Cryo electron microscopy of unstained, unfixed RecA-cssDNA complexes

Area of Science:

• Structural Biology
• Molecular Biology
• Biophysics

Background:

• The RecA protein plays a crucial role in DNA repair and recombination by binding to single-stranded DNA (ssDNA).
• Understanding the structural dynamics of RecA-DNA complexes is essential for elucidating its functional mechanisms.
• Previous studies using negative staining provided qualitative insights into RecA-DNA complex structures.

Purpose of the Study:

• To investigate the structural changes in RecA-protein complexes with phi X174 circular single-stranded DNA (cssDNA).
• To quantitatively analyze the helical parameters of RecA-cssDNA complexes in the presence and absence of ATP gamma S.
• To compare frozen-hydrated structures with previously obtained negative-stained data.

Main Methods:

• Formation of RecA-cssDNA complexes with and without ATP gamma S.
• Rapid freezing and embedding of complexes in vitreous ice for cryo-electron microscopy.
• Quantitative image analysis of electron micrographs to determine helical pitch and axial rise.

Main Results:

• Visible helicity was observed in the electron micrographs of frozen-hydrated RecA-cssDNA complexes.
• Quantitative analysis revealed significant increases in helical pitch and axial rise between DNA bases upon ATP gamma S addition.
• Structural parameters showed qualitative agreement but quantitative differences compared to negative-stained specimens.

Conclusions:

• The presence of ATP gamma S induces significant structural alterations in RecA-cssDNA complexes, characterized by increased helicity.
• These findings support the model that RecA-DNA interactions are modulated by the functional state of the complex.
• Cryo-electron microscopy of frozen-hydrated samples provides a more accurate structural representation compared to negative staining.