Studies on non-covalent interactions between strongly conelated biomolecules

逆转旋转酶是一种正向超螺旋DNA的酶，是所有嗜硫细菌中唯一存在的酶，但在所有中嗜酸细菌中都不存在。该酶由两个不同的结构域组成，一个是解旋酶样结构域，另一个是属于拓扑异构酶IA家族的结构域。有人指出，如果反向旋转酶是超嗜热生命的先决条件，那么生命的起源不可能是超嗜热性生命，因为在反向旋转酶出现之前，这两个不同的结构域必须在较不嗜热的生物体中独立进化。我们已经扰乱了一种极端嗜热的考古子--柯达卡拉热球菌KOD1的反向旋转酶基因。在trpE基因突变的宿主菌株中，通过双交叉重组，用trpE标记基因取代了基因组分析中鉴定出的单一反向旋转酶基因。经聚合酶链式反应和Southern印迹分析，证实转化子染色体上不存在反向旋转酶基因。与该基因缺失一致的是，在中断菌株中没有发现在宿主菌株中观察到的明显的正超卷曲活性。在不同温度下，比较了干扰菌和宿主菌的生长速率。我们发现，缺乏反向回旋酶会导致生长迟缓，这种趋势在较高的温度下会变得更严重。然而，反向旋转酶基因的破坏并没有导致90℃时的致死表型。这些结果提供了实验证据，证明反向旋转酶不是超高温生命的先决条件，反向旋转酶的结构及其在超嗜热性生物中的存在不再排除超嗜热性生物是最先进化的生物的可能性。研究结果还提供了证据，证明DNA(基因组)的拓扑结构是在高温下维持生命的关键因素。

Reverse gyrase, an enzyme that positively supercoils DNA, is the only enzyme present in all hyperthennophiles, but absent in all mesophiles. The enzyme is composed of two distinct domains, one helicase-like domain and another domain that belongs to the topoisomerase type IA family. It has been pointed out that if reverse gyrase were a prerequisite for hyperthermophilic life, the origin of life could not have been a hyperthermophile, as the two separate domains must have independently evolved in less thermophilic organisms before the occurrence of reverse gyrase. We have disrupted the reverse gyrase gene from a hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1. A single reverse gyrase gene, identified from genome analysis, was replaced with a trpE marker gene via double crossover recombination in a host strain disrupted of trpE. PCR and southern blot analyses were performed to confirm the absence of the reverse gyrase gene on the chromosome of the transformant. Consistent with the absence of the gene, an apparent positive supercoiling activity that was observed in the host strain was not found in the disruptant strain. Growth rates of the disruptant strain were compared with those of the host strain at various temperatures. We found that lack of reverse gyrase led to a retardation in growth, a tendency that became greater at higher temperatures. However, disruption of the reverse gyrase gene did not lead to a lethal phenotype at 90℃. The results provide experimental evidence that reverse gyrase is not a prerequisite for hyperthermophilic life, and that the structure of reverse gyrase and its presence in hyperthermophiles no longer rule out the possibility that hyperthermophiles were the first organisms to evolve. The results also provide evidence that the topology of DNA (the genome) is a key factor for maintaining life at high temperature.