Studies on non-covalent interactions between strongly conelated biomolecules

反旋酶是一种使DNA正向超旋的酶，是所有嗜中性粒细胞中唯一存在的酶，但在所有嗜中性粒细胞中不存在。该酶由两个不同的结构域组成，一个类似解旋酶的结构域和另一个属于IA型拓扑异构酶家族的结构域。有人指出，如果反回转酶是超嗜热生命的先决条件，那么生命的起源就不可能是超嗜热生物，因为在反回转酶出现之前，这两个分离的结构域必须在不那么嗜热的生物中独立进化。我们已经破坏了一个超嗜热古菌，柯达卡拉热球菌KOD1的逆转录酶基因。在trpE中断的宿主菌株中，通过双交叉重组将从基因组分析中鉴定出的单个反旋酶基因替换为trpE标记基因。PCR和southern blot分析证实了该转化体染色体上不存在逆转录酶基因。与该基因的缺失一致，在宿主菌株中观察到的明显的正超卷曲活性在干扰菌株中没有发现。在不同温度下，对干扰菌与宿主菌的生长速率进行了比较。我们发现，缺乏反向回转酶会导致生长迟缓，这种趋势在更高的温度下变得更大。然而，逆转录酶基因的破坏并没有导致90℃下的致死表型。这些结果提供了实验证据，证明反回转酶不是超嗜热生命的先决条件，并且反回转酶的结构及其在超嗜热生物中的存在不再排除超嗜热生物是最早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.