Real-time visualization of spatial and temporal coordination in resolvase-mediated Holliday junction binding

Area of Science:

• Molecular Biology
• Genetics
• Biophysics

Background:

• Holliday junctions (HJs) are critical intermediates in homologous recombination, essential for maintaining genome stability.
• The bacterial RuvC resolvase is a model enzyme for studying HJ processing.

Purpose of the Study:

• To directly visualize RuvC-DNA interactions at the single-molecule level using high-speed atomic force microscopy (HS-AFM).
• To investigate the role of magnesium ions in RuvC-HJ complex formation and stability.

Main Methods:

• High-speed atomic force microscopy (HS-AFM) for label-free, single-molecule visualization of RuvC-DNA interactions.
• Near-physiological ionic conditions were employed.
• Structural predictions were used to analyze ion effects.

Main Results:

• HS-AFM revealed RuvC exists as monomers and dimers, with dimers binding to HJ-mimicking cruciform DNA.
• Magnesium ions (Mg2+) significantly increased the persistence of RuvC-HJ complexes.
• Structural predictions indicated Mg2+ promotes a coordinated RuvC-HJ interface.

Conclusions:

• HS-AFM provides direct insights into the dynamics of DNA junction binding.
• Magnesium ions play a crucial structural role in stabilizing RuvC-HJ interactions before catalysis, contributing to genome stability.