Ff gene 5 protein has a high binding affinity for single-stranded phosphorothioate DNA.

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• The gene 5 protein (g5p) from Ff bacteriophages is a model ssDNA-binding protein.
• g5p exhibits cooperative binding to ssDNA, with affinity influenced by base composition.
• Previous studies highlight the impact of DNA sequence on g5p binding affinity.

Purpose of the Study:

• To investigate the effect of DNA backbone modification on g5p binding affinity.
• To determine if phosphorothioate modification alters g5p binding stoichiometry or site.
• To elucidate the mechanism behind altered binding affinity due to phosphorothioate modification.

Main Methods:

• Circular Dichroism (CD) titrations to assess protein-DNA binding.
• Comparison of binding affinities (K omega) for unmodified and phosphorothioate-modified DNA oligomers.
• Analysis of binding stoichiometry and spectral changes upon protein interaction.

Main Results:

• Phosphorothioate modification (S-d(A)(36)) increased g5p binding affinity by over 300-fold compared to unmodified DNA (P-d(A)(36)).
• g5p bound phosphorothioate DNA with similar stoichiometry and binding site as unmodified DNA.
• Binding affinity correlated with the extent of phosphorothioate modification, suggesting a sulfur-specific effect.

Conclusions:

• The DNA backbone, specifically phosphorothioate modification, significantly enhances g5p binding affinity.
• The increased affinity is likely attributed to the chemical properties of sulfur within the phosphorothioate group.
• This effect is independent of polyelectrolyte interactions or major alterations in DNA structure.