Probing DNA Bending Kinetics by yNhp6A with Ultrafast Temperature Jump Spectroscopy

Abstract

The yeast Nhp6A protein is a member of the eukaryotic HMGB family of chromatin factors that enhance apparent DNA flexibility in many cellular processes. yNhp6A exhibits no sequence-specificity but binds to DNA with 1-10 nM affinity, sharply bending the DNA by >60° at the binding site. The kinetic mechanism by which this DNA deformation occurs remains unclear. It is not known whether the protein first binds weakly to unbent DNA and then deforms the DNA or if partially bent DNA conformations are thermally accessible and are “captured” and stabilized by the bound protein. The limited (tens-of-milliseconds) time resolution of previous kinetics studies was insufficient to observe the dynamics of DNA deformations in this complex. Here, we report time-resolved FRET measurements on yNhp6A bound to an 18-bp DNA oligomer labeled at each end with Cy3/Cy5, in response to a laser temperature-jump (T-jump) perturbation. The us temporal resolution of T-jump, together with ionic-strength and concentration dependence of equilibrium FRET and anisotropy measurements, helps reveal some of the microscopic kinetics steps. Equilibrium measurements with varied Nhp6A-DNA concentrations indicate that, at 250mM NaCl, the decrease in FRET with increasing temperature, in the range 15-60°C, is from unimolecular unbending of DNA. Kinetics traces measured under these salt conditions are single-exponential with relaxation times ranging from ∼400 us (at 60°C) to ∼1ms (at 45°C). At lower [salt] 65°C, bi-exponential kinetics are observed, likely corresponding to unimolecular bending/unbending, followed by dissociation of the complex and concurrent DNA melting. These results represent the first observation of DNA bending/unbending dynamics in complex with a nonspecific DNA-binding protein, and are an important step towards a more comprehensive understanding of the kinetic mechanisms of DNA binding and bending interactions by this class of proteins.