Topoisomerase 3B (TOP3B) DNA and RNA Cleavage Complexes and Pathway to Repair TOP3B-linked RNA and DNA Breaks

Abstract

Genetic inactivation of TOP3B is linked with schizophrenia, autism, intellectual disability and cancer. The present study demonstrates that in vivo TOP3B forms both RNA and DNA cleavage complexes (TOP3Bccs) and reveals a pathway for repairing TOP3Bccs. For detecting cellular TOP3Bccs, we engineered a "self-trapping" mutant of TOP3B (R338W TOP3B) and to determine how human cells repair TOP3Bccs, we depleted tyrosyl-DNA phosphodiesterases (TDP1 and TDP2). TDP2-deficient cells produced elevated TOP3Bccs both in DNA and RNA. Conversely, overexpression of TDP2 lowered cellular TOP3Bccs. Using recombinant human TDP2, we demonstrate that TDP2 cannot excise the native form of TOP3Bccs. Hypothesizing that TDP2 cannot access phosphotyrosyl linkage unless TOP3B is either proteolyzed or denatured, we found that cellular TOP3Bccs are ubiquitinated by the E3 Ubiquitin Ligase TRIM41 before undergoing proteasomal degradation and excision by TDP2. HIGHLIGHTSO_LIMethod for in vivo detection of TOP3B cleavage complexes (TOP3Bccs) formed both in DNA and RNA, using a religation defective "self-trapping" R338W TOP3B mutant. C_LIO_LIFirst evidence that TDP2 excises TOPccs produced by a type IA topoisomerase. C_LIO_LITDP2 processes both RNA and DNA TOP3Bccs following their ubiquitylation and proteasomal degradation inside cell. C_LIO_LITRIM41 is the first reported E3 ubiquitin ligase for TOP3Bcc ubiquitylation and proteasomal degradation. C_LI