Title
Evidence for a bind-then-bend mechanism for architectural DNA binding protein yNhp6A
Author
Manas Kumar Sarangi, Viktoriya Zvoda, Molly Nelson Holte, Nicole A Becker, Justin P Peters, L James Maher III, Anjum Ansari
Year
2019
Journal
Nucleic Acids Research
Abstract
The yeast Nhp6A protein (yNhp6A) is a member of the eukaryotic HMGB family of chromatin factors that enhance apparent DNA flexibility. yNhp6A binds DNA nonspecifically with nM affinity, sharply bending DNA by >60°. It is not known whether the protein binds to unbent DNA and then deforms it, or if bent DNA conformations are ‘captured’ by protein binding. The former mechanism would be supported by discovery of conditions where unbent DNA is bound by yNhp6A. Here, we employed an array of conformational probes (FRET, fluorescence anisotropy, and circular dichroism) to reveal solution conditions in which an 18-base-pair DNA oligomer indeed remains bound to yNhp6A while unbent. In 100 mM NaCl, yNhp6A-bound DNA unbends as the temperature is raised, with no significant dissociation of the complex detected up to ∼45°C. In 200 mM NaCl, DNA unbending in the intact yNhp6A complex is again detected up to ∼35°C. Microseconds-resolved laser temperature-jump perturbation of the yNhp6a–DNA complex revealed relaxation kinetics that yielded unimolecular DNA bending/unbending rates on timescales of 500 μs−1 ms. These data provide the first direct observation of bending/unbending dynamics of DNA in complex with yNhp6A, suggesting a bind-then-bend mechanism for this protein.
Instrument
J-810
Keywords
Circular dichroism, Secondary structure, DNA binding, Thermal stability, DNA folding, Chemical stability, Thermodynamics, Biochemistry