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. ? ?
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