School of Life Science, Beijing Institute of Technology, Beijing, China.
PLoS One. 2010 Dec 9;5(12):e14271. doi: 10.1371/journal.pone.0014271.
The trinucleotide repeats AAT•ATT are simple DNA sequences that potentially form different types of non-B DNA secondary structures and cause genomic instabilities in vivo.
The molecular mechanism underlying the maintenance of a 24-triplet AAT•ATT repeat was examined in E. coli by cloning the repeats into the EcoRI site in plasmid pUC18 and into the attB site on the E. coli genome. Either the AAT or the ATT strand acted as lagging strand template in a replication fork. Propagations of the repeats in either orientation on plasmids did not affect colony morphology when triplet repeat transcription using the lacZ promoter was repressed either by supplementing LacI(Q)in trans or by adding glucose into the medium. In contrast, transparent colonies were formed by inducing transcription of the repeats, suggesting that transcription of AAT•ATT repeats was toxic to cell growth. Meanwhile, significant IS1E transposition events were observed both into the triplet repeats region proximal to the promoter side, the promoter region of the lacZ gene, and into the AAT•ATT region itself. Transposition reversed the transparent colony phenotype back into healthy, convex colonies. In contrast, transcription of an 8-triplet AAT•ATT repeat in either orientation on plasmids did not produce significant changes in cell morphology and did not promote IS1E transposition events. We further found that a role of IS1E transposition into plasmids was to inhibit transcription through the repeats, which was influenced by the presence of the H-NS protein, but not of its paralogue StpA.
Our findings thus suggest that the longer AAT•ATT triplet repeats in E. coli become vulnerable after transcription. H-NS and its facilitated IS1E transposition can silence long triplet repeats transcription and preserve cell growth and survival.
三核苷酸重复 AAT•ATT 是简单的 DNA 序列，它们可能形成不同类型的非 B 型 DNA 二级结构，并在体内引起基因组不稳定性。
通过将重复序列克隆到质粒 pUC18 的 EcoRI 位点和大肠杆菌基因组的 attB 位点，在大肠杆菌中检查了维持 24 个三联体 AAT•ATT 重复的分子机制。在复制叉中，AAT 或 ATT 链都可以作为滞后链模板。无论是正向还是反向扩增，当使用 lacZ 启动子抑制三核苷酸重复转录时（通过转染补充 LacI(Q)或在培养基中添加葡萄糖），对质粒上重复序列的传播都不会影响菌落形态。相反，当诱导重复序列转录时，形成透明菌落，表明 AAT•ATT 重复序列的转录对细胞生长有毒。同时，在靠近启动子侧的三核苷酸重复区域、lacZ 基因的启动子区域和 AAT•ATT 区域本身都观察到了显著的 IS1E 转位事件。转位将透明菌落表型逆转回健康、凸形菌落。相比之下，正向或反向在质粒上转录 8 个三核苷酸 AAT•ATT 重复序列不会引起细胞形态的显著变化，也不会促进 IS1E 转位事件。我们进一步发现，IS1E 转位到质粒上的作用是通过重复序列抑制转录，这受到 H-NS 蛋白的影响，但不受其同源物 StpA 的影响。
因此，我们的研究结果表明，大肠杆菌中较长的 AAT•ATT 三核苷酸重复在转录后变得脆弱。H-NS 及其促进的 IS1E 转位可以沉默长三核苷酸重复序列的转录，维持细胞生长和存活。
