Potaman V N, Oussatcheva E A, Lyubchenko Y L, Shlyakhtenko L S, Bidichandani S I, Ashizawa T, Sinden R R
Institute of Biosciences and Technology, Texas A&M University System Health Sciences Center, Houston, TX 77030, USA.
Nucleic Acids Res. 2004 Feb 20;32(3):1224-31. doi: 10.1093/nar/gkh274. Print 2004.
More than 15 human genetic diseases have been associated with the expansion of trinucleotide DNA repeats, which may involve the formation of non-duplex DNA structures. The slipped-strand nucleation of duplex DNA within GC-rich trinucleotide repeats may result in the changes of repeat length; however, such a mechanism seems less likely for the AT-rich (GAA)n*(TTC)n repeats. Using two-dimensional agarose gels, chemical probing and atomic force microscopy, we characterized the formation of non-B-DNA structures in the Friedreich ataxia-associated (GAA)n*(TTC)n repeats from the FRDA gene that were cloned with flanking genomic sequences into plasmids. For the normal genomic repeat length (n = 9) our data are consistent with the formation of a very stable protonated intramolecular triplex (H-DNA). Its stability at pH 7.4 is likely due to the high proportion of the T.A.T triads which form within the repeats as well as in the immediately adjacent AT-rich sequences with a homopurine. homopyrimidine bias. At the long normal repeat length (n = 23), a family of H-DNAs of slightly different sizes has been detected. At the premutation repeat length (n = 42) and higher negative supercoiling, the formation of a single H-DNA structure becomes less favorable and the data are consistent with the formation of a bi-triplex structure.
超过15种人类遗传疾病与三核苷酸DNA重复序列的扩增有关，这可能涉及非双链DNA结构的形成。富含GC的三核苷酸重复序列内双链DNA的滑链成核可能导致重复序列长度的改变；然而，对于富含AT的（GAA）n*（TTC）n重复序列，这种机制似乎不太可能。我们使用二维琼脂糖凝胶、化学探针和原子力显微镜，对从FRDA基因中克隆的与弗里德赖希共济失调相关的（GAA）n*（TTC）n重复序列中非B-DNA结构的形成进行了表征，这些重复序列与侧翼基因组序列一起克隆到质粒中。对于正常基因组重复长度（n = 9），我们的数据与非常稳定的质子化分子内三链体（H-DNA）的形成一致。它在pH 7.4时的稳定性可能是由于在重复序列内以及紧邻的富含AT且具有同型嘌呤-同型嘧啶偏向性的序列中形成的T.A.T三联体比例很高。在较长的正常重复长度（n = 23）时，检测到了一系列大小略有不同的H-DNA。在预突变重复长度（n = 42）和更高的负超螺旋条件下，单一H-DNA结构的形成变得不太有利，数据与双三链体结构的形成一致。
