Evidence for a bind-then-bend mechanism for architectural DNA binding protein yNhp6A

Area of Science:

• Molecular Biology
• Biophysics
• Chromatin Structure

Background:

• The yeast Nhp6A protein (yNhp6A) is a high-mobility group box (HMGB) protein known to bind DNA and induce sharp bends.
• The mechanism by which yNhp6A interacts with DNA, specifically whether it binds to unbent DNA and then bends it or captures pre-bent conformations, remains unclear.

Purpose of the Study:

• To investigate the conformational dynamics of DNA bound by yNhp6A under various solution conditions.
• To determine if yNhp6A can bind to DNA that is not significantly bent.
• To elucidate the kinetic mechanism of DNA bending and unbending by yNhp6A.

Main Methods:

• Utilized an array of conformational probes including Förster Resonance Energy Transfer (FRET), fluorescence anisotropy, and circular dichroism.
• Employed microsecond-resolved laser temperature-jump perturbation to study the yNhp6A-DNA complex dynamics.
• Analyzed relaxation kinetics to determine unimolecular DNA bending/unbending rates.

Main Results:

• Demonstrated that yNhp6A-bound DNA can remain unbent in solution under specific salt concentrations (100 mM and 200 mM NaCl) as temperature is increased.
• Observed DNA unbending within the intact yNhp6A complex up to approximately 45°C (100 mM NaCl) and 35°C (200 mM NaCl) without significant complex dissociation.
• Measured DNA bending/unbending rates on the microsecond to millisecond timescale, providing direct observation of these dynamics.

Conclusions:

• The findings provide the first direct evidence of DNA bending and unbending dynamics in complex with yNhp6A.
• The results support a 'bind-then-bend' mechanism, where yNhp6A initially binds to DNA and subsequently induces bending.
• This study offers critical insights into the molecular mechanisms of HMGB proteins in modulating DNA structure and chromatin organization.