Terminal residues modulate DNA-binding site size and stability of the SSB-like DNA-binding protein Gp13 from bacteriophage Phi11.

Gp13 is a homodimeric single-stranded DNA-binding protein (SSB) from bacteriophage Phi11, harboring a C-terminal oligonucleotide/oligosaccharide-binding (OB) fold. To study the functional contributions of its terminal regions, a series of N- and C-terminal deletion mutants were generated and characterized using biochemical and biophysical approaches. Deletions in the N-terminal (residues 2-8) and C-terminal (residues 177-184) residues impaired ssDNA binding and reduced the minimal ssDNA binding size from 25 to 20 nucleotides, revealing that both the terminal regions contribute to efficient DNA interaction. Further deletions in the N-terminus abolished DNA binding, highlighting the potential role of residues R6-E12 in ssDNA binding. Size-exclusion chromatography showed that most mutants retained their dimeric state, suggesting that the dimerization domain lies outside Gp13's terminal residues. Circular dichroism and intrinsic fluorescence analyses revealed localized secondary and tertiary changes in the mutants correlating with binding strength. In addition, Gp13 was found to associate with Staphylococcus aureus RecA in pull-down assays, suggesting a potential interaction between Phi11 SSB and host DNA recombination machinery. Together, these results elucidate the role of terminal residues of Gp13 in influencing the structure and ssDNA- binding properties of the protein.