Bacteriophages are viruses that infect and propagate within bacteria by making use of the host’s biosynthetic machinery. With a global population of 1031, phages pose a significant influence on microbial populations. Studies of bacteriophage proteins can elucidate the influence that bacteriophages play on the evolution of bacteria, as well as, providing the basis for the use of phage proteins as possible therapeutics and bioengineering solutions. This study aims to investigate the structural and functional role of the HK97 phage protein gp74. Sequence alignments indicate that gp74 is related to homing HNH endonucleases. Homing endonucleases are predominantly double-stranded DNases, suggesting that gp74 mediates integration of phage genes into the host genome or may target foreign phage DNA. DNA digestion experiments with gp74 reveals that gp74 mediates non-specific double-stranded cleavage of lambda phage DNA and single strand cleavage of plasmid DNA. Our initial work demonstrates that HK97 gp74 is an HNH endonuclease.

Bacteriophages are viruses that infect and propagate within bacteria by making use of the host’s biosynthetic machinery. With a global population of 1031, phages pose a significant influence on microbial populations. Studies of bacteriophage proteins can elucidate the influence that bacteriophages play on the evolution of bacteria, as well as, providing the basis for the use of phage proteins as possible therapeutics and bioengineering solutions. This study aims to investigate the structural and functional role of the HK97 phage protein gp74. Sequence alignments indicate that gp74 is related to homing HNH endonucleases. Homing endonucleases are predominantly double-stranded DNases, suggesting that gp74 mediates integration of phage genes into the host genome or may target foreign phage DNA. DNA digestion experiments with gp74 reveals that gp74 mediates non-specific double-stranded cleavage of lambda phage DNA and single strand cleavage of plasmid DNA. Our initial work demonstrates that HK97 gp74 is an HNH endonuclease.

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.