Developmental Therapeutics Branch & Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
Laboratory of Genetics and Genomics, National Institute on Aging, NIH, Baltimore, MD 21224, USA.
Cell Rep. 2022 Jul 12;40(2):111067. doi: 10.1016/j.celrep.2022.111067.
The present study demonstrates how TOP3B is involved in resolving R-loops. We observed elevated R-loops in TOP3B knockout cells (TOP3BKO), which are suppressed by TOP3B transfection. R-loop-inducing agents, the topoisomerase I inhibitor camptothecin, and the splicing inhibitor pladienolide-B also induce higher R-loops in TOP3BKO cells. Camptothecin- and pladienolide-B-induced R-loops are concurrent with the induction of TOP3B cleavage complexes (TOP3Bccs). RNA/DNA hybrid IP-western blotting show that TOP3B is physically associated with R-loops. Biochemical assays using recombinant TOP3B and oligonucleotides mimicking R-loops show that TOP3B cleaves the single-stranded DNA displaced by the R-loop RNA-DNA duplex. IP-mass spectrometry and IP-western experiments reveal that TOP3B interacts with the R-loop helicase DDX5 independently of TDRD3. Finally, we demonstrate that DDX5 and TOP3B are epistatic in resolving R-loops in a pathway parallel with senataxin. We propose a decatenation model for R-loop resolution by TOP3B-DDX5 protecting cells from R-loop-induced damage.
本研究展示了 TOP3B 如何参与解决 R 环。我们观察到 TOP3B 敲除细胞 (TOP3BKO) 中 R 环升高，而 TOP3B 转染可抑制 R 环。R 环诱导剂拓扑异构酶 I 抑制剂喜树碱和剪接抑制剂 pladienolide-B 也会在 TOP3BKO 细胞中诱导更高的 R 环。喜树碱和 pladienolide-B 诱导的 R 环与 TOP3B 切割复合物 (TOP3Bccs) 的诱导同时发生。RNA/DNA 杂交 IP-免疫印迹显示 TOP3B 与 R 环物理相关。使用重组 TOP3B 和模拟 R 环的寡核苷酸进行的生化分析表明，TOP3B 可切割 R 环 RNA-DNA 双链体取代的单链 DNA。IP-质谱和 IP-免疫印迹实验揭示 TOP3B 与 R 环解旋酶 DDX5 相互作用，与 TDRD3 无关。最后，我们证明 DDX5 和 TOP3B 在与 senataxin 平行的途径中对 R 环的解决具有上位性。我们提出了一个由 TOP3B-DDX5 保护细胞免受 R 环诱导损伤的解旋模型。
