Inactivation of PNKP by mutant ATXN3 triggers apoptosis by activating the DNA damage-response pathway in SCA3.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an untreatable autosomal dominant neurodegenerative disease, and the most common such inherited ataxia worldwide. The mutation in SCA3 is the expansion of a polymorphic CAG tri-nucleotide repeat sequence in the C-terminal coding region of the ATXN3 gene at chromosomal locus 14q32.1. The mutant ATXN3 protein encoding expanded glutamine (polyQ) sequences interacts with multiple proteins in vivo, and is deposited as aggregates in the SCA3 brain. A large body of literature suggests that the loss of function of the native ATNX3-interacting proteins that are deposited in the polyQ aggregates contributes to cellular toxicity, systemic neurodegeneration and the pathogenic mechanism in SCA3. Nonetheless, a significant understanding of the disease etiology of SCA3, the molecular mechanism by which the polyQ expansions in the mutant ATXN3 induce neurodegeneration in SCA3 has remained elusive. In the present study, we show that the essential DNA strand break repair enzyme PNKP (polynucleotide kinase 3'-phosphatase) interacts with, and is inactivated by, the mutant ATXN3, resulting in inefficient DNA repair, persistent accumulation of DNA damage/strand breaks, and subsequent chronic activation of the DNA damage-response ataxia telangiectasia-mutated (ATM) signaling pathway in SCA3. We report that persistent accumulation of DNA damage/strand breaks and chronic activation of the serine/threonine kinase ATM and the downstream p53 and protein kinase C-d pro-apoptotic pathways trigger neuronal dysfunction and eventually neuronal death in SCA3. Either PNKP overexpression or pharmacological inhibition of ATM dramatically blocked mutant ATXN3-mediated cell death. Discovery of the mechanism by which mutant ATXN3 induces DNA damage and amplifies the pro-death signaling pathways provides a molecular basis for neurodegeneration due to PNKP inactivation in SCA3, and for the first time offers a possible approach to treatment.

Visualization of recombination intermediates produced by RAD52-mediated single-strand annealing.

Double-strand breaks (DSBs) occur frequently during DNA replication. They are also caused by ionizing radiation, chemical damage or as part of the series of programmed events that occur during meiosis. In yeast, DSB repair requires RAD52, a protein that plays a critical role in homologous recombination. Here we describe the actions of human RAD52 protein in a model system for single-strand annealing (SSA) using tailed (i.e. exonuclease resected) duplex DNA molecules. Purified human RAD52 protein binds resected DSBs and promotes associations between complementary DNA termini. Heteroduplex intermediates of these recombination reactions have been visualized by electron microscopy, revealing the specific binding of multiple rings of RAD52 to the resected termini and the formation of large protein complexes at heteroduplex joints formed by RAD52-mediated annealing.

Radiation induced apoptosis and initial DNA damage are inversely related in locally advanced breast cancer patients.

Background. DNA-damage assays, quantifying the initial number of DNA double-strand breaks induced by radiation, have been proposed as a predictive test for radiation-induced toxicity. Determination of radiation-induced apoptosis in peripheral blood lymphocytes by flow cytometry analysis has also been proposed as an approach for predicting normal tissue responses following radiotherapy. The aim of the present study was to explore the association between initial DNA damage, estimated by the number of double-strand breaks induced by a given radiation dose, and the radio-induced apoptosis rates observed. Methods. Peripheral blood lymphocytes were taken from 26 consecutive patients with locally advanced breast carcinoma. Radiosensitivity of lymphocytes was quantified as the initial number of DNA double-strand breaks induced per Gy and per DNA unit (200 Mbp). Radio-induced apoptosis at 1, 2 and 8 Gy was measured by flow cytometry using annexin V/propidium iodide. Results. Radiation-induced apoptosis increased in order to radiation dose and data fitted to a semi logarithmic mathematical model. A positive correlation was found among radio-induced apoptosis values at different radiation doses: 1, 2 and 8 Gy (p < 0.0001 in all cases). Mean DSB/Gy/DNA unit obtained was 1.70 ± 0.83 (range 0.63-4.08; median, 1.46). A statistically significant inverse correlation was found between initial damage to DNA and radio-induced apoptosis at 1 Gy (p = 0.034). A trend toward 2 Gy (p = 0.057) and 8 Gy (p = 0.067) was observed after 24 hours of incubation. Conclusions. An inverse association was observed for the first time between these variables, both considered as predictive factors to radiation toxicity.

Combined low initial DNA damage and high radiation-induced apoptosis confers clinical resistance to long-term toxicity in breast cancer patients treated with high-dose radiotherapy

BACKGROUND. Either higher levels of initial DNA damage or lower levels of radiation-induced apoptosis in peripheral blood lymphocytes have been associated to increased risk for develop late radiation-induced toxicity. It has been recently published that these two predictive tests are inversely related. The aim of the present study was to investigate the combined role of both tests in relation to clinical radiation-induced toxicity in a set of breast cancer patients treated with high dose hyperfractionated radical radiotherapy. METHODS. Peripheral blood lymphocytes were taken from 26 consecutive patients with locally advanced breast carcinoma treated with high-dose hyperfractioned radical radiotherapy. Acute and late cutaneous and subcutaneous toxicity was evaluated using the Radiation Therapy Oncology Group morbidity scoring schema. The mean follow-up of survivors (n = 13) was 197.23 months. Radiosensitivity of lymphocytes was quantified as the initial number of DNA double-strand breaks induced per Gy and per DNA unit (200 Mbp). Radiation-induced apoptosis (RIA) at 1, 2 and 8 Gy was measured by flow cytometry using annexin V/propidium iodide. RESULTS. Mean DSB/Gy/DNA unit obtained was 1.70 ± 0.83 (range 0.63-4.08; median, 1.46). Radiation-induced apoptosis increased with radiation dose (median 12.36, 17.79 and 24.83 for 1, 2, and 8 Gy respectively). We observed that those "expected resistant patients" (DSB values lower than 1.78 DSB/Gy per 200 Mbp and RIA values over 9.58, 14.40 or 24.83 for 1, 2 and 8 Gy respectively) were at low risk of suffer severe subcutaneous late toxicity (HR 0.223, 95%CI 0.073-0.678, P = 0.008; HR 0.206, 95%CI 0.063-0.677, P = 0.009; HR 0.239, 95%CI 0.062-0.929, P = 0.039, for RIA at 1, 2 and 8 Gy respectively) in multivariate analysis. CONCLUSIONS. A radiation-resistant profile is proposed, where those patients who presented lower levels of initial DNA damage and higher levels of radiation induced apoptosis were at low risk of suffer severe subcutaneous late toxicity after clinical treatment at high radiation doses in our series. However, due to the small sample size, other prospective studies with higher number of patients are needed to validate these results.

Interaction between ATM and PARP-1 in response to DNA damage and sensitization of ATM deficient cells through PARP inhibition

ATM and PARP-1 are two of the most important players in the cell's response to DNA damage. PARP-1 and ATM recognize and bound to both single and double strand DNA breaks in response to different triggers. Here we report that ATM and PARP-1 form a molecular complex in vivo in undamaged cells and this association increases after gamma-irradiation. ATM is also modified by PARP-1 during DNA damage. We have also evaluated the impact of PARP-1 absence or inhibition on ATM-kinase activity and have found that while PARP-1 deficient cells display a defective ATM-kinase activity and reduced gamma-H2AX foci formation in response to gamma-irradiation, PARP inhibition on itself is able to activate ATM-kinase. PARP inhibition induced gamma H2AX foci accumulation, in an ATM-dependent manner. Inhibition of PARP also induces DNA double strand breaks which were dependent on the presence of ATM. As consequence ATM deficient cells display an increased sensitivity to PARP inhibition. In summary our results show that while PARP-1 is needed in the response of ATM to gamma irradiation, the inhibition of PARP induces DNA double strand breaks (which are resolved in and ATM-dependent pathway) and activates ATM kinase.

Temporal patterns of nucleotide misincorporations and DNA fragmentation in ancient DNA.

DNA that survives in museum specimens, bones and other tissues recovered by archaeologists is invariably fragmented and chemically modified. The extent to which such modifications accumulate over time is largely unknown but could potentially be used to differentiate between endogenous old DNA and present-day DNA contaminating specimens and experiments. Here we examine mitochondrial DNA sequences from tissue remains that vary in age between 18 and 60,000 years with respect to three molecular features: fragment length, base composition at strand breaks, and apparent C to T substitutions. We find that fragment length does not decrease consistently over time and that strand breaks occur preferentially before purine residues by what may be at least two different molecular mechanisms that are not yet understood. In contrast, the frequency of apparent C to T substitutions towards the 5'-ends of molecules tends to increase over time. These nucleotide misincorporations are thus a useful tool to distinguish recent from ancient DNA sources in specimens that have not been subjected to unusual or harsh treatments.

DNA structure-specific priming of ATR activation by DNA-PKcs.

Three phosphatidylinositol-3-kinase-related protein kinases implement cellular responses to DNA damage. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia-telangiectasia mutated respond primarily to DNA double-strand breaks (DSBs). Ataxia-telangiectasia and RAD3-related (ATR) signals the accumulation of replication protein A (RPA)-covered single-stranded DNA (ssDNA), which is caused by replication obstacles. Stalled replication intermediates can further degenerate and yield replication-associated DSBs. In this paper, we show that the juxtaposition of a double-stranded DNA end and a short ssDNA gap triggered robust activation of endogenous ATR and Chk1 in human cell-free extracts. This DNA damage signal depended on DNA-PKcs and ATR, which congregated onto gapped linear duplex DNA. DNA-PKcs primed ATR/Chk1 activation through DNA structure-specific phosphorylation of RPA32 and TopBP1. The synergistic activation of DNA-PKcs and ATR suggests that the two kinases combine to mount a prompt and specific response to replication-born DSBs.

Fission yeast rad51 and dmc1, two efficient DNA recombinases forming helical nucleoprotein filaments.

Homologous recombination is important for the repair of double-strand breaks during meiosis. Eukaryotic cells require two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, for meiotic recombination. To date, it is not clear, at the biochemical level, why two homologs of RecA are necessary during meiosis. To gain insight into this, we purified Schizosaccharomyces pombe Rad51 and Dmc1 to homogeneity. Purified Rad51 and Dmc1 form homo-oligomers, bind single-stranded DNA preferentially, and exhibit DNA-stimulated ATPase activity. Both Rad51 and Dmc1 promote the renaturation of complementary single-stranded DNA. Importantly, Rad51 and Dmc1 proteins catalyze ATP-dependent strand exchange reactions with homologous duplex DNA. Electron microscopy reveals that both S. pombe Rad51 and Dmc1 form nucleoprotein filaments. Rad51 formed helical nucleoprotein filaments on single-stranded DNA, whereas Dmc1 was found in two forms, as helical filaments and also as stacked rings. These results demonstrate that Rad51 and Dmc1 are both efficient recombinases in lower eukaryotes and reveal closer functional and structural similarities between the meiotic recombinase Dmc1 and Rad51. The DNA strand exchange activity of both Rad51 and Dmc1 is most likely critical for proper meiotic DNA double-strand break repair in lower eukaryotes.

Association of DNA repair inhibition and radiofrequency ablation in the treatment of xenografted colorectal cancer

Purpose: Most of the patients with advanced colorectal cancer will develop liver metastasis, even after primary tumor resection. Although surgical resection remains the gold standard treatment of hepatic metastases, only few patients are eligible to curative resection. Radiofrequency ablation (RFA) is the most common curative alternative. Dbait are new molecules that inhibit DNA double-strand breaks repair. In vitro, Dbait has shown to increase cell death after hyperthermia. Here, we have assessed the combination of Dbait and RFA in the treatment of human colorectal cancer model xenografted in nude mice.Materials: 98 mice were flank-grafted with HT29 (human colon adenocarcinoma). When tumor reached 500 mm3, mice were sham treated (n=19), treated by Dbait via local injections (n=20), treated by RFA using an incomplete ablation scheme (n=20) or treated by combination of Dbait and RFA (n=39 separated in two Dbait regimens). After RFA, 39 mice were sacrificed for blinded pathological study, and 59 others were followed for survival analysis.Results: Mice treated by RFA-Dbait had significantly longer survival as compared to RFA alone (median survival: 56 vs 39 days, p<0.05) while RFA improved survival as compared to controls (median survival: 39 vs 28 days, p<0.05). Pathological studies of tumor slice have demonstrated significant decrease of tumor area and cancer cell viability in the RFA-Dbait group.Conclusions: While the implication of DNA repair activity in heat sensitivity remains unclear, our results show that the addition of Dbait to RFA enhances the antitumor response in this model and provide an experimental basis for the use of Dbait as an additional therapy to RFA.

Reconstitution of the strand invasion step of double-strand break repair using human Rad51 Rad52 and RPA proteins.

The human Rad51 recombinase is essential for the repair of double-strand breaks in DNA that occur in somatic cells after exposure to ionising irradiation, or in germ line cells undergoing meiotic recombination. The initiation of double-strand break repair is thought to involve resection of the double-strand break to produce 3'-ended single-stranded (ss) tails that invade homologous duplex DNA. Here, we have used purified proteins to set up a defined in vitro system for the initial strand invasion step of double-strand break repair. We show that (i) hRad51 binds to the ssDNA of tailed duplex DNA molecules, and (ii) hRad51 catalyses the invasion of tailed duplex DNA into homologous covalently closed DNA. Invasion is stimulated by the single-strand DNA binding protein RPA, and by the hRad52 protein. Strikingly, hRad51 forms terminal nucleoprotein filaments on either 3' or 5'-ssDNA tails and promotes strand invasion without regard for the polarity of the tail. Taken together, these results show that hRad51 is recruited to regions of ssDNA occurring at resected double-strand breaks, and that hRad51 shows no intrinsic polarity preference at the strand invasion step that initiates double-strand break repair.

Mitochondrial DNA damage and animal longevity: insights from comparative studies

Chemical reactions in living cells are under strict enzyme control and conform to a tightly regulated metabolic program. However, uncontrolled and potentially deleterious endogenous reactions occur, even under physiological conditions. Aging, in this chemical context, could be viewed as an entropic process, the result of chemical side reactions that chronically and cumulatively degrade the function of biological systems. Mitochondria are a main source of reactive oxygen species (ROS) and chemical sidereactions in healthy aerobic tissues and are the only known extranuclear cellular organelles in animal cells that contain their own DNA (mtDNA). ROS can modify mtDNA directly at the sugar-phosphate backbone or at the bases, producing many different oxidatively modified purines and pyrimidines, as well as single and double strand breaks and DNA mutations. In this scenario, natural selection tends to decrease the mitochondrial ROS generation, the oxidative damage to mtDNA, and the mitochondrial mutation rate in long-lived species, in agreement with the mitochondrial oxidative stress theory of aging.

Visualizing the spatiotemporal dynamics of DNA damage in budding yeast.

Fluorescence microscopy has enabled the analysis of both the spatial distribution of DNA damage and its dynamics during the DNA damage response (DDR). Three microscopic techniques can be used to study the spatiotemporal dynamics of DNA damage. In the first part we describe how we determine the position of DNA double-strand breaks (DSBs) relative to the nuclear envelope. The second part describes how to quantify the co-localization of DNA DSBs with nuclear pore clusters, or other nuclear subcompartments. The final protocols describe methods for the quantification of locus mobility over time.

Plasmid DNA damage induced by singlet molecular oxygen released from the naphthalene endoperoxide DHPNO2 and photoactivated methylene blue

To investigate oxidative lesions and strand breaks induction by singlet molecular oxygen (¹O2), supercoiled-DNA plasmid was treated with thermo-dissociated DHPNO2 and photoactivated-methylene blue. DNA lesions were detected by Fpg that cleaves DNA at certain oxidized bases, and T4-endoV, which cleaves DNA at cyclobutane pyrimidine dimers and apurinic/apyrimidinic (AP) sites. These cleavages form open relaxed-DNA structures, which are discriminated from supercoiled-DNA. DHPNO2 or photoactivated-MB treatments result in similar plasmid damage profile: low number of single-strand breaks or AP-sites and high frequency of Fpg-sensitive sites; confirming that base oxidation is the main product for both reactions and that ¹O2 might be the most likely intermediate that reacts with DNA.

Photodynamic DNA damage induced by phycocyanin and its repair in Saccharomyces cerevisiae

In the present study, we analyzed DNA damage induced by phycocyanin (PHY) in the presence of visible light (VL) using a set of repair endonucleases purified from Escherichia coli. We demonstrated that the profile of DNA damage induced by PHY is clearly different from that induced by molecules that exert deleterious effects on DNA involving solely singlet oxygen as reactive species. Most of PHY-induced lesions are single strand breaks and, to a lesser extent, base oxidized sites, which are recognized by Nth, Nfo and Fpg enzymes. High pressure liquid chromatography coupled to electrochemical detection revealed that PHY photosensitization did not induce 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) at detectable levels. DNA repair after PHY photosensitization was also investigated. Plasmid DNA damaged by PHY photosensitization was used to transform a series of Saccharomyces cerevisiae DNA repair mutants. The results revealed that plasmid survival was greatly reduced in rad14 mutants, while the ogg1 mutation did not modify the plasmid survival when compared to that in the wild type. Furthermore, plasmid survival in the ogg1 rad14 double mutant was not different from that in the rad14 single mutant. The results reported here indicate that lethal lesions induced by PHY plus VL are repaired differently by prokaryotic and eukaryotic cells. Morever, nucleotide excision repair seems to play a major role in the recognition and repair of these lesions in Saccharomyces cerevisiae.