Age-dependent decline in rejoining of X-ray-induced DNA double-strand breaks in normal human lymphocytes

Unstimulated human peripheral blood lymphocytes (HPBL), separated by density centrifugation from anticoagulated whole blood, were X-irradiated (30 Gy) on ice and incubated in medium at 37°C for repair times of 15, 30, and 120 min. Blood donors were 18 normotensive, non-smoking Caucasians aged 23–78, free from overt pathology and not taking any medications. Neutral filter elution was used to assay DNA double-strand break (DSB) induction and completeness of DSB rejoining of any X-ray-induced alkali-labile sites converted to DSBs in vitro at pH 9.6). After 30 or 120 min repair incubation, the percentage of DSBs rejoined by cells from older donors (aged 66–78 years) was less than half the percentage of DSBs rejoined by cells from younger donors (aged 23–39 and 42–57). When data from the 3 age groups were pooled, the age-related decline in percent DSBs rejoined was significant for repair times 30 min (r = −0.63, p<0.005) and 120 min (r= −0.64, p<0.005) but not for 15 min (r=−0.04). These age-related declines were observed even though DNA from older donors sustained fewer strand breaks as demonstrated by the negative correlation between donor age and DSB induction (r=−0.65, p<0.005). These results suggest that the efficacy of X-ray-induced DSB repair diminishes with in vivo age in unstimulated HPBL.     

Autoantibodies that stabilize the molecular interaction of Ku antigen with DNA-dependent protein kinase catalytic subunit

DNA-dependent protein kinase (DNA-PK) consists of a DNA binding subunit (Ku autoantigen), and a catalytic subunit (DNA-PK_cs). In the present study, human autoantibodies that recognize novel antigenic determinants of DNA-PK were identified. One type of autoantibody stabilized the interaction of DNA-PK_cs with Ku and recognized the DNA-PK_cs–Ku complex, but not biochemically purified DNA-PK_cs. Another type recognized purified DNA-PK_cs. Autoantibodies to Ku (p70/p80 heterodimer), ‘stabilizing’ antibodies, and antibodies to DNA-PK_cs comprise a linked autoantibody set, since antibodies recognizing purified DNA-PK_cs were strongly associated with stabilizing antibodies, whereas stabilizing antibodies were strongly associated with anti-Ku. This hierarchical pattern of autoantibodies specific for components of DNA-PK (anti-Ku>stabilizing antibodies>anti-DNA-PK_cs) may have implications for the pathogenesis of autoimmunity to DNA-PK and other chromatin particles. The data raise the possibility that altered antigen processing and/or stabilization of the DNA-PK_cs–Ku complex due to autoantibody binding could play a role in spreading autoimmunity from Ku to the weakly associated antigen DNA-PK_cs.     

The molecular causes of low ATM protein expression in breast carcinoma; promoter methylation and levels of the catalytic subunit of DNA-dependent protein kinase

AIMS: To investigate whether aberrant methylation of the ATM promoter or loss of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) may be the underlying causes of reduced ATM protein levels often seen in breast tumours. METHODS AND RESULTS: Methylation-specific polymerase chain reaction was used to determine the ATM promoter status and DNA-PKcs levels were measured by immunohistochemistry. None of the 74 invasive carcinomas (ICs) studied showed ATM promoter hypermethylation, whereas promoter methylation of CDKN2A/p16 (1.8%) and GSTP1 (15.8%) was detected. Of 92 ICs examined, 68 had reduced DNA-PKcs levels, supporting previous findings that alterations in double-strand break repair are associated with breast cancer pathogenesis. Although no association was found between the DNA-PKcs and ATM scores for the series of 92 tissues and 22/24 tissues with normal DNA-PKcs had reduced ATM, 29 tumours showed low expression of both DNA-PKcs and ATM compared with normal tissues. CONCLUSIONS: No evidence was found that the reduction in ATM protein levels seen in breast carcinoma is the result of epigenetic silencing. However, cross-regulation between DNA-PKcs and ATM may be a possible cause in a subset of tumours and warrants further investigation.     

Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells

Chromosomal double-strand breaks (DSBs) in mammalian cells are repaired by either homology-directed repair (HDR), using a homologous sequence as a repair template, or nonhomologous end-joining (NHEJ), which often involves sequence alterations at the DSB site. To characterize the interrelationship of these two pathways, we analyzed HDR of a DSB in cells deficient for NHEJ components. We find that the HDR frequency is enhanced in Ku70(-/-), XRCC4(-/-), and DNA-PKcs(-/-) cells, with the increase being particularly striking in Ku70(-/-) cells. Neither sister-chromatid exchange nor gene-targeting frequencies show a dependence on these NHEJ proteins. A Ku-modulated two-ended versus one-ended chromosome break model is presented to explain these results.     

Photochemical production of double-strand breaks in cellular DNA

In a recent publication we described a novel route for the introductionof DNA double-strand breaks (DSBs) into cellular DNA. This involvedthe labelling of cellular DNA with bromodeoxyuridine (BrdU)and exposure to UVA light in the presence of Hoechst dye No.33258. Here, we report an extension of that work to the useof iododeoxyuridine (IdU); cells substituted with known levelsof IdU were subjected to a similar photolysis treatment andanalyzed for strand breaks by elution assays. Results indicatethat both single-strand breaks (SSBs) and DSBs depend linearlyon the level of IdU substitution and fluence of UVA light. Theyields of SSBs and DSBs were found to be 3.5x10^–5 and9.5 x10^–7/IdU moiety/ kJm^–2, respectively. Theseresults indicate that 15fold less SSBs and 5-fold less DSBsare produced per IdU than per BrdU moiety. ³To whom correspondence should be addressed     

Lack of detectable defect in DNA double-strand break repair and DNA-dependent protein kinase activity in radiosensitive human severe combined immunodeficiency fibroblasts

The initial step of the V(D)J recombination occurs through the generation of a DNA double-strand break (dsb). Defects in the DNA-dependent protein kinase complex (DNA-PK) result in an inability to perform either V(D)J recombination or any dsb repair effectively. The human autosomal T-B-severe combined immunodeficiency (SCID) condition is characterized by an absence of both B and T lymphocytes and is accompanied in some patients by an increase in γ-ray sensitivity (T-B-RS SCID) comparable to that found in mouse SCID cells. We show here that cells from six patients with T-B-RS SCID had normal DNA-dsb repair kinetics. Furthermore, DNA-PK activity was present in extracts from these human T-B-RS SCID fibroblasts. We therefore conclude that some human T-B-RS SCID disorders are not caused by a defect in an essential DNA-PK component.     

Early events in the mammalian response to DNA double-strand breaks

Physical and chemical agents that induce DNA double-strand breaks (DSBs) are among the most potent mutagens. The mammalian cell response to DSB comprises a highly co-ordinated, yet complex network of proteins that have been categorized as sensors, signal transducers, mediators and effectors of damage and repair. While this provides an accessible classification system, review of the literature indicates that many proteins satisfy the criteria of more than one category, pointing towards a series of highly co-operative pathways with overlapping function. In summary, the MRE11-NBS1-RAD50 complex is necessary for achieving optimal activation of ataxia-telangiectasia-mutated (ATM) kinase, which catalyses a phosphorylation-mediated signal transduction cascade. Among the subset of proteins phosphorylated by ATM are histone H2AX (H2AX), mediator of damage checkpoint protein 1, nibrin (NBS1), P53-binding protein 1 and breast cancer protein 1, all of which subsequently redistribute into DSB-containing sub-nuclear compartments. Post-translational modification of DSB responding proteins achieves a rapid and reversible change in protein behaviour and mediates damage-specific interactions, hence imparting a high degree of vigilance to the cell. This review highlights events fundamental in maintaining genetic integrity with emphasis on early stages of the DSB response.     

Cdc7-Drf1 is a developmentally regulated protein kinase required for the initiation of vertebrate DNA replication

Cdc7, a protein kinase required for the initiation of eukaryotic DNA replication, is activated by a regulatory subunit, Dbf4. A second activator of Cdc7 called Drf1 exists in vertebrates, but its function is unknown. Here, we report that in Xenopus egg extracts, Cdc7-Drf1 is far more abundant than Cdc7-Dbf4, and removal of Drf1 but not Dbf4 severely inhibits phosphorylation of Mcm4 and DNA replication. After gastrulation, when the cell cycle acquires somatic characteristics, Drf1 levels decline sharply and Cdc7-Dbf4 becomes the more abundant kinase. These results identify Drf1 as a developmentally regulated, essential activator of Cdc7 in Xenopus.     

Thymidine in the micromolar range promotes rejoining of UVC-induced DNA strand breaks and prevents azidothymidine from inhibiting the rejoining in quiescent human lymphocytes

The effect and inter-individual variation in the effect of exogenously added deoxynucleosides (2×10⁻⁶ M) on rejoining of UVC-induced DNA strand breaks was examined in quiescent human lymphocytes from 25 healthy persons. Thymidine at concentrations below 2×10⁻⁶ M, effectively and with statistically extreme significance, increased rejoining of UVC-induced DNA strand breaks in the lymphocytes of every one of the 25 persons tested (p<0.0001, Wilcoxon's signed ranks test). The mean stimulation after 20 h of postirradiation repair was 48% (range 18–78%) with an inter-individual variation of 30% (coefficient of variation, CV). Deoxyguanosine stimulated rejoining in 16, but inhibited in three of 19 test persons (mean stimulation 28%, range −31 to 71%). The stimulating effect of deoxyguanosine was also extremely significant (p<0.0004). Deoxycytidine and deoxyadenosine stimulated rejoining in some persons and inhibited it in others, and without statistical significance (p values above 0.5). The stimulating effect of thymidine was significantly inhibited by deoxycytidine (p<0.05, n=12) whereas deoxyguanosine neither promoted or inhibited the stimulation by thymidine (p=1, n=12). Rejoining of DNA strand breaks induced by methyl methanesulfonate did not appear significantly stimulated or inhibited by any of the four deoxynucleosides. Finally, the inhibiting effect of azidothymidine (AZT) on rejoining of UVC-induced DNA strand breaks was nullified by the addition of thymidine. In three donors examined, 10⁻⁴ M AZT inhibited the rejoining by about 40–50%. The presence of less than 10⁻⁵ M thymidine reduced the level of UVC-induced DNA strand breaks to below the level in control lymphocytes allowed to repair without AZT. These results indicate that among the four deoxynucleoside triphosphates, dTTP has a crucial role on the repair of UVC-induced DNA damage in quiescent lymphocytes. The results also indicate that an expansion of the dTTP pool may counteract the inhibiting effect of AZT on DNA repair in quiescent lymphocytes.     

Mitogen-activated protein kinase phosphatase is required for genotoxic stress relief in Arabidopsis

Genotoxic stress activates complex cellular responses allowing for the repair of DNA damage and proper cell recovery. Although plants are exposed constantly to increasing solar UV irradiation, the signaling cascades activated by genotoxic environments are largely unknown. We have identified an Arabidopsis mutant (mkp1) hypersensitive to genotoxic stress treatments (UV-C and methyl methanesulphonate) due to disruption of a gene that encodes an Arabidopsis homolog of mitogen-activated protein kinase phosphatase (AtMKP1). Growth of the mkp1 mutant under standard conditions is indistinguishable from wild type, indicating a stress-specific function of AtMKP1. MAP kinase phosphatases (MKPs), the potent inactivators of MAP kinases, are considered important regulators of MAP kinase signaling. Although biochemical data from mammalian cell cultures suggests an involvement of MKPs in cellular stress responses, there is no in vivo genetic support for this view in any multicellular organism. The genetic and biochemical data presented here imply a central role for a MAP kinase cascade in genotoxic stress signaling in plants and indicate AtMKP1 to be a crucial regulator of the MAP kinase activity in vivo, determining the outcome of the cellular reaction and the level of genotoxic resistance.     

The DNA-dependent protein kinase: the director at the end

Summary: Efficient repair of DNA double‐strand breaks is essential for the maintenance of chromosomal integrity. In higher eukaryotes, non‐homologous end‐joining (NHEJ) DNA is the primary pathway that repairs these breaks. NHEJ also functions in developing lymphocytes to repair strand breaks that occur during V(D)J recombination, the site‐specific recombination process that provides for the assembly of functional antigen‐receptor genes. If V(D)J recombination is impaired, B‐ and T‐lymphocyte development is blocked resulting in severe combined immunodeficiency disease. In the last decade, an intensive research effort has focused on NHEJ resulting in a reasonable understanding of how double‐strand breaks are resolved. Six distinct gene products have been identified that function in this pathway (Ku70, Ku86, XRCC4, DNA ligase IV, Artemis, and DNA‐PKcs). Three of these comprise one complex, the DNA‐dependent protein kinase (DNA‐PK). This protein complex is central during NHEJ, because DNA‐PK initially recognizes and binds to the damaged DNA and then targets the other repair activities to the site of DNA damage. In this review, we discuss recent developments that have provided insight into how DNA‐PK functions, once bound to DNA ends.     

Mutations induced by DNA double-strand breaks: The influence of genomic site

The transgenic CHO cell line PL61, carrying a recombinant SV40-gpt gene, was treated with restriction endonucleases to assess mutagenesis from defined DNA double-strand breaks. Mutations ingpt were measured under two conditions: a stringent condition where selection ensured that the closely-linkedneo gene was retained functionally intact, or a relaxed condition without the requirement forneo gene function. Despite testing 18 different restriction endonucleases with various numbers of potential break-sites within the transgene, mutations were only found under relaxed selection conditions. These mutations commonly led to complete loss of the transgene, suggesting that large deletions predominate when selection is relaxed. It is argued, in comparison to mutation data for other genomic sites in CHO cells, that variations in the ‘effective target size’ for mutagenesis may explain the response of the transgene under different conditions.     

Retrieval mediated by hippocampal extracellular signal-regulated kinase/mitogen-activated protein kinase is required for memory strengthening

Established memories can be strengthened by additional learning and rehearsal. However, the brain processes enabling memories to be updated by further information is unclear. We found that blockade of retrieval of a stabilized memory by inhibition of the extracellular signal-regulated kinase/mitogen-activated protein kinase signaling pathway in the hippocampus prevented memory enhancement induced by an additional learning trial in rats. The findings indicate that retrieval is critical for memory strengthening.     

Myotonic dystrophy protein kinase is expressed in embryonic myocytes and is required for myotube formation

Myotonic dystrophy (DM1) is a multi-systemic disease caused by a triplet nucleotide repeat expansion in the 3' untranslated region of the gene coding for myotonic dystrophy protein kinase (DMPK). The primary pathophysiology of DM1 is thought to result from RNA transport and processing defects. The function of DMPK in development or any potential role in DM1 remains unknown. Here we report a novel role for DMPK in myogenesis. We have discovered a specific expression pattern of DMPK in mouse and chick embryonic development. DMPK is expressed in postmitotic cardiac and skeletal myocytes and developmental signaling centers. During cardiac myocyte maturation, DMPK migrates from perinuclear to cellular membrane localization. Manipulating DMPK levels in cultured cardiac and skeletal myocytes has revealed a key role for DMPK in myocyte differentiation. Overexpression of DMPK induces cell rounding and apoptosis in myocytes. In addition, DMPK is necessary for myogenin expression in differentiating C2C12 myoblasts.