INVOLVEMENT OF DNA TOPOISOMERASE-I IN TRANSCRIPTION OF HUMAN RIBOSOMAL-RNA GENES
INVOLVEMENT OF DNA TOPOISOMERASE-I IN TRANSCRIPTION OF HUMAN RIBOSOMAL-RNA GENES
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DOI：
10.1073/pnas.85.4.1060
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发表时间：
1988-02-01
影响因子：
11.1
通讯作者：
LIU, LF
中科院分区：
文献类型：
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作者：
ZHANG, H;WANG, JC;LIU, LF
用DNA拓扑异构酶i特异性抑制剂喜树碱处理HeLa细胞，导致45S rRNA前体的合成迅速停止。rRNA合成的抑制在药物去除后是可逆的，并且与喜树碱捕获的拓扑异构酶I-DNA流产复合物的存在有关，可以通过十二烷基硫酸钠裂解拓扑异构酶I-DNA断裂来检测。这些断裂被发现集中在人类rRNA基因的转录区。在小鼠-人杂交细胞中，没有在失活的人rRNA基因中检测到这样的位点，这表明拓扑异构酶I与活跃转录的基因有优先关联。在喜树碱处理的HeLa细胞中，RNA聚合酶分子沿着人rRNA基因转录单元的分布，通过核运行转录检测，显示RNA聚合酶密度在转录单元的3”端呈梯度下降；在转录单元的5”起始处，密度受到的影响最小。这些结果表明，DNA拓扑异构酶I通常参与转录的延伸步骤，特别是当转录物很长时，喜树碱干扰了这一作用。
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.