Recombinant DNA topoisomerase 3 (topB), partial

What is DNA Topoisomerase 3 and what distinguishes it from other topoisomerases?

DNA Topoisomerase 3 belongs to the Type IA topoisomerase family, which is present in all living organisms. Type IA topoisomerases resolve DNA/RNA catenanes, knots, and supercoils by breaking and rejoining single-stranded DNA/RNA segments and allowing the passage of another nucleic acid segment through the break . Unlike Type II topoisomerases that cleave both strands of DNA, Type IA topoisomerases like Topoisomerase 3 make transient breaks in only one strand. The key distinguishing feature of Topoisomerase 3 is its ability to catalyze single-strand breaks and resolve topological issues without requiring ATP hydrolysis, making it energetically efficient for cells.

What are the structural components of Topoisomerase 3 that enable its catalytic activity?

Topoisomerase 3 consists of several structural domains that work together during catalysis. Based on recent cryo-EM studies of human TOP3B (a homolog of bacterial topB), the enzyme contains:

• A core domain (domains I-IV) that forms the catalytic center

• A C-terminal domain (CTD) with multiple zinc-finger-like motifs

• An active site containing a catalytic tyrosine residue (equivalent to Y336 in human TOP3B) that forms a transient covalent bond with the cleaved DNA/RNA

The enzyme transitions between open and closed conformations during its catalytic cycle. The opening between domains I and III and domains III and IV creates gates that allow the passage of the DNA T-segment during relaxation .

How does Topoisomerase 3 participate in DNA replication and repair processes?

Topoisomerase 3 plays crucial roles in maintaining genome stability by:

• Resolving DNA topological problems arising during replication

• Facilitating the unwinding of negative supercoils ahead of replication forks

• Participating in the resolution of recombination intermediates

• Preventing the accumulation of aberrant DNA structures that could lead to genomic instability

In eukaryotes, TOP3 homologs (TOP3α and TOP3β) ensure genome stability, proper neurodevelopment, and normal aging . TOP3α specifically functions in mitochondrial DNA maintenance, where it catalyzes single-strand breaks that can lead to linearization of mtDNA when a pre-existing nick is nearby .

What are the established protocols for purifying active recombinant Topoisomerase 3 (topB)?

For purifying active recombinant Topoisomerase 3, researchers typically follow these methodological steps:

• Cloning: The topB gene is PCR-amplified from bacterial genomic DNA and cloned into an expression vector with an appropriate tag (His-tag or GST-tag).

• Expression: The construct is transformed into an E. coli expression strain (BL21(DE3) or derivatives) and protein expression is induced with IPTG at optimal temperature and duration.

• Purification procedure:

  • Cell lysis in buffer containing protease inhibitors

  • Initial purification by affinity chromatography (Ni-NTA for His-tagged proteins)

  • Ion exchange chromatography to remove nucleic acid contaminants

  • Size exclusion chromatography for final polishing

  • Testing enzyme activity using supercoiled plasmid DNA relaxation assays

For enhanced stability, co-expression with cofactors like TDRD3 (as seen with human TOP3B) can be considered, as this has been shown to stabilize the enzyme complex .

What assays are most effective for measuring Topoisomerase 3 activity in vitro?

The following assays are most effective for measuring Topoisomerase 3 activity:

• DNA relaxation assay: This is the primary assay used to measure topoisomerase activity. Negatively supercoiled plasmid DNA (like pUC19) is incubated with the enzyme, and the conversion to relaxed forms is monitored by agarose gel electrophoresis . The addition of chloroquine or ethidium bromide during electrophoresis helps distinguish between different topological forms.

• DNA/RNA cleavage assay: Using specifically designed substrates with TOP3B cleavage sites, researchers can monitor the formation of covalent enzyme-nucleic acid intermediates .

• Strand passage assay: This measures the ability of the enzyme to allow one DNA segment to pass through another.

• Decatenation assay: This assesses the ability to separate interlinked DNA circles.

• FRET-based assays: For real-time monitoring of topoisomerase activity using fluorescently labeled DNA substrates.

How can researchers design optimal DNA substrates for Topoisomerase 3 activity studies?

To design optimal DNA substrates for Topoisomerase 3 studies, researchers should consider:

• Gapped DNA substrates: Create substrates with an 11-nt single-stranded segment flanked by double-stranded regions, similar to the 43-mer gapped substrate described in recent studies .

• Cleavage site inclusion: Ensure the single-stranded segment contains a known topoisomerase cleavage site.

• Mismatch bubbles: For studying CTD interactions, design substrates with mismatched nucleotides opposite to the cleavage strand, which mimics structures found in negatively supercoiled DNA .

• R-loop substrates: For studying RNA-DNA hybrid interactions, especially relevant for TOP3B which acts on both DNA and RNA .

• Fluorescent labeling: Consider adding fluorescent labels at strategic positions for FRET-based assays or visualization.

• Structural variants: Prepare substrates representing different topological challenges (supercoiled, knotted, or catenated) depending on the specific activity being studied.

How does the catalytic mechanism of Topoisomerase 3 differ when processing DNA versus RNA substrates?

Recent structural studies have revealed important insights into the dual DNA/RNA processing capabilities of Topoisomerase 3:

• Common catalytic mechanism: The catalytic sites of TOP3B are superimposable in pre-cleavage DNA and RNA complexes, indicating a shared mechanism for cleaving both nucleic acids .

• Substrate recognition differences: While DNA is typically B-form, RNA is A-form with shorter base-to-base distances due to different sugar pucker conformations and the presence of 2'-OH groups. TOP3B accommodates these differences while maintaining catalytic activity .

• Rejoining efficiency: DNA-rejoining-deficient mutants (K10M) can still slowly rejoin RNA, suggesting that the ribose 2'-OH group adjacent to the nucleophile 3'-OH makes it more susceptible to deprotonation .

• Metal ion cofactors: Mn²⁺ ions play a crucial role in both DNA and RNA processing, helping reposition protein domains and nucleic acid ends for rejoining .

The shared mechanism explains why TOP3B can process both substrates, while the enhanced RNA rejoining activity may relate to the unique chemical properties of RNA.

What is the role of Topoisomerase 3 in viral replication and potential antiviral applications?

Topoisomerase 3β (TOP3B) has been identified as a critical host factor required for efficient replication of positive-sense single-stranded RNA viruses:

• Viral dependency: Genome-scale loss-of-function screens have revealed that TOP3B is essential for yellow fever virus and dengue virus-2 replication .

• Broad spectrum requirement: TOP3B is required for efficient replication of all positive-sense single-stranded RNA viruses tested, including SARS-CoV-2 .

• Potential mechanism: TOP3B likely helps resolve topological problems in viral RNA structures during replication, though the exact mechanism requires further investigation.

• Antiviral target potential: TOP3B presents an attractive anti-viral target, though specific inhibitors are currently lacking .

• Research implications: Developing specific TOP3B inhibitors could lead to broad-spectrum antiviral therapies effective against multiple RNA viruses, including coronaviruses.

Further research into the exact mechanism of TOP3B involvement in viral replication could reveal new therapeutic strategies for a range of viral infections.

How does the cofactor TDRD3 enhance Topoisomerase 3β activity, and what are the implications for experimental design?

TDRD3 enhances Topoisomerase 3β activity through several mechanisms that should be considered in experimental designs:

• Activity stimulation: TDRD3 stimulates both DNA and RNA topoisomerase catalytic activities of TOP3β by binding and stabilizing single-stranded regions of nucleic acids .

• Complex stabilization: TOP3B is more stable when bound to TDRD3, making co-expression of these proteins advantageous for structural and functional studies .

• Targeting mechanism: TDRD3 guides TOP3B to its cellular targets through direct interactions with domain II of TOP3B .

• Retention of negative supercoiling: TDRD3 can retain negative supercoiling in plasmid DNA, potentially creating an environment that enhances TOP3β activity .

• Structural basis: The N-terminal region of TDRD3 (residues 1-190) establishes intramolecular contacts with both the core and CTD of TOP3B, stabilizing the heterodimer .

For optimal experimental design, co-expression and co-purification of TOP3B with TDRD3 is recommended for studying the physiologically relevant complex .

What are the key structural transitions during the catalytic cycle of Topoisomerase 3, and how can they be captured experimentally?

The catalytic cycle of Topoisomerase 3 involves several key structural transitions that can be captured using various experimental approaches:

• Gate-opening mechanism:

  • TOP3B transitions between open and closed conformations

  • The opening involves a dramatic swinging motion of domains II and III relative to domains I and IV

  • This creates gates between domains I and III and domains III and IV for strand passage

• Experimental capture approaches:

  • Cryo-EM with substrate variants: Using different DNA/RNA substrates to capture distinct states

  • Active site mutations: Y336F mutation to disable cleavage while preserving DNA/RNA interactions

  • Rejoining-deficient mutations: K10M mutation to study the rejoining complex

  • Metal ion manipulation: Controlling the presence of divalent metal ions (Mg²⁺, Mn²⁺) to stabilize specific states

  • Crosslinking strategies: To stabilize transient conformations

  • Time-resolved studies: To capture the dynamic transitions between states

• Catalytic states observed:

  • Pre-cleavage complex

  • Cleavage complex

  • Post-cleavage complex

  • Rejoining complex

  • Open-gate conformation

Each state represents a distinct step in the catalytic mechanism, and understanding these transitions is crucial for developing inhibitors or enhancers of topoisomerase activity.

What are the common challenges in expressing and purifying active recombinant Topoisomerase 3, and how can they be addressed?

Researchers face several challenges when working with recombinant Topoisomerase 3:

• Protein instability:

  • Challenge: Topoisomerase 3 can be unstable when expressed alone

  • Solution: Co-express with stabilizing partners like TDRD3 for human TOP3B

• Nucleic acid contamination:

  • Challenge: Topoisomerases bind DNA/RNA tightly, leading to nucleic acid contamination

  • Solution: Include high-salt washes and nuclease treatments during purification

• Loss of activity:

  • Challenge: Activity loss during purification or storage

  • Solution: Add reducing agents (DTT, β-mercaptoethanol), optimize buffer conditions, and ensure proper storage at -80°C with glycerol

• Expression toxicity:

  • Challenge: Overexpression in bacterial hosts can be toxic

  • Solution: Use tightly controlled expression systems, lower induction temperatures (16-18°C), and shorter induction times

• Proper folding:

  • Challenge: Ensuring correct folding, especially of the zinc finger domains

  • Solution: Supplement growth media with zinc, use chaperone co-expression systems

The most effective approach combines the expression of TOP3B with its cofactor TDRD3 in HEK293 cells, as demonstrated in recent structural studies, where TOP3B was found to be more stable when bound to TDRD3 .

How can researchers distinguish between DNA and RNA topoisomerase activities when characterizing Topoisomerase 3 variants?

To distinguish between DNA and RNA topoisomerase activities:

• Substrate-specific assays:

  • Use pure DNA substrates (supercoiled plasmids, oligonucleotides)

  • Use pure RNA substrates (in vitro transcribed RNAs with similar topology)

  • Compare activity rates and substrate preferences under identical conditions

• Comparative mutational analysis:

  • The K10M mutation affects DNA rejoining more than RNA rejoining in TOP3B

  • Create targeted mutations and assess differential effects on DNA vs. RNA processing

• Competition assays:

  • Perform activity assays with mixed DNA/RNA substrates

  • Measure preferential activity when both substrates are available

• Structural probing:

  • Use nuclease protection assays or chemical probing to map enzyme-substrate interactions

  • Identify differential binding sites or conformational changes

• Reaction condition optimization:

  • DNA and RNA activities may have different optimal conditions (salt, pH, metal ions)

  • Systematically vary conditions to identify differential optima

These approaches enable researchers to characterize the dual functionality of Topoisomerase 3 variants and identify mutations or conditions that selectively affect one activity over the other.

What approaches can resolve contradictory data regarding Topoisomerase 3 function in different experimental systems?

Resolving contradictory data regarding Topoisomerase 3 function requires systematic investigation:

• Cross-validation with multiple techniques:

  • Combine biochemical assays, structural studies, genetic approaches

  • Use both in vitro and in vivo systems to validate findings

  • Employ both gain-of-function and loss-of-function approaches

• Careful evaluation of experimental conditions:

  • Different buffer compositions, salt concentrations, and pH can affect activity

  • Temperature and incubation times may reveal condition-dependent effects

  • Presence/absence of cofactors like TDRD3 significantly impacts function

• Substrate considerations:

  • DNA vs. RNA substrates yield different results

  • Topology of substrates (supercoiled, relaxed, linear) affects activity

  • Sequence context around cleavage sites influences efficiency

• Species-specific differences:

  • Bacterial topB may function differently than eukaryotic TOP3α/β

  • Compare orthologous enzymes to identify conserved vs. species-specific functions

• Cofactor interactions:

  • Test activities with and without known cofactors

  • Screen for novel interacting partners that may modulate function

When contradictions arise, systematic exploration of these variables often reveals that the enzyme functions differently under specific conditions rather than truly contradictory behavior.

What are the emerging approaches for targeting Topoisomerase 3 in therapeutic applications, particularly for viral diseases?

Several promising approaches are emerging for targeting Topoisomerase 3 therapeutically:

• Structure-guided inhibitor design:

  • Recent cryo-EM structures of TOP3B in different catalytic states provide templates for rational drug design

  • Virtual screening against active site and gate-opening conformations

  • Fragment-based approaches targeting specific protein domains

• Antiviral applications:

  • TOP3B is required for efficient replication of all positive-sense single-stranded RNA viruses tested, including SARS-CoV-2

  • Developing TOP3B inhibitors as broad-spectrum antivirals

  • Screening of existing compound libraries for TOP3B inhibition

• Allosteric modulators:

  • Targeting the TOP3B-TDRD3 interface to disrupt complex formation

  • Designing compounds that prevent gate-opening required for strand passage

  • Developing inhibitors that interfere with the zinc-finger motifs in the CTD

• RNA-specific targeting:

  • Creating compounds that selectively inhibit RNA topoisomerase activity while sparing DNA activity

  • Exploiting structural differences in RNA vs. DNA substrate binding

• Combination approaches:

  • Combining TOP3B inhibitors with existing antivirals for synergistic effects

  • Developing dual-targeting compounds that affect both viral proteins and TOP3B

The lack of specific TOP3B inhibitors currently presents both a challenge and an opportunity for developing novel therapeutic agents against RNA viral diseases .

How might recent structural insights into Topoisomerase 3β function inform new experimental approaches?

Recent structural insights into TOP3B function open several new experimental avenues:

• Mechanism-based assay development:

  • Design assays specifically targeting gate-opening or strand passage

  • Create fluorescent reporters to monitor conformational changes during catalysis

  • Develop high-throughput screening methods based on structural transitions

• Engineering enhanced or specialized variants:

  • Target modifications to enhance stability without sacrificing activity

  • Create variants with altered substrate specificity (DNA vs. RNA preference)

  • Engineer topoisomerases with novel functions by modifying domains

• Interaction studies:

  • Investigate how TOP3B-TDRD3 interactions with other proteins are structurally mediated

  • Examine the role of the CTD in recruiting TOP3B to different cellular compartments

  • Study how zinc finger motifs contribute to nucleic acid recognition

• Single-molecule approaches:

  • Visualize individual steps in the catalytic cycle using FRET or optical tweezers

  • Measure kinetics of conformational changes during substrate processing

  • Directly observe strand passage events

• In vivo structural biology:

  • Apply cryo-electron tomography to visualize TOP3B in cellular contexts

  • Use proximity labeling to map the interactome in different cellular conditions

  • Develop biosensors to monitor TOP3B activity in living cells

The open-gate conformation captured by cryo-EM provides particularly valuable insights into the strand-passage mechanism, which can now be targeted for further experimental investigation.

What are the implications of Topoisomerase 3's dual DNA/RNA activity for understanding cellular RNA metabolism?

The dual DNA/RNA activity of Topoisomerase 3 has significant implications for RNA metabolism:

• R-loop regulation:

  • TOP3B promotes R-loop disassembly, impacting transcription and genomic stability

  • This activity affects gene expression patterns and may be particularly important in neurons

  • R-loops represent a critical interface between DNA and RNA metabolism

• mRNA translation effects:

  • TOP3B associates with polyribosomes and regulates translation

  • This activity is linked to neurodevelopment through complexes with FMRP

  • Topoisomerase activity may resolve structural impediments to translation

• RNA structural diversity:

  • Non-canonical RNA structures (knots, supercoils) may be more common than previously recognized

  • TOP3B may regulate RNA secondary and tertiary structures that affect function

  • This introduces a new dimension to RNA metabolism regulation

• Viral RNA replication:

  • TOP3B is required for efficient replication of positive-sense RNA viruses

  • This suggests critical roles in resolving topological problems during viral RNA synthesis

  • Understanding these mechanisms could reveal new antiviral strategies

• Evolutionary perspectives:

  • RNA topoisomerase activity has been identified across all three domains of life

  • This suggests ancient and fundamental roles in RNA metabolism

  • Differential activity (TOP3B acts on both DNA and RNA, while TOP3A acts only on DNA) points to specialized functions

These insights suggest that RNA topology regulation is a critical but understudied aspect of RNA metabolism, with implications for gene expression, neurodevelopment, and viral replication.