Introduction

Static Kinks or Flexible Hinges: Multiple Conformations of Bent DNA Bound to Integration Host Factor Revealed by Fluorescence Lifetime Measurements

“…At 100 mM KCl, the average FRET efficiency in the H' DNA construct alone was found to be 0.022 ± 0.001, which increased to 0.50 ± 0.03 upon binding of IHF under 1:1 binding conditions. These FRET efficiency values are consistent with steady-state values reported previously for these constructs, 61,[69][70] and reflect the decrease in the end-to-end distance when H' DNA is bent into a U-turn in the complex, as illustrated in the crystal structure of the IHF-H' complex (Figure 1).…”

“…89 Further kinetics studies resolved a two-step "interrogation-then-recognition" process: nonspecific interrogation on ~100-500 µs timescale prior to recognition on 1-10 ms timescale. 70 However, these "ensemble" approaches could only provide an average picture of the dynamics along the reaction trajectory. What remained elusive was whether multiple conformations of the complex could co-exist in solution, as was recently shown for damaged DNA specifically bound to NER damage-recognition protein XPC/Rad4.…”

“…In the case of IHF-DNA complexes, FRET between labels attached at the ends of 35-mer DNA substrates has proven to be exceptionally useful in measuring not only equilibrium changes from unbent (straight) to bent conformations upon binding of IHF, 68 but also the dynamics of these conformational changes along the recognition trajectory of the IHF binding site. 59,62,[69][70] Here, we take advantage of the sub-nanosecond time-resolution of the fluorescence lifetime studies that enables fluorescence decays to be measured over a wide temporal range and with high temporal resolution. A single DNA conformation in the ensemble of molecules, with a well-defined separation and relative orientation between the donor and acceptor of the FRET pair, is characterized by a unique FRET efficiency, and is expected to yield one (faster) exponential decay for the donor fluorescence in the presence of the acceptor for every exponential decay observed in the absence of the acceptor.…”

“…The 4-nt mismatch bubble is expected to enhance the "kinkability" of the H1 sequence on that side and increase the IHF binding affinity, as was shown previously for similar mismatched sequences in the H' context. 56,59,70 Binding affinity measurements with end-labeled H1_CTloop showed a ~125-fold decrease in K d compared with matched H1…”

“…(measured at 300 mM KCl; SI Figure S5 and Table 1), a result consistent with previous K d measurements on H' sequence with a TT/TT mismatch introduced at the same kink site. 56,59,70 We anticipated that the insertion of the mismatch in H1 to lower the energetic penalty for kinking should help compensate for the lack of stabilizing interactions afforded by the A-tract. In other words, we expected to recover some of the lost high FRET state in IHF-H1 in the presence of the mismatch.…”