Enhanced DNA dynamics due to cationic reagents, topological states of dsDNA and high mobility group box 1 as probed by PicoGreen.
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-DeltaA (deletion of 98 N-terminal amino acids) and HMGB1-DeltaC (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.
Noothi
Sunil K
SK
Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India.
Kombrabail
Mamata
M
Kundu
Tapas K
TK
Krishnamoorthy
G
G
Rao
Basuthkar J
BJ
D002412:Cations / Q000737:chemistry; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry; D024243:HMGB1 Protein / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D009930:Organic Chemicals / Q000737:chemistry; D013816:Thermodynamics
D002412:Cations; D005456:Fluorescent Dyes; D024243:HMGB1 Protein; D009930:Organic Chemicals; C099476:PicoGreen; D004247:DNA