Department of Pathology, Keck School of Medicine of University of Southern California, Los Angeles, CA, 90089, USA; Norris Comprehensive Cancer Center, Keck School of Medicine of University of Southern California, Los Angeles, CA, 90089, USA.
Department of Pathology, Keck School of Medicine of University of Southern California, Los Angeles, CA, 90089, USA; Norris Comprehensive Cancer Center, Keck School of Medicine of University of Southern California, Los Angeles, CA, 90089, USA; Department of Biological Sciences, Molecular and Computational Biology Section, University of Southern California, Los Angeles, CA, 90089, USA.
DNA Repair (Amst). 2019 Nov;83:102676. doi: 10.1016/j.dnarep.2019.102676. Epub 2019 Jul 26.
The Artemis nuclease recognizes and endonucleolytically cleaves at single-stranded to double-stranded DNA (ss/dsDNA) boundaries. It is also a key enzyme in the non-homologous end joining (NHEJ) DNA double-strand break repair pathway. Previously, a truncated form, Artemis-413, was developed that is constitutively active both in vitro and in vivo. Here, we use this constitutively active form of Artemis to detect DNA structures with ss/dsDNA boundaries that arise under topological stress. Topoisomerases prevent abnormal levels of torsional stress through modulation of positive and negative supercoiling. We show that overexpression of Artemis-413 in yeast cells carrying genetic mutations that ablate topoisomerase activity have an increased frequency of DNA double-strand breaks (DSBs). Based on the biochemical activity of Artemis, this suggests an increase in ss/dsDNA-containing structures upon increased torsional stress, with DSBs arising due to Artemis cutting at these ss/dsDNA structures. Camptothecin targets topoisomerase IB (Top1), and cells treated with camptothecin show increased DSBs. We find that expression of Artemis-413 in camptothecin-treated cells leads to a reduction in DSBs, the opposite of what we find with topoisomerase genetic mutations. This contrast between outcomes not only confirms that topoisomerase mutation and topoisomerase poisoning have distinct effects on cells, but also demonstrates the usefulness of Artemis-413 to study changes in DNA structure.
Artemis 核酸酶识别并在单链到双链 DNA（ss/dsDNA）边界处进行内切核酸酶切割。它也是非同源末端连接（NHEJ）DNA 双链断裂修复途径中的关键酶。以前，开发了一种截短形式的 Artemis-413，它在体外和体内均保持持续激活状态。在这里，我们使用这种持续激活的 Artemis 形式来检测拓扑结构压力下出现的具有 ss/dsDNA 边界的 DNA 结构。拓扑异构酶通过调节正超螺旋和负超螺旋来防止扭转应力的异常水平。我们表明，在携带消除拓扑异构酶活性的遗传突变的酵母细胞中过表达 Artemis-413 会增加 DNA 双链断裂（DSB）的频率。基于 Artemis 的生化活性，这表明在扭转应力增加时，含有 ss/dsDNA 的结构增加，由于 Artemis 在这些 ss/dsDNA 结构处切割，因此会出现 DSB。喜树碱靶向拓扑异构酶 IB（Top1），用喜树碱处理的细胞显示出增加的 DSB。我们发现，在喜树碱处理的细胞中表达 Artemis-413 会导致 DSB 减少，与拓扑异构酶遗传突变的情况相反。这种结果的对比不仅证实了拓扑异构酶突变和拓扑异构酶中毒对细胞有不同的影响，而且还证明了 Artemis-413 用于研究 DNA 结构变化的有用性。
