Department of Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
Chem Res Toxicol. 2012 Jul 16;25(7):1531-40. doi: 10.1021/tx3002115. Epub 2012 Jun 15.
Sulfolobus solfataricus DNA Polymerase IV (Dpo4), a prototype Y-family DNA polymerase, has been well characterized biochemically and biophysically at 37 °C or lower temperatures. However, the physiological temperature of the hyperthermophile S. solfataricus is approximately 80 °C. With such a large discrepancy in temperature, the in vivo relevance of these in vitro studies of Dpo4 has been questioned. Here, we employed circular dichroism spectroscopy and fluorescence-based thermal scanning to investigate the secondary structural changes of Dpo4 over a temperature range from 26 to 119 °C. Dpo4 was shown to display a high melting temperature characteristic of hyperthermophiles. Unexpectedly, the Little Finger domain of Dpo4, which is only found in the Y-family DNA polymerases, was shown to be more thermostable than the polymerase core. More interestingly, Dpo4 exhibited a three-state cooperative unfolding profile with an unfolding intermediate. The linker region between the Little Finger and Thumb domains of Dpo4 was found to be a source of structural instability. Through site-directed mutagenesis, the interactions between the residues in the linker region and the Palm domain were identified to play a critical role in the formation of the unfolding intermediate. Notably, the secondary structure of Dpo4 was not altered when the temperature was increased from 26 to 87.5 °C. Thus, in addition to providing structural insights into the thermal stability and an unfolding intermediate of Dpo4, our work also validated the relevance of the in vitro studies of Dpo4 performed at temperatures significantly lower than 80 °C.
嗜热硫化叶菌 DNA 聚合酶 IV（Dpo4）是 Y 家族 DNA 聚合酶的原型，其在 37°C 或更低温度下的生物化学和生物物理性质已得到很好的描述。然而，嗜热菌嗜热硫化叶菌的生理温度约为 80°C。由于温度差异如此之大，人们对这些在 Dpo4 体外研究中的体内相关性提出了质疑。在这里，我们使用圆二色性光谱和基于荧光的热扫描技术，在 26 至 119°C 的温度范围内研究了 Dpo4 的二级结构变化。结果表明，Dpo4 显示出高温嗜热菌的高熔点特征。出乎意料的是，Dpo4 中仅存在于 Y 家族 DNA 聚合酶中的小指结构域，其热稳定性比聚合酶核心更高。更有趣的是，Dpo4 表现出具有展开中间态的三态协同展开轮廓。Dpo4 的小指和拇指结构域之间的连接区被发现是结构不稳定的来源。通过定点突变，确定了连接区和 Palm 结构域之间残基的相互作用在展开中间态的形成中起关键作用。值得注意的是，当温度从 26°C 升高到 87.5°C 时，Dpo4 的二级结构没有改变。因此，除了为 Dpo4 的热稳定性和展开中间态提供结构见解外，我们的工作还验证了在远低于 80°C 的温度下进行的 Dpo4 体外研究的相关性。
