HU Induced Perturbation to the Structure and Dynamics of Flexible DNA Substrate Construct

Abstract

Many proteins modulate the structure and dynamics of the bacterial chromosome. One of the most abundant of such proteins is HU (Heat_Unstable) protein, a member of the DNABII family of DNA-binding proteins. An architectural protein, HU introduces sharp bends (120-160 degrees) into the DNA upon binding in a site-specific manner. DNA bending induced by these proteins is important for transcriptional regulation, initiation of replication, Mu transposition, base excision repair, recombination, and negative supercoiling in bacteria. HU exhibits a strong preference for various distortions in DNA, such as nicks, gaps, cruciforms, and sticky ends, all of which share a common motif of a flexible junction. There is little structural or dynamical information related to the perturbation of these DNA motifs upon HU binding. Characterization of HU binding to these motifs provides insight into HU's role in the regulation of DNA structure and also gives insight into the general mode of site-specific recognition and binding for non-sequence-specific DNA binding proteins found in both prokaryotes and eukaryotes. We have employed various fluorescence spectroscopic techniques to address this problem. Through the use of fluorescence anisotropy we have determined the dissociation constant in solution for HU binding to DNA overhang constructs to be 0.3 nM, which is a 1000-fold lower than binding to duplex DNA. The stoichiometry of HU binding specifically to these substrates is one-to-one, while non-specific binding generally leads to larger protein to DNA ratios in bound complexes. Forster resonance energy transfer (FRET) results are indicative of protein-induced bending of the DNA substrate at the duplex/single strand junction and in the single stand region upon HU binding. We further explore the mechanism of HU-induced bending, structural recognition and binding through the use of FRET mapping techniques and time-resolved fluorescence methods.