Kreuzer K N, Cozzarelli N R
Cell. 1980 May;20(1):245-54. doi: 10.1016/0092-8674(80)90252-4.
We have discovered that DNA gyrase interlocks duplex DNA circles to form catenanes and resolves catenanes into component monomers. The reactions were inhibited by novobiocin and oxolinic acid and required ATP, Mg++ and spermidine. DNA sequence homology is not involved in catenation, since hybrid catenanes were formed efficiently between supercoiled phi X174 and Col E1 DNA. Strikingly different results were obtained with native and relaxed Col E1 DNA substrates. Up to 50-60% of input native DNA was converted into oligomeric catenanes, predominantly dimers and trimers. Relaxed substrates were instead converted into vast interlocked networks and were occasionally knotted. Optimal catenation occurred only in the narrow range of 20-35 mM KCl; increased ionic strength blocked catenation completely but activated the back reaction of decatenation. Gyrase resolved both the oligomeric catenanes and interlocked networks it produced, as well as naturally occurring catenanes. These results imply that the mechanism of gyrase involves a transient double-strand break and passage of a DNA segment through the resulting gap. Gyrase is representative of a general class of enzymes, found in both procaryotic and eucaryotic cells, that facilitate diffusion of duplex DNA segments through each other and may thereby solve topological problems arising from the replication, recombination and condensation of DNA.
我们发现，DNA促旋酶可使双链DNA环相互连锁形成连环体，并将连环体分解为组成单体。这些反应受到新生霉素和恶喹酸的抑制，且需要ATP、Mg++和亚精胺。由于超螺旋φX174和Col E1 DNA之间能高效形成杂合连环体，所以DNA序列同源性不参与连环化过程。使用天然和松弛的Col E1 DNA底物时，得到了截然不同的结果。高达50 - 60%的输入天然DNA被转化为寡聚连环体，主要是二聚体和三聚体。相反，松弛的底物被转化为庞大的互锁网络，偶尔还会打结。最佳连环化仅发生在20 - 35 mM KCl的狭窄范围内；离子强度增加会完全阻止连环化，但会激活解连环的逆反应。促旋酶能分解它产生的寡聚连环体和互锁网络，以及天然存在的连环体。这些结果表明，促旋酶的作用机制涉及瞬时双链断裂以及DNA片段通过由此产生的缺口。促旋酶是一类普遍存在的酶的代表，在原核细胞和真核细胞中都能找到，这类酶能促进双链DNA片段相互扩散，从而可能解决DNA复制、重组和凝聚过程中出现的拓扑问题。
