Two binding modes in Escherichia coli single strand binding protein-single stranded DNA complexes. Modulation by NaCl...

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Escherichia coli single-strand binding protein (SSB) is crucial for DNA replication, recombination, and repair.
• SSB's interaction with single-stranded nucleic acids is fundamental to its cellular functions.
• Previous studies reported variable binding site sizes for SSB, leading to apparent discrepancies.

Purpose of the Study:

• To investigate the binding properties of E. coli SSB to various single-stranded nucleic acids.
• To determine the influence of NaCl concentration on SSB binding site size and mode.
• To propose a model explaining the observed binding behaviors of SSB.

Main Methods:

• Monitoring SSB binding to synthetic homopolynucleotides and M13 DNA using fluorescence quenching of tryptophan residues.
• Analyzing the effect of varying NaCl concentrations (from mM to M range) on SSB binding.
• Correlating changes in fluorescence quenching with alterations in the occluded binding site size.

Main Results:

• SSB binding site size (n) is highly dependent on NaCl concentration, exhibiting two distinct states: n = 33 ± 3 (low salt) and n = 65 ± 5 (high salt).
• Fluorescence quenching by poly(dT) shows two-state behavior (51% in low salt, 83% in high salt), correlating with site size changes.
• A model is proposed where SSB binds in two modes: (SSB)33 (two protomers) and (SSB)65 (four protomers), with intermediate concentrations showing a mixture.

Conclusions:

• E. coli SSB utilizes at least two distinct binding modes to single-stranded nucleic acids, modulated by physiological NaCl concentrations.
• The reversible transition between these modes, (SSB)33 and (SSB)65, suggests a mechanism for regulating SSB's diverse roles in DNA metabolism.
• In vivo ionic strength fluctuations may play a significant role in controlling SSB's functions during DNA replication, repair, and recombination.