Darby M K, Schmitt B, Jongstra-Bilen J, Vosberg H P
EMBO J. 1985 Aug;4(8):2129-34. doi: 10.1002/j.1460-2075.1985.tb03903.x.
The effect of poly(ADP-ribosylation) on calf thymus topoisomerase type II reactions has been investigated. Unknotting of phage P4 head DNA, and relaxation and catenation of supercoiled PM2 DNA are inhibited. We conclude that the inhibition results from poly(ADP-ribosylation) on the following grounds. Firstly, the enzyme poly(ADP-ribose) (PADPR) synthetase and NAD are required, secondly, the competitive synthetase inhibitor nicotinamide abolishes topoisomerase inhibition, and thirdly, the polymer alone is not inhibitory. The mechanism of inhibition appears to be disruption of the strand cleavage reaction. A topoisomerase-DNA complex can be formed that upon treatment with protein denaturant at low ionic strength results in strand cleavage. The amount of DNA present in such a cleavable-complex progressively decreased following pretreatment of topoisomerase type II with PADPR synthetase and increasing concentrations of NAD. Treatment of the pre-formed complex with NAD and PADPR synthetase had no effect on its salt-induced dissociation. This suggests that either poly(ADP-ribosylation) has no influence on dissociation of topoisomerase, in contrast to association, or topoisomerase is not accessible to the synthetase when bound to DNA. Similar data were obtained with calf thymus type I topoisomerase.
研究了聚（ADP-核糖基化）对小牛胸腺拓扑异构酶II反应的影响。噬菌体P4头部DNA的解结以及超螺旋PM2 DNA的松弛和连环化均受到抑制。基于以下理由，我们得出抑制作用是由聚（ADP-核糖基化）引起的结论。首先，需要聚（ADP-核糖）（PADPR）合成酶和NAD；其次，竞争性合成酶抑制剂烟酰胺可消除拓扑异构酶的抑制作用；第三，单独的聚合物没有抑制作用。抑制机制似乎是链断裂反应的破坏。可以形成拓扑异构酶-DNA复合物，在低离子强度下用蛋白质变性剂处理该复合物会导致链断裂。在用PADPR合成酶和增加浓度的NAD对拓扑异构酶II进行预处理后，这种可裂解复合物中存在的DNA量逐渐减少。用NAD和PADPR合成酶处理预先形成的复合物对其盐诱导的解离没有影响。这表明，与缔合相反，聚（ADP-核糖基化）对拓扑异构酶的解离没有影响，或者当与DNA结合时，合成酶无法作用于拓扑异构酶。用小牛胸腺I型拓扑异构酶也获得了类似的数据。
