The mechanism of Single strand binding protein-RecG binding: Implications for SSB interactome function

Area of Science:

• Molecular Biology
• Genetics
• Protein Structure

Background:

• Escherichia coli single-strand DNA binding protein (SSB) is crucial for DNA replication, repair, and recombination.
• SSB interacts with numerous proteins, forming a complex interactome essential for cellular viability.
• The intrinsically disordered linker region of SSB plays a key role in mediating interactions with both DNA and partner proteins.

Purpose of the Study:

• To elucidate the molecular mechanisms underlying SSB interactome function.
• To investigate the role of conserved PXXP motifs and the C-terminal acidic tip in SSB-partner protein interactions.
• To characterize the structural and functional significance of the SSB interactome as a family of genome guardians.

Main Methods:

• Site-directed mutagenesis to alter PXXP motifs and RecG OB-fold.
• Deletion analysis of partner OB-fold domains.
• Biochemical assays to assess SSB binding to DNA and interactome partners.

Main Results:

• Single point mutations in PXXP motifs or the RecG OB-fold abolished SSB binding.
• Deletion of partner OB-fold domains also eliminated SSB binding.
• Contrary to existing views, the C-terminal acidic tip of SSB is not required for interactome partner binding; it regulates conformation and serves as a secondary binding domain.

Conclusions:

• The PXXP motifs within the SSB linker are critical for mediating interactions with OB-fold domains in SSB and its partners.
• The C-terminal acidic tip has a regulatory role in maintaining SSB structure and acts as an additional binding site.
• The SSB interactome represents the first identified family of OB-fold genome guardians in prokaryotes, highlighting a conserved mechanism for genome maintenance.