The C‐terminus of the ssDNA binding protein of phage T7 modulates its ssDNA‐binding activity

Abstract

The gene 2.5 (gp2.5), the ssDNA-binding protein of phage T7, is indispensable for phage growth and a key component of the T7 DNA replication fork. Gp2.5 binds ssDNA, interacts with T7 DNA polymerase, and T7 helicase/primase, and is essential for coordination of leading and lagging strand DNA synthesis. Similar to other prokaryotic ssDNA-binding 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. The presence of an aromatic amino acid as the C-terminal residue of the protein is essential for gp2.5 function and correlates with its ability to physically interact with the gene 5 DNA polymerase. Moderate shortening 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 higher affinities for ssDNA and unmasks dsDNA-binding ability. Mutant proteins, in which the C-terminal phenylalanine is replaced with tyrosine and cysteine, bind long oligonucleotides with much higher affinity than the wild type gp2.5, whereas the affinity for short oligonucleotides is comparable. Models for the biological significance of the modulation of gp2.5 DNA binding properties by the acidic C-terminus are explored. This research is supported from NIH grants GM54397-41 and F32GM072305.