Developmental Therapeutics Branch & Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
Biomolecular Sciences Institute, Florida International University, Miami, FL 33199, USA; Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, USA.
Cell Rep. 2020 Dec 29;33(13):108569. doi: 10.1016/j.celrep.2020.108569.
The present study demonstrates that topoisomerase 3B (TOP3B) forms both RNA and DNA cleavage complexes (TOP3Bccs) in vivo and reveals a pathway for repairing TOP3Bccs. For inducing and detecting cellular TOP3Bccs, we engineer a "self-trapping" mutant of TOP3B (R338W-TOP3B). Transfection with R338W-TOP3B induces R-loops, genomic damage, and growth defect, which highlights the importance of TOP3Bcc repair mechanisms. To determine how cells repair TOP3Bccs, we deplete tyrosyl-DNA phosphodiesterases (TDP1 and TDP2). TDP2-deficient cells show elevated TOP3Bccs both in DNA and RNA. Conversely, overexpression of TDP2 lowers cellular TOP3Bccs. Using recombinant human TDP2, we demonstrate that TDP2 can process both denatured and proteolyzed TOP3Bccs. We also show that cellular TOP3Bccs are ubiquitinated by the E3 ligase TRIM41 before undergoing proteasomal processing and excision by TDP2.
本研究表明，拓扑异构酶 3B（TOP3B）在体内形成 RNA 和 DNA 切割复合物（TOP3Bccs），并揭示了修复 TOP3Bccs 的途径。为了诱导和检测细胞内的 TOP3Bccs，我们构建了一个“自捕获”的 TOP3B 突变体（R338W-TOP3B）。用 R338W-TOP3B 转染可诱导 R 环、基因组损伤和生长缺陷，这突出了 TOP3Bcc 修复机制的重要性。为了确定细胞如何修复 TOP3Bccs，我们耗尽了酪氨酸-DNA 磷酸二酯酶（TDP1 和 TDP2）。TDP2 缺陷细胞在 DNA 和 RNA 中均显示出升高的 TOP3Bccs。相反，TDP2 的过表达会降低细胞内的 TOP3Bccs。使用重组人 TDP2，我们证明 TDP2 可以处理变性和蛋白水解的 TOP3Bccs。我们还表明，细胞内的 TOP3Bccs 在被 TDP2 进行蛋白酶体处理和切除之前，被 E3 连接酶 TRIM41 泛素化。
