Thesis (M.S.)--California State University, Fullerton, 2020
Includes bibliographical references
Terminase enzymes are responsible for packaging viral DNA into empty procapsids. Bacteriophage lambda (λ), a double-stranded DNA virus (dsDNA), utilizes a terminase holoenzyme that is composed of two subunits to package DNA. GpNu1, the smaller subunit, is responsible for site-specific assembly of viral DNA. GpA, the large subunit, is responsible for all the catalytic activity for the enzyme to begin viral assembly. To stabilize the gpA catalytic subunit, a dual point mutation was produced in the DNA packaging ATPase domain (D178E/E179D) and two truncations were made to remove 50 N terminal amino acids and 19 C terminal amino acids. The D178E/E179D mutations inactivate the ATPase domain and stabilize the protein for crystallography. The truncation at the N-terminus prevents interactions with the small subunit, while the truncation at the C-terminus prevents interactions with the viral capsid. Nuclease and helicase studies suggest that the N-terminal truncation destabilized the protein unless the D178E/E179D mutation was performed, which stabilized the mutant. DNA binding studies suggest that in the presence of viral DNA, the wild-type mutant binds to DNA much tighter than the D178E/E179D mutant. Preliminary crystallization structure solutions will confirm logical hypotheses formed about the gpA subunit and its interactions with DNA during viral assembly
Electronic reproduction. Ann Arbor, Mich. : ProQuest, 2020
