Department of Applied Chemistry, National Defense Academy, Hashirimizu 1-10-20, Yokosuka, Kanagawa, 239-8686, Japan.
College of Liberal Arts, International Christian University, Osawa 3-10-2, Mitaka, Tokyo, 181-8585, Japan.
Mol Genet Genomics. 2019 Aug;294(4):1085-1093. doi: 10.1007/s00438-019-01565-9. Epub 2019 Apr 9.
Thermus thermophilus (T. thermophilus) HB27 is an extreme thermophile that grows optimally at 65-72 °C. Heat-induced DNA lesions are expected to occur at a higher frequency in the genome of T. thermophilus than in those of mesophiles; however, the mechanisms underlying the maintenance of genome integrity at high temperatures remain poorly understood. The study of mutation spectra has become a powerful approach to understanding the molecular mechanisms responsible for DNA repair and mutagenesis in mesophilic species. Therefore, we developed a supF-based system to detect a broad spectrum of mutations in T. thermophilus. This system was validated by measuring spontaneous mutations in the wild type and a udgA, B double mutant deficient in uracil-DNA glycosylase (UDG) activity. We found that the mutation frequency of the udgA, B strain was 4.7-fold higher than that of the wild type and G:C→A:T transitions dominated, which was the most reasonable for the mutator phenotype associated with the loss of UDG function in T. thermophilus. These results show that this system allowed for the rapid analysis of mutations in T. thermophilus, and may be useful for studying the molecular mechanisms responsible for DNA repair and mutagenesis in this extreme thermophile.
嗜热栖热菌（T. thermophilus）HB27 是一种极端嗜热菌，最适生长温度为 65-72°C。与中温菌相比，热诱导的 DNA 损伤预计会在 T. thermophilus 的基因组中更频繁地发生；然而，高温下维持基因组完整性的机制仍知之甚少。突变谱的研究已成为理解中温生物中 DNA 修复和诱变分子机制的有力方法。因此，我们开发了一种基于 supF 的系统来检测嗜热栖热菌中的广泛突变。该系统通过测量野生型和缺乏尿嘧啶-DNA 糖基化酶（UDG）活性的 udgA、B 双突变体的自发突变来验证。我们发现 udgA、B 菌株的突变频率比野生型高 4.7 倍，且以 G:C→A:T 颠换为主，这与 T. thermophilus 中 UDG 功能丧失相关的突变体表型最为合理。这些结果表明，该系统允许快速分析嗜热栖热菌中的突变，可能有助于研究该极端嗜热菌中 DNA 修复和诱变的分子机制。
