Manzini G, Yathindra N, Xodo L E
Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Italy.
Nucleic Acids Res. 1994 Nov 11;22(22):4634-40. doi: 10.1093/nar/22.22.4634.
The structural behaviour of repetitive cytosine DNA is examined in the oligodeoxynucleotide sequences of (CCCTAA)3CCCT (HTC4), GC(TCCC)3TCCT(TCCC)3 (KRC6) and the methylated (CCCT)3TCCT(CCCT)3C (KRM6) by circular dichroism (CD), gel electrophoresis (PAGE), and ultra violet (UV) absorbance studies. All the three sequences exhibit a pH-induced cooperative structural transition as monitored by CD. An intense positive CD band around 285 nm develops on lowering the pH from 8 to slightly acidic condition, indicative of the formation of base pairs between protonated cytosines. The oligomers are found to melt in a fully reversible and cooperative fashion, with a melting temperature (Tm) of around 50 degrees C at pH 5.5. The melting temperatures are independent from DNA concentration, indicative of an intramolecular process involved in the structural formation. PAGE experiments performed with 32P-labeled samples as well as with normal staining procedures show a predominantly single band migration for all the three oligomers suggestive of a unimolecular structure. From pH titrations the number of protons required for generating the structures formed by HTC4, KRC6 and KRM6 results to be around six. These findings strongly suggest that all the three sequences adopt an intramolecular i-motif structure. The demonstration of i-motif structure for KRC6, a critical functional stretch of the c-ki-ras promoter proto-oncogene, besides the human telomeric sequence HTC4, may be suggestive of larger significance in the functioning of DNA.
通过圆二色性（CD）、凝胶电泳（PAGE）和紫外（UV）吸收研究，对（CCCTAA）3CCCT（HTC4）、GC（TCCC）3TCCT（TCCC）3（KRC6）以及甲基化的（CCCT）3TCCT（CCCT）3C（KRM6）的寡脱氧核苷酸序列中重复胞嘧啶DNA的结构行为进行了研究。如通过CD监测的那样，所有这三个序列都表现出pH诱导的协同结构转变。当pH从8降低到略呈酸性的条件时，在285nm左右出现一个强烈的正CD带，这表明质子化胞嘧啶之间形成了碱基对。发现这些寡聚物以完全可逆和协同的方式解链，在pH 5.5时的解链温度（Tm）约为50℃。解链温度与DNA浓度无关，这表明结构形成过程涉及分子内过程。用32P标记的样品以及常规染色程序进行的PAGE实验表明，所有这三种寡聚物的迁移主要为单一条带，这表明其为单分子结构。通过pH滴定，生成由HTC4、KRC6和KRM6形成的结构所需的质子数约为6个。这些发现有力地表明，所有这三个序列都采用分子内i-基序结构。除了人类端粒序列HTC4外，c-ki-ras启动子原癌基因的关键功能片段KRC6的i-基序结构的证明，可能暗示了其在DNA功能方面具有更大的意义。
