Nakagama Hitoshi, Higuchi Kumiko, Tanaka Etsuko, Tsuchiya Naoto, Nakashima Katsuhiko, Katahira Masato, Fukuda Hirokazu
Biochemistry Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Tokyo 104-0045, Japan.
Mutat Res. 2006 Jun 25;598(1-2):120-31. doi: 10.1016/j.mrfmmm.2006.01.014. Epub 2006 Mar 2.
Mammalian genomes contain several types of repetitive sequences. Some of these sequences are implicated in various specific cellular events, including meiotic recombination, chromosomal breaks and transcriptional regulation, and also in several human disorders. In this review, we document the formation of DNA secondary structures by the G-rich repetitive sequences that have been found in several minisatellites, telomeres and in various triplet repeats, and report their effects on in vitro DNA synthesis. d(GGCAG) repeats in the mouse minisatellite Pc-1 were demonstrated to form an intra-molecular folded-back quadruplex structure (also called a G4' structure) by NMR and CD spectrum analyses. d(TTAGGG) telomere repeats and d(CGG) triplet repeats were also shown to form G4' and other unspecified higher order structures, respectively. In vitro DNA synthesis was substantially arrested within the repeats, and this could be responsible for the preferential mutability of the G-rich repetitive sequences. Electrophoretic mobility shift assays using NIH3T3 cell extracts revealed heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and A3, which were tightly and specifically bound to d(GGCAG) and d(TTAGGG) repeats with K(d) values in the order of nM. HnRNP A1 unfolded the G4' structure formed in the d(GGCAG)(n) and d(TTAGGG)(n) repeat regions, and also resolved the higher order structure formed by d(CGG) triplet repeats. Furthermore, DNA synthesis arrest at the secondary structures of d(GGCAG) repeats, telomeres and d(CGG) triplet repeats was efficiently repressed by the addition of hnRNP A1. High expression of hnRNPs may contribute to the maintenance of G-rich repetitive sequences, including telomere repeats, and may also participate in ensuring the stability of the genome in cells with enhanced proliferation. Transcriptional regulation of genes, such as c-myc and insulin, by G4 sequences found in the promoter regions could be an intriguing field of research and help further elucidate the biological functions of the hnRNP family of proteins in human diseases.
哺乳动物基因组包含几种类型的重复序列。其中一些序列与各种特定的细胞事件有关，包括减数分裂重组、染色体断裂和转录调控，也与几种人类疾病有关。在本综述中，我们记录了在几个小卫星、端粒和各种三联体重复序列中发现的富含G的重复序列形成DNA二级结构的情况，并报告了它们对体外DNA合成的影响。通过核磁共振（NMR）和圆二色光谱（CD）分析表明，小鼠小卫星Pc-1中的d(GGCAG)重复序列形成了分子内折叠回文四重结构（也称为G4'结构）。d(TTAGGG)端粒重复序列和d(CGG)三联体重复序列也分别显示形成了G4'和其他未明确的高阶结构。体外DNA合成在重复序列内基本停滞，这可能是富含G的重复序列优先发生突变的原因。使用NIH3T3细胞提取物进行的电泳迁移率变动分析显示，异质性核核糖核蛋白（hnRNP）A1和A3与d(GGCAG)和d(TTAGGG)重复序列紧密且特异性结合，解离常数（K(d)）值在纳摩尔级别。hnRNP A1展开了在d(GGCAG)(n)和d(TTAGGG)(n)重复区域形成的G4'结构，还解析了由d(CGG)三联体重复序列形成的高阶结构。此外，通过添加hnRNP A1可有效抑制d(GGCAG)重复序列、端粒和d(CGG)三联体重复序列二级结构处的DNA合成停滞。hnRNPs的高表达可能有助于维持包括端粒重复序列在内的富含G的重复序列，也可能参与确保增殖增强的细胞中基因组的稳定性。启动子区域中发现的G4序列对c-myc和胰岛素等基因的转录调控可能是一个有趣的研究领域，并有助于进一步阐明hnRNP蛋白家族在人类疾病中的生物学功能。
