Biochemical Characterization of Phage Lambda Assembly

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The assembly of an infectious virus particle requires the coordinated action of a number of proteins, and complex interactions with viral DNA. Terminase enzymes are common to many double-stranded (ds) DNA viruses, including poxvirus, the herpesvirus groups, and most dsDNA bacteriophage. Terminases "package" viral DNA into the capsid, a critical step in virus assembly. This project centers on the terminase enzyme from bacteriophage lambda and attempts to define the biochemical, biophysical, and structural characteristics of nucleoprotein intermediates involved in DNA packaging. Three approaches designed to provide mechanistic insight into virus assembly are examined. (I) One of the most fascinating and least understood aspects of the packaging pathway is the transition from complex I, an extremely stable packaging precursor, to a mobile packaging complex. Biophysical, kinetic, and structural experiments are used to provide a mechanistic underpinning for this transition. (II) Current models describing the packaging pathway are based on genetic and crude in vitro packaging studies. Biochemical studies are used that will define, at a molecular level, the role of terminase, and inancillaryls packaging proteins at each step of the packaging pathway. (III) Genetic studies suggest a domain organization for gpA, the large terminase subunit. Biochemical and biophysical experiments will rigorously define functional domains in gpA. These three experimental approaches represent discrete research directions that are intertwined in their attempt to provide a detailed description of lambda terminase, and its role in virus assembly. This work will provide a paradigm for genome packaging on other dsDNA viruses. Moreover, mechanistic similarities exist between genome packaging and complex cellular processes such as DNA replication, transcription, and site-specific recombination. Thus, an understanding of the structural and catalytic properties of this packaging apparatus has broad implications in our understanding of DNA manipulation by large multiprotein enzyme complexes in biology.