A two-state conformational equilibrium for alternating (A-T)n sequences in negatively supercoiled DNA

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Alternating adenine-thymine ((A-T)n) tracts in supercoiled plasmids can adopt non-B DNA structures, such as cruciforms.
• Understanding these structural transitions is crucial for comprehending DNA replication, repair, and gene regulation.

Purpose of the Study:

• To investigate the modification patterns of osmium tetroxide in supercoiled plasmids with alternating (A-T)n tracts.
• To elucidate the influence of salt concentration, temperature, and cations on DNA structural transitions.

Main Methods:

• Treatment of supercoiled plasmids with alternating (A-T)n tracts using osmium tetroxide.
• Analysis of modification patterns under varying conditions of salt concentration, temperature, and cation presence.
• Interpretation of results based on DNA structural models, including cruciforms and perturbed helical structures.

Main Results:

• Two distinct osmium tetroxide modification patterns were observed, dependent on experimental conditions.
• At moderate salt or low temperatures, modification was localized to the center of (A-T)n tracts, indicating cruciform formation.
• At higher temperatures without cations, uniform modification was observed, suggesting a perturbed helical structure.
• Cation effectiveness in stabilizing cruciforms varied with charge; some transition metals were ineffective.
• A third modification pattern emerged for longer (A-T)n tracts at intermediate salt concentrations, suggesting a disrupted four-way junction in the cruciform.

Conclusions:

• DNA structural transitions in (A-T)n tracts are sensitive to environmental factors like temperature and salt.
• A two-state equilibrium model explains the interconversion between cruciform and perturbed helical structures.
• The stability and structure of cruciforms are modulated by cation charge, with implications for DNA-protein interactions.