M. Lorenz et al., Global structure similarities of intact and nicked DNA complexed with IHF measured in solution by fluorescence resonance energy transfer, NUCL ACID R, 27(23), 1999, pp. 4619-4625
We have analyzed the structure of two related protein-DNA complexes consist
ing 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 re
ported 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 fro
m 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 compl
ex with nicked DNA was in agreement with the expected value from the crysta
l structure. Although we found that the two structures were distinguishable
, the global shape induced by IHF was retained between the two DNA molecule
s. Furthermore, our FRET and modeling techniques have sufficiently high res
olution to distinguish subtle changes in nucleoprotein complexes with biolo
gical relevance.