Dna Bending/Unbending Rates Revealed For Nonspecific Architectural Dna-Binding Protein Ynhp6a

The yeast Nhp6A protein (yNhp6A) is a nonspecific DNA-binding member of the eukaryotic HMGB family of chromatin factors that promote apparent DNA flexibility. yNhp6A sharply bends DNA by >60°. However, it remains unclear whether the protein first binds to unbent DNA and then deforms it, or if pre-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 examine the possibility of a “bind-then-bend” mechanism for HMGB proteins. This key question touches on the fundamental mechanism by which DNA bending is accomplished by architectural proteins, an area illuminated by few experimental results. To explore conditions under which DNA could be unbent within a stable yNhp6A complex, we combined an array of experimental probes: FRET on end-labeled DNA (to monitor DNA unbending), fluorescence anisotropy (to monitor complex dissociation), and circular dichroism (to monitor protein stability), at various ionic and temperature conditions. Our studies reveal that at 100 mM NaCl, an 18-base-pair DNA oligomer remains bound to yNhp6A but starts to unbend, at temperatures up to ∼45 °C; above that temperature, dissociation of the complex and unfolding of the protein is detected. In 200 mM NaCl, DNA straightening in the intact yNhp6A complex is again detected up to ∼35 °C. Microseconds-resolved laser temperature-jump perturbation of the yNhp6A-DNA complex reveal single-exponential FRET loss kinetics that, under conditions dominated by DNA unbending within complexes (below 45 °C at 100 mM NaCl), yield unimolecular DNA bending/unbending rates on timescales of 500 us - 1 ms. These data provide the first direct observation of bending/unbending dynamics of DNA bound to yNhp6A, suggesting a bind-then-bend mechanism for this protein, and the first measurement of these rates for a nonspecific DNA-bending protein.