Staphylococcus aureus single-stranded DNA-binding protein SsbA can bind but cannot stimulate PriA helicase.

Area of Science:

• Molecular Biology
• Structural Biology
• Bacterial DNA Replication

Background:

• Single-stranded DNA-binding proteins (SSBs) and PriA helicases are crucial for bacterial DNA replication restart.
• The interaction mechanism between SSB and PriA in Escherichia coli (Ec) is well-understood, but data for Gram-positive bacteria are scarce.
• This study investigates the interaction between Staphylococcus aureus SSB (SaSsbA) and PriA (SaPriA).

Purpose of the Study:

• To characterize the properties and interaction of SaSsbA with SaPriA.
• To elucidate the molecular basis for the lack of stimulation of SaPriA by SaSsbA, contrasting with Gram-negative bacteria.
• To identify key structural and sequence differences contributing to species-specific interactions.

Main Methods:

• Purification and gel filtration chromatography of SaSsbA.
• X-ray crystallography of SaSsbA to determine its structure.
• In vitro assays to analyze the interaction and functional effects of SaSsbA and SaPriA, including mutant analysis and cross-species complementation with KpSSB.
• Structure-sequence comparisons between SaPriA/SaSsbA and their E. coli/K. pneumoniae counterparts.

Main Results:

• SaSsbA forms a stable tetramer in solution, with a disordered C-terminal domain.
• SaSsbA does not stimulate SaPriA activity, and SaPriA binds SaSsbA but not its C-terminus.
• SaPriA is stimulated by KpSSB, indicating functional differences in PriA binding sites; SaPriA lacks conserved PriA residues (e.g., Arg697) crucial for Gram-negative SSB interaction, possessing a negatively charged Glu767 instead.
• A mutation in SaSsbA (S161F) significantly enhances SaPriA activity, suggesting this residue's role in modulating the interaction.

Conclusions:

• The mechanism of PriA helicase stimulation by SSB differs significantly between Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli, Klebsiella pneumoniae) bacteria.
• Absence of conserved binding sites in SaPriA and SaSsbA, and the presence of a negatively charged residue (Glu767) in SaPriA, preclude stimulation by SaSsbA.
• Specific residues, like S161 in SaSsbA, play a critical role in the interaction, and altering them can restore or enhance PriA activity, underscoring species-specific adaptations in DNA replication restart pathways.