PMID:12627977
| Citation |
Wojtuszewski, K and Mukerji, I (2003) HU binding to bent DNA: a fluorescence resonance energy transfer and anisotropy study. Biochemistry 42:3096-104 |
|---|---|
| Abstract |
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. |
| Links |
PubMed Online version:10.1021/bi0264014 |
| Keywords |
Adenine/chemistry; Bacterial Proteins/chemistry; DNA/chemistry; DNA-Binding Proteins/chemistry; Escherichia coli Proteins/chemistry; Fluorescence Polarization/methods; Fluorescence Resonance Energy Transfer/methods; Nucleic Acid Conformation; Nucleic Acid Heteroduplexes/chemistry; Oligonucleotides/chemistry; Protein Binding; Thymine/chemistry |
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