HNH proteins are a widespread component of phage DNA packaging machines

The genome packaging reactions of tailed bacteriophages and herpes viruses require the activity of a terminase enzyme, which is comprised of large and small subunits. Phage genomes are replicated as linear concatemers composed of multiple copies of the genome joined end to end. As the terminase enzyme packages the genome into the phage capsid, it cleaves the DNA into single genome-length units. In this work, we show that the phage HK97 HNH protein, gp74, is required for the specific endonuclease activity of HK97 terminase and is essential for phage head morphogenesis. HNH proteins are a very common family of proteins generally associated with nuclease activity that are found in all kingdoms of life. We show that the activity of gp74 in terminase-mediated cleavage of the phage cos site relies on the presence of an HNH motif active-site residue, and that the large subunit of HK97 terminase physically interacts with gp74. Bioinformatic analysis reveals that the role of HNH proteins in terminase function is widespread among long-tailed phages and is uniquely required for the activity of the Terminase_1 family of large terminase proteins.Tailed bacteriophages and herpes viruses package their large double-stranded DNA genomes into a preformed protein shell, known as the “prohead,” using terminase enzymes. In both types of viruses, the genome is synthesized as concatemers composed of multiple copies of the genome joined end to end. This concatemeric DNA is packaged into the prohead and cleaved into genome-length units by terminase in an ATP-dependent reaction. Phage terminases are composed of two proteins: the large subunit harbors an endonuclease domain and an ATPase that powers the DNA packaging reaction, and the small subunit mediates specific DNA-binding required for recognition of packaging sites in the phage genome. A variety of elegant structural and biophysical studies have recently provided insight into the molecular mechanisms of terminase function (1, 2). However, the factors that affect the action of terminase enzymes in vivo have been less well characterized.Terminase enzymes perform several functions. They specifically recognize and bind the viral genome, interact with the prohead, then drive the DNA into the head through the narrow entry channel formed by the portal protein that is positioned at a single vertex of the head. During this process terminases also cleave the viral DNA, either nonspecifically upon head filling or at a specific site known as “cos.” The efficient packaging of a phage genome in vivo may require phage-encoded cofactors in addition to the terminase enzyme. For example, Escherichia coli phage λ gpFI facilitates interaction of the terminase–DNA complex with proheads (3–6). A wide variety of phages appear to encode proteins with a function similar to λ gpFI (7). Additionally, the activity of Bacillus subtilis phage phi29 terminase requires a phage-encoded RNA molecule bound to its portal protein (8), and in vivo packaging of the E. coli phage T4 genome can only be completed with the participation of the phage-encoded endonuclease, gp49 (9). The general prevalence and importance of terminase cofactors is difficult to evaluate because few studies have addressed this issue.We recently reported that phage genomes often encode proteins possessing an HNH motif near their terminase genes (10). The HNH motif is ∼35 aa long, and is characterized by the presence of two highly conserved His residues and one Asn residue. These HNH motifs, as defined by the large (∼7,400-member) HNH Pfam (11) protein sequence family (PF01844), are often found in proteins that possess endonuclease activity, such as site-specific homing endonucleases (12, 13), colicins (14, 15), S pyocins (16), and restriction enzymes (17–19). HNH motif-containing proteins comprised of primarily an HNH motif as found in E. coli colicins, usually possess nonspecific endonuclease activity. Conversely, HNH motif-containing proteins may contain DNA-recognition domains in addition to the HNH motif and thus possess high sequence specificity, as found in the homing endonucleases.The frequent juxtaposition of HNH and phage terminase genes (10, 20) suggests a unique role for HNH proteins in the endonuclease and/or packaging activities of the terminases. To address this issue, we investigated the function of E. coli phage HK97 gp74, a 119-residue protein containing an HNH motif. The gene encoding gp74 is located at the extreme 3′ end of the mature linear HK97 genome, adjacent to the cos site. In both the lysogen and replicative form of the HK97 genome gene 74 is immediately adjacent to genes 1 and 2, which encode the small and large subunits of terminase (TerS and TerL), respectively. Whereas gp74 was previously found to possess endonuclease activity (10), its role in the HK97 replication cycle remained uncharacterized. In this study we used functional and bioinformatic analyses to investigate its function.