McCarthy J G, Frederick C A, Nicolas A
Institut de Génétique et Microbiologie, Université Paris-Sud, Orsay, France.
Nucleic Acids Res. 1993 Jul 11;21(14):3309-17. doi: 10.1093/nar/21.14.3309.
The chemical probes potassium permanganate (KMnO4) and diethylpyrocarbonate (DEPC) have been used to study the conformation of bent kinetoplast DNA from Crithidia fasciculata at different temperatures. Chemical reactivity data shows that the numerous short A-tracts of this bent DNA adopt a similar structure at 43 degrees C. This conformation appears to be very similar to the conformation of A-tracts in DNA exhibiting normal gel mobility. The A-tract structure detected by chemical probing is characterized by a high degree of base stacking on the thymine strand, and by an abrupt conformational change at the 3' end of the adenine strand. In general, no major alteration of this A-tract specific structure was detected between 4-53 degrees C. However, probing with KMnO4 revealed two unusual features of the C. fasciculata sequence that may contribute to the highly aberrant gel mobility of this DNA: 1) the B DNA/A-tract junction 5' dC/A3-6 3'. 5' dT3-6/G 3' is disproportionately represented and is conformationally distinct from other 5' end junctions, and 2) low temperature favors a novel strand-specific conformational distortion over a 20 base pair region of the bent kinetoplast DNA. Presence of the minor groove binding drug distamycin had little detectable effect on the A-tract conformation. However, distamycin did inhibit formation of the novel KMnO4 sensitive low temperature structure and partially eliminated the anomalous gel mobility of the kinetoplast DNA. Finally, we describe a simple and reproducible procedure for the production of an adenine-specific chemical DNA sequence ladder.
化学探针高锰酸钾（KMnO4）和焦碳酸二乙酯（DEPC）已被用于研究不同温度下来自克氏锥虫的弯曲动质体DNA的构象。化学反应性数据表明，这种弯曲DNA的众多短A序列在43摄氏度时采用相似的结构。这种构象似乎与在正常凝胶迁移率的DNA中的A序列构象非常相似。通过化学探测检测到的A序列结构的特征是胸腺嘧啶链上高度的碱基堆积，以及腺嘌呤链3'端的突然构象变化。一般来说，在4-53摄氏度之间未检测到这种A序列特定结构的重大改变。然而，用KMnO4探测揭示了克氏锥虫序列的两个不寻常特征，这可能导致这种DNA高度异常的凝胶迁移率：1）B型DNA/A序列连接处5'dC/A3-6 3'。5'dT3-6/G 3'的比例过高，并且在构象上与其他5'端连接处不同，2）低温有利于在弯曲的动质体DNA的20个碱基对区域上形成一种新的链特异性构象畸变。小沟结合药物偏端霉素的存在对A序列构象几乎没有可检测到的影响。然而，偏端霉素确实抑制了新的对KMnO4敏感的低温结构的形成，并部分消除了动质体DNA异常的凝胶迁移率。最后，我们描述了一种简单且可重复的方法来产生腺嘌呤特异性化学DNA序列阶梯。
