Induction Of DNA Repair Genes Research Articles

Tardigrades: Trained to be hardy in the face of DNA damage

Tardigrades withstand ionizing irradiation levels ∼500 times higher than humans can tolerate. Two recent papers shed light on how this might be achieved- via the transcriptional induction of DNA repair genes, the induction of a radioprotective DNA-binding protein, and possibly also the heightened capacity of repair proteins.

Transcriptomic and genomic effects of gamma-radiation exposure on strains of the black yeast Exophiala dermatitidis evolved to display increased ionizing radiation resistance.

Ionizing radiation poses a significant threat to living organisms and human health, given its destructive nature and widespread use in fields such as medicine and the potential for nuclear disasters. Melanized fungi exhibit remarkable survival capabilities, enduring doses up to 1,000-fold higher than mammals. Through adaptive laboratory evolution, we validated the protective role of constitutive upregulation of DNA repair genes in the black yeast Exophiala dermatitidis, enhancing survival after radiation exposure. Surprisingly, we found that evolved strains lacking melanin still achieved high levels of radioresistance. Our study unveiled the significance of robust activation and enhancement of redox homeostasis, as evidenced by the profound transcriptional changes and increased accumulation of mutations, in substantially improving ionizing radiation resistance in the absence of melanin. These findings underscore the delicate balance between DNA repair and redox homeostasis for an organism's ability to endure and recover from radiation exposure.

Assessment of DNA damage profile and oxidative /antioxidative biomarker level in patients with inflammatory bowel disease.

<b>Aim:</b> The purpose of this study was to investigate the oxidative DNA damage, pro-antioxidant status in Polish patients with inflammatory bowel disease (IBD). <br><b>Methods:</b> Oxidative DNA damage was measured by comet assay techniques; nitric oxide (NO) and plasmatic lipid peroxidation (MDA) as oxidative stress were valuated by colometric methods; superoxide dismutase (SOD1), catalase (CAT) and glutathione peroxidase (GPx1) as antioxidative defense were determined by spectrophotometric methods. <br><b>Results:</b> The level of oxidative DNA damage in IBD patients was significantly higher in relation to controls (P = 0.01). Alike, in control subject as well as in patients with IBD, lymphocytes are characterized by complete repair of DNA damage. A significant decrease of SOD (P = 0.031), CAT (P = 0.006), GPx1 (P = 0.001) activity was seen in IBD patients vs control. MDA (P = 0.001) and NO (P = 0.001) concentrations were significantly increased in IBD patients as compared to healthy subjects. <br><b>Conclusions:</b> Our results may be due to the induction of DNA repair genes which may occur at the stage of the pathological changes (IBD) that may be caused by excessive oxidative stress. However, the cause of this relationship, and whether it is direct or indirect, remains to be explored.

Molecular analysis of the dual targeting of the epidermal growth factor receptor and the O6-methylguanine-DNA methyltransferase with a double arm hybrid molecule.

Disordered expression of the epidermal growth factor receptor (EGFR) has been associated with induction of DNA repair genes (e.g. XRCC1, ERCC1) and resistance to radiation and genotoxic drugs. However, our previous work showed that EGFR inhibition did not affect O6-methylguanine-DNA methyltransferase (MGMT)-mediated resistance. In order to block uncoupled events associated with EGFR and MGMT, we designed MR30, a single molecule termed “combi-molecule” that contains a quinazoline arm targeted to EGFR and an O6-benzylguanine (O6-BG) moiety to block MGMT. Molecular analysis of the mechanism of action of its two arms showed that: (a) it could block EGFR phosphorylation, (b) down-regulate the RAF-MAPK and the PI3K-AKT pathways, and (c) covalently modify MGMT through S-benzylation, as confirmed by MALDI analysis of a direct binding assay with isolated MGMT, (d) it induced a dose-dependent down-regulation of MGMT in lung and melanoma cells. The pleiotropic mechanism of action of MR30 culminated into strong growth inhibition (IC50: 0.018-6.02 μM), with superior activity when compared with an equimolar combination of gefitinib (a clinical EGFR inhibitor) and O6-BG (a known MGMT inhibitor). Pulse exposure experiments were required to attenuate the contribution of EGFR inhibition to the strong potency of MR30, thereby allowing to achieve the dose level required to sensitize cells to temozolomide (TMZ). Indeed, MR30 significantly sensitized EGFR-MGMT co-expressing cells to TMZ (p<0.05-0.0001). The results in toto suggest that MR30 is the first prototype of agents that may be used against tumours addicted to EGFR and to sensitize resistant tumours co-expressing EGFR and MGMT to TMZ.

Synthesis of PLGA nanoparticles of tea polyphenols and their strong in vivo protective effect against chemically induced DNA damage

In spite of proficient results of several phytochemicals in preclinical settings, the conversion rate from bench to bedside is not very encouraging. Many reasons are attributed to this limited success, including inefficient systemic delivery and bioavailability under in vivo conditions. To achieve improved efficacy, polyphenolic constituents of black (theaflavin [TF]) and green (epigallocatechin-3-gallate [EGCG]) tea in poly(lactide-co-glycolide) nanoparticles (PLGA-NPs) were entrapped with entrapment efficacy of ~18% and 26%, respectively. Further, their preventive potential against 7,12-dimethylbenzanthracene (DMBA)-induced DNA damage in mouse skin using DNA alkaline unwinding assay was evaluated. Pretreatment (topically) of mouse skin with either TF or EGCG (100 μg/mouse) doses exhibits protection of 45.34% and 28.32%, respectively, against DMBA-induced DNA damage. However, pretreatment with TF-loaded PLGA-NPs protects against DNA damage 64.41% by 1/20th dose of bulk, 71.79% by 1/10th dose of bulk, and 72.46% by 1/5th dose of bulk. Similarly, 51.28% (1/20th of bulk), 57.63% (1/10th of bulk), and 63.14% (1/5th of bulk) prevention was noted using EGCG-loaded PLGA-NP doses. These results showed that tea polyphenol-loaded PLGA-NPs have ~30-fold dose-advantage than bulk TF or EGCG doses. Additionally, TF- or EGCG-loaded PLGA-NPs showed significant potential for induction of DNA repair genes (XRCC1, XRCC3, and ERCC3) and suppression of DNA damage responsive genes (p53, p21, MDM2, GADD45α, and COX-2) as compared with respective bulk TF or EGCG doses. Taken together, TF- or EGCG-loaded PLGA-NPs showed a superior ability to prevent DMBA-induced DNA damage at much lower concentrations, thus opening a new dimension in chemoprevention research.

Cobalt-induced genotoxicity in male zebrafish (Danio rerio), with implications for reproduction and expression of DNA repair genes

Although cobalt (Co) is an environmental contaminant of surface waters in both radioactive (e.g. 60Co) and non-radioactive forms, there is relatively little information about Co toxicity in fishes. The objective of this study was to investigate acute and chronic toxicity of Co in zebrafish, with emphasis on male genotoxicity and implications for reproductive success. The lethal concentration for 50% mortality (LC50) in larval zebrafish exposed (96h) to 0–50mgl−1 Co was 35.3±1.1 (95%C.I.)mgl−1 Co. Adult zebrafish were exposed (13d) to sub-lethal (0–25mgl−1) Co and allowed to spawn every 4d and embryos were collected. After 12-d exposure, fertilisation rate was reduced (6% total eggs fertilised, 25mgl−1) and embryo survival to hatching decreased (60% fertilised eggs survived, 25mgl−1). A concentration-dependent increase in DNA strand breaks was detected in sperm from males exposed (13d) to Co, and DNA damage in sperm returned to control levels after males recovered for 6d in clean water. Induction of DNA repair genes (rad51, xrcc5, and xrcc6) in testes was complex and not directly related to Co concentration, although there was significant induction in fish exposed to 15 and 25mgl−1 Co relative to controls. Induction of 4.0±0.9, 2.5±0.7, and 3.1±0.7-fold change (mean±S.E.M. for rad51, xrcc5, and xrcc6, respectively) was observed in testes at the highest Co concentration (25mgl−1). Expression of these genes was not altered in offspring (larvae) spawned after 12-d exposure. Chronic exposure to Co resulted in DNA damage in sperm, induction of DNA repair genes in testes, and indications of reduced reproductive success.

Tau Ser262 phosphorylation is critical for A 42-induced tau toxicity in a transgenic Drosophila model of Alzheimer's disease

The amyloid-beta 42 (Abeta42) peptide has been suggested to promote tau phosphorylation and toxicity in Alzheimer's disease (AD) pathogenesis; however, the underlying mechanisms are not fully understood. Using transgenic Drosophila expressing both human Abeta42 and tau, we show here that tau phosphorylation at Ser262 plays a critical role in Abeta42-induced tau toxicity. Co-expression of Abeta42 increased tau phosphorylation at AD-related sites including Ser262, and enhanced tau-induced neurodegeneration. In contrast, formation of either sarkosyl-insoluble tau or paired helical filaments was not induced by Abeta42. Co-expression of Abeta42 and tau carrying the non-phosphorylatable Ser262Ala mutation did not cause neurodegeneration, suggesting that the Ser262 phosphorylation site is required for the pathogenic interaction between Abeta42 and tau. We have recently reported that the DNA damage-activated Checkpoint kinase 2 (Chk2) phosphorylates tau at Ser262 and enhances tau toxicity in a transgenic Drosophila model. We detected that expression of Chk2, as well as a number of genes involved in DNA repair pathways, was increased in the Abeta42 fly brains. The induction of a DNA repair response is protective against Abeta42 toxicity, since blocking the function of the tumor suppressor p53, a key transcription factor for the induction of DNA repair genes, in neurons exacerbated Abeta42-induced neuronal dysfunction. Our results demonstrate that tau phosphorylation at Ser262 is crucial for Abeta42-induced tau toxicity in vivo, and suggest a new model of AD progression in which activation of DNA repair pathways is protective against Abeta42 toxicity but may trigger tau phosphorylation and toxicity in AD pathogenesis.

Expression of base excision DNA repair genes as a biomarker of oxidative DNA damage

Oxidative stress induced DNA damage is considered to be the most common insult affecting the genome. Moreover, it is recognized as a common pathway to mutations and is suggested to play a major role in the development of chronic diseases such as cancer. However, current analytical methods used to detect oxidative DNA damage have been hampered by both technical and biological obstacles. These include spurious oxidation during DNA isolation and processing, and the inherent removal of damaged bases by numerous operating DNA repair systems. The removal of oxidized bases is performed predominantly by the base excision repair (BER) pathway and it has been shown that induction of DNA repair genes occurs in response to oxidative stress. Here, we demonstrate the utility of measuring changes in expression of BER genes as a sensitive in vivo biomarker for oxidative DNA damage.

The transcriptome of prematurely aging yeast cells is similar to that of telomerase-deficient cells.

To help define the pathologies associated with yeast cells as they age, we analyzed the transcriptome of young and old cells isolated by elutriation, which allows isolation of biochemical quantities of old cells much further advanced in their life span than old cells prepared by the biotin-streptavidin method. Both 18-generation-old wild-type yeast and 8-generation-old cells from a prematurely aging mutant (dna2-1), with a defect in DNA replication, were evaluated. Genes involved in gluconeogenesis, the glyoxylate cycle, lipid metabolism, and glycogen production are induced in old cells, signifying a shift toward energy storage. We observed a much more extensive generalized stress response known as the environmental stress response (ESR), than observed previously in biotin-streptavidin-isolated cells, perhaps because the elutriated cells were further advanced in their life span. In addition, there was induction of DNA repair genes that fall in the so-called DNA damage "signature" set. In the dna2-1 mutant, energy production genes were also induced. The response in the dna2-1 strain is similar to the telomerase delete response, genes whose expression changes during cellular senescence in telomerase-deficient cells. We propose that these results suggest, albeit indirectly, that old cells are responding to genome instability.

The Fission Yeast spSet1p is a Histone H3-K4 Methyltransferase that Functions in Telomere Maintenance and DNA Repair in an ATM Kinase Rad3-dependent Pathway

We have characterized spSet1p, the Schizosaccharomyces pombe ortholog of the budding yeast histone H3 methyltransferase Set1p. SpSet1p catalyzes methylation of H3 at K4, in vivo and in vitro. Deleting spset1 partially affects telomeric and centromeric silencing. Strikingly, lack of spSet1p causes elongation of telomeres in wild-type cells and in most DNA damage checkpoint rad mutant cells, but not in cells lacking the ATM kinase Rad3 or its associated protein Rad26. Interestingly, spset1 deletion specifically causes a reduction in sensitivity to ultraviolet radiation of the PCNA-like checkpoint mutants hus1 and rad1, but not of cells devoid of Rad3. This partial suppression was not due to restoration of checkpoint function or to transcriptional induction of DNA repair genes. Moreover, spset1 allows recovery specifically of the crb2 checkpoint mutant upon treatment with the replication inhibitor hydroxyurea but not upon UV irradiation. Nevertheless, the pathway induced in spset1 cells cannot substitute for the Mus81/Rqh1 DNA damage tolerance pathway. Our results suggest that SpSet1p and the ATM kinase Rad3 function in a common genetic pathway linking chromatin to telomere length regulation and DNA repair.

The budding yeast Rad9 checkpoint protein is subjected to Mec1/Tel1-dependent hyperphosphorylation and interacts with Rad53 after DNA damage.

The Saccharomyces cerevisiae RAD9 checkpoint gene is required for transient cell-cycle arrests and transcriptional induction of DNA repair genes in response to DNA damage. Polyclonal antibodies raised against the Rad9 protein recognized several polypeptides in asynchronous cultures, and in cells arrested in S or G2/M phases while a single form was observed in G1-arrested cells. Treatment with various DNA damaging agents, i.e. UV, ionizing radiation or methyl methane sulfonate, resulted in the appearance of hypermodified forms of the protein. All modifications detected during a normal cell cycle and after DNA damage were sensitive to phosphatase treatment, indicating that they resulted from phosphorylation. Damage-induced hyperphosphorylation of Rad9 correlated with checkpoint functions (cell-cycle arrest and transcriptional induction) and was cell-cycle stage- and progression-independent. In asynchronous cultures, Rad9 hyperphosphorylation was dependent on MEC1 and TEL1, homologues of the ATR and ATM genes. In G1-arrested cells, damage-dependent hyperphosphorylation required functional MEC1 in addition to RAD17, RAD24, MEC3 and DDC1, demonstrating cell-cycle stage specificity of the checkpoint genes in this response to DNA damage. Analysis of checkpoint protein interactions after DNA damage revealed that Rad9 physically associates with Rad53.