DNA and RNA Cleavage Complexes and Repair Pathway for TOP3B RNA- and DNA-Protein Crosslinks

Abstract

SUMMARY 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.

A dual-activity Topoisomerase Complex Regulates both Transcription and Translation

“…Our ndings that multiple genes regulated by the TOP3B-TDRD3 are related to autism or schizophrenia support the notion that TOP3B mutations contribute to the pathogenesis of psychiatric disorders (2,15,16). In addition, the data that TOP3B topoisomerase activity is needed for translation imply that TOP3B may form covalent cleavage complexes on RNA in cells, which is supported by data that a self-cleavage mutant of TOP3B can form such complexes in cells (11). We propose that TOP3B-RNA cleavage complexes could be a target of anti-viral drugs.…”

“…In addition, TOP3B possesses a nuclear export sequence that allows it to shuttle between nucleus and cytoplasm (2). Moreover, overexpression of a TOP3B mutant can produce cleavage complexes on both DNA and RNA in cells (11), suggesting that TOP3B can catalyze reactions on both types of nucleic acid in vivo. Notably, TOP3B has been recently shown to be required for replication of positive-strand RNA viruses including SARS-CoV2, raising the possibility that TOP3B could be a feasible antiviral target (12).…”

Human topoisomerases and their roles in genome stability and organization

“…a | DPCs form either at 3′ DNA ends (topoisomerase 1 (TOP1) or mitochondrial TOP1 (TOP1MT)) or 5′ DNA ends (TOP2A, TOP2B, TOP3A or TOP3B). They also form at 5′ RNA ends for TOP3B 2 . TOP1, TOP1MT, TOP2A and TOP2B bind double-stranded DNA; TOP3A and TOP3B bind single-stranded DNA or RNA.…”

“…Human topoisomerases and the associated repair enzymes tyrosyl-DNA phosphodiesterases (TDPs; TDP1 and TDP2) are located both in the nucleus and in mitochondria; in addition, cytoplasmic RNAs are handled by TOP3B and TDP2 (refs 1 , 2 ) (Supplementary Table 1 ).…”

Topoisomerase Assays

“…The RADAR assay is readily adapted for studies of specific adducts and cell types for diverse applications, including analyses of the roles of SUMO (Sun et al, 2020) and SPRTN (Fielden et al, 2020) in resolution of topoisomerase I−DNA adducts; detection of endogenous adducts resolved by GCNA (Bhargava et al, 2020); characterization of covalent DNA and RNA adducts formed by TOP3B (Saha et al, 2020); identification of roles of adducts formed by HMCES in protection of abasic sites in single-stranded DNA (Mohni et al, 2019); and establishment of cell cycle−dependent crosslinking of the Epstein-Barr virus EBNA1 regulatory protein to viral episomes (Dheekollu et al, 2021).…”