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首页> 美国卫生研究院文献>Molecular Biology International >5′CAG and 5′CTG Repeats Create Differential Impediment to the Progression of a Minimal Reconstituted T4 Replisome Depending on the Concentration of dNTPs
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5′CAG and 5′CTG Repeats Create Differential Impediment to the Progression of a Minimal Reconstituted T4 Replisome Depending on the Concentration of dNTPs

机译：5CAG和5CTG重复产生最小化重组T4复制体进展的差异性障碍具体取决于dNTPs的浓度

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Instability of repetitive sequences originates from strand misalignment during repair or replicative DNA synthesis. To investigate the activity of reconstituted T4 replisomes across trinucleotide repeats (TNRs) during leading strand DNA synthesis, we developed a method to build replication miniforks containing a TNR unit of defined sequence and length. Each minifork consists of three strands, primer, leading strand template, and lagging strand template with a 5′ single-stranded (ss) tail. Each strand is prepared independently, and the minifork is assembled by hybridization of the three strands. Using these miniforks and a minimal reconstituted T4 replisome, we show that during leading strand DNA synthesis, the dNTP concentration dictates which strand of the structure-forming 5′CAG/5′CTG repeat creates the strongest impediment to the minimal replication complex. We discuss this result in the light of the known fluctuation of dNTP concentration during the cell cycle and cell growth and the known concentration balance among individual dNTPs.
机译：重复序列的不稳定性源自修复或复制性DNA合成过程中的链错位。为了研究在前导链DNA合成过程中跨三核苷酸重复（TNR）的重组T4复制体的活性，我们开发了一种构建包含定义序列和长度的TNR单元的复制叉的方法。每个迷你叉都由三条链组成，分别是引物，前导链模板和带有5'单链（ss）尾巴的后随链模板。每条链独立制备，并且迷你叉通过三链的杂交组装。使用这些小叉和最小的重组T4复制体，我们显示出在前导链DNA合成过程中，dNTP浓度决定了形成5'CAG / 5'CTG重复序列的哪条链对最小复制复合物产生了最强的阻碍。我们根据细胞周期和细胞生长期间dNTP浓度的已知波动以及各个dNTP之间的已知浓度平衡来讨论此结果。

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