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1.
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. 相似文献
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Zeng Z Sharma A Ju L Murai J Umans L Vermeire L Pommier Y Takeda S Huylebroeck D Caldecott KW El-Khamisy SF 《Nucleic acids research》2012,40(17):8371-8380
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. 相似文献
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Burhans WC Weinberger M Marchetti MA Ramachandran L D'Urso G Huberman JA 《Mutation research》2003,532(1-2):227-243
In budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast and other unicellular organisms, DNA damage and other stimuli can induce cell death resembling apoptosis in metazoans, including the activation of a recently discovered caspase-like molecule in budding yeast. Induction of apoptotic-like cell death in yeasts requires homologues of cell cycle checkpoint proteins that are often required for apoptosis in metazoan cells. Here, we summarize these findings and our unpublished results which show that an important component of metazoan apoptosis recently detected in budding yeast-reactive oxygen species (ROS)-can also be detected in fission yeast undergoing an apoptotic-like cell death. ROS were detected in fission and budding yeast cells bearing conditional mutations in genes encoding DNA replication initiation proteins and in fission yeast cells with mutations that deregulate cyclin-dependent kinases (CDKs). These mutations may cause DNA damage by permitting entry of cells into S phase with a reduced number of replication forks and/or passage through mitosis with incompletely replicated chromosomes. This may be relevant to the frequent requirement for elevated CDK activity in mammalian apoptosis, and to the recent discovery that the initiation protein Cdc6 is destroyed during apoptosis in mammals and in budding yeast cells exposed to lethal levels of DNA damage. Our data indicate that connections between apoptosis-like cell death and DNA replication or CDK activity are complex. Some apoptosis-like pathways require checkpoint proteins, others are inhibited by them, and others are independent of them. This complexity resembles that of apoptotic pathways in mammalian cells, which are frequently deregulated in cancer. The greater genetic tractability of yeasts should help to delineate these complex pathways and their relationships to cancer and to the effects of apoptosis-inducing drugs that inhibit DNA replication. 相似文献
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《Cell cycle (Georgetown, Tex.)》2013,12(6):767-771
Increasing evidence indicates that postmitotic, terminally differentiated neurons activate the cell cycle before death. The purpose of this cell cycle activation, however, remains elusive. In proliferating cells, cell cycle machinery is a major contributor to the DNA damage response, which is comprised of growth arrest. In quiescent cells such as terminally differentiated neurons, cell cycle-associated events may also be part of the DNA damage response. A link between DNA damage and repair, cell cycle regulation and cell death is becoming increasingly recognized for cycling cells but remains elusive for quiescent cells. Neurons are particularly susceptible to oxidative stress due to the high rate of oxidative metabolism in the brain and the low level of antioxidant enzymes compared to other somatic tissues. This is supported by fact that the intracellular end point of many neurotoxic stimuli is oxidative stress, which also represents a major cause of the neuropathology underlying a variety of neurodegenerative diseases. DNA is perhaps the major target of oxyradicals. Thus, oxidative stress may cause DNA damage, which is countered by a complex defense mechanism, the DNA damage response, which involves not only the elimination of DNA damage, but its coordination with other cellular processes such as cell-cycle progression, together directing to preserve genomic integrity. The function of such response is the removal of DNA damage by DNA repair pathways, or the elimination of damaged cells via apoptosis. The present review discusses the idea that the cell cycle machinery is a critical element of the DNA damage response not only in cycling, but also quiescent cells, and may bear the same function: to repair the damage or initiate apoptosis if the damage is too extensive to be repaired. 相似文献
6.
Processing of nucleopeptides mimicking the topoisomerase I-DNA covalent complex by tyrosyl-DNA phosphodiesterase
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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. 相似文献
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Yeasts have proven to be invaluable, genetically tractable systems to study various fundamental biological processes including programmed cell death. Recent advances in the elucidation of the molecular pathways underlying apoptotic cell death in yeasts have revealed remarkable similarities to mammalian apoptosis at cellular, organelle and macromolecular levels, thus making a strong case for the relevance of yeast models of regulated cell death. Programmed cell death has been reported in fission yeast Schizosaccharomyces pombe, primarily in the contexts of perturbed intracellular lipid metabolism, defective DNA replication, improper mitotic entry, chronological and replicative aging. Here we review the current understanding of the programmed cell death in fission yeast, paying particular attention to lipid-induced cell death. We discuss our recent findings that fission yeast exhibits plasticity of apoptotic and non-apoptotic modes of cell death in response to different lipid stimuli and growth conditions, and that mitochondria, reactive oxygen species and novel cell death mediators including metacaspase Pca1, SpRad9 and Pck1 are involved in the lipotoxic cell death. We also present perspectives on how various aspects of the cell and molecular biology of this organism can be explored to shed light on the governing principles underlying lipid-mediated signaling and cell demise. 相似文献
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Programmed cell death in fission yeast 总被引:2,自引:0,他引:2
Recently a metacaspase, encoded by YCA1, has been implicated in a primitive form of apoptosis or programmed cell death in yeast. Previously it had been shown that over-expression of mammalian pro-apoptotic proteins can induce cell death in yeast, but the mechanism of how cell death occurred was not clearly established. More recently, it has been shown that DNA or oxidative damage, or other cell cycle blocks, can result in cell death that mimics apoptosis in higher cells. Also, in fission yeast deletion of genes required for triacylglycerol synthesis leads to cell death and expression of apoptotic markers. A metacaspase sharing greater than 40% identity to budding yeast Yca1 has been identified in fission yeast, however, its role in programmed cell death is not yet known. Analysis of the genetic pathways that influence cell death in yeast may provide insights into the mechanisms of apoptosis in all eukaryotic organisms. 相似文献
10.
Abasic (AP) sites are formed spontaneously and are inevitably intermediates during base excision repair of DNA base damages. AP sites are both mutagenic and cytotoxic and key enzymes for their removal are AP endonucleases. However, AP endonuclease independent repair initiated by DNA glycosylases performing β,δ-elimination cleavage of the AP sites has been described in mammalian cells. Here, we describe another AP endonuclease independent repair pathway for removal of AP sites in Schizosaccharomyces pombe that is initiated by a bifunctional DNA glycosylase, Nth1 and followed by cleavage of the baseless sugar residue by tyrosyl phosphodiesterase Tdp1. We propose that repair is completed by the action of a polynucleotide kinase, a DNA polymerase and finally a DNA ligase to seal the gap. A fission yeast double mutant of the major AP endonuclease Apn2 and Tdp1 shows synergistic increase in MMS sensitivity, substantiating that Apn2 and Tdp1 process the same substrate. These results add new knowledge to the complex cellular response to AP sites, which could be exploited in chemotherapy where synthetic lethality is a key strategy of treatment. 相似文献
11.
We previously reported that Schizosaccharomyces pombe pnk1 cells are more sensitive than wild-type cells to γ-radiation and camptothecin, indicating that Pnk1 is required for DNA repair. Here, we report that pnk1pku70 and pnk1rhp51 double mutants are more sensitive to γ-radiation than single mutants, from which we infer that Pnk1's primary role is independent of either homologous recombination or non-homologous end joining mechanisms. We also report that pnk1 cells are more sensitive than wild-type cells to oxidizing and alkylating agents, suggesting that Pnk1 is involved in base excision repair. Mutational analysis of Pnk1 revealed that the DNA 3'-phosphatase activity is necessary for repair of DNA damage, whereas the 5'-kinase activity is dispensable. A role for Pnk1 in base excision repair is supported by genetic analyses which revealed that pnk1apn2 is synthetically lethal, suggesting that Pnk1 and Apn2 may function in parallel pathways essential for the repair of endogenous DNA damage. Furthermore, the nth1pnk1apn2 and tdp1pnk1apn2 triple mutants are viable, implying that single-strand breaks with 3'-blocked termini produced by Nth1 and Tdp1 contribute to synthetic lethality. We also examined the sensitivity to methyl methanesulfonate of all single and double mutant combinations of nth1, apn2, tdp1 and pnk1. Together, our results support a model where Tdp1 and Pnk1 act in concert in an Apn2-independent base excision repair pathway to repair 3'-blocked termini produced by Nth1; and they also provide evidence that Pnk1 has additional roles in base excision repair. 相似文献
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Schizosaccharomyces pombe Nthpl, an ortholog of the endonuclease III family, is the sole bifunctional DNA glycosylase encoded in its genome. The enzyme removes oxidative pyrimidine and incises 3' to the apurinic/apyrimidinic (AP) site, leaving 3'-alpha,beta-unsaturated aldehyde. Analysis of nth1 cDNA revealed an intronless structure including 5'- and 3'-untranslated regions. An Nth1p-green fluorescent fusion protein was predominantly localized in the nuclei of yeast cells, indicating a nuclear function. Deletion of nth1 confirmed that Nth1p is responsible for the majority of activity for thymine glycol and AP site incision in the absence of metal ions, while nth1 mutants exhibit hypersensitivity to methylmethanesulfonate (MMS). Complementation of sensitivity by heterologous expression of various DNA glycosylases showed that the methyl-formamidopyrimidine (me-fapy) and/or AP sites are plausible substrates for Nth1p in repairing MMS damage. Apn2p, the major AP endonuclease in S. pombe, also greatly contributes to the repair of MMS damage. Deletion of nth1 from an apn2 mutant resulted in tolerance to MMS damage, indicating that Nth1p-induced 3'-blocks are responsible for MMS sensitivity in apn2 mutants. Overexpression of Apn2p in nth1 mutants failed to suppress MMS sensitivity. These results indicate that Nth1p, not Apn2p, primarily incises AP sites and that the resultant 3'-blocks are removed by the 3'-phosphodiesterase activity of Apn2p. Nth1p is dispensable for cell survival against low levels of oxidative stress, but wild-type yeast became more sensitive than the nth1 mutant at high levels. Overexpression of Nth1p in heavily damaged cells probably induced cell death via the formation of 3'-blocked single-strand breaks. 相似文献
13.
Murai J Huang SY Das BB Dexheimer TS Takeda S Pommier Y 《The Journal of biological chemistry》2012,287(16):12848-12857
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) repairs topoisomerase I cleavage complexes (Top1cc) by hydrolyzing their 3'-phosphotyrosyl DNA bonds and repairs bleomycin-induced DNA damage by hydrolyzing 3'-phosphoglycolates. Yeast Tdp1 has also been implicated in the repair of topoisomerase II-DNA cleavage complexes (Top2cc). To determine whether vertebrate Tdp1 is involved in the repair of various DNA end-blocking lesions, we generated Tdp1 knock-out cells in chicken DT40 cells (Tdp1-/-) and Tdp1-complemented DT40 cells with human TDP1. We found that Tdp1-/- cells were not only hypersensitive to camptothecin and bleomycin but also to etoposide, methyl methanesulfonate (MMS), H(2)O(2), and ionizing radiation. We also show they were deficient in mitochondrial Tdp1 activity. In biochemical assays, recombinant human TDP1 was found to process 5'-phosphotyrosyl DNA ends when they mimic the 5'-overhangs of Top2cc. Tdp1 also processes 3'-deoxyribose phosphates generated from hydrolysis of abasic sites, which is consistent with the hypersensitivity of Tdp1-/- cells to MMS and H(2)O(2). Because recent studies established that CtIP together with BRCA1 also repairs topoisomerase-mediated DNA damage, we generated dual Tdp1-CtIP-deficient DT40 cells. Our results show that Tdp1 and CtIP act in parallel pathways for the repair of Top1cc and MMS-induced lesions but are epistatic for Top2cc. Together, our findings reveal a broad involvement of Tdp1 in DNA repair and clarify the role of human TDP1 in the repair of Top2-induced DNA damage. 相似文献
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So-Young Lee Hoyeun Kim Hyun-Ju Hwang Young-Min Jeong Sang Hyeon Na Je-Chang Woo Sang-Gu Kim 《Plant physiology》2010,154(3):1460-1469
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. 相似文献
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Oda K Kawasaki N Fukuyama M Ikeda S 《Journal of biochemistry and molecular biology》2007,40(6):1095-1099
Endonuclease G (EndoG) is a mitochondrial non-specific nuclease that is highly conserved among the eukaryotes. Although the precise role of EndoG in mitochondria is not yet known, the enzyme is released from the mitochondria and digests nuclear DNA during apoptosis in mammalian cells. Schizosaccharomyces pombe has an EndoG homolog Pnu1p (previously named SpNuc1) that is produced as a precursor protein with a mitochondrial targeting sequence. During the sorting into mitochondria the signal sequence is cleaved to yield the functionally active endonuclease. From the analogy to EndoG, active extramitochondrial Pnu1p may trigger cell killing by degrading nuclear DNA. Here, we tested this possibility by expressing a truncated Pnu1p lacking the signal sequence in the extramitochondrial region of pnu1-deleted cells. The truncated Pnu1p was localized in the cytosol and nuclei of yeast cells. And ectopic expression of active Pnu1p led to cell death with fragmentation of nuclear DNA. This suggests that the Pnu1p is possibly involved in a certain type of yeast cell death via DNA fragmentation. Although expression of human Bak in S. pombe was lethal, Pnu1p nuclease is not necessary for hBak-induced cell death. 相似文献
16.
He X van Waardenburg RC Babaoglu K Price AC Nitiss KC Nitiss JL Bjornsti MA White SW 《Journal of molecular biology》2007,372(4):1070-1081
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. 相似文献
17.
Katyal S el-Khamisy SF Russell HR Li Y Ju L Caldecott KW McKinnon PJ 《The EMBO journal》2007,26(22):4720-4731
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. 相似文献
18.
Porcedda P Turinetto V Lantelme E Fontanella E Chrzanowska K Ragona R De Marchi M Delia D Giachino C 《DNA Repair》2006,5(8):904-913
The repair of DNA double-strand breaks is critical for genome integrity and tumor suppression. Here we show that following treatment with the DNA-intercalating agent actinomycin D (ActD), normal quiescent T cells accumulate double-strand breaks and die, whereas T cells from ataxia telangiectasia (AT) and Nijmegen breakage syndrome (NBS) patients are resistant to this death pathway despite a comparable amount of DNA damage. We demonstrate that the ActD-induced death pathway in quiescent T lymphocytes follows DNA damage and H2AX phosphorylation, is ATM- and NBS1-dependent and due to p53-mediated cellular apoptosis. In response to genotoxic 2-Gy gamma-irradiation, on the other hand, quiescent T cells from normal donors survive following complete resolution of the damage thus induced. T cells from AT and NBS patients also survive, but retain foci of phosphorylated H2AX due to a subtle double-strand break (DSB) repair defect. A common consequence of these two genetic defects in the DSB response is the apparent tolerance of cells containing DNA breaks. We suggest that this tolerance makes a major contribution to the oncogenic risk of patients with chromosome instability syndromes. 相似文献
19.
Reactive oxygen species (ROS) have been implicated as one of the agents responsible for many neurodegenerative diseases. A critical target for ROS is DNA. Most oxidative stress-induced DNA damage in the nucleus and mitochondria is removed by the base excision repair pathway. Apn1 is a yeast enzyme in this pathway which possesses a wider substrate specificity and greater enzyme activity than its mammalian counterpart for removing DNA damage, making it a good therapeutic candidate. For this study we targeted Apn1 to mitochondria in a neuronal cell line derived from the substantia nigra by using a mitochondrial targeting signal (MTS) in an effort to hasten the removal of DNA damage and thereby protect these cells. We found that following oxidative stress, mitochondrial DNA (mtDNA) was repaired more efficiently in cells containing Apn1 with the MTS than controls. There was no difference in nuclear repair. However, cells that expressed Apn1 without the MTS showed enhanced repair of both nuclear and mtDNA. Both Apn1-expressing cells were more resistant to cell death following oxidative stress compared with controls. Therefore, these results reveal that the expression of Apn1 in neurons may be of potential therapeutic benefit for treating patients with specific neurodegenerative diseases. 相似文献
20.
Heidi M. Blank Jason M. Sheltzer Colleen M. Meehl Angelika Amon 《Molecular biology of the cell》2015,26(8):1440-1451
Genetic instability is a hallmark of aneuploidy in budding and fission yeast. All aneuploid yeast strains analyzed to date harbor elevated levels of Rad52-GFP foci, a sign of DNA damage. Here we investigate how continuously elevated levels of DNA damage affect aneuploid cells. We show that Rad52-GFP foci form during S phase, consistent with the observation that DNA replication initiation and elongation are impaired in some aneuploid yeast strains. We furthermore find that although DNA damage is low in aneuploid cells, it nevertheless has dramatic consequences. Many aneuploid yeast strains adapt to DNA damage and undergo mitosis despite the presence of unrepaired DNA leading to cell death. Wild-type cells exposed to low levels of DNA damage exhibit a similar phenotype, indicating that adaptation to low levels of unrepaired DNA is a general property of the cell''s response to DNA damage. Our results indicate that by causing low levels of DNA damage, whole-chromosome aneuploidies lead to DNA breaks that persist into mitosis. Such breaks provide the substrate for translocations and deletions that are a hallmark of cancer. 相似文献