Unusual Structures Are Present in DNA Fragments Containing Super-Long Huntingtin CAG Repeats

BackgroundIn the R6/2 mouse model of Huntington's disease (HD), expansion of the CAG trinucleotide repeat length beyond about 300 repeats induces a novel phenotype associated with a reduction in transcription of the transgene.
Methodology/Principal FindingsWe analysed the structure of polymerase chain reaction (PCR)-generated DNA containing up to 585 CAG repeats using atomic force microscopy (AFM). As the number of CAG repeats increased, an increasing proportion of the DNA molecules exhibited unusual structural features, including convolutions and multiple protrusions. At least some of these features are hairpin loops, as judged by cross-sectional analysis and sensitivity to cleavage by mung bean nuclease. Single-molecule force measurements showed that the convoluted DNA was very resistant to untangling. In vitro replication by PCR was markedly reduced, and TseI restriction enzyme digestion was also hindered by the abnormal DNA structures. However, significantly, the DNA gained sensitivity to cleavage by the Type III restriction-modification enzyme, EcoP15I.
Conclusions/Significance“Super-long” CAG repeats are found in a number of neurological diseases and may also appear through CAG repeat instability. We suggest that unusual DNA structures associated with super-long CAG repeats decrease transcriptional efficiency in vitro. We also raise the possibility that if these structures occur in vivo, they may play a role in the aetiology of CAG repeat diseases such as HD.

研究背景：在亨廷顿舞蹈症（Huntington's disease, HD）的R6/2小鼠模型中，当CAG三核苷酸重复序列长度扩增至约300个重复以上时，会诱导出一种与转基因转录水平降低相关的新型表型。
研究方法与主要结果：我们利用原子力显微镜（atomic force microscopy, AFM）分析了经聚合酶链式反应（polymerase chain reaction, PCR）扩增得到的、包含至多585个CAG重复序列的DNA分子结构。随着CAG重复序列数量增加，越来越多的DNA分子展现出异常结构特征，包括卷曲构象与多处凸起。通过横截面分析以及对绿豆核酸酶（mung bean nuclease）切割的敏感性判断，至少部分此类异常结构为发夹环。单分子力学测量结果显示，这种卷曲的DNA极难解缠。体外PCR扩增过程显著受到抑制，且TseI限制性内切酶的酶切反应也因异常DNA结构而受阻。值得注意的是，此类DNA对III型限制性修饰酶（Type III restriction-modification enzyme）EcoP15I的切割敏感性显著提升。
结论与意义：超长CAG重复序列在多种神经系统疾病中均有发现，且也可通过CAG重复不稳定性产生。我们提出，与超长CAG重复序列相关的异常DNA结构会在体外降低转录效率。此外我们还推测，若此类结构在体内出现，则可能在亨廷顿舞蹈症等CAG重复疾病的发病机制中发挥作用。