Castora F J, Vissering F F, Simpson M V
Biochim Biophys Acta. 1983 Sep 9;740(4):417-27. doi: 10.1016/0167-4781(83)90090-8.
Using isolated rat liver mitochondria, which have previously been shown to carry out true replicative DNA synthesis, we have obtained results which are in accord with the presence and functioning of a DNA gyrase in this organelle. The effects of the Escherichia coli DNA gyrase inhibitors, novobiocin, coumermycin, nalidixic acid and oxolinic acid, upon mtDNA replication suggest the involvement of the putative mitochondrial enzyme in various aspects of this process. First, the preferential inhibition of [3H]dATP incorporation into highly supercoiled DNA together with the appearance of labeled, relaxed DNA are consistent with the involvement of a gyrase in the process of generating negative supercoils in mature mtDNA. Second, the overall depression of incorporation of labeled dATP into mtDNA, including the reduction of radioactivity incorporated into replicative intermediates, suggests a 'swivelase' role for the putative gyrase, and this hypothesis is further supported by results obtained on sucrose gradient centrifugation of heat-denatured, D-loop mtDNA. Here, the synthesis of the completed clean circles is inhibited while 9 S initiator strand synthesis is not, suggesting that chain elongation is blocked by the gyrase inhibitors.
利用先前已被证明能进行真正复制性DNA合成的离体大鼠肝线粒体，我们获得的结果与该细胞器中存在DNA促旋酶并发挥作用相一致。大肠杆菌DNA促旋酶抑制剂新生霉素、香豆霉素、萘啶酸和恶喹酸对线粒体DNA复制的影响表明，推测的线粒体酶参与了这一过程的各个方面。首先，[3H]dATP优先抑制掺入高度超螺旋DNA，同时出现标记的松弛DNA，这与促旋酶参与在成熟线粒体DNA中产生负超螺旋的过程一致。其次，标记的dATP掺入线粒体DNA总体减少，包括掺入复制中间体的放射性降低，这表明推测的促旋酶具有“旋转酶”作用，热变性D环线粒体DNA蔗糖梯度离心得到的结果进一步支持了这一假设。在此，完整闭环的合成受到抑制，而9S起始链的合成不受抑制，这表明链延伸被促旋酶抑制剂阻断。
