The structural ensemble of a Holliday junction determined by X-ray scattering interference

Area of Science:

• Molecular Biology
• Biophysics
• Structural Biology

Background:

• The DNA four-way (Holliday) junction is crucial for genetic recombination.
• Its complete conformational and thermodynamic properties, especially under low ionic conditions, are not fully understood.
• Existing methods like smFRET have limitations in resolving conformational ensembles.

Purpose of the Study:

• To fully characterize the conformational ensemble of the Holliday junction across various ionic conditions.
• To investigate the Holliday junction's structure at low and intermediate salt concentrations.
• To assess the utility of X-ray scattering interferometry (XSI) for probing conformational ensembles.

Main Methods:

• Single-molecule Förster resonance energy transfer (smFRET) measurements.
• X-ray scattering interferometry (XSI) across a wide range of ionic conditions.

Main Results:

• smFRET revealed two states under high salt but could not resolve dynamics under low/intermediate salt.
• XSI demonstrated an out-of-plane geometry for the Holliday junction under low ionic conditions.
• XSI identified a previously undetected conformational state at intermediate ionic conditions.

Conclusions:

• The study provides critical insights into the Holliday junction's conformational landscape.
• XSI is a powerful technique for probing conformational ensembles under diverse solution conditions.
• Understanding Holliday junction dynamics is key for comprehending genetic recombination.