SUNY at Albany
June 19-23, 2001
HU Binding to Bent DNA: A Fluorescence Resonance Energy Transfer Study
HU, a multi-functional DNA-binding protein, acts primarily as an architectural protein. The proposed function of HU is stabilization of DNA in a bent conformation or bending of duplex DNA to allow access for other proteins to bind to the DNA and carry out their respective function. Previous fluorescence intensity and anisotropy results from our lab have shown that binding of HU to linear DNA is accompanied by a decrease in stacking interactions indicative of local unwinding and bending of the DNA, supporting the proposal that HU is an architectural protein. These results were observed using an intrinsic, fluorescent guanosine analog placed at various positions in the duplex (1).
Several recent studies have suggested that HU binding affinity is enhanced for nicked, gapped or cruciform DNA, indicating that its binding affinity may be regulated by DNA structure (2, 3). In this study the goal is to assess the affinity of HU for A-tract DNA, which has been shown to exhibit bending, and to determine the degree of bending induced upon HU binding. Fluorescence resonance energy transfer (FRET) was used to quantitatively determine the end-to-end distance of the DNA upon HU binding to a 20 bp duplex containing phased A-tracts (5'-CAAAATTTTGGAAAATTTTC-3') relative to a linear non-A-tract (5'-CTTTTAAAAGGTTTTAAAAC-3') sequence. DNA molecules were labeled at the 5'-end with either fluorescein as the donor or rhodamine as the acceptor. These results show that in the absence of HU, the end-to-end distance in the A-tract duplex is shorter than that predicted from the structural parameters of B-form DNA, indicative of sequence-induced bending. Interestingly, HU binding to the A-tract duplex does not significantly change the end-to-end distance. Fluorescence anisotropy measurements reveal almost a 20-fold increase in HU binding affinity for the A-tract and non-A-tract duplex relative to a random linear duplex (Ka= 6.1 /mM vs. 0.33 /mM). Stoichiometry measurements indicate that one HU dimer binds to one 20 bp A-tract duplex and two HU dimers binding to one 20 bp non-A-tract duplex. We suggest that HU binding exhibits a structural specificity that is demonstrated by the A-tract sequence as shown by the 1:1 binding stoichiometry. These two sequences will be studied further to determine key structural differences between the two binding interactions.References and Footnotes
Kristi Wojtuszewski and Ishita Mukerji;
Wesleyan University, Molecular Biology and Biochemistry Department, Molecular Biophysics Program, Middletown, CT 06459