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首页> 外文期刊>Journal of Molecular Biology >Conformation and rigidity of DNA microcircles containing waf1 responseelement for p53 regulatory protein
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Conformation and rigidity of DNA microcircles containing waf1 responseelement for p53 regulatory protein

机译：包含waf1反应元件的p53调控蛋白的DNA微环的构象和刚性

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The tuner-suppressor activity of p53 is closely related to its DNA-binding properties. It binds a number of DNA response-elements and it is likely that these share a common structural feature. Here, we present a new, general method to determine the absolute twist of flexible DNA promoter sequences based on direct imaging of the topology of microcircles containing the sequences. We have used magnetically driven dynamic force microscopy ("MacMode" AFM) to observe, in solution, the conformation of 168 base-pair DNA microcircles, each containing four equally spaced copies of the waf1/cip1/p21 p53 response-element. Analysis of the images showed that the microcircles are markedly puckered with a small excess of negatively writhed molecules. The average measured values of writhe are 0.109 +/- 0.013 (for 60 positively writhed molecules) and -0.098 +/- 0.011 (for 65 negatively writhed molecules). These values lead directly to a difference in Linking number for the positively and negatively writhed molecules prior to ligation, from which we derive a twist mismatch of 178 degrees (overtwist). This is 44.5 degrees for each 42-mer precursor containing a single waf1/cip1/p21 p53 response-element, in good agreement with the range of values deduced by indirect biochemical techniques. The two values of writhe may also be used to determine the ratio of the bending (B) to twisting (C) rigidity, yielding B/C = 0.23. This is about one-third of the value for long, random-sequence DNA, suggesting that the waf1/cip1/p21 p53 response-element is extremely flexible, a result that is also consistent with indirect biochemical experiments. These results support the idea, proposed by us earlier, that torsional stress may play a role in the regulation of p53 binding through modulation of twist at the binding site.
机译：p53的调谐抑制活性与其DNA结合特性密切相关。它结合了许多DNA反应元件，并且这些可能具有相同的结构特征。在这里，我们提出了一种新的通用方法，可以基于包含序列的微圆拓扑的直接成像来确定柔性DNA启动子序列的绝对扭曲。我们已经使用磁力驱动的动态力显微镜（“ MacMode” AFM）在溶液中观察了168个碱基对DNA微圆的构象，每个微圆包含waf1 / cip1 / p21 p53应答元件的四个等距拷贝。对图像的分析表明，微圆形明显皱褶，并带有少量过量的负面扭曲分子。扭曲的平均测量值为0.109 +/- 0.013（对于60个正向扭曲的分子）和-0.098 +/- 0.011（对于65个负向扭曲的分子）。这些值直接导致连接前正向和负向扭动的分子的连接数不同，由此可得出178度的扭曲不匹配（过度扭曲）。对于包含单个waf1 / cip1 / p21 p53反应元素的每个42-mer前体，这是44.5度，与通过间接生化技术推导的值范围非常一致。扭曲的两个值还可用于确定弯曲刚度（B）与扭曲（C）的比率，得出B / C = 0.23。这大约是长随机序列DNA值的三分之一，这表明waf1 / cip1 / p21 p53反应元件非常灵活，这一结果也与间接生化实验一致。这些结果支持了我们先前提出的想法，即扭转应力可能通过调节结合位点的扭曲来调节p53结合。

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