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 A4T4 tracts in a 20 bp duplex. Binding affinity for 20 bp duplexes containing two phased A-tracts in either a 5'-3' or 3'-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 microM(-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 +/- 7 degrees in the absence of HU and 70 +/- 3 degrees in the presence of HU. The bend angle calculated for the T4A4 tract duplex was 62 +/- 4 degrees after binding two HU dimers. Fluorescence anisotropy measurements reveal that HU binds in a 1:1 stoichiometry to the A4T4 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.
Wojtuszewski
Kristi
K
Molecular Biology and Biochemistry Department, Molecular Biophysics Program, Wesleyan University, Middletown, Connecticut 06459-0175, USA.
Mukerji
Ishita
I
D000225:Adenine / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry; D005454:Fluorescence Polarization / Q000379:methods; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding; D013941:Thymine / Q000737:chemistry
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides; C040330:histone-like protein HU, bacteria; D004247:DNA; D000225:Adenine; D013941:Thymine