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.
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.
|Hardies, Stephen C; Thomas, Julie A; Black, Lindsay et al. (2016) Identification of structural and morphogenesis genes of Pseudoalteromonas phage Ï†RIO-1 and placement within the evolutionary history of Podoviridae. Virology 489:116-27|
|Thomas, Julie A; BenÃtez Quintana, Andrea Denisse; Bosch, Martine A et al. (2016) Identification of Essential Genes in the Salmonella Phage SPN3US Reveals Novel Insights into Giant Phage Head Structure and Assembly. J Virol 90:10284-10298|
|Black, Lindsay W (2015) Old, new, and widely true: The bacteriophage T4 DNA packaging mechanism. Virology 479-480:650-6|
|Liu, Jinny L; Dixit, Aparna Banerjee; Robertson, Kelly L et al. (2014) Viral nanoparticle-encapsidated enzyme and restructured DNA for cell delivery and gene expression. Proc Natl Acad Sci U S A 111:13319-24|
|Mullaney, Julienne M; Black, Lindsay W (2014) Bacteriophage T4 capsid packaging and unpackaging of DNA and proteins. Methods Mol Biol 1108:69-85|
|Thomas, Julie A; Black, Lindsay W (2013) Mutational analysis of the Pseudomonas aeruginosa myovirus KZ morphogenetic protease gp175. J Virol 87:8713-25|
|Black, Lori Williams; Fraser, Debbie; Goodall, Heather et al. (2013) Academy news. Neonatal Netw 32:75-80|
|Dixit, Aparna Banerjee; Ray, Krishanu; Thomas, Julie A et al. (2013) The C-terminal domain of the bacteriophage T4 terminase docks on the prohead portal clip region during DNA packaging. Virology 446:293-302|
|Robertson, Kelly; Furukawa, Yoko; Underwood, Alison et al. (2012) Deletion of the Hoc and Soc capsid proteins affects the surface and cellular uptake properties of bacteriophage T4 derived nanoparticles. Biochem Biophys Res Commun 418:537-40|
|Black, Lindsay W; Thomas, Julie A (2012) Condensed genome structure. Adv Exp Med Biol 726:469-87|
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