Essential Residues in the C Terminus of the Bacteriophage T7 Gene 2.5 Single-stranded DNA-binding Protein

Abstract

Gene 2.5 of bacteriophage T7 encodes a single-stranded DNA (ssDNA)-binding protein (gp2.5) that is an essential component of the phage replisome. Similar to other prokaryotic ssDNAbinding proteins, gp2.5 has an acidic C terminus that is involved in protein-protein interactions at the replication fork and in modulation of the ssDNA binding properties of the molecule. We have used genetic and biochemical approaches to identify residues critical for the function of the C terminus of gp2.5. The presence of an aromatic residue in the C-terminal position is essential for gp2.5 function. Deletion of the C-terminal residue, phenylalanine, is detrimental to its function, as is the substitution of this residue with non-aromatic amino acids. Placing the C-terminal phenylalanine in the penultimate position also results in loss of function. Moderate shortening of the length of the acidic portion of the C terminus is tolerated when the aromatic nature of the C-terminal residue is preserved. Gradual removal of the acidic C terminus of gp2.5 results in a higher affinity for ssDNA and a decreased ability to interact with T7 DNA polymerase/thioredoxin. The replacement of the charged residues in the C terminus with neutral amino acids abolishes gp2.5 function. Our data show that both the C-terminal aromatic residue and the overall acidic charge of the C terminus of gp2.5 are critical for its function.

Acidic C-terminal tail of the ssDNA-binding protein of bacteriophage T7 and ssDNA compete for the same binding surface

“…Similar to ssDNA, the removal of the acidic C-terminal tail results in higher affinities for these substrates. Deletion analysis of the C-terminal tail of gp2.5 demonstrated that the gradual removal of charged residues results in the gradual increase of the affinity for ssDNA (31). This observation provides further evidence that the C-terminal tail shields the ssDNA-binding cleft.…”

“…gp2.5 lacking its C-terminal phenylalanine is defective in DNA replication and is unable to grow in E. coli (31). In vitro, gp2.5 is defective in its interaction with T7 DNA polymerase in vitro.…”

Molecular Genetic and Biochemical Characterization of the Vaccinia Virus I3 Protein, the Replicative Single-Stranded DNA Binding Protein

“…Interestingly, it was the acidic C= terminus of I3 that was shown to bind to the small subunit of the ribonucleotide reductase. Many SSBs have an acidic C terminus, and this region has been shown to coordinate interactions with a plethora of core components of the replication apparatus as well as auxiliary proteins involved in diverse aspects of DNA metabolism (20,31,48). I3 has very recently been shown to bind to the cellular translation initiation factor eIF4G, which relocalizes to viral factories after infection (60).…”

Flap Endonuclease Activity of Gene 6 Exonuclease of Bacteriophage T7

“…gp2.5 can stably bind to the 20-nt 5Ј-ssDNA tail, but the 5 nt ssDNA is not sufficient for binding. gp2.5 can bind to the 10-nt 5Ј-ssDNA, but the association is not stable (49). The flap endonuclease activity of gp6 on any of these substrates was not stimulated by gp2.5 over a range of concentrations (40,200, and 1000 nM) (Fig.…”