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Many double-stranded DNA (dsDNA) viruses utilize terminase enzymes, which package viral DNA into empty procapsids. Bacteriophage lambda (λ) catalyzes viral DNA packaging with the functional terminase holoenzyme, composed of subunits gpNu1, which is involved in viral genome recognition, and gpA, which cleaves the concatemeric viral DNA into a single genome and then translocates the now mature viral genome into a procapsid. Using sequence alignment studies we mutated the N-terminal ATPase domain (D178E/E179D) and truncated the C terminus at residue 622 to stabilize the dynamic teriminase structure. The mutation impacted the fully assembled holoenzyme, increasing nuclease and helicase activity, while showing only minimal ATPase activity as compared to the wild type holoenzyme. DNA binding studies suggest that in the presence of viral DNA, the holoenzyme mutant is confined to a single conformation. Future crystallization studies with the holoenzyme mutant and the viral DNA may allow for structure solution of the DNA packaging complex. Here we present the biochemical characterization of the assembled terminase holoenzyme mutant and the individual gpA subunit of bacteriophage lambda terminase.
Hinrichs, Brandon D., "Biochemical and Structural Analysis of a Bacteriophage DNA Packaging Molecular Motor" (2017). Biology Honors Projects. 14.
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