Title
Two-step interrogation then recognition of DNA binding site by Integration Host Factor: an architectural DNA-bending protein
Author
Yogambigai Velmurugu, Paula Vivas, Mitchell Connolly, Serguei V Kuznetsov, Phoebe A Rice, Anjum Ansari
Year
2017
Journal
Nucleic Acids Research
Abstract
The dynamics and mechanism of how site-specific DNA-bending proteins initially interrogate potential binding sites prior to recognition have remained elusive for most systems. Here we present these dynamics for Integration Host factor (IHF), a nucleoid-associated architectural protein, using a μs-resolved T-jump approach. Our studies show two distinct DNA-bending steps during site recognition by IHF. While the faster (∼100 μs) step is unaffected by changes in DNA or protein sequence that alter affinity by >100-fold, the slower (1–10 ms) step is accelerated ∼5-fold when mismatches are introduced at DNA sites that are sharply kinked in the specific complex. The amplitudes of the fast phase increase when the specific complex is destabilized and decrease with increasing [salt], which increases specificity. Taken together, these results indicate that the fast phase is non-specific DNA bending while the slow phase, which responds only to changes in DNA flexibility at the kink sites, is specific DNA kinking during site recognition. Notably, the timescales for the fast phase overlap with one-dimensional diffusion times measured for several proteins on DNA, suggesting that these dynamics reflect partial DNA bending during interrogation of potential binding sites by IHF as it scans DNA.
Instrument
J-810
Keywords
Circular dichroism, Secondary structure, DNA binding, Thermal stability, Protein folding, Thermodynamics, Biochemistry