Mechanism of integration host factor, a DNA-bending protein, probed with laser temperature-jump

Many cellular processes such as DNA replication, transcription, recombination, gene regulation, and DNA repair involve interactions between proteins and DNA. These interactions often require proteins that recognize specific sites on the DNA, with thousand- or million-fold higher binding affinities than to random DNA sequences. Favorable interactions between protein and DNA are facilitated by localized distortions in DNA at these sites, as well as induced conformational rearrangements in the protein. These concerted changes in proteins and DNA are believed to be a key feature underlying the recognition mechanism.;To elucidate the underlying energetics that leads to the precise recognition of DNA binding sites by proteins, it is essential to study the dynamics of the conformational rearrangements that lead to the tight complex. These dynamics occur on microseconds-to-milliseconds time-scales, making them difficult to capture with conventional stopped-flow techniques. We use laser temperature jump (T jump) to perturb the complex on ∼10 nanosecond time-scale, and time-resolved fluorescence resonance energy transfer (FRET) to monitor the dynamics of the protein-DNA complex.;My research has focused on laser T-jump studies of DNA substrates in complex with Integration Host Factor (IHF), an architectural protein from E. coli that bends it cognate site by nearly 180°. IHF recognizes its specific binding sites primarily by the indirect readout mechanism, in which sequence-dependent DNA flexibility and "bendability" play an important role. I have carried out a series of measurements on IHF with cognate and modified DNA sequences (with inserted nicks and mismatches that enhance the DNA bendability) as well as measurements with IHF mutants that destabilize the complex, to probe the nature of the transition state ensemble separating the nonspecific from the specific complex. The picture that emerges is that the bottleneck in the recognition step is spontaneous bending/kinking of DNA, which is sequence-dependent. Specific protein-DNA interactions that stabilize the final complex do not affect this rate-limiting step. These measurements also reveal a previously undetected rapid phase in the bending kinetics, which remains unaffected by changes in the DNA sequence, and which may represent nonspecific bending of DNA by IHF in its attempt to find its target site in genomic DNA.