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.
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