DNA and RNA topoisomerase activities of Top3β are promoted by mediator protein Tudor domain-containing protein 3

Abstract

Topoisomerase 3β (Top3β) can associate with the mediator protein Tudor domain-containing protein 3 (TDRD3) to participate in two gene expression processes of transcription and translation. Despite the apparent importance of TDRD3 in binding with Top3β and directing it to cellular compartments critical for gene expression, the biochemical mechanism of how TDRD3 can affect the functions of Top3β is not known. We report here sensitive biochemical assays for the activities of Top3β on DNA and RNA substrates in resolving topological entanglements and for the analysis of TDRD3 functions. TDRD3 stimulates the relaxation activity of Top3β on hypernegatively supercoiled DNA and changes the reaction from a distributive to a processive mode. Both supercoil retention assays and binding measurement by fluorescence anisotropy reveal a heretofore unknown preference for binding single-stranded nucleic acids over duplex. Whereas TDRD3 has a structure-specific binding preference, it does not discriminate between DNA and RNA. This unique property for binding with nucleic acids can have an important function in serving as a hub to form nucleoprotein complexes on DNA and RNA. To gain insight into the roles of Top3β on RNA metabolism, we designed an assay by annealing two singlestranded RNA circles with complementary sequences. Top3β is capable of converting two such single-stranded RNA circles into a double-stranded RNA circle, and this strand-annealing activity is enhanced by TDRD3. These results demonstrate that TDRD3 can enhance the biochemical activities of Top3β on both DNA and RNA substrates, in addition to its function of targeting Top3β to critical sites in subcellular compartments.Tudor domain-containing domain 3 | DNA topoisomerases | RNA topoisomerases | RNA circles | RNA duplex H igher-order structural complexities in nucleic acids can be brought about by the folding and intertwining through interand intramolecular base pairing. They are impediments to the transactions of genetic information, including replication, transcription, recombination, and translation, which involve the unwinding and rewinding of base-paired regions to access encoded information (1-3). DNA topoisomerases are nature's tools to resolve the topological entanglements in nucleic acids. They are ubiquitous in nature, first discovered in bacteria in 1971 (4), and in mammalian cells in 1972 (5), and are characterized by a mechanism involving the formation of a covalent protein/DNA adduct to generate a transient and reversible break allowing for topological transformation (6). Based on whether the strand passage is through a protein-mediated single-stranded gate or a double-stranded gate, the enzymes are classified into type I or type II topoisomerases, respectively. Both types are further classified into A and B families based on their structural and mechanistic features (7). All topoisomerases are essential for the growth and development of an organism, suggesting that they have critical but distinct functions in all cells.Ever since their discovery, t...

Topoisomerase 3β is the major topoisomerase for mRNAs and linked to neurodevelopment and mental dysfunction

“…In fact, deletion of the Top3β-RGG domain or mutation of its catalytic residue can impair both RNA and DNA topoisomerase activity of the enzyme (4,10,13,27). Thus, it remains to be determined whether it is the DNA or RNA topoisomerase activity of Top3β that is important for neurodevelopment.…”

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

“…TOP3B localizes to both the nucleus and cytoplasm under steady-state condition (more abundant in the cytosol) as it facilitates both DNA and RNA metabolic processes inside cells (Stoll et al, 2013;Xu et al, 2013). Inside the nucleus, TOP3B in association with the scaffolding protein TDRD3 (Tudor Domain Containing 3) is recruited to promoter regions of target genes (NRAS, DDX5 and c-MYC) and has been proposed to facilitate transcription by relaxing hypernegatively supercoiled DNA and resolving R-loops (non-canonical nucleic acid structure consisting of RNA-DNA hybrids and displaced single-stranded DNA) (Goto-Ito et al, 2017;Huang et al, 2018a;Siaw et al, 2016;Yang et al, 2014;Zhang et al, 2019). TDRD3 also associates with the other well-known mRNA-binding protein FMRP (Fragile X Mental Retardation Protein) having roles in neuronal translation via its C-terminal FMRP Interacting Motif (FIM).…”

Topoisomerase III-ß is required for efficient replication of positive-sense RNA viruses

“…A well-known function of TDRD3 is to bind and stabilize Topoisomerase III-ß (TOP3B) [6][7][8] , a type IA topoisomerase and the only human topoisomerase known to act on both DNA and RNA 6,9 . Knockout of TDRD3 in HuH-7 cells led to levels of TOP3B that were almost as low as those obtained with knockout of TOP3B itself ( Fig 1C).…”