Experiments are proposed to investigate the mechanism of virus DNA packaging, using bacteriophage lambda as a model system. lambda, like phages T3 and T7 and the herpesviruses, generates virion chromosomes by introducing specific cuts in multimeric precursor DNA. For each of these viruses, a complex array of DNA sites instructs the packaging machinery to cut and package the viral DNA. The lambda DNA packaging system is the best understood of the group. To initiate DNA packaging terminase, the lambda DNA packaging enzyme, binds to the cosB site, and introduces staggered nicks in the adjacent site, cosN, to generate the cohesive ends of virion DNA. Terminase then binds an empty protein shell, the prohead, and translocation of the DNA into the head ensues, accompanied by ATP hydrolysis. Termination of packaging occurs when the translocating packaging machinery encounters the next cosN, which is nicked to generate the terminus of the virion DNA. Termination requires cosN, cosB and the newly discovered site, cosQ. The proposed experiments will examine in detail the interactions of terminase with the cos sites during initiation and termination of packaging. These interactions will be defined at the molecular level, giving information about DNA recognition by terminase, and about the domains of terminase involved in DNA recognition. A series of intermediates in the packaging process, including a terminase-DNA complex, a paused translocating complex, and a paused termination complex will be examined in detail to determine the protein composition and structures of the complexes. This information should lead to an understanding of structural changes within the packaging machinery during the packaging process. A model for how cosQ participates in changing the translocating complex into a termination complex will be tested. ATP has multiple roles in DNA packaging as an effector molecule and an energy source. Studies of mutant terminases altered for the two ATPase activities will define the role of ATP in the nicking, cohesive end separation, and translocation activities of terminase. DNA packaging, like other DNA-protein transactions such as transcription, recombination and replication, involves complex interactions between a protein assembly and the DNA. Understanding these processes at the molecular level is a major goal of biology.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM051611-01
Application #
2190264
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1994-08-01
Project End
1998-07-31
Budget Start
1994-08-01
Budget End
1995-07-31
Support Year
1
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Iowa
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
041294109
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Feiss, Michael; Young Min, Jea; Sultana, Sawsan et al. (2015) DNA Packaging Specificity of Bacteriophage N15 with an Excursion into the Genetics of a Cohesive End Mismatch. PLoS One 10:e0141934
Rao, Venigalla B; Feiss, Michael (2015) Mechanisms of DNA Packaging by Large Double-Stranded DNA Viruses. Annu Rev Virol 2:351-78
Feiss, Michael; Geyer, Henriette; Klingberg, Franco et al. (2015) Novel DNA packaging recognition in the unusual bacteriophage N15. Virology 482:260-8
Sippy, Jean; Patel, Priyal; Vahanian, Nicole et al. (2015) Genetics of critical contacts and clashes in the DNA packaging specificities of bacteriophages ? and 21. Virology 476:115-123
Feiss, Michael; Rao, Venigalla B (2012) The bacteriophage DNA packaging machine. Adv Exp Med Biol 726:489-509
Giri, Lopamudra; Feiss, Michael G; Bonning, Bryony C et al. (2012) Production of baculovirus defective interfering particles during serial passage is delayed by removing transposon target sites in fp25k. J Gen Virol 93:389-99
Giri, Lopamudra; Li, Huarang; Sandgren, David et al. (2010) Removal of transposon target sites from the Autographa californica multiple nucleopolyhedrovirus fp25k gene delays, but does not prevent, accumulation of the few polyhedra phenotype. J Gen Virol 91:3053-64
Zeng, Lanying; Skinner, Samuel O; Zong, Chenghang et al. (2010) Decision making at a subcellular level determines the outcome of bacteriophage infection. Cell 141:682-91
Feiss, Michael; Reynolds, Erin; Schrock, Morgan et al. (2010) DNA packaging by lambda-like bacteriophages: mutations broadening the packaging specificity of terminase, the lambda-packaging enzyme. Genetics 184:43-52
Tsay, James M; Sippy, Jean; DelToro, Damian et al. (2010) Mutations altering a structurally conserved loop-helix-loop region of a viral packaging motor change DNA translocation velocity and processivity. J Biol Chem 285:24282-9

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