Replication protein A unfolds G-quadruplex structures with varying degrees of efficiency

Area of Science:

• Biochemistry
• Molecular Biology
• Genomics

Background:

• Replication protein A (RPA) interacts with G-rich sequences forming G-quadruplexes (GQs).
• Previous studies focused on homogeneous GQs, leaving RPA's unfolding capabilities on diverse GQs unexplored.
• Heterogeneous GQs in the genome, like those in the tyrosine hydroxylase gene promoter, are biologically relevant.

Purpose of the Study:

• To compare the thermal stability of three potential G-quadruplex-forming sequences (PQSs) with their stability against RPA-mediated unfolding.
• To investigate the correlation between GQ thermal stability and RPA unfolding efficiency.
• To determine the time scale of RPA-mediated GQ unfolding using single-molecule techniques.

Main Methods:

• Single-molecule fluorescence resonance energy transfer (smFRET).
• Bulk biophysical and biochemical experiments.
• Single-molecule stopped-flow method for kinetic analysis.

Main Results:

• GQ thermal stabilities varied significantly (Tm = 69 °C to 86 °C).
• RPA unfolding stability did not correlate directly with thermal stability; the least stable GQ was most resistant to RPA.
• All three GQs were unfolded by RPA within approximately 0.30 ± 0.10 seconds, irrespective of their thermal or RPA unfolding stability.

Conclusions:

• Thermal stability alone is insufficient to predict the physiological viability of G-quadruplex structures.
• The accessibility of the GQ structure to RPA appears to be the rate-limiting step in unfolding.
• RPA unfolds GQs rapidly, suggesting a consistent mechanism across different GQ structures in a cellular context.