Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208-3500, USA.
Nucleic Acids Res. 2011 Mar;39(6):2249-59. doi: 10.1093/nar/gkq1140. Epub 2010 Nov 21.
The multistep kinetics through which DNA-binding proteins bind their targets are heavily studied, but relatively little attention has been paid to proteins leaving the double helix. Using single-DNA stretching and fluorescence detection, we find that sequence-neutral DNA-binding proteins Fis, HU and NHP6A readily exchange with themselves and with each other. In experiments focused on the Escherichia coli nucleoid-associated protein Fis, only a small fraction of protein bound to DNA spontaneously dissociates into protein-free solution. However, if Fis is present in solution, we find that a concentration-dependent exchange reaction occurs which turns over the bound protein, with a rate of k(exch) = 6 × 10(4) M(-1)s(-1). The bacterial DNA-binding protein HU and the yeast HMGB protein NHP6A display the same phenomenon of protein in solution accelerating dissociation of previously bound labeled proteins as exchange occurs. Thus, solvated proteins can play a key role in facilitating removal and renewal of proteins bound to the double helix, an effect that likely plays a major role in promoting the turnover of proteins bound to DNA in vivo and, therefore, in controlling the dynamics of gene regulation.
DNA 结合蛋白与其靶标结合的多步动力学过程受到了广泛研究,但相对较少关注蛋白质离开双螺旋的过程。使用单链 DNA 拉伸和荧光检测,我们发现序列中性的 DNA 结合蛋白 Fis、HU 和 NHP6A 很容易与自身和彼此进行交换。在针对大肠杆菌核相关蛋白 Fis 的实验中,只有一小部分结合到 DNA 上的蛋白质会自发地离解到无蛋白质的溶液中。然而,如果 Fis 存在于溶液中,我们发现会发生浓度依赖性的交换反应,从而使结合的蛋白质发生周转,周转率为 k(exch) = 6 × 10(4) M(-1)s(-1)。细菌 DNA 结合蛋白 HU 和酵母 HMGB 蛋白 NHP6A 都显示出相同的现象,即溶液中的蛋白质会加速先前结合的标记蛋白质的解离,从而发生交换。因此,溶解的蛋白质可以在促进双螺旋上结合的蛋白质的去除和更新方面发挥关键作用,这种效应可能在促进体内与 DNA 结合的蛋白质的周转率方面发挥主要作用,从而控制基因调控的动力学。