Double-strand Breaks In Cells Research Articles

Two faces of TEMPO (2,2,6,6-tetramethylpiperidinyl-1-oxyl) – An antioxidant or a toxin?

2,2,6,6-Tetramethylpiperidine-1-oxyl, commonly known as TEMPO, is one of the compounds called nitroxides that are used in the chemical industry for synthesis of many organic compounds as well as for electrodes in all-organic radical batteries. Additionally, TEMPO is a widely used antioxidant in scientific studies. Technological progress and simultaneous care for the environment leads to resorting to new industrial methods which require the use of compounds that have not been fully tested for their impact on living organisms. Therefore, TEMPO may be an environmental pollutant and its effect on living organisms is not fully understood. The aim of our study was to determine the influence of TEMPO on the physiology, chronological lifespan and wide transcription changes of a eukaryotic model organism, namely the Saccharomyces cerevisiae yeast. For this purpose, we used the BY4741 wild-type and isogenic mutants with a disorder in the response to oxidative stress (sod1Δ, sod2Δ, yap1Δ) and repair of DNA damage (rad52Δ). We showed that supplementation with TEMPO inhibited the cell growth rate of all analyzed strains while simultaneously slowing down the aging of post-mitotic cells in the yeast population. In addition, TEMPO-treated yeast cells manifested a significantly increased level of metabolism in the wild-type and sod2Δ strains. TEMPO also displayed genoprotective effect by reducing the number of DNA double-strand breaks in cells. Here, we are the first to show the widespread effect of TEMPO on yeast. In conclusion, we have shown that, contrary to the commonly accepted notion, TEMPO has also a toxic effect, especially on active mitotic cells. We hypothesize that translation impairment or ribosome biogenesis disorder is likely to be considered secondary effects of TEMPO toxicity related to cell cycle arrest. Therefore, despite the growing interest in the use of this compound in the chemical industry, its toxic effect on the environment, especially biosphere, should be taken into account.

OGG1 Inhibitor TH5487 Alters OGG1 Chromatin Dynamics and Prevents Incisions.

8-oxoguanine DNA glycosylase (OGG1) is the main DNA glycosylase responsible for the excision of 7,8-dihydro-8-oxoguanine (8-oxoG) from duplex DNA to initiate base excision repair. This glycosylase activity is relevant in many pathological conditions including cancer, inflammation, and neurodegenerative diseases. To have a better understanding of the role of OGG1, we previously reported TH5487, a potent active site inhibitor of OGG1. Here, we further investigate the consequences of inhibiting OGG1 with TH5487. TH5487 treatment induces accumulation of genomic 8-oxoG lesions. Furthermore, it impairs the chromatin binding of OGG1 and results in lower recruitment of OGG1 to regions of DNA damage. Inhibiting OGG1 with TH5487 interferes with OGG1′s incision activity, resulting in fewer DNA double-strand breaks in cells exposed to oxidative stress. This study validates TH5487 as a potent OGG1 inhibitor that prevents the repair of 8-oxoG and alters OGG1–chromatin dynamics and OGG1′s recruitment kinetics.

Dissimilar DNA Damage to Blood Lymphocytes After 177Lu-Labeled DOTATOC or Prostate-Specific Membrane Antigen Therapy.

DNA double-strand breaks in cells of radionuclide-treated patients are quantifiable by immunofluorescence microscopy, using phosphorylation of histone-variant H2AX (γ-H2AX) to mark radiation-induced foci (RIFs). Using this method, we compared excess RIFs side by side in recipients of 177Lu-DOTATOC or 177Lu-prostate specific membrane antigen-617 (PSMA) radioligands. We also examined relations between blood dose and dose rate, RIFs, and platelet counts. Methods: Venous blood samples were obtained from 48 patients subjected to 177Lu-labeled radioligand therapy (177Lu-DOTATOC, 26; 177Lu-PSMA, 22) to quantify blood lymphocyte RIFs and blood activity concentrations at various time points, including baseline (before injection) and postinjection readings (5 min, 30 min, 4 h, 24 h, 48 h, and 72 h). Absorbed doses and dose rates to blood were derived from sequentially assessed blood activity concentrations and γ-camera imaging. Platelet levels in routine blood tests were monitored for 3 d after injection to assess responses. Results: RIF counts averaged 0.25 ± 0.15 at baseline. Postinjection RIF counts were significantly higher than baseline values, peaking at 5 min (average, 3.93 ± 2.51 min) and declining thereafter. Compared with RIF counts of 177Lu-DOTATOC, those of 177Lu-PSMA were significantly higher at 5 min after injection and significantly lower at 72 h after injection. These differences could not be fully explained by blood doses and dose rates, which were significantly higher for 177Lu-PSMA than for 177Lu-DOTATOC treatment at every time point. RIF counts overall correlated with dose rates across all time points (Pearson r = 0.78; P < 0.01) and with absorbed dose until 4 h after injection only (Pearson r = 0.42; P < 0.01). Declines in platelet concentration correlated significantly with RIFs at 72 h after injection (Pearson r = -0.34; P < 0.05). Conclusion: Although values generated by the currently used blood dosimetry model correlated with RIF counts, the difference observed in 177Lu-DOTATOC and 177Lu-PSMA treatment groups was unexplained. Significantly more RIFs were found in 177Lu-DOTATOC recipients by comparison, despite lower dose rates and blood doses, exposing a potential limitation.

Druggable binding sites in the multicomponent assemblies that characterise DNA double-strand-break repair through non-homologous end joining.

Non-homologous end joining (NHEJ) is one of the two principal damage repair pathways for DNA double-strand breaks in cells. In this review, we give a brief overview of the system including a discussion of the effects of deregulation of NHEJ components in carcinogenesis and resistance to cancer therapy. We then discuss the relevance of targeting NHEJ components pharmacologically as a potential cancer therapy and review previous approaches to orthosteric regulation of NHEJ factors. Given the limited success of previous investigations to develop inhibitors against individual components, we give a brief discussion of the recent advances in computational and structural biology that allow us to explore different targets, with a particular focus on modulating protein–protein interaction interfaces. We illustrate this discussion with three examples showcasing some current approaches to developing protein–protein interaction inhibitors to modulate the assembly of NHEJ multiprotein complexes in space and time.

DNA double-strand breaks of human peripheral blood lymphocyte induced by CT examination of oral and maxillofacial region.

To explore whether a computed tomography (CT) examination of the head and neck region induces biological damage and whether the damage was correlated with the radiation dose. Peripheral blood was taken from 33 individuals who received head and neck CT examinations. Blood samples were divided into three groups: the control group and the in vivo and in vitro irradiation groups. The number of DNA double-strand breaks was estimated by comparing the changes in the rates of γ-H2AX foci formation in the peripheral blood before and after CT examination. The absorbed dose and effective dose were calculated with the software VirtualDose based on the Monte Carlo method, and the absorbed doses in blood were estimated accordingly. The γ-H2AX foci rates were increased in the in vivo (p < 0.001) and in vitro irradiation groups (p < 0.001) after CT examination when compared with those in the control group. The rate of γ-H2AX foci formation showed linear dose-responses for the CT dose index volume (CTDIvol), dose-length product (DLP), and blood dose after CT examination. A CT examination of the head and neck region provides a high enough radiation dose to induce DNA double-strand breaks in cells in the peripheral blood. There was a linear correlation between the formation of DNA double-strand breaks and radiation doses after CT examination. In addition to ensuring image quality, in a real clinical situation, the scanning area should be strictly administered, and repeated operations should be avoided to minimise the patient's radiation dose.

Persistent 3′-phosphate termini and increased cytotoxicity of radiomimetic DNA double-strand breaks in cells lacking polynucleotide kinase/phosphatase despite presence of an alternative 3′-phosphatase

Polynucleotide kinase/phosphatase (PNKP) has been implicated in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). To assess the consequences of PNKP deficiency for NHEJ of 3′-phosphate-ended DSBs, PNKP-deficient derivatives of HCT116 and of HeLa cells were generated using CRISPR/CAS9. For both cell lines, PNKP deficiency conferred sensitivity to ionizing radiation as well as to neocarzinostatin (NCS), which specifically induces DSBs bearing protruding 3′-phosphate termini. Moreover, NCS-induced DSBs, detected as 53BP1 foci, were more persistent in PNKP −/− HCT116 cells compared to their wild-type (WT) counterparts. Surprisingly, PNKP-deficient whole-cell and nuclear extracts were biochemically competent in removing both protruding and recessed 3′-phosphates from synthetic DSB substrates, albeit much less efficiently than WT extracts, suggesting an alternative 3′-phosphatase. Measurements by ligation-mediated PCR showed that PNKP-deficient HeLa cells contained significantly more 3′-phosphate-terminated and fewer 3′-hydroxyl-terminated DSBs than parental cells 5–15 min after NCS treatment, but this difference disappeared by 1 h. These results suggest that, despite presence of an alternative 3′-phosphatase, loss of PNKP significantly sensitizes cells to 3′-phosphate-terminated DSBs, due to a 3′-dephosphorylation defect.

Comparison of DSB effects of the beta particles of iodine-131 and 6 MV X-ray at a dose of 2 Gy in the presence of 2-Methoxyestradiol, IUdR, and TPT in glioblastoma spheroids

Glioblastoma is one of the lethal brain tumors and one of the resistant tumors against radiotherapy. Multiple treatment methods and different types of radiation and Radiosensitizers drugs have been combined to optimize the efficacy of radiotherapy. Radiosensitizers are employed to reinforce tumor cell killing and have much fewer effects on the normal tissue. Inducing DNA double strand break in tumoral cells is a major goal of radiation sensitivity. In this study, the level of DNA double strand break in glioblastoma spheroids irradiated by 2Gy beta particles of iodine-131 and 6MV X-rays in the presence of 2-Methoxyestradiol (2ME2), iodo-deoxy-uridine (IUdR) and Topotecan (TPT) was measured using the PicoGreen method.Spheroids of the U87MG cell line were cultured to reach a 300µm diameter. In the phase one of the study, the spheroids were treated in four groups individually, including 2Gy of iodine-131, TPT+iodine-131, IUdR+iodine-131, IUdR+2ME2+iodine-131. In the next phase, the cells were treated with 2Gy of 6MV X-ray, TPT+6MV X-ray, IUdR+6MV X-ray, TPT+IUdR+6MV X-ray. DSB lesions were measured by the Pico Green assay. The amount of DSB lesions in groups irradiated with iodine-131 individually was greater than the group irradiated with 6MV X-ray (p<0.05). DNA double strand breaks became more significant in combination with TPT. However, the amount of DSBs in the two independent groups of TPT+IUdR+2ME2+iodine-131 and TPT+IUdR+2ME2+6MV X-ray was approximately in the same range (P>0.05). The level of DNA double strand breaks in cells irradiated with Iodine-131 was higher than cells irradiated with 6MV X-ray at the same dose and Topotecan had a positive effect on inducing the damage. The role of 2ME2+IUdR in increasing the damage caused by beta particles of iodine-131 was not significant. Iodine-131 could lead to major DSB damage than 6MV X-ray at the same dose due to its cross fire effect and spatial distribution of energy in different angels. This study showed that a combination of chemotherapy and iodine-131 had better efficacy than radiotherapy with 6MV X-ray in the treatment of glioblastoma.

MiroRNA-127-3p targets XRCC3 to enhance the chemosensitivity of esophageal cancer cells to a novel phenanthroline-dione derivative

MicroRNAs are small non-coding RNAs with 18–22 nucleotides in length and have been proposed to function in various biological processes by targeting genes for post-transcriptional degradation via their 3′ untranslated region. Moreover, they have been suggested to improve the chemosensitivity in a panel of tumors. However, the biological functions of microRNA-127-3p in esophageal carcinogenesis are still enigmatic. Thus, in the study, we firstly analyzed the roles of microRNA-127-3p in regulating the growth of esophageal cancer cells both in vitro and in vivo. Afterwards, using the microRNA-targeted gene prediction software and the dual-luciferase reporter assays, we confirmed that microRNA-127-3p specifically reduced the expression of X-ray repair complementing defective repair in Chinese hamster cells 3, one of RAD51 recombinase paralogs, at both mRNA and protein levels. Furthermore, using the homologous recombination repair and non-homologous end joining repair reporter systems, we found that microRNA-127-3p specifically compromised the homologous recombination repair and significantly increased DNA double strand breaks in cells. Besides, it statistically increased the chemosensitivity of esophageal cancer cells to a novel phenanthroline-dione derivative in vivo by mechanistically impairing the recruitment of RAD51 to the damage sites. In summary, our findings not only suggest that microRNA-127-3p can be used as a predictor for evaluating the development of esophageal carcinoma, but also show that it can be used to increase the chemosensitivity of esophageal cancer patients to the phenanthroline-dione derivative, which might be a potential anticancer candidate in the future.

Disruption of SLX4-MUS81 Function Increases the Relative Biological Effectiveness of Proton Radiation

Clinical proton beam therapy has been based on the use of a generic relative biological effectiveness (RBE) of ∼1.1. However, emerging data have suggested that Fanconi anemia (FA) and homologous recombination pathway defects can lead to a variable RBE, at least in vitro. We investigated the role of SLX4 (FANCP), which acts as a docking platform for the assembly of multiple structure-specific endonucleases, in the response to proton irradiation. Isogenic cell pairs for the study of SLX4, XPF/ERCC1, MUS81, and SLX1 were irradiated at the mid-spread-out Bragg peak of a clinical proton beam (linear energy transfer 2.5 keV/μm) or with 250 kVp x-rays, and the clonogenic survival fractions were determined. To estimate the RBE of the protons relative to cobalt-60 photons (Co60Eq), we assigned a RBE(Co60Eq) of 1.1 to x-rays to correct the physical dose measured. Standard DNA repair foci assays were used to monitor the damage responses, and the cell cycle distributions were assessed by flow cytometry. The poly(ADP-ribose) polymerase inhibitor olaparib was used for comparison. Loss of SLX4 function resulted in an enhanced proton RBE(Co60Eq) of 1.42 compared with 1.11 for wild-type cells (at a survival fraction of 0.1; P<.05), which correlated with increased persistent DNA double-strand breaks in cells in the S/G2 phase. Genetic analysis identified the SLX4-binding partner MUS81 as a mediator of resistance to proton radiation. Both proton irradiation and olaparib treatment resulted in a similar prolonged accumulation of RAD51 foci in SLX4/MUS81-deficient cells, suggesting a common defect in the repair of DNA replication fork-associated damage. A defect in the FA pathway at the level of SLX4 results in hypersensitivity to proton radiation, which is, at least in part, due to impaired MUS81-mediated processing of replication forks that stall at clustered DNA damage. In vivo and clinical studies are needed to confirm these findings in human cancers.

Cellular Recognition and Repair of Monofunctional-Intercalative Platinum--DNA Adducts.

The cellular recognition and processing of monofunctional-intercalative DNA adducts formed by [PtCl(en)(L)](NO3)2 (P1-A1; en = ethane-1,2-diamine; L = N-[2-(acridin-9-ylamino)ethyl]-N-methylpropionamidine, acridinium cation), a cytotoxic hybrid agent with potent anticancer activity, was studied. Excision of these adducts and subsequent DNA repair synthesis were monitored in plasmids modified with platinum using incubations with mammalian cell-free extract. On the basis of the levels of [α-(32)P]-dCTP incorporation, P1-A1-DNA adducts were rapidly repaired with a rate approximately 8 times faster (t1/2 ≈ 18 min at 30 °C) than the adducts (cross-links) formed by the drug cisplatin. Cellular responses to P1-A1 and cisplatin were also studied in NCI-H460 lung cancer cells using immunocytochemistry in conjunction with confocal fluorescence microscopy. At the same dose, P1-A1, but not cisplatin, elicited a distinct requirement for DNA double-strand break repair and stalled replication fork repair, which caused nuclear fluorescent staining related to high levels of MUS81, a specialized repair endonuclease, and phosphorylated histone protein γ-H2AX. The results confirm previous observations in yeast-based chemical genomics assays. γ-H2AX fluorescence is observed as a large number of discrete foci signaling DNA double-strand breaks, pan-nuclear preapoptotic staining, and unique circularly shaped staining around the nucleoli and nuclear rim. DNA cleavage assays indicate that P1-A1 does not act as a typical topoisomerase poison, suggesting the high level of DNA double-strand breaks in cells is more likely a result of topoisomerase-independent replication fork collapse. Overall, the cellular response to platinum-acridines shares striking similarities with that reported for DNA adduct-forming derivatives of the drug doxorubicin. The results of this study are discussed in light of the cellular mechanism of action of platinum-acridines and their ability to overcome resistance to cisplatin.

Abstract PL04-02: ATM: Bridging DNA damage responses and metabolic regulation

Abstract Much of our understanding of the mechanisms involved in cellular DNA damage response pathways have come from studies of human cancer susceptibility syndromes. ATM, the gene mutated in the cancer-prone, radiosensitive disorder, Ataxia-telangiectasia (A-T), is a protein kinase that is a central mediator of responses to DNA double strand breaks in cells. Once activated, ATM phosphorylates numerous substrates in the cell that modulate the cell's response to the DNA damage. p53, one of the many targets of the ATM kinase, is a critical mediator of cell cycle changes and cell death signaling following DNA damage and other stresses. Though ATM clearly plays a central role in mediating cellular responses to DNA breakage, some of the clinical abnormalities in A-T are difficult to attribute directly to alterations in DNA damage signaling. The broad-based symptoms in A-T and recently acquired data suggest that ATM has physiologic roles that extend beyond its regulation of DNA damage responses. In particular, ATM appears to affect insulin signaling pathways and to have a direct role in regulating mitochondrial function. Interestingly, we found in mouse models of A-T that these mitochondrial defects associated with ATM deficiency appear to be a contributing factor to cancer development in A-T. These new insights suggest that Ataxia-Telangiectasia should be considered, at least in part, as a mitochondrial dysfunction disease and provide links between cancer-related DNA damage signaling, general metabolic signaling pathways, and cancer development. Citation Format: Michael B. Kastan, Yasmine Valentin-Vega, Adam Brown, Peter Scarbrough, Donald Fleenor. ATM: Bridging DNA damage responses and metabolic regulation. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr PL04-02.

Abstract IA08: Ataxia-telangiectasia: Broad implications from the study of a rare disease

Abstract DNA damage signaling pathways are critical mediators of both tumor development and tumor responses to chemotherapy and radiation therapy. Significant progress has been made in recent years in elucidating the molecular controls of cellular responses to DNA damage in mammalian cells. Much of what we have learned about damage signaling pathways has come from the study of human diseases, particularly cancer predisposition syndromes. Ataxia-Telangiectasia (A-T) is a pleiotropic disorder with multiple phenotypic abnormalities, including progressive neurodegeneration, immunodeficiencies, cancer predisposition, insulin resistance, telangiectasia development, infertility, and extreme radiosensitivity. ATM, the gene mutated in A-T, is a central mediator of responses to DNA double strand breaks in cells. Once activated, ATM phosphorylates numerous substrates in the cell that modulate the cell's response to the DNA damage. Though ATM clearly plays a central role in mediating cellular responses to DNA breakage, some of the clinical abnormalities in A-T are difficult to attribute directly to alterations in DNA damage signaling. The broad-based symptoms in A-T and recently acquired data suggest that ATM may have roles that extend beyond its regulation of DNA damage responses. In particular, ATM appears to affect insulin signaling pathways and evidence has been generated supporting a direct role for ATM in mitochondrial function. These new insights suggest that Ataxia-Telangiectasia should be considered, at least in part, as a mitochondrial dysfunction disease and provide links between cancer-related DNA damage signaling and more general metabolic signaling pathways. Some of these new insights are also providing opportunities for considerations of novel ways to modulate ATM function for clinical benefit. Citation Format: Michael B. Kastan. Ataxia-telangiectasia: Broad implications from the study of a rare disease. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr IA08.

Arsenic is cytotoxic and genotoxic to primary human lung cells

Arsenic originates from both geochemical and numerous anthropogenic activities. Exposure of the general public to significant levels of arsenic is widespread. Arsenic is a well-documented human carcinogen. Long-term exposure to high levels of arsenic in drinking water has been linked to bladder, lung, kidney, liver, prostate, and skin cancers. Among them, lung cancer is of great public concern. However, little is known about how arsenic causes lung cancer and few studies have considered effects in normal human lung cells. The purpose of this study was to determine the cytotoxicity and genotoxicity of arsenic in human primary bronchial fibroblast and epithelial cells. Our data show that arsenic induces a concentration-dependent decrease in cell survival after short (24h) or long (120h) exposures. Arsenic induces concentration-dependent but not time-dependent increases in chromosome damage in fibroblasts. No chromosome damage is induced after either 24h or 120h arsenic exposure in epithelial cells. Using neutral comet assay and gamma-H2A.X foci forming assay, we found that 24h or 120h exposure to arsenic induces increases in DNA double strand breaks in both cell lines. These data indicate that arsenic is cytotoxic and genotoxic to human lung primary cells but lung fibroblasts are more sensitive to arsenic than epithelial cells. Further research is needed to understand the specific mechanisms involved in arsenic-induced genotoxicity in human lung cells.

Post-irradiation chemical processing of DNA damage generates double-strand breaks in cells already engaged in repair

In cells exposed to ionizing radiation (IR), double-strand breaks (DSBs) form within clustered-damage sites from lesions disrupting the DNA sugar–phosphate backbone. It is commonly assumed that these DSBs form promptly and are immediately detected and processed by the cellular DNA damage response (DDR) apparatus. This assumption is questioned by the observation that after irradiation of naked DNA, a fraction of DSBs forms minutes to hours after exposure as a result of temperature dependent, chemical processing of labile sugar lesions. Excess DSBs also form when IR-exposed cells are processed at 50°C, but have been hitherto considered method-related artifact. Thus, it remains unknown whether DSBs actually develop in cells after IR exposure from chemically labile damage. Here, we show that irradiation of ‘naked’ or chromatin-organized mammalian DNA produces lesions, which evolve to DSBs and add to those promptly induced, after 8–24 h in vitro incubation at 37°C or 50°C. The conversion is more efficient in chromatin-associated DNA, completed within 1 h in cells and delayed in a reducing environment. We conclude that IR generates sugar lesions within clustered-damage sites contributing to DSB formation only after chemical processing, which occurs efficiently at 37°C. This subset of delayed DSBs may challenge DDR, may affect the perceived repair kinetics and requires further characterization.

ATM Regulates a RASSF1A-Dependent DNA Damage Response

Hypermethylation of CpG islands in the RASSF1 promoter is one of the most frequent events identified in human cancer. The epigenetic-driven loss of RASSF1A protein expression is observed more often in tumors of higher grade and correlates with a decreased responsiveness to DNA-damaging therapy. Ras association domain-containing family 1A (RASSF1A) promotes apoptosis by signaling through the MST2 and LATS1 kinases, leading to stabilization of the YAP1/p73 transcriptional complex. Here we provide evidence for a new pathway linking DNA damage signaling to RASSF1A via the main sensor of double-strand breaks in cells, ataxia telangiectasia mutated (ATM). We show that, upon DNA damage, RASSF1A is phosphorylated by ATM on Ser131 and is involved in the activation of both MST2 and LATS1, leading to the stabilization of p73. Furthermore, lung and ovarian tumor cell lines that retain RASSF1A expression commonly harbor polymorphisms in the region of Ser131, and our analysis shows that the S131F polymorphism conveys resistance to DNA-damaging agents. Thus, we present a novel DNA damage pathway emanating from ATM that is frequently disabled in tumors via epigenetic silencing of RASSF1 or mutation of an ATM phosphorylation site.

Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination

Eukaryotes possess mechanisms to limit crossing over during homologous recombination, thus avoiding possible chromosomal rearrangements. We show here that budding yeast Mph1, an ortholog of human FancM helicase, utilizes its helicase activity to suppress spontaneous unequal sister chromatid exchanges and DNA double-strand break-induced chromosome crossovers. Since the efficiency and kinetics of break repair are unaffected, Mph1 appears to channel repair intermediates into a noncrossover pathway. Importantly, Mph1 works independently of two other helicases-Srs2 and Sgs1-that also attenuate crossing over. By chromatin immunoprecipitation, we find targeting of Mph1 to double-strand breaks in cells. Purified Mph1 binds D-loop structures and is particularly adept at unwinding these structures. Importantly, Mph1, but not a helicase-defective variant, dissociates Rad51-made D-loops. Overall, the results from our analyses suggest a new role of Mph1 in promoting the noncrossover repair of DNA double-strand breaks.

Our cells get stressed too! Implications for human disease

Significant progress has been made in recent years elucidating the molecular controls of cellular responses to DNA damage in mammalian cells. Many of the insights that we have gained into the mechanisms involved in cellular DNA damage response pathways have come from studies of human cancer susceptibility syndromes that are altered in DNA damage responses. ATM, the gene mutated in the cancer-prone disorder, ataxia telangiectasia, is a protein kinase that is a central mediator of responses to DNA double strand breaks in cells. Such insights provide us with opportunities to develop new approaches to benefit patients. For example, inhibitors of the ATM pathway have the potential to act as sensitizers to chemotherapy or radiation therapy and could even have anti-neoplastic effects on their own. Conversely, activators of ATM could improve responses to cellular stresses such as oxidative damage. The potential benefits of ATM modulation in disease settings ranging from metabolic syndrome to cancer will be discussed.

DNA damage response pathways in cancer causation and treatment

DNA damage response pathways in cancer causation and treatment

The ATM-dependent DNA Damage Signaling Pathway

Many of the insights that we have gained into the mechanisms involved in cellular DNA damage response pathways have come from studies of human cancer susceptibility syndromes that are altered in DNA damage responses. ATM, the gene mutated in the disorder, ataxia-telangiectasia, is a protein kinase that is a central mediator of responses to DNA double-strand breaks in cells. Recent studies have elucidated the mechanism by which DNA damage activates the ATM kinase and initiates these critical cellular signaling pathways. The SMC1 protein appears to be a particularly important target of the ATM kinase, playing critical roles in controlling DNA replication forks and DNA repair after the damage. A major role for the NBS1 and BRCA1 proteins appears to be in the recruitment of an activated ATM kinase molecule to the sites of DNA breaks so that ATM can phosphorylate SMC1. Generation of mice and cells that are unable to phosphorylate SMC1 demonstrated the importance of SMC1 phosphorylation in the DNA-damage-induced S-phase checkpoint, in determining rates of repair of chromosomal breaks, and in determining cell survival after DNA damage. Focusing on ATM and SMC1, the molecular controls of these pathways is discussed.

7 Repair of DNA double strand breaks in cells of higher eukaryotes

7 Repair of DNA double strand breaks in cells of higher eukaryotes