Binding of Nucleoid-Associated Protein Fis to DNA Is Regulated by DNA Breathing Dynamics
Figure 6
Fis binding site modifications, Langevin dynamics simulations of DNA breathing, and EMSA experiments, the second set.
(A) Langevin dynamics simulations reinforcing the local DNA breathing dynamics in FIS2m3 via three O6-methylguanine modifications in the bubble formation region of FIS2 (Table 1). The direct points-of-contacts remain unchanged. (B) Polyacrylamide gel electrophoresis of dsDNA oligonucleotides sequences - FIS2, FIS2m2, and FIS2m3 - demonstrating gel migratory effects due to possible bubble formation (gel at 15%). (C) Langevin dynamics simulations demonstrating local disruption of the hydrogen bonds in the super-enhanced DNA local openings of the FIS1–FIS2 sequence (Table 1) caused by the presence of five mismatches at the FIS1 bubble formation region. (D) EMSA demonstrating the lack in complex formation in FIS1–FIS2. Concentration of the FIS1 and FIS1–FIS2 oligomers were constant at 100 nM and Fis protein ranged from 0 to 0.75 µM. Sonicated salmon sperm DNA at 0.5–1 µg/µl was added to the binding reactions to eliminate non-specific binding. In Langevin dynamics simulations (panels A and C) the probability of bubble openings is represented by the same color map as in Figure 5; red denotes high probability and blue denotes low probability of opening. The probability is determined from the lifetimes of all open states with a given length (bp) and above amplitude of 1.0 (Å), normalized over the complete time of the simulation. The length of the transient bubbles, given in base pairs [bp], is shown along the vertical axis. The horizontal axis depicts base pair position; the bubble formation region is highlighted in blue while the points-of-contact are highlighted in yellow. The names of each of the sequences are shown in the panels while the complete nucleotide sequences could be found in Table 1.