Herring Petra, Roedgaard Morten, Holst Camilla Myrup, Christensen Helene, Knudsen Birgitta R, Bjergbaek Lotte, Andersen Anni Hangaard
Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark.
FEBS Lett. 2025 Jun;599(12):1749-1759. doi: 10.1002/1873-3468.70053. Epub 2025 May 1.
We present a transcription-coupled Flp-nick system enabling a stable protein-bound nick mimicking a topoisomerase I-DNA cleavage complex. The nick is introduced at a single site within a controllable LacZ gene inserted into the Saccharomyces cerevisiae genome. This system allows unique single-site studies of a frequently occurring damage within a transcription unit in vivo. As proof of principle, we demonstrate RNA polymerase II accumulation at the damage site when MG132 inhibits the proteasome. Similarly, accumulation occurs when polymerase ubiquitination is abolished by deletion of the ubiquitinase ELC1 gene. This indicates that a topoisomerase I-DNA mimicking cleavage complex per se induces RNA polymerase II ubiquitination and degradation. These findings advance understanding of cellular responses to topoisomerase I-targeting drugs used in cancer chemotherapy.
我们展示了一种转录偶联的Flp-切口系统，该系统能够形成一种稳定的与蛋白质结合的切口，模拟拓扑异构酶I-DNA裂解复合物。该切口在插入酿酒酵母基因组的可控LacZ基因内的单个位点引入。该系统允许在体内对转录单元中频繁出现的损伤进行独特的单位点研究。作为原理验证，我们证明当MG132抑制蛋白酶体时，RNA聚合酶II在损伤位点积累。同样，当通过缺失泛素酶ELC1基因消除聚合酶泛素化时也会发生积累。这表明模拟裂解复合物的拓扑异构酶I-DNA本身会诱导RNA聚合酶II泛素化和降解。这些发现推动了对细胞对癌症化疗中使用的拓扑异构酶I靶向药物反应的理解。
