On the kinetics of distamycin binding to its target sites on duplex DNA
On the kinetics of distamycin binding to its target sites on duplex DNA
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DOI：
10.1073/pnas.97.14.7814
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发表时间：
2000-07-05
影响因子：
11.1
通讯作者：
Crothers, DM
中科院分区：
文献类型：
--
作者：
Baliga, R;Crothers, DM
偏端霉素 A 是一种众所周知的聚酰胺抗生素，可以作为单体或并排反平行二聚体结合在双链 DNA 的小沟中，主要结合在富含 AT 的序列上。由于在监测快速双分子反应所需的低浓度下缺乏足够的 UV 或 CD 信号，偏端霉素结合反应的缔合相尚未以其任何一种结合模式进行研究。我们报告称，偏端霉素与其特定靶位点结合时，荧光振幅显着增加，并伴有小红移。该信号可用于监测稳态和时间分辨过程中的药物结合。偏端霉素与 1:1 结合位点的结合速度极快，双分子速率为 7 x 10(7) M-1.s(-1)，并且解离速度相当快（大约 3 s(-1)）。当 DNA 过量时，缔合反应中有一个缓慢的成分，其速率随着 DNA 浓度的增加而急剧下降。药物与 2:1 位点的结合分两个不同的步骤进行：每个药物分子以与 1:1 位点相当的双分子速率快速、连续地与 DNA 结合，然后缓慢（大约 4 S-1）与最终群体平衡。 2:1 位点的解离速度比 1:1 位点的解离速度慢大约 40 倍。这项研究为分析较长聚酰胺和共价连接聚酰胺的结合动力学奠定了基础，最近已证明这些聚酰胺可以抑制体内转录。
Distamycin A is a well known polyamide antibiotic that can bind in the minor groove of duplex DNA primarily at AT-rich sequences both as a monomer or as a side-by-side antiparallel dimer. The association phase of the distamycin binding reaction has not been studied in either of its binding modes, because of the lack of an adequate UV or CD signal at the low concentrations needed to monitor the fast bimolecular reaction. We report a significant increase in fluorescence amplitude, accompanied by a small red shift, on binding distamycin to its specific target sites. This signal can be used to monitor drug binding in steady-state and time-resolved processes. Distamycin shows extremely fast association with the 1:1 binding site, with a bimolecular rate of 7 x 10(7) M-1.s(-1) and also fairly rapid dissociation (approximate to 3 s(-1)). When DNA is in excess, there is a slow component in the association reaction whose rate decreases strongly with increasing DNA concentration. Binding of the drug to the 2:1 site occurs in two distinct steps: fast, sequential binding of each drug molecule to the DNA with a bimolecular rate comparable to that at the 1:1 site, followed by a slow (approximate to 4 S-1) equilibration to the final population. Dissociation from the 2:1 site is approximate to 40-fold slower than from the 1:1 site. This study provides the groundwork for analysis of the binding kinetics of longer polyamides and covalently linked polyamides that have recently been shown to inhibit transcription in vivo.