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...

Small molecules that can enhance the DNA cleavage activity of type IA DNA topoisomerase can be identified and may provide leads for development of novel antibacterial compounds.

Escherichia coli expressing SOS-inducing mutant topoisomerase I was utilized to demonstrate that covalent protein-DNA complex accumulation results in oxidative damage. Hydroxyl radicals were detected following mutant topoisomerase induction. The presence of the Fe 2؉ chelator 2,2-dipyridyl and an iscS mutation affecting Fe-S cluster formation protect against topoisomerase I cleavage complex-mediated cell killing.

Bacterial topoisomerase I function is actively involved in the SOS response to antibiotics and stress challenge. Cell killing initiated by the topoisomerase I cleavage complex would be enhanced by antibiotics and the host response. These findings provide further support for bacterial topoisomerase I as a therapeutic target.

Accumulation of mutant topoisomerase I cleavage complex can lead to SOS induction and cell death in Escherichia coli. The single-stranded break associated with mutant topoisomerase I cleavage complex is converted to double-stranded break, which then is processed by the RecBCD pathway, followed by association of RecA with the single-stranded DNA.