TDP2 Human

What is TDP2 and what is its primary biochemical function?

TDP2 is an enzyme that repairs irreversible topoisomerase II (TOP2)-mediated cleavage complexes generated by anticancer topoisomerase-targeted drugs and during normal cellular processes . Its primary biochemical function is to specifically cleave off tyrosine residues covalently linked to the 5′-end of DNA, leaving a phosphate group that enables direct DNA repair . Beyond this canonical role, TDP2 also processes replication intermediates for picornaviruses (acting as a VPg unlinkase) and hepatitis B virus .

What are the different isoforms of TDP2 in human cells and how are they distributed?

Human cells express two main TDP2 isoforms:

• Full-length TDP2: Contains a nuclear localization signal and ubiquitin-associated domain in the N-terminus, present predominantly in the nucleus but also found in mitochondria and cytosol .

• TDP2S (short isoform): Expressed from an alternative transcription start site, contains a mitochondrial targeting sequence (MTS) that contributes to its enrichment in mitochondria and cytosol while being excluded from the nucleus .

Both isoforms maintain similar biochemical activity despite their different N-terminal sequences. This differential localization appears to be functionally important, as demonstrated by fractionation studies in multiple human cell lines including H226 lung cancer cells, HEK293T kidney cells, and HCT116 colon carcinoma cells .

How does TDP2 protect genome integrity at the molecular level?

TDP2 protects genome integrity through several mechanisms:

• Efficient processing of TOP2-induced DNA double-strand breaks (DSBs) by removing covalently bound TOP2 from DNA ends .

• Generation of clean 5′-phosphate termini with 4-bp cohesive overhangs that facilitate rapid and accurate repair through the canonical non-homologous end joining (NHEJ) pathway .

• Suppression of chromosomal translocations by reducing the likelihood of incorrect end joining during DNA repair .

Research with TDP2-deficient human cells demonstrates that loss of TDP2 significantly increases genome instability markers following treatment with TOP2 poisons, including elevated levels of micronuclei, nucleoplasmic bridges, and approximately 10-fold more chromosome aberrations per cell compared to normal cells .

What methodologies are effective for studying TDP2 subcellular localization?

Researchers employ several complementary approaches to investigate TDP2 subcellular localization:

Cellular Fractionation and Immunoblotting:

• Separate nuclear, cytosolic, and mitochondrial fractions using established protocols

• Treat samples with digitonin (0.1 mg/ml) prior to fractionation to minimize cross-contamination

• Verify fraction purity using compartment-specific markers:

  • Nuclear: TOP1

  • Mitochondrial: COX4

  • Cytosolic: GAPDH

• Detect TDP2 isoforms using specific antibodies

Proteinase K Protection Assay:
This technique confirms the presence of TDP2 inside mitochondria rather than merely associated with the outer membrane:

• Isolate intact mitochondria

• Treat with Proteinase K in the presence or absence of SDS

• Proteins within mitochondrial membranes remain resistant to Proteinase K unless membranes are disrupted by SDS

• Use appropriate controls:

  • TOM70 (outer membrane protein): degraded by Proteinase K

  • Cytochrome C (intermembrane space): protected from Proteinase K

  • TFAM (matrix protein): protected from Proteinase K

This methodological approach has conclusively demonstrated that both TDP2 isoforms are present inside human mitochondria, not merely associated with the outer membrane .

How can researchers effectively generate and validate TDP2-deficient models?

Researchers have successfully employed multiple strategies to develop TDP2-deficient experimental models:

Cell Line Development:

• CRISPR-Cas9 gene editing in human cell lines:

  • RPE-1 hTERT cells with disrupted TDP2 gene show hypersensitivity to etoposide and reduced repair of etoposide-induced DSBs

• Avian DT40 cell models:

  • TDP2-/- DT40 cells serve as a clean genetic background for complementation studies

  • These can be reconstituted with human TDP2 to assess functional rescue

Validation Methods:

• Functional characterization:

  • Hypersensitivity to topoisomerase II poisons (etoposide)

  • Reduced repair of TOP2-induced DNA damage measured by γH2AX immunofoci

  • Increased chromosomal aberrations following genotoxic stress

• Biochemical confirmation:

  • Loss of TDP2 catalytic activity in cell extracts

  • Absence of protein by immunoblotting

  • Complementation studies with wild-type or catalytically inactive mutants (e.g., TDP2 D262A)

• Isoform-specific analysis:

  • CRISPR-engineered human cells expressing only the TDP2S isoform can be used to dissect isoform-specific functions

What are effective approaches for measuring TDP2 enzymatic activity?

Multiple approaches have been developed to assess TDP2 enzymatic activity:

Biochemical Assays:

• 5′-phosphotyrosyl cleavage assay:

  • Uses synthetic DNA substrates with tyrosine residues covalently linked to 5′-end

  • Measures removal of tyrosine leaving a phosphate group

  • Can compare apparent rates of cleavage between different TDP2 isoforms or mutants

Cellular Functional Assays:

• DNA damage repair kinetics:

  • Measure repair of etoposide-induced DNA damage by quantifying γH2AX foci resolution over time

  • TDP2-deficient cells show delayed resolution of DNA damage

• Mitochondrial DNA (mtDNA) protection assay:

  • Treatment with mitochondrial-targeted doxorubicin (mtDox)

  • Measurement of mtDNA depletion, which is more severe in TDP2-deficient cells

• Mitochondrial transcription analysis:

  • Quantification of mitochondrial transcription levels

  • TDP2 deficiency leads to reduced mitochondrial transcription in human cell lines

How does TDP2 suppress TOP2-induced chromosomal translocations?

TDP2 plays a critical role in suppressing chromosomal translocations through several mechanisms:

• Efficient processing of TOP2-induced DSBs to create clean, directly ligatable DNA ends

• Generation of 4-bp cohesive 5′-overhangs that enable rapid ligation of adjacent termini without further processing

• Reduction in the chance of joining incorrect chromosomal termini through preservation of these cohesive ends

Sequence analysis of translocation junctions in MLL (a common translocation site) revealed that approximately 80% of translocation partners were transcriptionally active regions, with a strong bias toward partners located on the same chromosome as MLL. This suggests that the proximity of TOP2-induced DSBs influences the likelihood of incorrect rejoining .

Notably, TDP2 patient cells exhibited approximately 5-fold more abnormal metaphases and 10-fold more chromosome aberrations per cell than control cells following etoposide treatment, with a particularly high frequency of complex events involving multiple chromosomes .

What is the specific role of TDP2 in transcription-associated DNA damage repair?

TDP2 has a specialized role in repairing TOP2-induced DNA damage during gene transcription:

• Protection during transcription:

  • TDP2 repairs TOP2-induced DSBs arising during gene transcription, which can occur independently of cell cycle status

  • This function is particularly important in non-cycling cells that lack alternative error-free repair pathways

• Experimental evidence:

  • In quiescent RPE-1 cells, the elevated accumulation of etoposide-induced DSBs in TDP2-deficient cells was prevented by pre-incubation with the RNA polymerase II transcription inhibitor DRB

  • This demonstrates that TDP2 is specifically important for repairing TOP2-induced DSBs arising during transcription

• Translocation patterns:

  • Approximately 84% of MLL translocation breakpoints identified after etoposide treatment involved partner loci located in actively transcribed regions

  • This further supports the connection between transcription, TOP2-induced damage, and TDP2 repair activity

How does TDP2 activity differ between repair of nuclear and mitochondrial DNA damage?

TDP2 functions in both nuclear and mitochondrial compartments, but with some notable differences:

Nuclear TDP2 Activity:

• Predominantly involves the full-length TDP2 isoform containing the nuclear localization signal and ubiquitin-associated domain

• Focuses on repairing TOP2-induced DSBs to maintain chromosomal stability and prevent translocations

• Works in conjunction with the canonical NHEJ pathway to facilitate error-free repair

Mitochondrial TDP2 Activity:

• Involves both full-length TDP2 and the enriched TDP2S isoform

• Protects mitochondrial DNA from damage induced by topoisomerase poisons

• Prevents selective depletion of mitochondrial DNA when cells are exposed to mitochondrial-targeted doxorubicin (mtDox)

• Supports mitochondrial transcription, as loss of TDP2 in mitochondria reduces transcription levels

The presence of both TDP2 isoforms in mitochondria has been conclusively demonstrated through:

• Cellular fractionation studies showing TDP2 in mitochondrial fractions

• Proteinase K protection assays confirming that TDP2 is located inside mitochondria rather than merely associated with the outer membrane

• Functional studies showing that TDP2-deficient cells are hypersensitive to mitochondrial-targeted doxorubicin

How might TDP2 inhibitors enhance the efficacy of TOP2-targeting chemotherapeutics?

TDP2 inhibitors show significant promise for enhancing the efficacy of TOP2-targeting chemotherapeutics through several mechanisms:

• Selective inhibition:

  • Deazaflavin derivatives selectively inhibit human TDP2 enzyme in a competitive manner

  • These compounds work with both recombinant and native TDP2

• Synergistic activity:

  • Deazaflavin derivatives show potent synergy in combination with the topoisomerase II inhibitor etoposide in:

    • Human prostate cancer DU145 cells

    • TK6 human lymphoblast cells

    • Avian DT40 cells

  • This synergy is specifically TDP2-dependent, as demonstrated in genetic models

• Enhanced DNA damage:

  • By inhibiting TDP2, these compounds prevent the repair of TOP2-induced DNA damage

  • This potentially converts reversible DNA damage into irreversible lesions, increasing cancer cell death

• Mitochondrial targeting potential:

  • Given TDP2's role in protecting mitochondrial DNA, TDP2 inhibitors might also disrupt mitochondrial function in cancer cells

  • This provides a potential dual mechanism for cancer cell killing

The table below summarizes the key features of deazaflavin TDP2 inhibitors:

These deazaflavin derivatives represent the first suitable platform for the development of potent and selective TDP2 inhibitors with potential clinical applications .

What is known about TDP2 expression and function in different cancer types?

Research on TDP2 in cancer contexts has revealed several important aspects:

• TDP2 expression has been studied in various cancer cell lines:

  • Human prostate cancer DU145 cells (used in synergy studies with TDP2 inhibitors)

  • Human lung cancer H226 cells (used in subcellular localization studies)

  • Human colon carcinoma HCT116 cells (express lower levels of TDP2S compared to other cell lines)

• Functional role in cancer cell survival:

  • TDP2 protects cancer cells from TOP2 poison-induced DNA damage

  • This protection extends to both nuclear and mitochondrial DNA

  • TDP2-dependent repair may contribute to chemotherapy resistance

• Potential impact on treatment response:

  • Sensitivity to TOP2 poisons (etoposide, doxorubicin) is increased in TDP2-deficient cells

  • TDP2 activity may be a determinant of clinical response to these widely used anticancer drugs

  • The protective role of TDP2 in mitochondria may be particularly relevant, as mitochondrial dysfunction is an important mechanism of cancer cell death

While current research provides valuable insights into TDP2's role in cancer biology, comprehensive studies across different cancer types are still needed to fully understand the clinical implications of TDP2 expression and activity in cancer.

What are the potential resistance mechanisms to TDP2 inhibitors?

Understanding potential resistance mechanisms to TDP2 inhibitors is crucial for their development as therapeutics:

• Species-specific structural differences:

  • Studies have identified that mouse, fish, and C. elegans TDP2 enzymes are highly resistant to deazaflavin inhibitors

  • Key protein residues responsible for this resistance include human L313 and T296, which when mutated to their mouse counterparts confer high resistance

  • These differences provide insight into possible mutation-based resistance mechanisms

• Alternative DNA repair pathways:

  • In the absence of TDP2 activity, cells may upregulate alternative repair mechanisms

  • Research with TDP2-deficient mice shows a lack of spontaneous genome instability, suggesting that alternative mechanisms (such as Mre11-dependent NHEJ or homologous recombination) can compensate for TDP2 loss in processing TOP2-induced DNA breaks

• Isoform-specific functions:

  • The existence of multiple TDP2 isoforms (full-length and TDP2S) may provide redundancy

  • Inhibitors targeting only one isoform or one subcellular pool might be insufficient

  • Comprehensive inhibition strategies targeting both nuclear and mitochondrial TDP2 may be required

• Post-translational modifications:

  • Potential alterations in TDP2 regulation through post-translational modifications could affect inhibitor binding or efficacy

  • Understanding these regulatory mechanisms will be important for developing robust inhibition strategies

How does mitochondrial TDP2 contribute to cellular metabolism and energy production?

The discovery of TDP2's presence and activity in mitochondria opens important questions about its contribution to cellular metabolism:

• Mitochondrial DNA integrity:

  • Both TDP2 isoforms (full-length and TDP2S) are present and active in mitochondria

  • TDP2-deficient cells are hypersensitive to mitochondrial-targeted doxorubicin (mtDox)

  • mtDox selectively depletes mitochondrial DNA in TDP2-deficient cells

  • Complementing TDP2-deficient cells with human TDP2 restores resistance to mtDox

• Mitochondrial transcription:

  • Lack of TDP2 in mitochondria reduces mitochondrial transcription levels

  • This has been demonstrated in multiple human cell lines

  • Reduced mitochondrial gene expression could impact oxidative phosphorylation and energy production

• Specialized function of TDP2S:

  • The short isoform (TDP2S) contains a mitochondrial targeting sequence

  • TDP2S is enriched in mitochondria compared to the full-length isoform

  • CRISPR-engineered human cells expressing only TDP2S show protection against mtDox

  • This suggests TDP2S has evolved specifically to protect mitochondrial function

The mitochondrial role of TDP2 represents an important area for future research, particularly regarding its impact on cellular metabolism, energy production, and potential implications for diseases involving mitochondrial dysfunction.

What is the three-dimensional structure of TDP2 and how does it inform inhibitor design?

The structural understanding of TDP2 provides crucial insights for inhibitor development:

• Key structural features:

  • The catalytic domain contains the enzymatic activity responsible for cleaving tyrosyl-DNA phosphodiester bonds

  • The N-terminal region differs between isoforms:

    • Full-length TDP2: Contains nuclear localization signal and ubiquitin-associated domain

    • TDP2S: Lacks these features but includes a mitochondrial targeting sequence

• Inhibitor binding:

  • Deazaflavin derivatives inhibit TDP2 in a competitive manner

  • This suggests they interact with the enzyme's active site

  • The competitive nature of inhibition has been demonstrated with both recombinant and native TDP2

• Species specificity determinants:

  • Structural studies have identified key residues responsible for species-specific inhibitor sensitivity

  • Human residues L313 and T296 are critical determinants of inhibitor sensitivity

  • When these residues are mutated to their mouse counterparts, high resistance to inhibitors is conferred

  • This structural information is valuable for designing inhibitors with improved specificity and potency

• Catalytic mechanism insights:

  • Biochemical studies show that truncated TDP2 (amino acids 110-362) maintains similar activity to full-length protein

  • This indicates that the N-terminal leading sequences do not impact the core biochemical activity

  • This knowledge allows for focused inhibitor design targeting the catalytic domain

What are the outstanding research questions regarding TDP2 in neurological disorders?

Several critical research questions remain regarding TDP2's role in neurological disorders:

• Clinical relevance:

  • Patients with TDP2 mutations have been identified with clinical features including intellectual disability, seizures, and ataxia

  • These symptoms suggest a crucial role for TDP2 in neuronal function and development

  • The precise mechanisms linking TDP2 deficiency to these neurological manifestations remain incompletely understood

• Mitochondrial connection:

  • Neurons are particularly dependent on mitochondrial function due to their high energy demands

  • TDP2's role in mitochondrial DNA protection and transcription may be especially important in neurons

  • The contribution of mitochondrial versus nuclear TDP2 deficiency to neurological phenotypes remains to be clarified

• Transcription-related neuronal vulnerability:

  • Neurons are post-mitotic cells that rely heavily on transcription

  • TDP2 protects gene transcription from TOP2-induced DSBs, which is particularly important in non-cycling cells

  • The specific transcriptional programs affected by TDP2 deficiency in neurons warrant further investigation

• Therapeutic opportunities:

  • Understanding TDP2's role in neurological disorders could reveal new therapeutic approaches

  • Targeting the specific DNA repair pathways affected in TDP2-deficient neurons might provide neuroprotective strategies

  • The potential for gene therapy approaches to restore TDP2 function in affected tissues represents an important area for future research

The continued investigation of these questions will advance our understanding of TDP2's role in neurological function and may lead to new therapeutic approaches for patients with TDP2-related disorders.