The protein gp74 from the bacteriophage HK97 functions as a HNH endonuclease

Abstract

The last gene in the genome of the bacteriophage HK97 encodes the protein gp74. We present data in this article that demonstrates, for the first time, that gp74 possesses HNH endonuclease activity. HNH endonucleases are small DNA binding and digestion proteins characterized by two His residues and an Asn residue. We demonstrate that gp74 cleaves lambda phage DNA at multiple sites and that gp74 requires divalent metals for its endonuclease activity. We also present intrinsic tryptophan fluorescence data that show direct binding of Ni2+ to gp74. The activity of gp74 in the presence of Ni2+ is significantly decreased below neutral pH, suggesting the presence of one or more His residues in metal binding and/or DNA digestion. Surprisingly, this pH‐dependence of activity is not seen with Zn2+, suggesting a different mode of binding of Zn2+ and Ni2+. This difference in activity may result from binding of a second Zn2+ ion by a putative zinc finger in gp74 in addition to binding of a Zn2+ ion by the HNH motif. These studies define the biochemical function of gp74 as an HNH endonuclease and provide a platform for determining the role of gp74 in life cycle of the bacteriophage HK97.

HNH proteins are a widespread component of phage DNA packaging machines

“…The requirement for the metal-binding residue His82 of gp74 for function supports this idea. We previously demonstrated a nonspecific endonuclease activity for gp74 (10), but this activity does not appear to be involved in its terminase-related function as the in vitro assays in this study were performed under conditions where this nonspecific activity was not strong (low gp74 concentration and lack of Ni 2+ or Zn 2+ ions). One possible role that gp74 may play is to nick a single strand of the DNA genome at the cos site, with the TerL-TerS complex providing the nick on the other strand.…”

“…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.…”

“…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.…”

“…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 Protein Interactome of Mycobacteriophage Giles Predicts Functions for Unknown Proteins

“…gp76 has similarity to HNH endonucleases. HNH motifcontaining endonuclease proteins may interact with phage terminase proteins to promote phage DNA packaging and maturation (31). For example, a recent report showed that gp74 (an HNH endonuclease) of phage HK97 interacts with its terminase protein (32) and that the HNH motif of endonuclease is essential for interacting with terminase and completion of DNA packaging.…”