McCarthy J G, Williams L D, Rich A
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Biochemistry. 1990 Jun 26;29(25):6071-81. doi: 10.1021/bi00477a027.
We have examined the reactivity of B DNA with two chemical probes of DNA structure, potassium permanganate (KMnO4; thymine specific) and diethyl pyrocarbonate (DEPC; purine specific, A greater than G). The DNA probed is from the beta-lactamase promoter region of the vector pBR322, and from the 3' noncoding region of a chicken embryonic myosin heavy chain gene. The chemical probes display variable reactivity with the susceptible bases in these fragments, suggesting that modification of these bases by KMnO4 and DEPC is quite sequence dependent. In contrast, these probes react with the short A-tracts present in these DNA fragments in a reproducible fashion, generating two related patterns of reactivity. In the majority of the A-tracts, all but the 3'-terminal thymine are protected from KMnO4 attack, while DEPC reacts significantly with all but the 3'-terminal adenine of the A-tracts. Some A-tracts also display a very high DEPC reactivity at the adenine adjacent to the 3'-terminal unreactive adenine. Little qualitative difference in the KMnO4 reactivity of the A-tracts was found between 12 and 43 degrees C. However, at lower temperatures the elevated KMnO4 reactivity at the 3'-terminal A-tract thymine is sometimes lost. Raising the temperature of the KMnO4 reaction can cause relatively large increases in the reactivity of some single thymines, suggesting that significant local changes in stacking occur at these thymines at elevated temperatures. The data presented suggest that many short A-tracts embedded in long fragments of DNA can assume a number of related structures in solution, each of which possess distinct junctions with the flanking DNA. This result is consistent with high-resolution structural studies on oligonucleotides containing short A-tracts. The relevance of these results to current models of A-tract structure and DNA bending is discussed. Our data also indicate that KMnO4 and DEPC are potentially useful reagents for the study of sequence-dependent variations in B DNA structure.
我们用两种DNA结构化学探针——高锰酸钾（KMnO4；对胸腺嘧啶具有特异性）和焦碳酸二乙酯（DEPC；对嘌呤具有特异性，对腺嘌呤的反应性大于鸟嘌呤）检测了B型DNA的反应活性。被检测的DNA来自载体pBR322的β-内酰胺酶启动子区域，以及鸡胚胎肌球蛋白重链基因的3'非编码区。这些化学探针与这些片段中易被作用的碱基呈现出不同的反应活性，这表明KMnO4和DEPC对这些碱基的修饰具有相当强的序列依赖性。相比之下，这些探针以可重复的方式与这些DNA片段中存在的短A序列发生反应，产生两种相关的反应模式。在大多数A序列中，除了3'-末端的胸腺嘧啶外，所有其他胸腺嘧啶都受到保护不被KMnO4攻击，而DEPC与A序列中除3'-末端腺嘌呤外的所有腺嘌呤都有显著反应。一些A序列在与3'-末端无反应性腺嘌呤相邻的腺嘌呤处也表现出非常高的DEPC反应活性。在12至43摄氏度之间，未发现A序列的KMnO4反应活性有明显的定性差异。然而，在较低温度下，3'-末端A序列胸腺嘧啶处升高的KMnO4反应活性有时会丧失。提高KMnO4反应的温度会导致一些单个胸腺嘧啶的反应活性相对大幅增加，这表明在高温下这些胸腺嘧啶处的堆积发生了显著的局部变化。所呈现的数据表明，许多嵌入DNA长片段中的短A序列在溶液中可以呈现多种相关结构，每种结构与侧翼DNA都有独特的连接。这一结果与对含有短A序列的寡核苷酸的高分辨率结构研究一致。讨论了这些结果与当前A序列结构和DNA弯曲模型的相关性。我们的数据还表明，KMnO4和DEPC是研究B型DNA结构中序列依赖性变异的潜在有用试剂。
