Tyrosyl-DNA-phosphodiesterase I (TDP1) participates in the removal and repair of stabilized-Top2α cleavage complexes in human cells

Borda, Miguel Angel; Palmitelli, Micaela; Verón, Gustavo Luis; González-Cid, Marcela; Nebel, Marcelo de Campos

doi:10.1016/j.mrfmmm.2015.09.003

Cited by 20 publications

(15 citation statements)

References 56 publications

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“…Tdp1-depleted and overexpressing cells have provided mixed answers. Cells from Tdp1 knockout mice and MEFs showed no hypersensitivity to etoposide (Hirano et al, 2007b), while down-regulation of TDP1 in HeLa cells did confer hypersensitivity (Borda et al, 2015). Furthermore, overexpression of TDP1 in HEK293 cells induced resistance to etoposide (Barthelmes et al, 2004).…”

Section: Spinocerebellar Ataxia With Axonal Neuropathy (Scan1) Andmentioning

confidence: 99%

Neurological disorders associated with DNA strand-break processing enzymes

Jiang

Glover

Weinfeld

2017

Mechanisms of Ageing and Development

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The termini of DNA strand breaks induced by reactive oxygen species or by abortive DNA metabolic intermediates require processing to enable subsequent gap filling and ligation to proceed. The three proteins, tyrosyl DNA-phosphodiesterase 1 (TDP1), aprataxin (APTX) and polynucleotide kinase/phosphatase (PNKP) each act on a discrete set of modified strand-break termini. Recently, a series of neurodegenerative and neurodevelopmental disorders have been associated with mutations in the genes coding for these proteins. Mutations in TDP1 and APTX have been linked to Spinocerebellar ataxia with axonal neuropathy (SCAN1) and Ataxia-ocular motor apraxia 1 (AOA1), respectively, while mutations in PNKP are considered to be responsible for Microcephaly with seizures (MCSZ) and Ataxia-ocular motor apraxia 4 (AOA4). Here we present an overview of the mechanisms of these proteins and how their impairment may give rise to their respective disorders.

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Section: Spinocerebellar Ataxia With Axonal Neuropathy (Scan1) Andmentioning

confidence: 99%

Neurological disorders associated with DNA strand-break processing enzymes

Jiang

Glover

Weinfeld

2017

Mechanisms of Ageing and Development

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“…Subsequently, chromatid exchanges as well as total chromosome breaks per cell were greater in the TDP1-knockdown than in parental cells, in both the presence and absence of the DNA-PK inhibitor NU7026. These results suggest that TDP1 plays a role in both the canonical as well as alternative NHEJ pathways [164].…”

Section: Tdp1 and Tdp2mentioning

confidence: 81%

End-processing nucleases and phosphodiesterases: An elite supporting cast for the non-homologous end joining pathway of DNA double-strand break repair

Menon

Povirk

2016

DNA Repair

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“…In order to determine whether Top2α and Top2β isoforms bound to chromatin of HL‐60 cells can be recognized by flow cytometry following protein extraction with detergent and heparin, we immunolabeled either isoforms with specific antibodies that have been previously validated in immunofluorescence experiments . As shown in Figure A and B, both antibodies recognized their antigens providing sufficient signal intensities following extraction to differentiate them from their isotype controls and from samples that were not heparin‐extracted.…”

Section: Resultsmentioning

confidence: 99%

“…Drug‐stabilized Top2cc are removed by a complementary network of hydrolytic , nucleolytic , and proteolytic pathways through diverse cellular processes. Genetic knock‐down of proteins involved in Top2cc removal pathways can lead to increased susceptibility to Top2 poisons . Proteosomal inhibition‐mediated stabilization of Top2 was also shown to revert the resistant phenotype to Top2 poisons on solid and hematological tumors .…”

Section: Discussionmentioning

confidence: 99%

“…The removal of the protein covalently bound to DNA is an important task for the repair of Top2‐mediated damage. So far, the evidence points to different mechanisms for its removal including activities of endonucleolytic cleavage , enzyme‐mediated hydrolysis of the phosphotyrosyl linkage , and proteolysis by a proteasome‐dependent pathway . In any case, their relative contribution during different cellular processes is not clear yet.…”

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confidence: 99%

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A flow cytometry‐based method for a high‐throughput analysis of drug‐stabilized topoisomerase II cleavage complexes in human cells

2016

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Topoisomerase II (Top2) is an important target for anticancer therapy. A variety of drugs that poison Top2, including several epipodophyllotoxins, anthracyclines, and anthracenediones, are widely used in the clinic for both hematologic and solid tumors. The poisoning of Top2 involves the formation of a reaction intermediate Top2-DNA, termed Top2 cleavage complex (Top2cc), which is persistent in the presence of the drug and involves a 5 0 end of DNA covalently bound to a tyrosine from the enzyme through a phosphodiester group. Drug-induced Top2cc leads to Top2 linked-DNA breaks which are the major responsible for their cytotoxicity. While biochemical detection is very laborious, quantification of drug-induced Top2cc by immunofluorescence-based microscopy techniques is time consuming and requires extensive image segmentation for the analysis of a small population of cells. Here, we developed a flow cytometry-based method for the analysis of drug-induced Top2cc. This method allows a rapid analysis of a high number of cells in their cell cycle phase context. Moreover, it can be applied to almost any human cell type, including clinical samples. The methodology is useful for a high-throughput analysis of drugs that poison Top2, allowing not just the discrimination of the Top2 isoform that is targeted but also to track its removal. Key terms flow cytometry; topoisomerase II; Top2 poison; high throughput analysis TOPOISOMERASE II (Top2) is a dimeric enzyme that catalyzes topological changes in the DNA, thus being essential in several metabolic processes of the genome of eukaryotic cells (1,2). In mammals, there have been characterized two variants of Top2, a and b isoforms, showing different activities and regulation of their expression. While Top2a expression and activity increase throughout the cell cycle, Top2b remains at similar levels (3,4). Top2a is essential for cell proliferation, participating in several processes such as replication, transcription, and chromosome condensation and segregation (5). In contrast, Top2b is dispensable for cell proliferation, but contributes to transcription and is required during development (6,7). Top2 acts by nicking both strands of the DNA to allow the passage through it of other doublestranded segment of DNA from the same or a different molecule. Following the breakage of the double-stranded DNA, each monomer of Top2 remains covalently linked to each 5 0 terminus of DNA by a transient phosphotyrosyl linkage, a reaction intermediate termed cleavage complex (Top2cc) (8,9). Certain natural compounds, spontaneous DNA lesions and several chemotherapeutic agents can stabilize the Top2cc increasing their half-life (10-12). These agents interfere with the breakagereunion reaction that leads to a reversible DNA damage, which becomes irreversible

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Tyrosyl-DNA-phosphodiesterase I (TDP1) participates in the removal and repair of stabilized-Top2α cleavage complexes in human cells

Cited by 20 publications

References 56 publications

Neurological disorders associated with DNA strand-break processing enzymes

Neurological disorders associated with DNA strand-break processing enzymes

End-processing nucleases and phosphodiesterases: An elite supporting cast for the non-homologous end joining pathway of DNA double-strand break repair

A flow cytometry‐based method for a high‐throughput analysis of drug‐stabilized topoisomerase II cleavage complexes in human cells

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