Department of Chemistry, Furman University, Greenville, South Carolina 29613, USA.
Biochemistry. 2010 Apr 13;49(14):3024-30. doi: 10.1021/bi902043u.
Long repeated sequences of DNA and their associated secondary structure govern the development and severity of a significant class of neurological diseases. Utilizing the effect of base stacking on fluorescence quantum yield, 2-aminopurine substitutions for adenine previously demonstrated sequestered bases in the stem and exposed bases in the loop for an isolated (CAG)(8) sequence. This study evaluates (CAG)(8) that is incorporated into a duplex, as this three-way junction is a relevant model for intermediates that lead to repeat expansion during DNA replication and repair. From an energetic perspective, thermally induced denaturation indicates that the duplex arms dictate stability and that the secondary structure of the repeated sequence is disrupted. Substitutions with 2-aminopurine probe base exposure throughout this structure, and two conclusions about secondary structure are derived. First, the central region of (CAG)(8) is more solvent-exposed than single-stranded DNA, which suggests that hairpin formation in the repeated sequence is disrupted. Second, base stacking becomes compromised in the transition from the duplex to (CAG)(8), resulting in bases that are most similar to single-stranded DNA at the junction. Thus, an open (CAG)(8) loop and exposed bases in the arms indicate that the strand junction profoundly influences repeated sequences within three-way junctions.
长重复序列的 DNA 及其相关的二级结构控制着一大类神经疾病的发展和严重程度。利用碱基堆积对荧光量子产率的影响，2-氨基嘌呤取代腺嘌呤，以前曾证明在茎部隔离（CAG）（8）序列中隔离碱基，并在环部暴露碱基。本研究评估了（CAG）（8）掺入双链体，因为这种三链结是导致 DNA 复制和修复过程中重复扩展的中间产物的相关模型。从能量的角度来看，热诱导变性表明双链体臂决定稳定性，并且重复序列的二级结构被破坏。用 2-氨基嘌呤探针碱基暴露整个结构的取代物，并得出关于二级结构的两个结论。首先，（CAG）（8）的中心区域比单链 DNA 更暴露于溶剂，这表明重复序列中的发夹形成被破坏。其次，从双链体到（CAG）（8）的转变中，碱基堆积受到损害，导致在连接点处与单链 DNA 最相似的碱基。因此，开放的（CAG）（8）环和臂中的暴露碱基表明链连接点深刻影响三链结中的重复序列。
