title: Global structure similarities of intact and nicked DNA complexed with IHF measured in solution by fluorescence resonance energy transfer.
abstract: We have analyzed the structure of two related protein-DNA complexes consisting of integration host factor (IHF) bound to two different versions of the H' site of bacteriophage lambda. Both DNA substrates were 55 bp in length. While one was native duplex the other possessed a nick in one strand at a crucial position within the IHF consensus at the same position as in the reported crystal structure of the DNA-IHF complex. By labeling the 5'-ends of these DNA molecules with donor and acceptor fluorescent dyes, we were able to measure the distance between the dyes by fluorescence resonance energy transfer (FRET) and model DNA distortion. The FRET efficiency decreased from 0.49 +/- 0.01 (nicked DNA) to 0.37 +/- 0.01 (intact DNA) when the gap in the DNA strand was closed. The measured dye-to-dye distance of IHF in complex with nicked DNA was in agreement with the expected value from the crystal structure. Although we found that the two structures were distinguishable, the global shape induced by IHF was retained between the two DNA molecules. Furthermore, our FRET and modeling techniques have sufficiently high resolution to distinguish subtle changes in nucleoprotein complexes with biological relevance.
authors.affiliation: Department of Molecular Biology, Institute for Molecular Biotechnology, PO Box 100813, D-07708 Jena, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Lorenz
authors.forename: A
authors.initials: A
authors.lastname: Hillisch
authors.forename: S D
authors.initials: SD
authors.lastname: Goodman
authors.forename: S
authors.initials: S
authors.lastname: Diekmann
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D034982:Integration Host Factors; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D013050:Spectrometry, Fluorescence
chemical_list: D001426:Bacterial Proteins; D034982:Integration Host Factors; D004247:DNA