Tuntiwechapikul Wirote, Salazar Miguel
Division of Medicinal Chemistry, College of Pharmacy, and Institute for Cellular and Molecular Biology, The University of Texas at Austin, 78712, USA.
Biochemistry. 2002 Jan 22;41(3):854-60. doi: 10.1021/bi0110950.
Studies of sequence repeat expansions from duplexes consisting of DNA repeat sequences greater than three bases are currently lacking. These studies are needed in order to gain a better understanding of DNA expansions in general and as a first step in understanding expansions of longer sequence repeats that have been implicated in human diseases. We have undertaken an in vitro study of tetranucleotide, hexanucleotide, and octanucleotide repeat expansions from short DNA duplexes using Taq DNA polymerase. Expansions of hexanucleotide repeats were also studied with the Klenow fragment of DNA polymerase I and with T4 DNA polymerase. Studies with Taq DNA polymerase show that expansions occur more readily as the length of the repeat sequence decreases but are generally more efficient at reaction temperatures closer to the melting point of the starting duplex. A mechanism for the observed expansions with Taq DNA polymerase is proposed that does not invoke strand slippage or DNA structure. Studies at 37 degrees C with Klenow pol I and T4 DNA polymerase indicate that the template-switching and/or strand-displacement activities of the polymerases used can play a major role in the apparent in vitro expansions of short repetitive DNA duplexes.
目前缺乏对由大于三个碱基的DNA重复序列组成的双链体中序列重复扩增的研究。为了更全面地了解DNA扩增，并作为理解与人类疾病相关的较长序列重复扩增的第一步，需要进行这些研究。我们使用Taq DNA聚合酶对短DNA双链体中的四核苷酸、六核苷酸和八核苷酸重复扩增进行了体外研究。还使用DNA聚合酶I的Klenow片段和T4 DNA聚合酶对六核苷酸重复扩增进行了研究。用Taq DNA聚合酶进行的研究表明，随着重复序列长度的减少，扩增更容易发生，但在更接近起始双链体熔点的反应温度下通常更有效。提出了一种Taq DNA聚合酶观察到的扩增机制，该机制不涉及链滑动或DNA结构。在37℃下用Klenow pol I和T4 DNA聚合酶进行的研究表明，所用聚合酶的模板切换和/或链置换活性在短重复DNA双链体的明显体外扩增中可能起主要作用。
