Nucleic Acids Research, Vol 27, Issue 23 4619-4625, Copyright © 1999 by Oxford University Press
M Lorenz, A Hillisch, SD Goodman and S Diekmann
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.
ARTICLES
Global structure similarities of intact and nicked DNA complexed with IHF measured in solution by fluorescence resonance energy transfer
Department of Molecular Biology, Institute for Molecular Biotechnology, PO Box 100813, D-07708 Jena, Germany.
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