Pausing of DNA polymerases on duplex DNA templates due to ligand binding in vitro.
Pausing of DNA polymerases on duplex DNA templates due to ligand binding in vitro.
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DOI：
10.1016/s0022-2836(03)00044-5
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发表时间：
2003-02
影响因子：
5.6
通讯作者：
I. Smolina;V. Demidov;M. Frank-Kamenetskii
中科院分区：
文献类型：
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作者：
I. Smolina;V. Demidov;M. Frank-Kamenetskii
利用最近发展的肽核酸(PNA)辅助分析方法，我们研究了DNA结合配体对DNA聚合酶沿双链DNA模板移动的序列特异性抑制。为此，我们制备了带有两个侧翼引发点的多连接体为特征的质粒载体，以双向启动相互之间的引物延伸反应。在这个质粒中，我们克隆了一组随机的DNA序列，并分析了这些反应的产物与几个能够进行链置换合成的噬菌体和细菌DNA聚合酶。在DNA结合配体存在的情况下，发现其中的两种酶，？29和改良的T7(Sequenase 2.0)酶对扩增的作用最强。我们使用这些酶来详细研究配体诱导的停顿效应，其中四种配体与DNA双螺旋的结合方式不同。GC专一性的插层剂放线菌素D和三种次要的沟槽粘结剂，色霉素A3(GC专一性)、地塞米松A和Netropsin(都是AT专一性的)已经被选择。在每个配体的存在下，两个选定的DNA聚合酶都经历了多次明确的停顿。对于特定的DNA序列，每个配体都产生了自己特有的停顿模式。大多数停顿位点可以单核苷酸分辨定位，并对应于文献中所研究的配体的首选结合位点。此外，DNA聚合酶正好停滞在PNA寡聚体所占据的位置，而PNA寡聚体被用来启动引物延伸。这些发现为了解DNA聚合酶的性能提供了重要的见解。此外，我们首次获得的双链DNA上DNA聚合酶延长的高分辨率配体诱导的暂停模式可能成为现有用于监测双链DNA与各种DNA结合配体(包括药物)相互作用的方法的有价值的补充。
Using the recently developed peptide nucleic acid (PNA)-assisted assay, which makes it possible to extend a primer on duplex DNA, we study the sequence-specific inhibition of the DNA polymerase movement along double-stranded DNA templates imposed by DNA-binding ligands. To this end, a plasmid vector has been prepared featuring the polylinker with two flanking priming sites to bi-directionally initiate the primer-extension reactions towards each other. Within this plasmid, we have cloned a set of random DNA sequences and analyzed the products of these reactions with several phage and bacterial DNA polymerases capable of strand-displacement synthesis. Two of them, ø29 and modified T7 (Sequenase 2.0) enzymes, were found to be most potent for primer extension in the presence of DNA-binding ligands. We used these enzymes for a detailed study of ligand-induced pausing effects with four ligands differing in modes of binding to the DNA double-helix. GC-specific intercalator actinomycin D and three minor groove-binders, chromomycin A3(GC-specific), distamycin A and netropsin (both AT-specific), have been chosen. In the presence of each ligand both selected DNA polymerases experienced multiple clear-cut pauses. Each ligand yielded its own characteristic pausing pattern for a particular DNA sequence. The majority of pausing sites could be located with a single-nucleotide resolution and corresponded to the preferred binding sites known from the literature for the ligands under study. Besides, DNA polymerases stalled exactly at the positions occupied by PNA oligomers that were employed to initiate the primer extension. These findings provide an important insight into the DNA polymerase performance. In addition, the high-resolution ligand-induced pausing patterns we obtained for the first time for DNA polymerase elongation on duplex DNA may become a valuable addition to the existing arsenal of methods used to monitor duplex DNA interactions with various DNA-binding ligands, including drugs.