Stacked-unstacked equilibrium at the nick site of DNA
Stacked-unstacked equilibrium at the nick site of DNA
复制标题
DOI：
10.1016/j.jmb.2004.07.075
复制
发表时间：
2004-09-17
影响因子：
5.6
通讯作者：
Frank-Kamenetskii, MD
中科院分区：
文献类型：
--
作者：
Protozanova, E;Yakovchuk, P;Frank-Kamenetskii, MD
双链DNA在互补链分离方面的稳定性对于DNA在细胞中执行其主要功能至关重要，并且它在DNA的主要生物技术应用中也起着核心作用：DNA测序，聚合酶链式反应和DNA微阵列。众所周知，两种类型的相互作用有助于DNA的稳定性：相邻碱基对之间的堆叠和互补碱基之间的配对。然而，它们对双链体稳定性的贡献尚未确定。现在，我们填补了DNA双螺旋结构知识中的这一基本空白。我们制备了一系列32，300 bp长的DNA片段，这些片段在同一位置上具有单独的切口，不同之处仅在于切口两侧的碱基对。这些片段在凝胶中的电泳迁移率进行了研究。假设在缺口位置的堆叠和非堆叠构象之间的平衡，已经获得了所有32个堆叠自由能参数。其中只有10个是必需的，它们控制着完整DNA双螺旋中相邻碱基对之间的堆积相互作用。首次确定了一套完整的DNA堆积参数。根据这些数据和众所周知的DNA解链温度对G-C含量的依赖性，估计了碱基配对对双链体稳定性的贡献。所获得的能量参数的DNA双螺旋是至关重要的理解序列依赖的DNA的灵活性和许多生物技术的应用。(C)2004爱思唯尔有限公司保留所有权利。
Stability of duplex DNA with respect to separation of complementary strands is crucial for DNA executing its major functions in the cell and it also plays a central role in major biotechnology applications of DNA: DNA sequencing, polymerase chain reaction, and DNA microarrays. Two types of interaction are well known to contribute to DNA stability: stacking between adjacent base-pairs and pairing between complementary bases. However, their contribution into the duplex stability is yet to be determined. Now we fill this fundamental gap in our knowledge of the DNA double helix. We have prepared a series of 32, 300 bp-long DNA fragments with solitary nicks in the same position differing only in basepairs flanking the nick. Electrophoretic mobility of these fragments in the gel has been studied. Assuming the equilibrium between stacked and unstacked conformations at the nick site, all 32 stacking free energy parameters have been obtained. Only ten of them are essential and they govern the stacking interactions between adjacent base-pairs in intact DNA double helix. A full set of DNA stacking parameters has been determined for the first time. From these data and from a well-known dependence of DNA melting temperature on G - C content, the contribution of base-pairing into duplex stability has been estimated. The obtained energy parameters of the DNA double helix are of paramount importance for understanding sequence-dependent DNA flexibility and for numerous biotechnology applications. (C) 2004 Elsevier Ltd. All rights reserved.