Insight into the Mechanism of Hu-Induced DNA Bending

Abstract

The structure and dynamics of the bacterial chromosome are maintained by many proteins. One of the most abundant proteins, a member of the DNABII family of DNA-binding proteins, is HU (Heat_Unstable). An architectural protein is a term used to describe proteins that introduce sharp bends (120-160 degrees) into DNA upon binding. 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; nicks, gaps, cruciform, and sticky ends. The distorted DNA structures seem to share a similar binding motif of a flexible junction. There is little structural or dynamical information related to perturbation of these DNA motifs upon HU binding. A better understanding of HU binding to these motifs would provide insight into HU’s role in the regulation of DNA structure. This information could also give insight into the general mode of recognition and binding for all non-sequence-specific DNA binding proteins found in both prokaryotes and eukaryotes. We have employed various fluorescence spectroscopy techniques to help answer questions related to this problem. Through the use of fluorescence anisotropy we have determined the dissociation constant in solution for HU binding to 3’overhang DNA substrate to be 3.41E-09+/- 1.2E-09 M, which is in good agreement with literature results. The stoichiometry for the specific binding of HU to these substrates is one to one, while non-specific binding may lead to higher order complexes. Fluorescence resonance energy transfer (FRET) results suggest bending of the DNA substrate upon HU binding. We hope to gain further information about the mechanism of HU-induced bending, structural recognition and binding through the use of such techniques as FRET mapping, Time-resolved fluorescence, and stop flow kinetics.