Phase T4 Head Assembly and Initiation of Infection

描述（由申请人提供）：本申请提出继续研究两个主要目标：1）了解末端酶催化的病毒DNA易位进入原衣壳的机制，以及2）确定导致由T-偶联物注入的CTS-（衣壳靶向序列）启动的一组多态性内部蛋白进化的宿主抗性机制。通过开发一种新的噬菌体衍生技术来评估蛋白质相互作用，将进一步实现这一目标。 作为第一个目标的一部分，DNA包装机制将集中在末端酶和门户十二聚体的结构和功能进行调查。可以通过与末端酶小亚基和针对变性末端酶大亚基的抗体孵育来产生末端酶大亚基的多聚体活化ATP酶形式。与门户或功能性门户-GFP（绿色荧光蛋白）融合一起，将对重建的包装体马达（末端酶+门户+ DNA）进行表征。将portaI-GFP和HOC-门户融合到功能性prohead和噬菌体中的工作表明，prohead的门户十二聚体可以被固定而不阻断DNA包装。这项工作的继续可以确定是否有利的门户网站旋转模型的DNA包装是有效的。初步工作表明，NMR可以解决的小末端酶亚基的结构。DNA结合和大亚基ATP酶刺激小亚基的部分将进行生物化学和NMR表征。 T4展示技术已经成功地鉴定出一种新的终止酶相互作用物T4晚期a因子gp 55，它是复制偶联T4晚期转录所必需的。最近的工作已经确定了DNA包装对该组分的依赖性;我们相信这种相互作用有助于将大末端酶亚基装载到DNA上。这种相互作用也可以用于紧密耦合包装DNA修复在T4发育的后期复制依赖性转录阶段。活性体外T4 DNA包装的重建将使用分离的基因55缺陷体内多联体以及T4转录测定质粒。这些DNA将通过加载晚转录-复制组分而被激活用于包装。 在第二个目标，一个不同的家庭的CTS注射蛋白质已经演变的基因扩增的IPI位点的T-甚至是挑战相应的一组抗性基因在这些病毒的天然宿主。通过对从一株大肠杆菌中克隆的两个抗性基因IBEGs和IBEGd的分析，确定了宿主的抗性机制。大肠杆菌中，这是必要的和足够的噬菌体T4 IPI-和其他T-甚至噬菌体的阻力。这两个克隆的基因编码一种新的双亚基限制性内切酶，其靶向含葡糖基化羟甲基胞嘧啶（HmC）的T4 DNA。IPI保护DNA不受这种核酸酶的影响。IPI基因座基因在T-偶联蛋白中的进化扩展和多态性可能对应于HmC加合物的多态性，HmC加合物是这种以前未发现的限制性内切酶的特异性靶点。

DESCRIPTION (provided by applicant): This application proposes continuation of research toward two main objectives: 1) understanding the mechanism of terminase catalyzed viral DNA translocation into the procapsid, and 2) determining the host resistance mechanism leading to the evolution of the CTS- (capsid targeting sequence) initiated set of polymorphic internal proteins injected by the T-even phages. The objectives will be furthered by development of a new phage-derived technique for assessing protein interactions. As part of the first objective, the DNA packaging mechanism will be investigated by focusing on terminase and portal dodecamer structure and function. A multimeric activated ATPase form of the terminase large subunit can be produced by incubation with the terminase small subunit and with antibody against denatured terminase large subunit. Together with portal or functional portaI-GFP (green fluorescent protein) fusion, the reconstituted packasome motor (terminase + portal + DNA) will be characterized. Work incorporating portaI-GFP and HOC-portal fusions into functional proheads and phage suggests that the portal dodecamer of the prohead can be immobilized without blocking DNA packaging. Continuation of this work can determine whether a favored portal rotation model for DNA packaging is valid. Preliminary work reveals that NMR can solve the structure of the small terminase subunit. DNA binding and large subunit ATPase stimulating portions of the small subunit will be characterized biochemically and by NMR. T4 display has successfully identified a new terminase interactant, T4 late a factor gp55, required for replication-coupled T4 late transcription. Recent work has established a dependence of DNA packaging on this component; we believe the interaction serves to load the large terminase subunit onto DNA. This interaction could also serve to tightly couple packaging to DNA repair at the late replication-dependent transcription stage of T4 development. Reconstitution of active in vitro T4 DNA packaging will employ isolated gene 55 defective in vivo concatemers as well as T4 transcription assay plasmids. These DNAs will be activated for packaging by loading late transcription-replication components. In the second objective, a diverse family of CTS-injected proteins has evolved by gene expansion at the IPI locus of the T-even phages to challenge a corresponding set of resistance genes in natural hosts of these viruses. We have determined the host resistance mechanism by analysis of two resistance gene(s), IBEGs and IBEGd, cloned from one pathogenic strain of E. coli that are necessary and sufficient for phage T4 IPI- and other T-even phage resistance. These two cloned genes encode a novel two-subunit restriction endonuclease that targets glucosylated hydroxymethylcytosine (HmC)-containing T4 DNA. IPI shields the DNA from this nuclease. Evolutionary expansion and polymorphism of IPI locus genes among the T-even phages likely correspond to polymorphism of HmC adducts as specific targets for this type of previously undiscovered restriction endonuclease.