Spinocerebellar ataxia with axonal neuropathy: consequence of a Tdp1 recessive neomorphic mutation?

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) cleaves the phosphodiester bond between a covalently stalled topoisomerase I (Topo I) and the 3' end of DNA. Stalling of Topo I at DNA strand breaks is induced by endogenous DNA damage and the Topo I-specific anticancer drug camptothecin (CPT). The H493R mutation of Tdp1 causes the neurodegenerative disorder spinocerebellar ataxia with axonal neuropathy (SCAN1). Contrary to the hypothesis that SCAN1 arises from catalytically inactive Tdp1, Tdp1-/- mice are indistinguishable from wild-type mice, physically, histologically, behaviorally, and electrophysiologically. However, compared to wild-type mice, Tdp1-/- mice are hypersensitive to CPT and bleomycin but not to etoposide. Consistent with earlier in vitro studies, we show that the H493R Tdp1 mutant protein retains residual activity and becomes covalently trapped on the DNA after CPT treatment of SCAN1 cells. This result provides a direct demonstration that Tdp1 repairs Topo I covalent lesions in vivo and suggests that SCAN1 arises from the recessive neomorphic mutation H493R. This is a novel mechanism for disease since neomorphic mutations are generally dominant.     

Analysis of the active-site mechanism of tyrosyl-DNA phosphodiesterase I: a member of the phospholipase D superfamily

Tyrosyl-DNA phosphodiesterase I (Tdp1) is a member of the phospholipase D superfamily that hydrolyzes 3'-phospho-DNA adducts via two conserved catalytic histidines-one acting as the lead nucleophile and the second acting as a general acid/base. Substitution of the second histidine specifically to arginine contributes to the neurodegenerative disease spinocerebellar ataxia with axonal neuropathy (SCAN1). We investigated the catalytic role of this histidine in the yeast protein (His432) using a combination of X-ray crystallography, biochemistry, yeast genetics, and theoretical chemistry. The structures of wild-type Tdp1 and His432Arg both show a phosphorylated form of the nucleophilic histidine that is not observed in the structure of His432Asn. The phosphohistidine is stabilized in the His432Arg structure by the guanidinium group that also restricts the access of nucleophilic water molecule to the Tdp1-DNA intermediate. Biochemical analyses confirm that His432Arg forms an observable and unique Tdp1-DNA adduct during catalysis. Substitution of His432 by Lys does not affect catalytic activity or yeast phenotype, but substitutions with Asn, Gln, Leu, Ala, Ser, and Thr all result in severely compromised enzymes and DNA topoisomerase I-camptothecin dependent lethality. Surprisingly, His432Asn did not show a stable covalent Tdp1-DNA intermediate that suggests another catalytic defect. Theoretical calculations revealed that the defect resides in the nucleophilic histidine and that the pK(a) of this histidine is crucially dependent on the second histidine and on the incoming phosphate of the substrate. This represents a unique example of substrate-activated catalysis that applies to the entire phospholipase D superfamily.     

The crystal structure of human tyrosyl-DNA phosphodiesterase, Tdp1

Tyrosyl-DNA phosphodiesterase (Tdp1) catalyzes the hydrolysis of a phosphodiester bond between a tyrosine residue and a DNA 3' phosphate. The enzyme appears to be responsible for repairing the unique protein-DNA linkage that occurs when eukaryotic topoisomerase I becomes stalled on the DNA in the cell. The 1.69 A crystal structure reveals that human Tdp1 is a monomer composed of two similar domains that are related by a pseudo-2-fold axis of symmetry. Each domain contributes conserved histidine, lysine, and asparagine residues to form a single active site. The structure of Tdp1 confirms that the protein has many similarities to the members of the phospholipase D (PLD) superfamily and indicates a similar catalytic mechanism. The structure also suggests how the unusual protein-DNA substrate binds and provides insights about the nature of the substrate in vivo.     

Insights into substrate binding and catalytic mechanism of human tyrosyl-DNA phosphodiesterase (Tdp1) from vanadate and tungstate-inhibited structures

Tyrosyl-DNA phosphodiesterase (Tdp1) is a DNA repair enzyme that catalyzes the hydrolysis of a phosphodiester bond between a tyrosine residue and a DNA 3'-phosphate. The only known example of such a linkage in eukaryotic cells occurs normally as a transient link between a type IB topoisomerase and DNA. Thus human Tdp1 is thought to be responsible for repairing lesions that occur when topoisomerase I becomes stalled on the DNA in the cell. Tdp1 has also been shown to remove glycolate from single-stranded DNA containing a 3'-phosphoglycolate, suggesting a role for Tdp1 in repair of free-radical mediated DNA double-strand breaks. We report the three-dimensional structures of human Tdp1 bound to the phosphate transition state analogs vanadate and tungstate. Each structure shows the inhibitor covalently bound to His263, confirming that this residue is the nucleophile in the first step of the catalytic reaction. Vanadate in the Tdp1-vanadate structure has a trigonal bipyramidal geometry that mimics the transition state for hydrolysis of a phosphodiester bond, while Tdp1-tungstate displays unusual octahedral coordination. The presence of low-occupancy tungstate molecules along the narrow groove of the substrate binding cleft is suggestive evidence that this groove binds ssDNA. In both cases, glycerol from the cryoprotectant solution became liganded to the vanadate or tungstate inhibitor molecules in a bidentate 1,2-diol fashion. These structural models allow predictions to be made regarding the specific binding mode of the substrate and the mechanism of catalysis.     

TDP1 facilitates chromosomal single-strand break repair in neurons and is neuroprotective in vivo

Defective Tyrosyl-DNA phosphodiesterase 1 (TDP1) can cause spinocerebellar ataxia with axonal neuropathy (SCAN1), a neurodegenerative syndrome associated with marked cerebellar atrophy and peripheral neuropathy. Although SCAN1 lymphoblastoid cells show pronounced defects in the repair of chromosomal single-strand breaks (SSBs), it is unknown if this DNA repair activity is important for neurons or for preventing neurodegeneration. Therefore, we generated Tdp1-/- mice to assess the role of Tdp1 in the nervous system. Using both in vitro and in vivo assays, we found that cerebellar neurons or primary astrocytes derived from Tdp1-/- mice display an inability to rapidly repair DNA SSBs associated with Top1-DNA complexes or oxidative damage. Moreover, loss of Tdp1 resulted in age-dependent and progressive cerebellar atrophy. Tdp1-/- mice treated with topotecan, a drug that increases levels of Top1-DNA complexes, also demonstrated significant loss of intestinal and hematopoietic progenitor cells. These data indicate that TDP1 is required for neural homeostasis, and reveal a widespread requisite for TDP1 function in response to acutely elevated levels of Top1-associated DNA strand breaks.     

TDP1 serine 81 promotes interaction with DNA ligase IIIα and facilitates cell survival following DNA damage

Tyrosyl DNA phosphodiesterase (TDP1) is a DNA 3'-end processing enzyme that preferentially hydrolyses the bond between the 3'-end of DNA and stalled DNA topoisomerase 1. The importance of TDP1 is highlighted by its association with the human genetic disease spinocerebellar ataxia with axonal neuropathy (SCAN1). TDP1 comprises of a highly conserved C-terminus phosphodiesterase domain and a less conserved N-terminus tail. The importance of the N-terminus domain was suggested by its interaction with Lig3α. Here we show that this interaction is promoted by serine 81 that is located within a putative S/TQ site in the N-terminus domain of TDP1. Although mutation of serine 81 to alanine had no impact on TDP1 activity in vitro and had little impact on the ability of TDP1 to mediate the rapid repair of CPT- or IR-induced DNA breaks in vivo, it led to marked reduction of protein stability. Moreover, it reduced the ability of TDP1 to promote cell survival following genotoxic stress. Together, our findings identify a novel mechanism for regulating TDP1 function in mammalian cells that is not directly related to its enzymatic activity.     

Tdp1 protects against oxidative DNA damage in non‐dividing fission yeast

In humans, a mutation in the tyrosyl‐DNA phosphodiesterase (Tdp1) is responsible for the recessively inherited syndrome spinocerebellar ataxia with axonal neuropathy (SCAN1). Tdp1 is a well‐conserved DNA repair enzyme, which processes modified 3′ phospho‐DNA adducts in vitro. Here, we report that in the yeast Schizosaccharomyces pombe, tdp1 mutant cells progressively accumulate DNA damage and rapidly lose viability in a physiological G0/quiescent state. Remarkably, this effect is independent of topoisomerase I function. Moreover, we provide evidence that Tdp1, with the polynucleotide kinase (Pnk1), processes the same naturally occurring 3′‐ends, produced from oxidative DNA damage in G0. We also found that one half of the dead cells lose their nuclear DNA. Nuclear DNA degradation is genetically programmed and mainly depends on the two DNA damage checkpoint responses, ATM/Tel1 and ATR/Rad3, reminiscent to programmed cell death. Diminishing the respiration rate or treating cells with a low concentration of antioxidants rescues the quiescent tdp1 mutant cells. These findings suggest that mitochondrial respiration causes neuronal cell death in the SCAN1 syndrome and in other neurological disorders.     

Identification of Tyrosyl-DNA Phosphodiesterase as a Novel DNA Damage Repair Enzyme in Arabidopsis

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a key enzyme that hydrolyzes the phosphodiester bond between tyrosine of topoisomerase and 3′-phosphate of DNA and repairs topoisomerase-mediated DNA damage during chromosome metabolism. However, functional Tdp1 has only been described in yeast and human to date. In human, mutations of the Tdp1 gene are involved in the disease spinocerebellar ataxia with axonal neuropathy. In plants, we have identified the functional nuclear protein AtTDP, homolog to human Tdp1 from Arabidopsis (Arabidopsis thaliana). The recombinant AtTDP protein certainly hydrolyzes the 3′-phosphotyrosyl DNA substrates related to repairing in vivo topoisomerase I-DNA-induced damage. The loss-of-function AtTDP mutation displays developmental defects and dwarf phenotype in Arabidopsis. This phenotype is substantially caused by decreased cell numbers without any change of individual cell sizes. The tdp plants exhibit hypersensitivities to camptothecin, a potent topoisomerase I inhibitor, and show rigorous cell death in cotyledons and rosette leaves, suggesting the failure of DNA damage repair in tdp mutants. These results indicate that AtTDP plays a clear role in the repair of topoisomerase I-DNA complexes in Arabidopsis.In all living organisms, a variety of DNA damage is constantly caused by replication errors, UV light, ionizing radiation, DNA damage agents, etc. Once DNA damage has occurred, specific DNA repair proteins, such as AP endonuclease, RAD1 (for radiation sensitive), RAD9, RAD51, XRCC2 (for x-ray repair cross-complementing), Ku80 (XRCC6), and ligase, initiate to act through the repair pathways (Wood et al., 2001). Defects in DNA damage repair have evolved into cancer or genetic diseases in mammals and affect productivity or growth in plants (Tuteja et al., 2001; Wood et al., 2001).In the repair of DNA-protein cross-links, tyrosyl-DNA phosphodiesterase 1 (Tdp1) is known as a unique protein. Tdp1 was initially reported as an active enzyme in Saccharomyces cerevisiae that specifically removes the Tyr group from the covalent intermediate between the Tyr residue and the terminal 3′- phosphate of the oligonucleotide (Yang et al., 1996). Subsequently, the yeast TDP1 gene was identified and showed highly conserved sequences with other organisms, such as Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, and Homo sapiens (Pouliot et al., 1999). The Tdp1 homologs of these species are members of the phospholipase D (PLD) superfamily (Pouliot et al., 1999; Interthal et al., 2001). Yeast Tdp1 is mainly studied concerning the topoisomerase I-repair pathway using double or triple mutants. The deletion mutations of yeast Tdp1 were shown lacking in the repair of DNA damage induced by a topoisomerase inhibitor, the anticancer drug camptothecin (CPT; Pouliot et al., 2001; Liu et al., 2002; Vance and Wilson, 2002). Tdp1 has been further implicated in multiple repair pathways, including the damage repair of topoisomerase II-DNA in yeast (Nitiss et al., 2006).In multicellular eukaryotes, the defect of human Tdp1 has resulted in the neurodisorder disease spinocerebellar ataxia with axonal neuropathy (SCAN1; Takashima et al., 2002). SCAN1 is a rare autosomal recessive neurodegenerative disease, and the patients present distal muscle weakness and peripheral neuropathy (Interthal et al., 2001; Takashima et al., 2002). SCAN1 is caused by a missense mutation (His-493Arg) in the Tdp1 catalytic site. As in yeast, the human Tdp1 protein plays a role in the repair of topoisomerase I-DNA complex lesions in SCAN1 cells (El-Khamisy et al., 2005; Miao et al., 2006). SCAN1 cells are hypersensitive to CPT (Interthal et al., 2005; Miao et al., 2006) and accumulate single-strand break and double-strand break DNAs by CPT (El-Khamisy et al., 2005).At present, although the functional analysis of Tdp1 has been widely conducted in yeast and human cell lines, its role in the overall growth and development of higher plants remains unknown. Here, we investigate the function of a novel Arabidopsis (Arabidopsis thaliana) TDP, a human and yeast Tdp1 homolog. The AtTDP protein shows the DNA damage-repairing activity and substrate specificities in biochemical assay. The dwarf phenotype of the Arabidopsis tdp mutant may be due to the reduced cell number caused by the accumulation of DNA damage and progressive cell death during Arabidopsis development.     

Structural impact of the leukemia drug 1-beta-D-arabinofuranosylcytosine (Ara-C) on the covalent human topoisomerase I-DNA complex

1-beta-d-Arabinofuranosylcytosine (Ara-C) is a potent antineoplastic drug used in the treatment of acute leukemia. Previous biochemical studies indicated the incorporation of Ara-C into DNA reduced the catalytic activity of human topoisomerase I by decreasing the rate of single DNA strand religation by the enzyme by 2-3-fold. We present the 3.1 A crystal structure of human topoisomerase I in covalent complex with an oligonucleotide containing Ara-C at the +1 position of the non-scissile DNA strand. The structure reveals that a hydrogen bond formed between the 2'-hydroxyl of Ara-C and the O4' of the adjacent -1 base 5' to the damage site stabilizes a C3'-endo pucker in the Ara-C arabinose ring. The structural distortions at the site of damage are translated across the DNA double helix to the active site of human topoisomerase I. The free sulfhydryl at the 5'-end of the nicked DNA strand in this trapped covalent complex is shifted out of alignment with the 3'-phosphotyrosine linkage at the catalytic tyrosine 723 residue, producing a geometry not optimal for religation. The subtle structural changes caused by the presence of Ara-C in the DNA duplex may contribute to the cytotoxicity of this leukemia drug by prolonging the lifetime of the covalent human topoisomerase I-DNA complex.     

Unrepaired oxidative DNA damage induces an ATR/ATM apoptotic-like response in quiescent fission yeast

Programmed cell death is a term which refers to a genetic decision of self-killing or suicide of a cell. Programmed cell death is not restricted to multicellular organisms and was described in a wide range of unicellular eukaryotes, indicating phylogenetically conserved functions, that participate in an adaptive response to cellular stress. Here we review and discuss our observations recently published in the EMBO Journal1, that non-dividing fission yeast, Schizosaccharomyces pombe, exhibits a DNA damage response leading to cell death. We found that Tdp1 protects quiescent S. pombe cells against oxidative DNA damage. Tdp1 is a well-conserved tyrosyl-DNA phosphodiesterase required for single-strand break DNA repair, the mutation of Tdp1 is responsible for the recessively inherited syndrome spinocerebellar ataxia with axonal neuropathy (SCAN1) in humans. We found that tdp1 mutant yeast cells grow, as well as the wild-type cells, during the vegetative state, but progressively die in the quiescent state. We showed that, in the absence of Tdp1, the accumulation of unrepaired oxidative DNA damage triggers a genetic response, leading to checkpoint-dependent (ATM/ATR) nuclear DNA degradation, reminiscent of apoptosis. Our results indicate that the reactive oxygen species (ROS) produced during mitochondrial respiration are the main DNA damaging agents in the physiological quiescent state.     

Processing of nucleopeptides mimicking the topoisomerase I-DNA covalent complex by tyrosyl-DNA phosphodiesterase

Tyrosyl-DNA phosphodiesterase-1 (Tdp1) is the only known enzyme to remove tyrosine from complexes in which the amino acid is linked to the 3′-end of DNA fragments. Such complexes can be produced following DNA processing by topoisomerase I, and recent studies in yeast have demonstrated the importance of TDP1 for cell survival following topoisomerase I-mediated DNA damage. In the present study, we used synthetic oligodeoxynucleotide–peptide conjugates (nucleopeptides) and recombinant yeast Tdp1 to investigate the molecular determinants for Tdp1 activity. We find that Tdp1 can process nucleopeptides with up to 13 amino acid residues but is poorly active with a 70 kDa fragment of topoisomerase I covalently linked to a suicide DNA substrate. Furthermore, Tdp1 was more effective with nucleopeptides with one to four amino acids than 15 amino acids. Tdp1 was also more effective with nucleopeptides containing 15 nt than with homolog nucleopeptides containing 4 nt. These results suggest that DNA binding contributes to the activity of Tdp1 and that Tdp1 would be most effective after topoisomerase I has been proteolyzed in vivo.     

In vitro complementation of Tdp1 deficiency indicates a stabilized enzyme-DNA adduct from tyrosyl but not glycolate lesions as a consequence of the SCAN1 mutation

A homozygous H493R mutation in the active site of tyrosyl-DNA phosphodiesterase (TDP1) has been implicated in hereditary spinocerebellar ataxia with axonal neuropathy (SCAN1), an autosomal recessive neurodegenerative disease. However, it is uncertain how the H493R mutation elicits the specific pathologies of SCAN1. To address this question, and to further elucidate the role of TDP1 in repair of DNA end modifications and general physiology, we generated a Tdp1 knockout mouse and carried out detailed behavioral analyses as well as characterization of repair deficiencies in extracts of embryo fibroblasts from these animals. While Tdp1^?/? mice appear phenotypically normal, extracts from Tdp1^?/? fibroblasts exhibited deficiencies in processing 3′-phosphotyrosyl single-strand breaks and 3′-phosphoglycolate double-strand breaks (DSBs), but not 3′-phosphoglycolate single-strand breaks. Supplementing Tdp1^?/? extracts with H493R TDP1 partially restored processing of 3′-phosphotyrosyl single-strand breaks, but with evidence of persistent covalent adducts between TDP1 and DNA, consistent with a proposed intermediate-stabilization effect of the SCAN1 mutation. However, H493R TDP1 supplementation had no effect on phosphoglycolate (PG) termini on 3′ overhangs of double-strand breaks; these remained completely unprocessed. Altogether, these results suggest that for 3′-phosphoglycolate overhang lesions, the SCAN1 mutation confers loss of function, while for 3′-phosphotyrosyl lesions, the mutation uniquely stabilizes a reaction intermediate.     

TDP2 promotes repair of topoisomerase I-mediated DNA damage in the absence of TDP1

The abortive activity of topoisomerases can result in clastogenic and/or lethal DNA damage in which the topoisomerase is covalently linked to the 3'- or 5'-terminus of a DNA strand break. This type of DNA damage is implicated in chromosome translocations and neurological disease and underlies the clinical efficacy of an important class of anticancer topoisomerase 'poisons'. Tyrosyl DNA phosphodiesterase-1 protects cells from abortive topoisomerase I (Top1) activity by hydrolyzing the 3'-phosphotyrosyl bond that links Top1 to a DNA strand break and is currently the only known human enzyme that displays this activity in cells. Recently, we identified a second tyrosyl DNA phosphodiesterase (TDP2; aka TTRAP/EAPII) that possesses weak 3'-tyrosyl DNA phosphodiesterase (3'-TDP) activity, in vitro. Herein, we have examined whether TDP2 contributes to the repair of Top1-mediated DNA breaks by deleting Tdp1 and Tdp2 separately and together in murine and avian cells. We show that while deletion of Tdp1 in wild-type DT40 cells and mouse embryonic fibroblasts decreases DNA strand break repair rates and cellular survival in response to Top1-induced DNA damage, deletion of Tdp2 does not. However, deletion of both Tdp1 and Tdp2 reduces rates of DNA strand break repair and cell survival below that observed in Tdp1(-)(/)(-) cells, suggesting that Tdp2 contributes to cellular 3'-TDP activity in the absence of Tdp1. Consistent with this idea, over-expression of human TDP2 in Tdp1(-)(/)(-)/Tdp2(-)(/)(-)(/)(-) DT40 cells increases DNA strand break repair rates and cell survival above that observed in Tdp1(-)(/)(-) DT40 cells, suggesting that Tdp2 over-expression can partially complement the defect imposed by loss of Tdp1. Finally, mice lacking both Tdp1 and Tdp2 exhibit greater sensitivity to Top1 poisons than do mice lacking Tdp1 alone, further suggesting that Tdp2 contributes to the repair of Top1-mediated DNA damage in the absence of Tdp1. In contrast, we failed to detect a contribution for Tdp1 to repair Top2-mediated damage. Together, our data suggest that Tdp1 and Tdp2 fulfil overlapping roles following Top1-induced DNA damage, but not following Top2-induced DNA damage, in vivo.     

Mutation of a conserved active site residue converts tyrosyl-DNA phosphodiesterase I into a DNA topoisomerase I-dependent poison

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the resolution of 3' and 5' phospho-DNA adducts. A defective mutant, associated with the recessive neurodegenerative disease SCAN1, accumulates Tdp1-DNA complexes in vitro. To assess the conservation of enzyme architecture, a 2.0 A crystal structure of yeast Tdp1 was determined that is very similar to human Tdp1. Poorly conserved regions of primary structure are peripheral to an essentially identical catalytic core. Enzyme mechanism was also conserved, because the yeast SCAN1 mutant (H(432)R) enhanced cell sensitivity to the DNA topoisomerase I (Top1) poison camptothecin. A more severe Top1-dependent lethality of Tdp1H(432)N was drug-independent, coinciding with increased covalent Top1-DNA and Tdp1-DNA complex formation in vivo. However, both H(432) mutants were recessive to wild-type Tdp1. Thus, yeast H(432) acts in the general acid/base catalytic mechanism of Tdp1 to resolve 3' phosphotyrosyl and 3' phosphoamide linkages. However, the distinct pattern of mutant Tdp1 activity evident in yeast cells, suggests a more severe defect in Tdp1H(432)N-catalyzed resolution of 3' phospho-adducts.     

Tyrosyl-DNA Phosphodiesterase I Catalytic Mutants Reveal an Alternative Nucleophile That Can Catalyze Substrate Cleavage

Tyrosyl-DNA phosphodiesterase I (Tdp1) catalyzes the repair of 3′-DNA adducts, such as the 3′-phosphotyrosyl linkage of DNA topoisomerase I to DNA. Tdp1 contains two conserved catalytic histidines: a nucleophilic His (His^nuc) that attacks DNA adducts to form a covalent 3′-phosphohistidyl intermediate and a general acid/base His (His^gab), which resolves the Tdp1-DNA linkage. A His^nuc to Ala mutant protein is reportedly inactive, whereas the autosomal recessive neurodegenerative disease SCAN1 has been attributed to the enhanced stability of the Tdp1-DNA intermediate induced by mutation of His^gab to Arg. However, here we report that expression of the yeast His^nucAla (H182A) mutant actually induced topoisomerase I-dependent cytotoxicity and further enhanced the cytotoxicity of Tdp1 His^gab mutants, including H432N and the SCAN1-related H432R. Moreover, the His^nucAla mutant was catalytically active in vitro, albeit at levels 85-fold less than that observed with wild type Tdp1. In contrast, the His^nucPhe mutant was catalytically inactive and suppressed His^gab mutant-induced toxicity. These data suggest that the activity of another nucleophile when His^nuc is replaced with residues containing a small side chain (Ala, Asn, and Gln), but not with a bulky side chain. Indeed, genetic, biochemical, and mass spectrometry analyses show that a highly conserved His, immediately N-terminal to His^nuc, can act as a nucleophile to catalyze the formation of a covalent Tdp1-DNA intermediate. These findings suggest that the flexibility of Tdp1 active site residues may impair the resolution of mutant Tdp1 covalent phosphohistidyl intermediates and provide the rationale for developing chemotherapeutics that stabilize the covalent Tdp1-DNA intermediate.     

Conversion of topoisomerase I cleavage complexes on the leading strand of ribosomal DNA into 5'-phosphorylated DNA double-strand breaks by replication runoff

Topoisomerase I cleavage complexes can be induced by a variety of DNA damages and by the anticancer drug camptothecin. We have developed a ligation-mediated PCR (LM-PCR) assay to analyze replication-mediated DNA double-strand breaks induced by topoisomerase I cleavage complexes in human colon carcinoma HT29 cells at the nucleotide level. We found that conversion of topoisomerase I cleavage complexes into replication-mediated DNA double-strand breaks was only detectable on the leading strand for DNA synthesis, which suggests an asymmetry in the way that topoisomerase I cleavage complexes are metabolized on the two arms of a replication fork. Extension by Taq DNA polymerase was not required for ligation to the LM-PCR primer, indicating that the 3' DNA ends are extended by DNA polymerase in vivo closely to the 5' ends of the topoisomerase I cleavage complexes. These findings suggest that the replication-mediated DNA double-strand breaks generated at topoisomerase I cleavage sites are produced by replication runoff. We also found that the 5' ends of these DNA double-strand breaks are phosphorylated in vivo, which suggests that a DNA 5' kinase activity acts on the double-strand ends generated by replication runoff. The replication-mediated DNA double-strand breaks were rapidly reversible after cessation of the topoisomerase I cleavage complexes, suggesting the existence of efficient repair pathways for removal of topoisomerase I-DNA covalent adducts in ribosomal DNA.     

Deficiency in 3'-phosphoglycolate processing in human cells with a hereditary mutation in tyrosyl-DNA phosphodiesterase (TDP1)

Tyrosyl-DNA phosphodiesterase (TDP1) is a DNA repair enzyme that removes peptide fragments linked through tyrosine to the 3′ end of DNA, and can also remove 3′-phosphoglycolates (PGs) formed by free radical-mediated DNA cleavage. To assess whether TDP1 is primarily responsible for PG removal during in vitro end joining of DNA double-strand breaks (DSBs), whole-cell extracts were prepared from lymphoblastoid cells derived either from spinocerebellar ataxia with axonal neuropathy (SCAN1) patients, who have an inactivating mutation in the active site of TDP1, or from closely matched normal controls. Whereas extracts from normal cells catalyzed conversion of 3′-PG termini, both on single-strand oligomers and on 3′ overhangs of DSBs, to 3′-phosphate termini, extracts of SCAN1 cells did not process either substrate. Addition of recombinant TDP1 to SCAN1 extracts restored 3′-PG removal, allowing subsequent gap filling on the aligned DSB ends. Two of three SCAN1 lines examined were slightly more radiosensitive than normal cells, but only for fractionated radiation in plateau phase. The results suggest that the TDP1 mutation in SCAN1 abolishes the 3′-PG processing activity of the enzyme, and that there are no other enzymes in cell extracts capable of processing protruding 3′-PG termini. However, the lack of severe radiosensitivity suggests that there must be alternative, TDP1-independent pathways for repair of 3′-PG DSBs.     

Design and synthesis of fluorescent substrates for human tyrosyl-DNA phosphodiesterase I

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a DNA repair enzyme that acts upon protein–DNA covalent complexes. Tdp1 hydrolyzes 3′-phosphotyrosyl bonds to generate 3′-phosphate DNA and free tyrosine in vitro. Mutations in Tdp1 have been linked to patients with spinocerebellar ataxia, and over-expression of Tdp1 results in resistance to known anti-cancer compounds. Tdp1 has been shown to be involved in double-strand break repair in yeast, and Tdp1 has also been implicated in single-strand break repair in mammalian cells. Despite the biological importance of this enzyme and the possibility that Tdp1 may be a molecular target for new anti-cancer drugs, there are very few assays available for screening inhibitor libraries or for characterizing Tdp1 function, especially under pre-steady-state conditions. Here, we report the design and synthesis of a fluorescence-based assay using oligonucleotide and nucleotide substrates containing 3′-(4-methylumbelliferone)-phosphate. These substrates are efficiently cleaved by Tdp1, generating the fluorescent 4-methylumbelliferone reporter molecule. The kinetic characteristics determined for Tdp1 using this assay are in agreement with the previously published values, and this fluorescence-based assay is validated using the standard gel-based methods. This sensitive assay is ideal for kinetic analysis of Tdp1 function and for high-throughput screening of Tdp1 inhibitory molecules.