Noothi Sunil K, Kombrabail Mamata, Kundu Tapas K, Krishnamoorthy G, Rao Basuthkar J
Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India.
FEBS J. 2009 Jan;276(2):541-51. doi: 10.1111/j.1742-4658.2008.06802.x. Epub 2008 Dec 11.
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.
我们报告了一项关于动力学的研究，该研究使用一种名为PicoGreen的双链DNA特异性染料与质粒DNA（3.4 kb）结合，并表明在低染料/DNA磷酸比（1:100及以下）时，PicoGreen动力学反映了双链DNA的运动动力学。我们通过测量在影响DNA动力学的阳离子试剂[MgCl2和聚乙烯亚胺（PEI）]存在下与双链DNA结合的PicoGreen的时间分辨荧光动力学，以及不同拓扑状态的质粒DNA的时间分辨荧光动力学，进一步评估了该探针的实用性。在这些条件中，MgCl2、PEI和质粒的超螺旋形式导致旋转相关时间的极短成分（0.2 - 0.4 ns）增加。另外，从极短（0.2 - 0.4 ns）和短（2 - 4 ns）旋转相关时间尺度成分的旋转相关时间观察到，高迁移率族蛋白B1（HMGB1）增强了DNA动力学。通过研究特定缺失突变体HMGB1 - ΔA（缺失98个N端氨基酸）和HMGB1 - ΔC（缺失30个C端氨基酸）的作用，我们表明酸性C端尾巴是增强DNA动力学所必需的。然后，我们在大核蛋白复合物形成的背景下讨论了HMGB1介导的DNA灵活性的可能机制和意义。
