is, we will test the hypothesis that the topographical or positional localization of a DNA sequence within a superhelix can modulate DNA structural transitions. This is an important and novel concept in that it provides another level of regulation of DNA secondary structural transitions that are clearly involved in biological processes. This will be determined by positioning inverted repeats at an apical or non-apical position in a plasmid. The studies outlined above will utilize atomic force microscopy (AFM), 2-dimensional agarose gel electrophoresis, and chemical probe analysis for structural studies of DNA and nucleoprotein complexes.