TDP1 Human

What is TDP1 and what is its primary function in human cells?

TDP1 (Tyrosyl-DNA phosphodiesterase 1) is a DNA repair enzyme that belongs to the phospholipase D superfamily. Its primary function is to process 3′-end blocking lesions at DNA breaks, particularly those resulting from trapped topoisomerase I-DNA covalent complexes. TDP1 hydrolyzes DNA-adducts via two coordinated SN2 nucleophilic attacks mediated by paired catalytic histidine and lysine residues within two conserved motifs . This activity is crucial for maintaining genomic integrity, as it prevents the accumulation of DNA damage that could otherwise lead to cell death or genomic instability .

How does TDP1 contribute to DNA repair pathways?

TDP1 participates in multiple DNA repair pathways, with particularly important roles in single-strand break repair (SSBR) and double-strand break repair. In the SSBR pathway, TDP1 removes blocking lesions at 3′ ends, allowing subsequent processing by polymerases and ligases to complete repair. Research has demonstrated that TDP1 plays a crucial role in protecting against oxidative DNA damage, especially in non-dividing cells . Methodologically, this function can be studied using nitrogen starvation approaches to arrest cells in G0/quiescent states, where cells remain metabolically active and can efficiently repair DNA damage, similar to post-mitotic neuronal cells in humans .

What types of DNA lesions can TDP1 process?

TDP1 has a remarkably versatile substrate specificity. Beyond its canonical activity against 3′-phosphotyrosyl linkages (formed by trapped topoisomerase I), TDP1 can process:

• A wide range of synthetic DNA adducts

• 3′-phosphoglycolates and other oxidative DNA damage products

• Various functional groups linked to the 3′ end of DNA

• 3′-(4-methylumbelliferone)-phosphate groups in specially designed oligonucleotide substrates

This broad substrate specificity makes TDP1 a critical guardian of genome integrity against diverse types of DNA damage.

What is known about the catalytic mechanism of human TDP1?

Human TDP1 catalyzes the removal of 3′-DNA adducts through a two-step mechanism. First, a nucleophilic attack by His263 on the phosphodiester bond forms a covalent TDP1-DNA intermediate. Second, His493 activates a water molecule that hydrolyzes this intermediate, releasing the DNA 3′-phosphate and regenerating the enzyme. Crystal structure analyses have revealed that TDP1 uses a hydrophobic wedge to split DNA ends, directing the scissile strand through a channel towards the active site . The conserved phenylalanine (F259) stacks against the -3 base pair, delimiting the junction of duplexed and melted DNA, and fixes the scissile strand in the channel . This structural arrangement explains why TDP1 cleavage is non-processive and provides insights into DNA 3′-end processing mechanisms.

What is the "transphosphooligonucleotidation" reaction catalyzed by TDP1 and how can it be experimentally induced?

TDP1 can catalyze not only hydrolysis but also a "transphosphooligonucleotidation" reaction in which it transfers the substrate residue to primary alcohols instead of water . This reaction is analogous to the transphosphatidylation reaction observed in other phospholipase D enzymes. Experimentally, this can be induced by:

• Exposing TDP1 to DNA substrates in reaction conditions with high concentrations of primary alcohols

• Monitoring the formation of covalent DNA adducts with different alcohol residues

• Comparing the cleavage efficiency of these adducts by wild-type TDP1 versus disease-associated mutants

This reaction property has revealed that glycerol residues are efficiently cleaved from the 3′-end by wild-type TDP1 but not by the mutant form associated with spinocerebellar ataxia with axonal neuropathy .

How does TDP1 interact with topoisomerase I in cells?

TDP1 appears to have a different cellular distribution pattern than topoisomerase I. When expressed as biofluorescent chimera in human cells, TDP1 is more mobile than topoisomerase I, less accumulated in nucleoli, and not chromosome-bound during early mitosis . Upon exposure to camptothecin (a topoisomerase I poison), both proteins clear from nucleoli and become less mobile in the nucleoplasm, but this process occurs much more slowly with TDP1. These observations suggest that rather than maintaining a steady association with topoisomerase I, TDP1 is likely integrated into repair complexes that assemble after the stabilization of DNA-topoisomerase I intermediates . This dynamic relationship can be methodologically studied using fluorescent protein tagging and live cell imaging techniques.

What is the connection between TDP1 mutations and SCAN1?

A mutation in the TDP1 gene (H493R) is responsible for spinocerebellar ataxia with axonal neuropathy (SCAN1), a rare neurodegenerative disorder . This mutation affects a critical histidine residue in the enzyme's active site, compromising its catalytic activity. From a mechanistic perspective, this mutation impacts the second step of the TDP1 reaction, where His493 activates a water molecule to hydrolyze the TDP1-DNA covalent intermediate. Consequently, SCAN1 patients accumulate TDP1-DNA adducts and exhibit progressive cerebellar ataxia and peripheral neuropathy . Experimental comparisons between wild-type TDP1 and the SCAN1-associated mutant have shown that the mutant is deficient in cleaving glycerol residues from 3′-DNA ends , providing insights into the molecular basis of the disease.

Why does TDP1 dysfunction specifically affect non-dividing neuronal cells despite being expressed in all cells?

The specific neuronal impact of TDP1 dysfunction appears related to several factors:

• Neurons are post-mitotic cells with high oxygen consumption, making them particularly vulnerable to accumulated oxidative DNA damage

• Non-dividing cells lack DNA replication-associated repair pathways that could serve as alternatives to TDP1-mediated repair

• Studies using S. pombe (fission yeast) models arrested in G0 phase have demonstrated that Tdp1 has a crucial role in protecting against physiological oxidative DNA damage specifically in non-dividing cells

This explains why SCAN1 patients develop normally but manifest neurological symptoms later in life. Methodologically, researchers can model this neuronal vulnerability by studying TDP1 function in non-dividing cell models, such as nitrogen-starved yeast or growth-arrested human cells .

How does TDP1 overexpression affect sensitivity to anti-cancer therapies?

Cells overexpressing TDP1 at levels >100-fold in excess of endogenous levels exhibit significant resistance to DNA damage induced by both:

• The topoisomerase I poison camptothecin

• The topoisomerase II poison VP-16 (etoposide)

Interestingly, DNA damage independent of topoisomerases I or II is not affected by TDP1 overexpression. The protection is specifically dependent on catalytically active TDP1, as overexpression of the inactive mutant GFP-TDP1(H263A) does not confer resistance . Since TDP1 overexpression does not compromise cell proliferation, it could potentially serve as a pleiotropic resistance mechanism in cancer therapy. This knowledge informs therapeutic strategies for cancers where TDP1 expression is elevated and suggests that TDP1 inhibitors might sensitize resistant tumors to topoisomerase poisons.

What fluorescent assays are available for measuring human TDP1 activity?

Researchers have developed several fluorescence-based assays for TDP1, addressing the historical challenges in measuring its activity, especially under pre-steady-state conditions. Key methodological approaches include:

• Oligonucleotide substrates containing 3′-(4-methylumbelliferone)-phosphate (DNA-MUP), which are not fluorescent until cleaved by TDP1, releasing the fluorescent 4-methylumbelliferone reporter molecule

• Nucleotide substrates with similar 3′-modifications that can be used for high-throughput screening applications

• Real-time monitoring of TDP1 activity using these substrates, allowing for detailed kinetic analyses

These fluorescent substrates are efficiently cleaved by TDP1 and provide advantages over traditional gel-based assays that are labor-intensive and limited in providing real-time data .

How can researchers express and purify active human TDP1 for biochemical studies?

To obtain active human TDP1 for in vitro studies, researchers typically:

• Clone the human TDP1 cDNA into appropriate expression vectors, often with affinity tags (His, GST, or GFP)

• Express the protein in bacterial systems (E. coli) or eukaryotic systems (insect or mammalian cells) depending on the experimental requirements

• Use affinity chromatography followed by ion-exchange and size-exclusion chromatography for purification

• Verify enzyme activity using established assays such as the fluorescent DNA-MUP substrate

For studies requiring visualization of TDP1 in cells, GFP-tagged TDP1 constructs have been successfully used and shown to retain catalytic activity . When designing expression constructs, researchers should consider that TDP1 overexpression (>100-fold) can alter cellular responses to topoisomerase poisons without affecting cell proliferation .

What cellular models are most appropriate for studying TDP1 function?

Different cellular models offer complementary insights into TDP1 function:

• Human cell lines expressing tagged TDP1 constructs provide information about subcellular localization and mobility

• SCAN1 patient-derived lymphoblastoid cells offer a disease-relevant model with H493R mutant TDP1

• Non-dividing cell models (nitrogen-starved S. pombe or growth-arrested human cells) are particularly valuable for understanding TDP1's role in post-mitotic neurons

• Cancer cell lines with varying TDP1 expression levels help elucidate its impact on chemotherapy sensitivity

For studying TDP1's protective role against oxidative damage in non-dividing cells, the S. pombe model arrested in G0/quiescent state by nitrogen starvation has proven particularly informative . These cells remain metabolically active and viable for weeks while being able to efficiently repair DNA damage.

How does TDP1 interact with DNA structure beyond its active site?

Crystal structures of TDP1-DNA complexes have revealed sophisticated interactions between the enzyme and DNA. Beyond the active site, TDP1 employs a hydrophobic wedge that splits DNA ends and guides the scissile strand through a channel . The conserved phenylalanine (F259) plays a critical role by stacking against the -3 base pair, marking the boundary between duplexed and melted DNA . This structural arrangement explains why TDP1 cleavage is non-processive and provides insights into how the enzyme accommodates duplex DNA. To investigate these structural features, researchers can employ:

• Site-directed mutagenesis of key residues like F259

• Biochemical assays with various DNA substrates to assess how structural changes affect activity

• Advanced structural biology techniques combining site-specific DNA-protein cross-linking with mass spectrometry

What is the unexpected role of TDP1 in topoisomerase II-mediated DNA damage repair?

While TDP1 was initially characterized for its role in processing topoisomerase I-DNA adducts, research has revealed it also contributes to repairing topoisomerase II-mediated DNA damage. Cells overexpressing active TDP1 show reduced DNA damage from the topoisomerase II poison VP-16 (etoposide), while cells expressing the inactive mutant do not exhibit this protection . This suggests TDP1 has a broader role in DNA repair than initially thought, potentially processing a variety of 3′-blocking lesions including those generated by topoisomerase II poisons. Methodologically, researchers can investigate this phenomenon by:

• Comparing cellular sensitivity to topoisomerase I versus II poisons in cells with varied TDP1 expression

• Developing in vitro assays with topoisomerase II-DNA adducts as potential TDP1 substrates

• Using chromatin immunoprecipitation techniques to assess TDP1 recruitment to topoisomerase II-induced DNA damage sites

What is the relationship between TDP1 and SSB repair pathways in different cellular contexts?

TDP1's involvement in single-strand break (SSB) repair appears to be context-dependent:

• In non-dividing cells, TDP1 has a crucial role in processing endogenous DNA damage, particularly oxidative lesions

• In SCAN1 lymphoblastoid cells blocked in S phase (to mimic the postmitotic state), impaired SSB repair is observed following camptothecin treatments

• TDP1 likely functions as part of larger repair complexes rather than maintaining steady associations with topoisomerase I

Researchers investigating these relationships should consider experimental approaches that:

• Compare TDP1 function in dividing versus non-dividing cells

• Assess interactions between TDP1 and other SSB repair factors using co-immunoprecipitation or proximity ligation assays

• Utilize cell cycle synchronization techniques to study phase-specific repair activities

What approaches might lead to effective TDP1 inhibitors for enhancing cancer therapy?

Developing TDP1 inhibitors could potentially sensitize cancer cells to topoisomerase poisons. Strategic approaches include:

• High-throughput screening using fluorescent TDP1 substrates like DNA-MUP

• Structure-based drug design informed by crystal structures of TDP1-DNA complexes

• Development of combination therapies targeting both TDP1 and topoisomerases

• Exploiting the "transphosphooligonucleotidation" reaction to develop mechanism-based inhibitors

Since TDP1 overexpression can confer resistance to both topoisomerase I and II poisons without affecting cellular proliferation , TDP1 inhibitors could prove valuable in overcoming therapy resistance in certain cancers.

How might understanding TDP1 function inform therapeutic approaches for neurodegenerative diseases?

The connection between TDP1 dysfunction and neurodegeneration in SCAN1 suggests broader implications for neurodegenerative disorders associated with DNA damage:

• TDP1's protective role against oxidative DNA damage in non-dividing cells indicates potential relevance to other conditions where neuronal oxidative stress contributes to pathology

• The specific vulnerability of neurons to TDP1 dysfunction highlights the importance of DNA repair in post-mitotic cells

• Testing whether enhancing TDP1 activity could provide neuroprotection in models of oxidative stress-related neurodegeneration

Experimental models using non-dividing cells provide valuable platforms for investigating these potential therapeutic applications.

What methodological advances would enhance our ability to study TDP1 dynamics in living cells?

Future research would benefit from advanced techniques to study TDP1 in real-time within cellular environments:

• Development of activity-based probes that can report on TDP1 function in living cells

• Advanced imaging technologies to track TDP1 mobilization to sites of DNA damage with higher temporal and spatial resolution

• CRISPR-based approaches for endogenous tagging of TDP1 to avoid artifacts associated with overexpression

• Single-molecule techniques to observe individual TDP1 molecules processing DNA substrates in real-time

These methodological advances would provide deeper insights into the dynamic behavior of TDP1 in response to various DNA damage scenarios and its interactions with other repair factors.