Ishimi Y, Sugasawa K, Hanaoka F, Eki T, Hurwitz J
Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
J Biol Chem. 1992 Jan 5;267(1):462-6.
The effects of topoisomerases I and II on the replication of SV40 DNA were examined using an in vitro replication system of purified proteins that constitutes the monopolymerase system. In the presence of the two topoisomerases, two distinct nascent DNAs were formed. One product arising from the replication of the leading template strand was approximately half the size of the template DNA, whereas the other product derived from the lagging template strand consisted of short DNAs. These products were synthesized from both SV40 naked DNA and SV40 chromosomes. For the replication of SV40 naked DNA, either topoisomerase I or II maintained replication fork movement and supported complete leading strand synthesis. When SV40 chromosomes were replicated with the same proteins, reactions containing only topoisomerase I produced shorter leading strands. However, mature size DNA products accumulated in reactions supplemented with topoisomerase II, as well as in reactions containing only topoisomerase II. In the presence of crude extracts of HeLa cells, VP-16, a specific inhibitor of topoisomerase II, blocked elongation of the nascent DNA during the replication of SV40 chromosomes. These results indicate that topoisomerase II plays a crucial role as a swivelase in the late stage of SV40 chromosome replication in vitro.
使用由纯化蛋白构成的单聚合酶系统的体外复制体系，研究了拓扑异构酶I和II对SV40 DNA复制的影响。在两种拓扑异构酶存在的情况下，形成了两种不同的新生DNA。一种由前导模板链复制产生的产物大小约为模板DNA的一半，而另一种来自后随模板链的产物由短DNA组成。这些产物可从SV40裸DNA和SV40染色体中合成。对于SV40裸DNA的复制，拓扑异构酶I或II均可维持复制叉移动并支持完整的前导链合成。当用相同的蛋白复制SV40染色体时，仅含拓扑异构酶I的反应产生较短的前导链。然而，在补充了拓扑异构酶II的反应以及仅含拓扑异构酶II的反应中，积累了成熟大小的DNA产物。在存在HeLa细胞粗提物的情况下，拓扑异构酶II的特异性抑制剂VP-16在SV40染色体复制过程中阻断了新生DNA的延伸。这些结果表明，在体外SV40染色体复制后期，拓扑异构酶II作为解旋酶发挥着关键作用。
