dNTP imbalance and DNA double strand breaks in mouse FM3A cells and the mechanism of cell death

The mechanism of intracellular deoxyribonucleoside-triphosphate (dNTP) imbalance death of mouse mammary tumor FM3A cells was studied. When the cells were exposed to 5-fluorodeoxyuridine, deoxyadenosine, or 2-chlorodeoxyadenosine, dNTP pool imbalance resulted. The imbalance was followed by DNA double-strand breaks and subsequent cell death. The DNA double strand breaks were directly examined by means of orthogonal-field-alternation gel electrophoresis (OFAGE). Fragmented DNA band appeared to be approximately 100-200 kbp in size. The bases of 5'-termini in the DNA were cytosine and thymine. The imbalance induced endonuclease has been isolated by DEAE-agarose column chromatography.     

Deoxyribonucleoside triphosphate imbalance. 5-Fluorodeoxyuridine-induced DNA double strand breaks in mouse FM3A cells and the mechanism of cell death

The mechanism of cytotoxic action of 5-fluorodeoxyuridine (FdUrd) in mouse FM3A cells was investigated. We observed the FdUrd-induced imbalance of intracellular deoxyribonucleoside triphosphate (dNTP) pools and subsequent double strand breaks in mature DNA, accompanied by cell death. The imbalance of dNTP pools was maximal at 8 h after 1 microM FdUrd treatment; a depletion of dTTP and dGTP pools and an increase in the dATP pool were observed. The addition of FdUrd in culture medium induced strand breaks in DNA, giving rise to a 90 S peak by alkaline sucrose gradient sedimentation. The loss of cell viability and colony-forming ability occurred at about 10 h. DNA double strand breaks as measured by the neutral elution method were also observed in FdUrd-treated cells about 10 h after the addition. These results lead us to propose that DNA double strand breaks play an important role in the mechanism of FdUrd-mediated cell death. A comparison of the ratio of single and double strand breaks induced by FdUrd to that observed following radiation suggested that FdUrd produced double strand breaks exclusively. Cycloheximide inhibited both the production of DNA double strand breaks and the FdUrd-induced cell death. An activity that can induce DNA double strand breaks was detected in the lysate of FdUrd-treated FM3A cells but not in the untreated cells. This suggests that FdUrd induces the cellular DNA double strand breaking activity. The FdUrd-induced DNA strand breaks and cell death appear to occur in the S phase. Our results indicate that imbalance of the dNTP pools is a trigger for double strand DNA break and cell death.     

dNTP pool imbalance induced endonuclease: 5-fluorodeoxyuridine induced DNA double strand break in mouse FM3A cells and the mechanism of cell death.

The mechanism of intracellular deoxyribonucleotide triphosphates (dNTP) pool imbalance-induced cell death in mouse FM3A cells was studied. When the cells were treated with 1 microM 5-fluorodeoxyuridine (FdUrd), the imbalance of the cellular dNTP pool was induced. The imbalance was followed by DNA double stranded breaks and subsequent cell death. The endonuclease toward double stranded DNA has been found in a fraction of FdUrd treated cell lysate, and isolated using column chromatography. SDS-polyacrylamide gel electrophoresis showed a major protein species of approximate 45 kDa. The endonuclease was revealed, using electrophoretic separation in SDS-polyacrylamide gels containing DNA, by incubating the gels in buffer to remove SDS and to allow renaturation and enzyme activity.     

Accumulation of DNA strand breaks during thymineless death in thymidylate synthase-negative mutants of mouse FM3A cells

Thymidylate synthase-negative mutants of cultured mouse cells were immediately committed to cell death upon thymidine deprivation, especially when the cells were synchronized in the S phase. Thymidylate deprivation induced single strand breaks in chromosome-size DNA strands, as measured by alkaline sucrose gradient sedimentation, giving rise to two peaks, one with large and the other with small fragments, the latter about the size of T4 DNA. An increase in the small DNA fragments paralleled that of thymineless death. Thymidine deprivation also produced double strand DNA fragments as determined by a method of neutral filter elution, and their extent paralleled that of cell death. Double-stranded DNA eluted through the filter sedimented as a single peak both in a neutral and in an alkaline sucrose gradient that coincided with that of the above small DNA fragments. Therefore, the strand breaks seemed to occur in some defined portions of the genome and in a specific manner compared to breaks induced by x-rays, which occurred rather randomly. Cycloheximide blocked both thymineless death and the production of the small DNA fragments. The strand breaks induced by thymidine starvation were not repaired but instead advanced on subsequent incubation of the cells in growth medium containing thymidine.     

Detection of deoxyribonucleoside-triphosphate imbalance death-induced DNA double strand breakage in FM3A cells by orthogonal-field-alternation gel electrophoresis (OFAGE)

The mechanism of intracellular deoxyribonucleoside-triphosphate (dNTP) imbalance death of mouse mammary tumor FM3A cells was studied. When the cells were exposed to 5-fluorodeoxyuridine, deoxyadenosine, or 2-chlorodeoxyadenosine, dNTP pool imbalance resulted. The imbalance was followed by DNA double strand breaks and subsequent cell death. The DNA double strand breaks have been directly examined by means of orthogonal-field-alternation gel electrophoresis (OFAGE). Fragmented DNA band appeared to be approximately 100-200 kb in size.     

Methoxyamine potentiates DNA single strand breaks and double strand breaks induced by temozolomide in colon cancer cells

We have previously shown that human cancer cells deficient in DNA mismatch repair (MMR) are resistant to the chemotherapeutic methylating agent temozolomide (TMZ) and can be sensitized by the base excision repair (BER) blocking agent methoxyamine (MX) [21]. To further characterize BER-mediated repair responses to methylating agent-induced DNA damage, we have now evaluated the effect of MX on TMZ-induced DNA single strand breaks (SSB) by alkaline elution and DNA double strand breaks (DSB) by pulsed field gel electrophoresis in SW480 (O6-alkylguanine-DNA-alkyltransferase [AGT]+, MMR wild type) and HCT116 (AGT+, MMR deficient) colon cancer cells. SSB were evident in both cell lines after a 2-h exposure to equitoxic doses of temozolomide. MX significantly increased the number of TMZ-induced DNA-SSB in both cell lines. In contrast to SSB, TMZ-induced DNA-DSB were dependent on MMR status and were time-dependent. Levels of 50 kb double stranded DNA fragments in MMR proficient cells were increased after TMZ alone or in combination with O6-benzylguanine or MX, whereas, in MMR deficient HCT116 cells, only TMZ plus MX produced significant levels of DNA-DSB. Levels of AP endonuclease, XRCC1 and polymerase beta were present in both cell lines and were not significantly altered after MX and TMZ. However, cleavage of a 30-mer double strand substrate by SW480 and HCT116 crude cell extracts was inhibited by MX plus TMZ. Thus, MX potentiation of TMZ cytotoxicity may be explained by the persistence of apurinic/apyrimidinic (AP) sites not further processed due to the presence of MX. Furthermore, in MMR-deficient, TMZ-resistant HCT116 colon cancer cells, MX potentiates TMZ cytotoxicity through formation of large DS-DNA fragmentation and subsequent apoptotic signalling.     

DNA strand breaks, NAD metabolism, and programmed cell death

An intimate relationship exists between DNA single-strand breaks, NAD metabolism, and cell viability in quiescent human lymphocytes. Under steady-state conditions, resting lymphocytes continually break and rejoin DNA. The balanced DNA excision-repair process is accompanied by a proportional consumption of NAD for poly(ADP-ribose) synthesis. However, lymphocytes have a limited capacity to resynthesize NAD from nicotinamide. An increase in DNA strand break formation in lymphocytes, or a block in DNA repair, accelerates poly(ADP-ribose) formation and may induce lethal NAD and ATP depletion. In this way, the level of DNA single-strand breaks in the lymphocyte nucleus is linked to the metabolic activity of the cytoplasm. The programmed removal of lymphocytes (and perhaps of other cells) with damaged DNA, may represent a novel physiologic function for poly(ADP-ribose)-dependent NAD cycling.     

A nervous predisposition to unrepaired DNA double strand breaks

Ataxia-telangiectasia (A–T) has for a long time stood apart from most other human neurodegenerative syndromes by the characteristic failure of cells derived from these patients to properly repair DNA damage-induced by ionizing radiation. The discovery of mutations in the ATM gene as being the underlying cause for A–T and the demonstration that the ATM protein functions as a DNA damage-responsive kinase has defined current research focusing on decoding how the cell responds to genotoxic stress. Yet, despite significant advances in delineating the cellular DNA damage response pathways coordinated by ATM, very little headway has been made toward understanding how loss of ATM leads to progressive cerebellar ataxia and whether this can be attributed to an underlying defect in DNA double strand break repair (DSBR). Since its identification, A–T has been used as the archetypal model for how a deficiency in DNA repair affects both the development and maintenance of the nervous and immune systems in humans as well as contributing to the process of tumourigenesis. However, following the growing availability and cost effectiveness of next generation sequencing technologies, the increasing recognition of novel human disorders associated with abnormal DNA repair has demonstrated that the neuropathology typified by A–T is an ‘exception’ rather than the ‘rule’. As a consequence, this throws into doubt the longstanding hypothesis that the neurodegeneration seen in A–T is due to the progressive loss of damaged neurons that have acquired toxic levels of unrepaired DNA lesions over time. Therefore, this review aims to address the question: Is defective DNA double strand break repair an underlying cause of neurodegeneration?     

Processing of 3'-phosphoglycolate-terminated DNA double strand breaks by Artemis nuclease

The Artemis nuclease is required for V(D)J recombination and for repair of an as yet undefined subset of radiation-induced DNA double strand breaks. To assess the possibility that Artemis acts on oxidatively modified double strand break termini, its activity toward model DNA substrates, bearing either 3'-hydroxyl or 3'-phosphoglycolate moieties, was examined. A 3'-phosphoglycolate had little effect on Artemis-mediated trimming of long 3' overhangs (> or =9 nucleotides), which were efficiently trimmed to 4-5 nucleotides. However, 3'-phosphoglycolates on overhangs of 4-5 bases promoted Artemis-mediated removal of a single 3'-terminal nucleotide, while at least 2 nucleotides were trimmed from identical hydroxyl-terminated substrates. Artemis also efficiently removed a single nucleotide from a phosphoglycolate-terminated 3-base 3' overhang, while leaving an analogous hydroxyl-terminated overhang largely intact. Such removal was completely dependent on DNA-dependent protein kinase and ATP and was largely dependent on Ku, which markedly stimulated Artemis activity toward all 3' overhangs. Together, these data suggest that efficient Artemis-mediated cleavage of 3' overhangs requires a minimum of 2 nucleotides, or a nucleotide plus a phosphoglycolate, 3' to the cleavage site, as well as 2 unpaired nucleotides 5' to the cleavage site. Shorter 3'-phosphoglycolate-terminated overhangs and blunt ends were also processed by Artemis but much more slowly. Consistent with a role for Artemis in repair of terminally blocked double strand breaks in vivo, human cells lacking Artemis exhibited hypersensitivity to x-rays, bleomycin, and neocarzinostatin, which all induce 3'-phosphoglycolate-terminated double strand breaks.     

Non-homologous end-joining for repairing I-SceI-induced DNA double strand breaks in human cells

DNA double strand breaks (DSBs) are usually repaired through either non-homologous end-joining (NHEJ) or homologous recombination (HR). While HR is basically error-free repair, NHEJ is a mutagenic pathway that leads to deletion. NHEJ must be precisely regulated to maintain genomic integrity. To clarify the role of NHEJ, we investigated the genetic consequences of NHEJ repair of DSBs in human cells. Human lymphoblastoid cell lines TSCE5 and TSCE105 have, respectively, single and double I-SceI endonuclease sites in the endogenous thymidine kinase gene (TK) located on chromosome 17q. I-SceI expression generated DSBs at the TK gene. We used the novel transfection system (Amaxa Nucleofector) to introduce an I-SceI expression vector into the cells and randomly isolated clones. We found mutations involved in the DSBs in the TK gene in 3% of TSCE5 cells and 30% of TSCE105 cell clones. Most of the mutations in TSCE5 were small (1-30bp) deletions with a 0-4bp microhomology at the junction. The others consisted of large (>60) bp deletions, an insertion, and a rearrangement. Mutants resulting from interallelic HR also occurred, but infrequently. Most of the mutations in TSCE105, on the other hand, were deletions that encompassed the two I-SceI sites generated by NHEJ at DSBs. The sequence joint was similar to that found in TSCE5 mutants. Interestingly, some mutants formed a new I-SceI site by perfectly joining the two original I-SceI sites without deletion of the broken-ends. These results support the idea that NHEJ for repairing I-SceI-induced DSBs mainly results in small or no deletions. Thus, NHEJ must help maintain genomic integrity in mammalian cells by repairing DSBs as well as by preventing many deleterious alterations.     

Chromatin dynamics and the repair of DNA double strand breaks

DNA double-strand breaks (DSBs) arise through both replication errors and from exogenous events such as exposure to ionizing radiation. DSBs are potentially lethal, and cells have evolved a highly conserved mechanism to detect and repair these lesions. This mechanism involves phosphorylation of histone H2AX (γH2AX) and the loading of DNA repair proteins onto the chromatin adjacent to the DSB. It is now clear that the chromatin architecture in the region surrounding the DSB has a critical impact on the ability of cells to mount an effective DNA damage response. DSBs promote the formation of open, relaxed chromatin domains which are spatially confined to the area surrounding the break. These relaxed chromatin structures are created through the coupled action of the p400 SWI/SNF ATPase and histone acetylation by the Tip60 acetyltransferase. The resulting destabilization of nucleosomes at the DSB by Tip60 and p400 is required for ubiquitination of the chromatin by the RNF8 ubiquitin ligase, and for the subsequent recruitment of the brca1 complex. Chromatin dynamics at DSBs can therefore exert a powerful influence on the process of DSB repair. Further, there is emerging evidence that the different chromatin structures in the cell, such as heterochromatin and euchromatin, utilize distinct remodeling complexes and pathways to facilitate DSB. The processing and repair of DSB is therefore critically influenced by the nuclear architecture in which the lesion arises.Key words: p400, chromatin remodeling, DNA repair, NuA4, H2AX, acetylation, nucleosome, tip60Damage to cellular DNA can occur through multiple pathways, including exposure to genotoxic agents, the production of endogenous reactive oxygen species or errors which arise during DNA replication. To combat this continuous assault on the genome, mammalian cells have evolved multiple DNA repair pathways. The most challenging lesions to repair are DSBs, which physically cleave the DNA strand. DSBs can occur through exposure to IR, the collapse of replication forks or during the processing of certain types of DNA damage. Over the last 20 years, a clear picture of how the cell detects and repairs DSBs has emerged.^1,2 The earliest event in the cell''s response to DSBs is the rapid recruitment of the ATM kinase, followed by the phosphorylation of histone H2AX (termed γH2AX) on large chromatin domains which extend for 100''s of kilobases on either side of the DSB.³ The mdc1 scaffold protein is then recruited to γH2AX,⁴ providing a docking platform for the recruitment and retention of additional DNA repair proteins, including the MRN complex, the RNF8 ubiquitin ligase and the brca1 and 53BP1 proteins, onto the chromatin at DSBs.^5–7 Eventually, this spreading of DNA repair proteins along the chromatin from the DSB leads to the formation of IRIF, which can be visualized by immunofluorescent techniques. DSBs are then repaired by NHEJ, in which broken DNA ends are directly religated, or by HR, using the undamaged sister chromatid (present during S-phase) as a template. A defining characteristic of DSB repair is the dominant role that chromatin structure plays in the detection and repair of these lesions. In this review, we will examine recent work exploring how remodeling of the chromatin structure adjacent to DSBs plays a key role in the repair of DSBs.     

Joining of nonhomologous DNA double strand breaks in vitro.

Extracts of Xenopus laevis eggs can efficiently join ends of duplex DNA that differ in structure and sequence. This was analysed by recircularisation of linear plasmid DNA molecules with dissimilar termini, generated by successive cuts with two different restriction enzymes within the pSP65 polylinker. Use of various enzymes provided blunt ended or 4 nucleotides long 3' and 5' protruding single strand (PSS) termini which were successfully joined in vitro in any tested combination. Sequence analysis of numerous junctions from cloned reaction products of 7 terminus combinations reveal: apart from very rare base exchanges and single nucleotide insertions less than 10% deletions (1 to 18 nucleotides long) were detected. Blunt/PSS or 3'PSS/5'PSS terminus pairs undergo simple "blunt end" joining which preserves PSS ends by fill-in. In contrast, equally polar 3'PSS/3'PSS or 5'PSS/5'PSS terminus pairs are joined by a complex mode: PSS ends overlap by a defined number of nucleotides, set by matching basepairs. Even one basematch suffices to define the setting. This then determines the final mismatch repair and fill-in pattern. We propose that yet unknown terminal DNA-binding proteins stabilize the energetically highly unfavorable configuration of single matching basepairs and help to support defined overlap structures.     

Deciphering the chromatin landscape induced around DNA double strand breaks

DNA double strand breaks (DSBs) are among the most deleterious forms of lesions and deciphering the details of the chromatin landscape induced around DSBs represents a great challenge for molecular biologists.

Chromatin Immunoprecipitation, followed by microarray hybridisation (ChIP-chip) or high-throughput sequencing (ChIP-seq), are powerful techniques that provide high-resolution maps of protein-genome interactions. However, applying these techniques to study chromatin changes induced around DSBs was previously hindered due to a lack of suitable DSB induction techniques.

We have recently developed an experimental system utilizing a restriction enzyme fused to a modified oestrogen receptor ligand binding domain (AsiSI-ER), which generates multiple, sequence-specific and unambiguously positioned DSBs across the genome upon induction with 4-hydroxytamoxifen (4OHT) ¹. Cell lines expressing this construct represent a powerful tool to study specific chromatin changes during DSB repair, enabling high-resolution profiling of DNA repair complexes and chromatin modifications induced around DSBs. Using this system, we have recently produced the first map of gH2AX, a DSB-induced chromatin modification, on two human chromosomes and have investigated its spreading properties ¹. Here we provide additional data characterizing the cell lines, present a genome-wide profile of gH2AX obtained by ChIP-seq, and discuss the potential of our system towards investigations of previously uncharacterized aspects of DSB repair.     

Activity of ribonucleotide reductase helps determine how cells repair DNA double strand breaks

Mammalian cells can choose either nonhomologous end joining (NHEJ) or homologous recombination (HR) for repair of chromosome breaks. Of these two pathways, HR alone requires extensive DNA synthesis and thus abundant synthesis precursors (dNTPs). We address here if this differing requirement for dNTPs helps determine how cells choose a repair pathway. Cellular dNTP pools are regulated primarily by changes in ribonucleotide reductase activity. We show that an inhibitor of ribonucleotide reductase (hydroxyurea) hypersensitizes NHEJ-deficient cells, but not wild type or HR-deficient cells, to chromosome breaks introduced by ionizing radiation. Hydroxyurea additionally reduces the frequency of irradiated cells with a marker for an early step in HR, Rad51 foci, consistent with reduced initiation of HR under these conditions. Conversely, promotion of ribonucleotide reductase activity protects NHEJ-deficient cells from ionizing radiation. Importantly, promotion of ribonucleotide reductase activity also increases usage of HR in cells proficient in both NHEJ and HR at a targeted chromosome break. Activity of ribonucleotide reductase is thus an important factor in determining how mammalian cells repair broken chromosomes. This may explain in part why G1/G0 cells, which have reduced ribonucleotide reductase activity, rely more on NHEJ for DSB repair.     

DNA double strand breaks: Not all foci are created equal

Comment on: Processing of O6-methylguanine into DNA double-strand breaks requires two rounds of replication whereas apoptosis is also induced in subsequent cell cycles. Quiros S, et al. Cell Cycle 2010; 9:In press.     

Induction of DNA double strand breaks by arsenite: comparative studies with DNA breaks induced by X-rays

Chinese hamster ovary (CHO-K1) cell line and two of its DNA double strand break (DSB) repair deficient mutant cell lines, xrs-5 (Ku80 mutant) and irs-20 (DNA-PKcs mutant), were treated with various concentrations of sodium arsenite for 2.5h, and the colony forming abilities were studied. The wild type cells showed the highest cell survival, while xrs-5 cells showed the lowest survival, and irs-20 cells had an intermediate survival. These results are very similar to the cell survival curves induced by X-rays in these three cell lines. Our data also show the dose dependent induction of DNA-DSBs in these cell lines exposed to arsenite. However, in order to obtain a similar cell survival in wild type cells, twice as many DNA-DSBs are necessary with arsenite exposure when compared with X-rays, suggesting that the types of DNA lesions leading to DSB induced by arsenite are different from those by X-rays. Based on these data, further mechanistic investigations including the involvement of DNA-DSB repair proteins are warranted in the recovery process from arsenic (As) exposure.     

ATM-mediated response to DNA double strand breaks in human neurons derived from stem cells

Ataxia-telangiectasia (A-T) is a multi-system genomic instability syndrome that is caused by loss or inactivation of the ATM protein kinase. ATM is largely nuclear in proliferating cells, and activates an extensive network of pathways in response to double strand breaks (DSBs) in the DNA by phosphorylating key proteins in these pathways. The prominent symptom of A-T is neuronal degeneration, making the elucidation of ATM's functions in neurons essential to understanding the disease. It has been suggested that ATM is cytoplasmic in neurons and functions in processes that are not associated with the DNA damage response. Recently we showed that in human neuron-like cells obtained by in vitro differentiation of neuroblastomas, ATM was largely nuclear and mediated the DSB response as in proliferating cells. We have now extended these studies to two additional model systems: neurons derived from human embryonic stem cells, and cortical neurons derived from neural stem cells. The results substantiate the notion that ATM is nuclear in human neurons and mediates the DSB response, the same as it does in proliferating cells. We present here unique and powerful model systems to further study the ATM-mediated network in neurons.     

The role of DNA double strand breaks in ionizing radiation-induced killing of eukaryotic cells.

A widely accepted assumption in radiobiology is that ionizing radiation kills cells by inducing forms of damage in DNA structures that lead to the formation of lethal chromosome aberrations. One goal of radiation biology research is the identification of these forms of DNA damage, the characterization of the mechanisms involved in their repair and the elucidation of the processes involved in their transformation to chromosome damage. In recent years, evidence has accumulated implicating DNA double stranded breaks as lesions relevant for cell killing. Here, the available information on this topic is reviewed together with the methods most commonly used to quantitate induction and repair of this type of lesion. The presentation concludes with an outline of present research directions and future goals.     

Possible association of BLM in decreasing DNA double strand breaks during DNA replication

Bloom's syndrome (BS) is a rare genetic disorder and the cells from BS patients show genomic instability and an increased level of sister chromatid exchange (SCE). We generated BLM(-/-) and BLM(-/-)/RAD54(-/-) DT40 cells from the chicken B-lymphocyte line DT40. The BLM(-/-) DT40 cells showed higher sensitivity to methyl methanesulfonate and elevated levels of SCE as expected. The targeted integration frequency was also increased remarkably in BLM(-/-) cells. The SCE frequency increase in BLM(-/-) cells was considerably reduced and the enhanced targeted integration observed in BLM(-/-) cells was almost completely abolished in BLM(-/-)/RAD54(-/-) cells, indicating that a large portion of the SCE in BLM(-/-) cells occurs via homologous recombination, and homologous recombination events increase with the defect of BLM function. The BLM(-/-)/RAD54(-/-) cells showed a slow growth phenotype and an increased incidence of chromosome-type breaks/gaps while each single mutant showed relatively small numbers of chromosome-type breaks/gaps.     

Induction of DNA double strand breaks in cultured mammalian cells exposed to hydrogen peroxide and histidine

The amino acid histidine was found to increase the toxicity of H₂O₂ in cultured mammalian cells. Histidine also augmented the level of DNA single strand breaks (SSB) detectable in cells exposed to the oxidant and, in addition, resulted in the appearance of DNA double strand breaks (DSB), a lesion which is not produced by H₂O₂ alone.