Bacteriophages are viruses that infect bacteria. Phage DNA packaging is one process in the assembly of mature phages that is not well characterized. Elucidating the mechanism of this process would enhance our understanding of the biological significance of the machinery. A previous study suggested that the protein gp74 from bacteriophage HK97 functions as an HNH endonuclease and is required for phage DNA packaging. In this thesis the functionality was assessed for a protein from a phage that replicates under extreme conditions. Our data suggest that the endonuclease activity may not be essential for the role of gp74 in phage DNA packaging.

Bacteriophages are viruses that infect bacteria. This thesis describes studies of gp74 from the bacteriophage HK97, which functions as an HNH endonuclease. HNH endonucleases are DNA digestion proteins characterized by two highly conserved His residues and an Asn residue. Like other HNH endonucleases, the activity of gp74 is dependent on binding of divalent metal ions to the HNH motif. Current work focused on confirming the identity of conserved HNH motif residues of gp74. We hypothesized the catalytic His residue is H43, the structural Asn residue is N73, and that H82 is involved in metal–binding. Additional residues in the ββα–fold, such as D42, may also bind the metal. Our bound metal analysis and the sequence of gp74 also suggest the presence of a Zn2+–finger motif. Mutations of D42 and H82 decrease the activity of gp74, without affecting the structure. These studies advance our understanding of the gp74 activity.

Bacteriophages are viruses that infect bacteria. This thesis describes studies of gp74 from the bacteriophage HK97, which functions as an HNH endonuclease. HNH endonucleases are DNA digestion proteins characterized by two highly conserved His residues and an Asn residue. Like other HNH endonucleases, the activity of gp74 is dependent on binding of divalent metal ions to the HNH motif. Current work focused on confirming the identity of conserved HNH motif residues of gp74. We hypothesized the catalytic His residue is H43, the structural Asn residue is N73, and that H82 is involved in metal–binding. Additional residues in the ββα–fold, such as D42, may also bind the metal. Our bound metal analysis and the sequence of gp74 also suggest the presence of a Zn2+–finger motif. Mutations of D42 and H82 decrease the activity of gp74, without affecting the structure. These studies advance our understanding of the gp74 activity.