76 Protein–DNA binding and breathing dynamics of DNA

Abstract

In the living cell, the double-stranded DNA molecule experiences thermal motions that induce spontaneous openings and re-closings of the double helix known as “DNA breathing,” or “DNA transient bubbles” (Englander et al., 1980). The propensity for breathing is interconnected with DNA local stability and flexibility (Vafabakhsh & Ha, 2012), which play a key role in DNA biological function (Bishop et al., 2012). Here, we investigate the relationship between the DNA local propensity for breathing and binding of two transcription factors (TFs): (i) the human TF YY1, (see e.g. Usheva & Shenk, 1996), and (ii) the nucleoid-associated protein Fis in Escherichia coli (see e.g. Finkel & Johnson, 1992). Using a mesoscopic nonlinear model of double-stranded DNA (Peyrard & Bishop, 1989) that can be augmented for rational design of DNA breathing (Alexandrov et al., 2010), we have simulated the dynamics of known Fis- and YY1-binding sites, analyzed published in vitro and genomic data-sets, and conducted targeted experimental tests of our predictions (Alexandrov et al., 2012; Nowak-Lovato et al., in press). We found a strong correlation between the propensity for breathing (at the binding sites) and YY1/Fis binding. We identified a breathing profile that is characteristic for a strong Fis-binding site that is significantly enriched among the identified in vivo E. coli Fis-binding sites. To test our understanding of how Fis binding is influenced by the breathing, we designed base-pair substitutions, mismatch, and O6-Guanine methylation modifications of nucleotides, in sequences that are known to interact (or not interact) with Fis, seeking to make the breathing either closer to or farther from the breathing profile of a strong Fis-binding site. For the modified DNA segments, we found that Fis-DNA binding, as assessed by EMSA, changed in accordance with our expectations. Further, by using site-specific chromatin immunopecipitations, BIOBASE data, and simulations, we also found a specific breathing profile at the binding cites of YY1 in vivo. Our finding suggests that the genomic-flanking sequence variations and SNPs presence may exert long-range effects on DNA breathing and predetermine YY1 binding in cells.