Wittig B, Dorbic T, Rich A
Institut für Molekularbiologie und Biochemie, Freie Universität, Berlin, Federal Republic of Germany.
Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2259-63. doi: 10.1073/pnas.88.6.2259.
Mammalian cells have been encapsulated in agarose microbeads, and from these cells metabolically active permeabilized nuclei were prepared. Previously, we showed that biotin-labeled monoclonal antibodies against Z-DNA can be diffused into the nuclei and, over a specific concentration range, they will bind to Z-DNA within the nucleus in a concentration-independent manner. By using radiolabeled streptavidin, we showed that the amount of Z-DNA antibody bound is related to the torsional strain of the DNA in the nucleus. Relaxation of the DNA results in a decrease of Z-DNA formation, whereas increasing torsional strain through inhibiting topoisomerase I results in increased Z-DNA formation. Here we measure the influence of RNA transcription and DNA replication. Transcription is associated with a substantial increase in the binding of anti-Z-DNA antibodies, paralleling the increased level of RNA synthesized as the level of ribonucleoside triphosphate in the medium is increased. DNA replication yields smaller increases in the binding of Z-DNA antibodies. Stopping RNA transcription with inhibitors results in a large loss of Z-DNA antibody binding, whereas only a small decrease is associated with inhibition of DNA replication.
哺乳动物细胞已被包裹在琼脂糖微珠中，并从这些细胞中制备出代谢活跃的通透细胞核。此前，我们发现针对Z-DNA的生物素标记单克隆抗体可以扩散到细胞核中，并且在特定浓度范围内，它们将以浓度无关的方式与细胞核内的Z-DNA结合。通过使用放射性标记的链霉亲和素，我们发现结合的Z-DNA抗体的量与细胞核中DNA的扭转应变有关。DNA的松弛导致Z-DNA形成减少，而通过抑制拓扑异构酶I增加扭转应变则导致Z-DNA形成增加。在这里，我们测量RNA转录和DNA复制的影响。转录与抗Z-DNA抗体结合的大幅增加相关，随着培养基中三磷酸核糖核苷水平的增加，RNA合成水平也相应增加。DNA复制导致Z-DNA抗体结合的增加幅度较小。用抑制剂停止RNA转录会导致Z-DNA抗体结合大量丧失，而抑制DNA复制仅会导致少量减少。
