LOCAL AND GLOBAL STRUCTURES OF SUPERCOILED DNA

描述：(逐字摘自申请者的摘要)DNA超级卷曲发挥着 在几乎所有的遗传过程中扮演着关键的角色，然而我们对 超螺旋DNA的结构与其特殊作用的关系 生物学仍然相当有限。超级卷取的一个重要特征是 它极大地增加了相隔遥远的星球相互作用的可能性 DNA片段。许多遗传事件需要结合的蛋白质之间的通信 到DNA上遥远的位置。例如DNA复制、基因控制 表达、定点重组和其他基因组重排。 超螺旋DNA的另一个基本性质是负的超螺旋 促进DNA中的局部结构转变。某些短DNA区域可以 采用替代构象，在某些情况下，特定的酶或 调节蛋白以这些区域为靶点，发挥其调节作用。 这笔赠款将检验这样一种想法，即 超螺旋DNA被连接在一起，使得局部结构转变可以具有 对超螺旋DNA的整体几何或地形产生重大影响。 需要检验的一个假设是，局部替代构象可以定义 DNA分子的形状可能会影响两个分子的并置 沿着DNA分子的位置。我们将测试形成的影响 十字形、分子内三链DNA(H-DNA)和左手Z-DNA 质粒拓扑学。我们还将确定那些具有强大的 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，参与改变整体几何构型 分子。此外，我们将测试一种新的生物学想法--即我们将 检验DNA的地形或位置定位的假设 超螺旋中的序列可以调节DNA的结构转变。这是 一个重要而新颖的概念，因为它提供了另一个层面的监管 DNA二级结构转变显然与生物学有关 流程。这将通过在顶端放置反向重复来确定 或非顶端位置。以上概述的研究将利用 原子力显微镜(AFM)，双向琼脂糖凝胶电泳，以及 DNA和核蛋白结构研究中的化学探针分析 复合体。

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.