Visualization of RecA protein and its complexes with DNA by quick-freeze/deep-etch electron microscopy

Area of Science:

• Structural Biology
• Molecular Biophysics
• Biochemistry

Background:

• RecA protein plays a critical role in DNA repair and recombination.
• Understanding RecA protein's conformational states is key to elucidating its function.
• Previous studies suggested RecA protein forms helical structures, but distinct forms were not clearly defined.

Purpose of the Study:

• To clearly differentiate between the two helical polymer forms of RecA protein.
• To investigate the role of ATP and its analogs in RecA protein polymerization.
• To understand the structural basis of RecA protein's DNA binding and conformational changes.

Main Methods:

• Freeze-etch electron microscopy was employed to visualize RecA protein structures.
• Analysis included pure RecA protein aggregates and complexes with single- and double-stranded DNA.
• Experiments were conducted with various nucleotides, including ATP, non-hydrolyzable ATP analogs, and ADP.

Main Results:

• Two distinct RecA protein helices were identified: a 'long pitch' (9.5 nm pitch, 10 nm diameter) and a 'short pitch' (6 nm pitch, 12 nm diameter).
• The 'long pitch' helix is DNA-bound, while the 'short pitch' helix exists in pure RecA protein polymers and paracrystals.
• ATP binding, particularly with non-hydrolyzable analogs, induces dissociation of pure RecA protein polymers into monomer chains and rings, and stabilizes the 'long pitch' DNA-bound form.

Conclusions:

• ATP acts as a reversible allosteric effector, modulating RecA protein's polymeric structure.
• Cyclic interconversion between short- and long-pitch conformations likely mediates reversible DNA interactions during homology search.
• The findings provide structural insights into RecA protein's mechanism in DNA recombination and repair.