Abstract:
We report a study of dynamics with a dsDNA-specific dye called PicoGreen bound to plasmid DNA (3.4 kb), and show that at low dye/DNA phosphate ratios (1 : 100 and below), PicoGreen dynamics reflect the motional dynamics of dsDNA. We further evaluated the usefulness of this probe by measuring the time-resolved fluorescence dynamics of PicoGreen bound to dsDNA in the presence of cationic reagents that affect DNA dynamics [MgCl2 and polyethyleneimine (PEI)] and also with plasmid DNA in different topological states. Among these conditions, MgCl2, PEI and the supercoiled form of plasmid resulted in increases in the very short component (0.2-0.4 ns) of the rotational correlation time. Separately, HMGB1 protein enhanced DNA dynamics, as observed from the rotational correlation times of very short (0.2-0.4 ns) and short (2-4 ns) rotational correlation timescale components. By studying the effect of specific deletion mutants HMGB1-Delta A (deletion of 98 N-terminal amino acids) and HMGB1-Delta C (deletion of 30 C-terminal amino acids), we show that the acidic C-terminal tail is required for enhancement of DNA dynamics. We then discuss the possible mechanisms and implications of HMGB1-mediated flexibility of DNA in the context of formation of large nucleoprotein complexes.
