Phage T4 Head Assembly and Initiation of Infection

描述(由申请人提供)：拨款申请建议继续进行两个长期目标的研究：1)了解保守的病毒DNA包装马达的移位机制及其调节；2)为对抗新型DNA移位限制酶而注射的内部蛋白质的进化。包装马达由T4大末端亚基组成，作用于前头的入口十二聚体，以接近100%的效率转运任何序列的染料标记的短DNA。在单一复合体之后，我们将分析门户-GFP前向包装。通过FCS和核酸酶分析，与气泡不匹配的DNA不同，短缺口的DNA没有被包装，这表明发动机在DNA中引入扭转来使其移位。FRET将被用来用双染料DNA来确定DNA的尺寸被停滞的马达改变。此外，标签末端和入口之间和内部的FRET将建立包装过程中的距离和构象变化。FRET还将用于确定封装头中两端的接近程度。我们将确定终止酶的头部切割特异性。我们将完成我们的工作，展示晚期sigma因子(Gp55)-滑动钳(Gp45)复合体通过大末端酶亚单位gp17与一些调节蛋白的相互作用参与体内包装对Nike环状DNA的起始作用。在第二个目标中，CTS注射蛋白的不同家族已经通过在IPI基因座上的基因扩展而进化，以挑战由NTP驱动的DNA转位限制酶家族。我们对两个GMRS/gmrD限制性内切酶(CT和UT)、两个多态噬菌体基因拮抗剂(IPI*和IP5*)以及这些酶的不同目标糖HMC加合物的持续分析揭示了一条在纳米尺度上产生复杂性的进化途径。总体而言，致力于第一个DNA包装目标的努力将比第二个限制性内切酶注射抑制剂部分更重，这是我们之前拨款的两个研究目标。然而，在这两个研究领域研究DNA易位的方法将是互补的。公共卫生相关性：病毒核酸包装在dsDNA噬菌体和许多临床上重要的DNA病毒之间以高度保守的机制发生，包括疱疹和腺病毒。在许多临床重要的RNA病毒的包装中也可以看到共同的特征，例如呼肠孤病毒。更好地了解单一包装机制可以促进针对动物病毒(如疱疹病毒)的包装定向抗病毒药物的开发；此外，体外包装成与噬菌体相当的动物病毒可能被用于开发与真核基因转移剂相同的病毒。最后，了解病毒包装对基本DNA过程至关重要的密切相关酶的机制具有重要影响，例如解旋酶和NTP驱动的DNA转位限制性内切酶。

DESCRIPTION (provided by applicant): The grant application proposes continuation of research toward two long term objectives: understanding 1) the translocation mechanism of the conserved viral DNA packaging motor and its regulation; and 2) the evolution of the internal proteins injected to counter novel DNA translocating restriction enzymes. The packaging motor consists of the T4 large terminase subunit working at the portal dodecamer of the prohead to translocate dye labeled short DNAs of any sequence with near 100% efficiency. Following single complexes we will analyze portal-GFP prohead packaging. By FCS and nuclease assay short nicked DNAs are not packaged, unlike bubble mismatch DNAs, suggesting that the motor introduces torsion into the DNA to translocate it. FRET will be used to establish with double dye DNAs that the dimensions of the DNA are changed by the stalled motor. Additionally FRET between and within tagged terminase and portal will establish distance and conformational changes during packaging. FRET will also be used to establish the proximity of the two ends in packaged heads. We will determine the headful cutting specificity of the terminase. We will complete our work showing the participation of the late sigma factor (gp55)-sliding clamp (gp45) complex in packaging initiation in vivo on nicked circular DNAs by large terminase subunit gp17 interaction with a number of regulatory proteins. In the second objective, a diverse family of CTS-injected proteins has evolved by gene expansion at the IPI locus to challenge a novel family of NTP driven DNA translocating restriction enzymes. Our ongoing analysis of two of the gmrS/gmrD restriction endonucleases (CT and UT), two polymorphic phage gene antagonists (IPI* and IP5* whose structures we have determined), and diverse target sugar HMC adducts of these enzymes reveals an evolutionary pathway generating complexity on a nanoscale. Overall, effort devoted to the first DNA packaging objectives will be more heavily weighted than to the second restriction endonuclease-injected inhibitors portion, the two research objectives of our previous grant. However, approaches to studying DNA translocation in the two research areas will be complementary. PUBLIC HEALTH RELEVANCE: Viral nucleic acid packaging occurs by a highly conserved mechanism among dsDNA bacteriophages and many DNA viruses of clinical importance including Herpes and Adenoviruses. Common features are seen also in the packaging of many RNA viruses of clinical importance, such as Reoviruses. Better understanding of the unitary packaging mechanism could promote development of packaging directed antiviral agents of animal viruses such as Herpes; moreover packaging in vitro into such animal viruses with efficiencies comparable to the phages' might be exploited in developing these same viruses as eukaryotic gene transfer agents. Finally, understanding viral packaging has an important bearing on the mechanism of closely related enzymes of importance to fundamental DNA processes, e.g. helicases and NTP driven DNA translocating restriction endonucleases.