TOPOLOGICAL MODIFICATION OF VIRAL DNA

枯草芽孢杆菌噬菌体phi29是一种小井 具有特征的病毒，含有18kbp的双链DNA。 它的整个基因组已经测序，23个人中的每一个 基因被标记为抑制子敏感(Sus)或温度 敏感(Ts)突变体。大多数基因的产物都有 被鉴定出来，结构蛋白组装成一个单一的 形态发生途径。我们已经描述了一个完全定义的 Phi29与DNA共价的体外组装系统 绑定的GP3借助于单元格被封装成Prohead 蛋白gp16(PNAS，83,3505,1986)和高效 相当于体内组装。目前的提案是以 对包装的DNA-GP3的拓扑结构进行了分析，并对 DNA包装蛋白gp16，a的结构与功能 DNA依赖的ATPase从拓扑上修饰phi29 DNA- GP3作为包装的先决条件。我们将研究病毒内的 离子刻蚀制备DNA-GP3的拓扑结构 从外到内的病毒粒子成分，同时保存 总体结构。通过使用这种方法和病毒粒子 在体外组装，我们将确定1)左侧的位置 和基因组右端在衣壳中；2)GP3的位置； 3)存在平行的(180度)急转弯 DNA链。我们将研究DNA与GP3的结合 将gp16蛋白包装成DNA-GP3诱导拓扑 DNA在体外组装过程中的修饰。我们会研究 Gp16与DNA-gp3结合的化学计量学和特异性。 将研究gp16与特定dna片段的相互作用。 通过足迹或使用甲基化。负超螺旋 DNA/gp3-gp16复合体的密度将通过使用 补骨脂素光亲和探针的光结合率。我们 将寻找核小体类结构形成的证据 通过使用核酸酶消化改变DNA螺旋间距 实验。我们将研究gp16的分子结构。 我们将尝试确定gp16的功能结构域。 参与前列环素、DNA-GP3和ATP的相互作用。我们会 通过克隆改变的DNA和DNA来扰乱蛋白质的局部结构 基因改变的蛋白质在DNA中的功能特征 绑定和拓扑修改。我们将尝试生产 二维和三维晶体的gp16；二维晶体将分析 电子显微照片和3-D晶体的傅里叶滤波将被 经X-射线衍射仪分析。

Bacteriophage phi29 of Bacillus subtilis is a small, well characterized virus containing a double-stranded DNA of 18 Kbp. Its entire genome has been sequenced and each of twenty-three genes is marked with suppressor-sensitive (sus) or temperature sensitive (ts) mutants. The products of most of the genes have been identified, and structural proteins are assembled in a single morphogenetic pathway. We have described a completely defined in vitro assembly system in which the phi29 DNA with covalently bound gp3 is packaged into a prohead with the aid of the single protein gp16 (PNAS, 83, 3505, 1986) and with an efficiency equivalent to in vivo assembly. The present proposal is centered on an analysis of the topology of packaged DNA-gp3, and the structure and function of the DNA packaging protein gp16, a DNA-dependent ATPase that topologically modifies phi29 DNA- gp3 as a prerequisite for packaging. We will study the intravirion topology of DNA-gp3 by ion etching, which progressively erodes virion components from the outside to the inside while preserving the overall structure. By use of this method and virions assembled in vitro, we will determine 1) the location of the left and right ends of the genome in the capsid; 2) the location of gp3; and 3) the presence of sharp (180 degrees) bends in the parallel DNA strands. We will study the binding of the DNA-gp3 packaging protein gp16 to DNA-gp3 that induces topological modification of the DNA during in vitro assembly. We will study the stoichiometry and specificity of binding of gp16 to DNA-gp3. Interaction of gp16 with specific segments of DNA will be studied by footprinting or by use of methylation. Negative superhelical density in DNA/gp3-gp16 complexes will be measured by the use of psoralen photoaffinity probes and the rate of photobinding. We will seek evidence for formation of nucleosome-like structures with an altered DNA helical pitch by the use of nuclease digestion experiments. The molecular structure of gp16 will be studied. We will attempt to identify the functional domains of gp16 involved in prohead, DNA-gp3, and ATP interactions. We will perturb local structures of the protein by cloning altered DNA and characterizing genetically altered proteins for function in DNA binding and topological modification. We will attempt to produce 2-D and 3-D crystals of gp16; 2-D crystals will be analyzed by Fourier filtering of electron micrographs and 3-D crystals will be analyzed by X-ray diffraction.