DNA-ETHIDIUM REACTION-KINETICS - DEMONSTRATION OF DIRECT LIGAND TRANSFER BETWEEN DNA BINDING-SITES
DNA-ETHIDIUM REACTION-KINETICS - DEMONSTRATION OF DIRECT LIGAND TRANSFER BETWEEN DNA BINDING-SITES
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DOI：
10.1016/0022-2836(75)90339-3
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发表时间：
1975-01-01
影响因子：
5.6
通讯作者：
CROTHERS, DM
中科院分区：
文献类型：
--
作者：
BRESLOFF, JL;CROTHERS, DM
对乙锭与DNA相互作用的弛豫动力学的研究揭示了一种新的和潜在的重要的一般结合机制，即在DNA结合位点之间直接转移配体而不需要解离成游离配体。可测量的弛豫光谱显示三个弛豫时间，表明三种结合的染料物种存在于平衡状态;约80%的染料是在主要的插层形式。对于每个弛豫的倒数弛豫时间随浓度线性变化，直到非常高的DNA浓度。在高浓度下较长的弛豫时间到平台的失败可以通过包括从一种复合物形式转化为另一种的双分子途径来解释。我们设想这是将与一个DNA分子结合的乙锭分子直接转移到另一个DNA分子上的空结合位点。这种直接转移机制的其他证据来自一项实验，该实验显示DNA（其相对快速地结合乙锭）加速乙锭与poly（rA）· poly（rU）的结合，推测是通过首先形成DNA-乙锭复合物，然后将乙锭转移到RNA。转移的双分子速率常数被发现是约四倍大于插入的自由染料的常数。因此，转移途径提供了一种高效的手段，使配体在其DNA结合位点上平衡，特别是在高聚合物浓度下。DNA结合调节蛋白的直接转移的潜在重要性被强调。
Study of the relaxation kinetics of the interaction of ethidium and DNA reveals a novel and potentially important general binding mechanism, namely direct transfer of the ligand between DNA binding sites without requiring dissociation to free ligand. The measurable relaxation spectrum shows three relaxation times, indicating that three bound dye species are present at equilibrium; about 80% of the dye is in the major intercalated form. For each relaxation the reciprocal relaxation time varies linearly with concentration up to very high DNA concentrations. The failure of the longer relaxation times to plateau at high concentration can be accounted for by including a bimolecular pathway for conversion from one complex form to another. This we envisage as direct transfer of an ethidium molecule, bound to one DNA molecule, to an empty binding site on another DNA molecule. Additional evidence for this direct transfer mechanism was obtained from an experiment showing that DNA (which binds ethidium relatively rapidly) accelerates the binding of ethidium to poly(rA) · poly(rU), presumably by first forming a DNA-ethidium complex and then transferring the ethidium to RNA. The bimolecular rate constant for transfer is found to be about four times larger than the constant for intercalating the free dye. The transfer pathway thus provides a highly efficient means for the ligand to equilibrate over its DNA binding sites, especially at high polymer concentration. The potential importance of direct transfer for DNA-binding regulatory proteins is emphasized.