HU binding to bent DNA: A fluorescence resonance energy transfer and anisotropy study

HU, an architectural DNA-binding protein, either stabilizes DNA in a bent conformation or induces a bend upon binding to give other proteins access to the DNA. In this study, HU binding affinity for a bent DNA sequence relative to a linear sequence was investigated using fluorescence anisotropy measurements. A static bend was achieved by the introduction of two phased A(4)T(4) tracts in a 20 bp duplex. Binding affinity for 20 bp duplexes containing two phased A-tracts in either a 5'-3' or Y-5' orientation was found to be almost 10-fold higher than HU binding to a random sequence 20 bp duplex (6.1 vs 0.68 muM(-1)). The fluorescence technique of resonance energy transfer was used to quantitatively determine the static bend of the DNA duplexes and the HU-induced bend. DNA molecules were 5'-end labeled with fluorescein as the donor or rhodamine as the acceptor. From the efficiency of energy transfer, the end-to-end distance of the DNA duplexes was calculated. The end-to-end distance relative to DNA contour length (R/R-C) yields a bend angle for the A-tract duplex of 45 +/- 7degrees in the absence of HU and 70 +/- 3degrees in the presence of HU. The bend angle calculated for the T(4)A(4) tract duplex was 62 +/- 4degrees after binding two HU dimers. Fluorescence anisotropy measurements reveal that HU binds in a 1: 1 stoichiometry to the A(4)T(4) tract duplex but a 2:1 stoichiometry to the T4A4 tract and random sequence duplex. These findings suggest that HU binding and recognition of DNA may be governed by a structural mechanism.