STRUCTURAL STUDIES OF INTEGRASE/DNA INTERACTIONS

研究人员将研究Tn916中涉及的核蛋白复合体 换位。Tn916转座元件负责 抗生素耐药性基因在人类病原体中的接合转移， 并使用与多种不同的 原核和真核定点特异性重组反应。这个 转座的核酸化学是由一个单一的 转座子编码蛋白，整合酶。值得注意的是，共价整合酶 连接两个相隔超过18千碱基的DNA位点。关键的一步 在这个过程中是重组复合体的组装，其中 转座子的末端被转座子的四聚体并列和切割 整合酶蛋白。重组复合体的组装被认为是 需要多种整合酶-整合酶和整合酶-DNA相互作用。 为了理解转座的分子基础，我们将使用 核磁共振(核磁共振)光谱和生化工具 鉴定参与的基本整合酶-DNA相互作用 换位。我们将首先确定三维结构 以及单一整合酶-DNA复合体的化学性质 有利于重组复合体的形成。之后 成功地识别了保持 整合酶蛋白紧密结合到转座子上，我们将确定 这些相互作用如何作为核点 其他原核糖体的协同结合。最后，我们将在 原子分辨率，DNA结合的整合酶原体间的相互作用。 这将通过确定三维结构来实现 指由两个结合在一起的整合酶蛋白组成的蛋白质-DNA复合体 相邻的DNA结合位点。

Research will study the nucleoprotein complexes involved in Tn916 transposition. Tn916 transposable elements are responsible for the conjugative transfer of antibiotic resistance genes in human pathogens, and transpose using a mechanism that is homologous to a wide variety of prokaryotic and eukaryotic site-specific recombination reactions. The nucleic acid chemistry of transposition is mediated by a single transposon encoded protein, integrase. Remarkably, integrase covalently joins two DNA sites separated by greater than 18 kilobases. A key step in this process is the assembly of a recombination complex in which the ends of the transposon are juxtaposed and cleaved by a tetramer of the integrase protein. Assembly of the recombination complex is believed to require multiple integrase-integrase and integrase-DNA interactions. In order to understand the molecular basis of transposition we will use nuclear magnetic resonance (NMR) spectroscopy and biochemical tools to characterize essential integrase-DNA interactions involved in transposition. We will first determine the three dimensional structure and chemical properties of a single integrase-DNA complex that facilitates the formation of the recombination complex. After successfully identifying the molecular interactions that hold the integrase protein tightly onto the transposon sites, we will determine how these interactions might serve as nucleation points for the cooperative binding of other protomers. Finally, we will visualize, at atomic resolution, interactions between DNA-bound integrase protomers. This will be accomplished by determining the three dimensional structure of a protein-DNA complex consisting of two integrase proteins bound to adjacent DNA binding sites.