Generation of assays and antibodies to facilitate the study of human 5′-tyrosyl DNA phosphodiesterase

Thomson, Graeme; Watson, Amanda J.; Caldecott, Keith W.; Denneny, Olive; Depledge, Paul; Hamilton, Nicola; Jordan, Allan M.; Morrow, Christopher J.; Raoof, Ali; Waddell, Ian D.; Ogilvie, Donald

doi:10.1016/j.ab.2013.02.001

Cited by 17 publications

(36 citation statements)

References 15 publications

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“…3 F and G), in accord with recent reports (20,26,35). Given the interest in TDP2 inhibitors for cancer therapy (13), we note that our fluorescent TDP2 assay should be easily adaptable to high-throughput applications with potential advantages over assays using low-affinity chromogenic substrates (42,50).…”

Section: Discussionsupporting

confidence: 80%

Involvement of the host DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses

Königer

Wingert

Marsmann

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

207

193

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Hepatitis B virus (HBV), the causative agent of chronic hepatitis B and prototypic hepadnavirus, is a small DNA virus that replicates by protein-primed reverse transcription. The product is a 3-kb relaxed circular DNA (RC-DNA) in which one strand is linked to the viral polymerase (P protein) through a tyrosyl-DNA phosphodiester bond. Upon infection, the incoming RC-DNA is converted into covalently closed circular (ccc) DNA, which serves as a viral persistence reservoir that is refractory to current anti-HBV treatments. The mechanism of cccDNA formation is unknown, but the release of P protein is one mandatory step. Structural similarities between RC-DNA and cellular topoisomerase-DNA adducts and their known repair by tyrosyl-DNA-phosphodiesterase (TDP) 1 or TDP2 suggested that HBV may usurp these enzymes for its own purpose. Here we demonstrate that human and chicken TDP2, but only the yeast ortholog of TDP1, can specifically cleave the Tyr-DNA bond in virus-adapted model substrates and release P protein from authentic HBV and duck HBV (DHBV) RC-DNA in vitro, without prior proteolysis of the large P proteins. Consistent with TPD2's having a physiological role in cccDNA formation, RNAi-mediated TDP2 depletion in human cells significantly slowed the conversion of RC-DNA to cccDNA. Ectopic TDP2 expression in the same cells restored faster conversion kinetics. These data strongly suggest that TDP2 is a first, although likely not the only, host DNA-repair factor involved in HBV cccDNA biogenesis. In addition to establishing a functional link between hepadnaviruses and DNA repair, our results open new prospects for directly targeting HBV persistence.hepatitis B virus persistence | TDP substrate specificity | virus-DNA repair interface M ore than 250 million people worldwide are chronically infected with hepatitis B virus (HBV) (1) and are at a highly increased risk for developing end-stage liver disease (2). Current treatments with IFN-α or nucleos(t)ide analogs (NAs) are only partially effective (3, 4). Importantly, they do not directly target the viral persistence reservoir, an episomal covalently closed circular (ccc) DNA form of the viral genome that serves as template for all viral transcripts; hence a few cccDNA molecules present in the liver can reactivate full viral replication. Eliminating infection thus will require the elimination of cccDNA. cccDNA is generated, upon infection, from the viral polymerase (P protein)-linked relaxed circular (RC) DNA present in incoming virions (Fig. 1A). The mechanism by which RC-DNA is converted to cccDNA is poorly understood, but it must involve multiple steps (see below). Because the ∼3-kb genomes of HBV and the other hepadnaviruses [e.g., from ducks (DHBV)] are too small to encode all the activities required for this conversion, these activities must be provided by the host cell.RC-DNA from all hepadnaviruses bears several molecular peculiarities that result from its generation by protein-primed reverse transcription (for reviews, see refs. 5 and 6). The pregenomic RNA (p...

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Section: Discussionsupporting

confidence: 80%

Involvement of the host DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses

Königer

Wingert

Marsmann

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

207

193

View full text Add to dashboard Cite

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“…Anti-TDP2 antibody was used at 1:5000 in Western blotting and has been described previously. 10 Anti-Actin (Sigma A4700) was used at 1:2000. Western blot assessment of OXPHOS components in patient primary fibroblasts was conducted as described previously, 11 using antibodies against NDUFB8 (Abcam cat# ab110242), SDHA (Abcam cat# ab14715), UQCRC2 (Abcam cat# ab14745), COXI (Abcam cat# ab14705), ATP5A (Abcam cat# ab14748), SDHB (Abcam cat# ab14714), COX II (Abcam cat# 110258), and VDAC1 (Abcam cat# ab14734).…”

Section: Methodsmentioning

confidence: 99%

Confirming TDP2 mutation in spinocerebellar ataxia autosomal recessive 23 (SCAR23)

et al. 2018

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ObjectiveTo address the relationship between mutations in the DNA strand break repair protein tyrosyl DNA phosphodiesterase 2 (TDP2) and spinocerebellar ataxia autosomal recessive 23 (SCAR23) and to characterize the cellular phenotype of primary fibroblasts from this disease.MethodsWe have used exome sequencing, Sanger sequencing, gene editing and cell biology, biochemistry, and subcellular mitochondrial analyses for this study.ResultsWe have identified a patient in the United States with SCAR23 harboring the same homozygous TDP2 mutation as previously reported in 3 Irish siblings (c.425+1G>A). The current and Irish patients share the same disease haplotype, but the current patient lacks a homozygous variant present in the Irish siblings in the closely linked gene ZNF193, eliminating this as a contributor to the disease. The current patient also displays symptoms consistent with mitochondrial dysfunction, although levels of mitochondrial function in patient primary skin fibroblasts are normal. However, we demonstrate an inability in patient primary fibroblasts to rapidly repair topoisomerase-induced DNA double-strand breaks (DSBs) in the nucleus and profound hypersensitivity to this type of DNA damage.ConclusionsThese data confirm the TDP2 mutation as causative for SCAR23 and highlight the link between defects in nuclear DNA DSB repair, developmental delay, epilepsy, and ataxia.

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“…Anti-TDP2 antibody was employed at 1:5000 in western blotting and has been described previously 33 . Anti-Actin (Sigma A4700) was employed at 1 : 2000 and anti-Histone 1.2 (Abcam ab17677) at 1 : 20,000.…”

Section: Methodsmentioning

confidence: 99%

TDP2 suppresses chromosomal translocations induced by DNA topoisomerase II during gene transcription

et al. 2017

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DNA double-strand breaks (DSBs) induced by abortive topoisomerase II (TOP2) activity are a potential source of genome instability and chromosome translocation. TOP2-induced DNA double-strand breaks are rejoined in part by tyrosyl-DNA phosphodiesterase 2 (TDP2)-dependent non-homologous end-joining (NHEJ), but whether this process suppresses or promotes TOP2-induced translocations is unclear. Here, we show that TDP2 rejoins DSBs induced during transcription-dependent TOP2 activity in breast cancer cells and at the translocation ‘hotspot’, MLL. Moreover, we find that TDP2 suppresses chromosome rearrangements induced by TOP2 and reduces TOP2-induced chromosome translocations that arise during gene transcription. Interestingly, however, we implicate TDP2-dependent NHEJ in the formation of a rare subclass of translocations associated previously with therapy-related leukemia and characterized by junction sequences with 4-bp of perfect homology. Collectively, these data highlight the threat posed by TOP2-induced DSBs during transcription and demonstrate the importance of TDP2-dependent non-homologous end-joining in protecting both gene transcription and genome stability.

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Generation of assays and antibodies to facilitate the study of human 5′-tyrosyl DNA phosphodiesterase

Cited by 17 publications

References 15 publications

Involvement of the host DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses

Involvement of the host DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses

Confirming TDP2 mutation in spinocerebellar ataxia autosomal recessive 23 (SCAR23)

TDP2 suppresses chromosomal translocations induced by DNA topoisomerase II during gene transcription

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