Zhang H, Wang J C, Liu L F
Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD 21205.
Proc Natl Acad Sci U S A. 1988 Feb;85(4):1060-4. doi: 10.1073/pnas.85.4.1060.
Treatment of HeLa cells with a DNA topoisomerase I-specific inhibitor, camptothecin, results in rapid cessation of the synthesis of the 45S rRNA precursor. The inhibition of rRNA synthesis is reversible following drug removal and correlates with the presence of camptothecin-trapped topoisomerase I-DNA abortive complexes, which can be detected as topoisomerase I-linked DNA breaks upon lysis with sodium dodecyl sulfate. These breaks were found to be concentrated within the transcribed region of human rRNA genes. No such sites can be detected in the inactive human rRNA genes in mouse-human hybrid cells, suggesting a preferential association of topoisomerase I with actively transcribed genes. The distribution of RNA polymerase molecules along the transcription unit of human rRNA genes in camptothecin-treated HeLa cells, as assayed by nuclear run-on transcription, shows a graded decrease of the RNA polymerase density toward the 3' end of the transcription unit; the density is minimally affected near the 5' start of the transcription unit. These results suggest that DNA topoisomerase I is normally involved in the elongation step of transcription, especially when the transcripts are long, and that camptothecin interferes with this role.
用DNA拓扑异构酶I特异性抑制剂喜树碱处理HeLa细胞，会导致45S rRNA前体的合成迅速停止。去除药物后，rRNA合成的抑制作用是可逆的，且与喜树碱捕获的拓扑异构酶I-DNA流产复合物的存在相关，在用十二烷基硫酸钠裂解后，这些复合物可被检测为拓扑异构酶I连接的DNA断裂。发现这些断裂集中在人类rRNA基因的转录区域内。在小鼠-人杂交细胞的无活性人类rRNA基因中未检测到此类位点，这表明拓扑异构酶I优先与活跃转录的基因结合。通过核转录延伸分析测定，在喜树碱处理的HeLa细胞中，RNA聚合酶分子沿人类rRNA基因转录单元的分布显示，RNA聚合酶密度朝着转录单元的3'端呈梯度下降；在转录单元5'端起始附近，密度受影响最小。这些结果表明，DNA拓扑异构酶I通常参与转录的延伸步骤，尤其是当转录本较长时，并且喜树碱会干扰这一作用。
