To Kink or Not to Kink: Sequence-Dependent DNA Flexibility Unveiled in Complex with DNA-Bending Protein IHF

Abstract

Site-specific recognition by DNA-binding proteins often involves severe DNA deformations including sharp kinks. Yet, it has been unclear how rigid or flexible these protein-induced kinks are. Here we investigated the dynamic nature of DNA in complex with integration host factor (IHF), a nucleoid-associated architectural protein known to bend one of its cognate sites (35 base pair H’) into a U-turn by sharply kinking DNA at two sites by ∼90°. We utilized fluorescence lifetime-based FRET spectroscopy and revealed a surprisingly dynamic IHF-H’ specific complex: while 78% of the IHF-H’ population exhibited FRET efficiency consistent with the crystal structure, 22% exhibited FRET efficiency indicative of partially bent DNA, with one side or the other unkinked. This conformational flexibility was modulated by sequence variations in the cognate site. Notably, IHF bound to another cognate site, H1, that differs from H’ primarily by the lack of an A-tract on one side, exhibited a significantly higher population (68%) in the partially bent conformations. These differences in their dynamics reflect the different roles of these complexes in their natural context, in the phage lambda “intasome”. The population distribution between bent/unbent conformations could be further tuned by introducing mismatches in the DNA at one of the kink sites, to enhance DNA “kinkability” at that site. We propose to take advantage of this FRET assay to further investigate sequence-dependent flexibility at the kink sites by performing SELEX studies for tight-binders to IHF, selected from a pool of semi-randomized sequences, with segments that encompass the kink sites chosen to be randomized for selection of the most “kinkable” (by IHF) DNA sequences. These SELEX studies are expected to go beyond similar studies on nucleosomes and reveal sequence patterns that significantly enhance DNA flexibility.