PHAGE T4 HEAD ASSEMBLY AND INITIATION OF INFECTION

研究重点为病毒DNA包装机制 以及噬菌体T4体外包装衍生的构建 克隆载体。 我们的工作特别针对 1) 了解多功能的结构和功能 噬菌体 T4 包装（前头和终止酶）蛋白。 3- 酶促（DNA 易位）的维度基础和 主要衣壳的结构（前头形状决定）作用 蛋白质 gp23*)，是一种微加工酶产物 (gp23**)， 以及衣壳基因突变体的数量将被确定。 的组装 DNA 入口（也是前头起始）顶点作为 将使用过表达来检查整合膜蛋白 载体；催化基因产物和宿主系统的作用 将确定膜插入。 具体包装 这两个前头蛋白的突变都将被选择 基因定向诱变后。 DNA 终止酶 与 DNA 入口顶点蛋白相互作用的蛋白质 前头已被过表达和纯化-它们 DNA 易位和串联体的作用机制 切割将被调查。 lambda 和 lambda-pBR322 衍生的 DNA 可以在体外包装到 T4 头中。 因斯 和/或 P1 DNA 包装到 T4 中并用 同源位点特异性重组系统应允许 T4-杂交大粘粒载体的开发。 我们提出了 一种新颖的“螺旋折叠”包装噬菌体 DNA 模型。 形态学和（噬菌体）化学工作可以明确地 建立这个模型。 包装的扭结或非 B 型 DNA 将 检测到，及其与 DNA 结合位点的相互作用 主要衣壳蛋白及其次要加工酶产物 (gp23**) 将被探测。 我们将确定 DNA 结构 可体内、体外包装；例如 是否有刻痕和 异源双链体环从体外包装的 DNA 中排除，并且 终止酶蛋白是否起到区分这种作用 耦合 DNA 修复过程中的结构。

The research focuses on the mechanism of viral DNA packaging and on the construction of phage T4 in vitro packaging-derived cloning vectors. Our work is especially directed toward 1) understanding the structures and functions of the multifunctional phage T4 packaging (prohead and terminase) proteins. The 3- dimensional basis for enzymatic (DNA translocating) and structural (prohead form-determining) roles of the major capsid protein gp23*), of a minor processed enzymatic product (gp23**), and of capsid gene mutants will be determined. The assembly of the DNA entrance (also the prohead initiation) vertex as an integral membrane protein will be examined using overexpression vectors; the role of a catalytic gene product and host systems in the membrane insertion will be determined. Specific packaging mutations in both these prohead proteins will be selected following gene-directed mutagenesis. The DNA terminase proteins which interact with the DNA entrance vertex protein in the prohead have been overexpressed and purified-their mechanisms of action in DNA translocation and concatemer cutting will be investigated. lambda and lambda-pBR322 derivative DNAs can be packaged into T4 heads in vitro. cos and/or P1 DNAs packaged into T4 and recircularized with the homologous site-specific recombination systems should allow development of T4-hybrid megacosmid vectors. We have proposed a novel "spiral-fold" model for packaged phage DNA. Morphological and (with phage ) chemical work can definitively establish this model. Packaged kinked or non-B form DNA will be detected, and its interaction with the DNA binding site in the major capsid protein and its minor processed enzymatic product (gp23**) will be probed. We will determine what DNA structures can be packaged in vivo and in vitro; e.g. whether nicks and heteroduplex loops are excluded from packaged DNA in vitro, and whether the terminase proteins act to discriminate against such structures in coupled DNA repair processes.