Alcohol-induced One-carbon Metabolism Impairment Promotes Dysfunction of DNA Base Excision Repair in Adult Brain

The brain is one of the major targets of chronic alcohol abuse. Yet the fundamental mechanisms underlying alcohol-mediated brain damage remain unclear. The products of alcohol metabolism cause DNA damage, which in conditions of DNA repair dysfunction leads to genomic instability and neural death. We propose that one-carbon metabolism (OCM) impairment associated with long term chronic ethanol intake is a key factor in ethanol-induced neurotoxicity, because OCM provides cells with DNA precursors for DNA repair and methyl groups for DNA methylation, both critical for genomic stability. Using histological (immunohistochemistry and stereological counting) and biochemical assays, we show that 3-week chronic exposure of adult mice to 5% ethanol (Lieber-Decarli diet) results in increased DNA damage, reduced DNA repair, and neuronal death in the brain. These were concomitant with compromised OCM, as evidenced by elevated homocysteine, a marker of OCM dysfunction. We conclude that OCM dysfunction plays a causal role in alcohol-induced genomic instability in the brain because OCM status determines the alcohol effect on DNA damage/repair and genomic stability. Short ethanol exposure, which did not disturb OCM, also did not affect the response to DNA damage, whereas additional OCM disturbance induced by deficiency in a key OCM enzyme, methylenetetrahydrofolate reductase (MTHFR) in Mthfr^+/− mice, exaggerated the ethanol effect on DNA repair. Thus, the impact of long term ethanol exposure on DNA repair and genomic stability in the brain results from OCM dysfunction, and MTHFR mutations such as Mthfr 677C→T, common in human population, may exaggerate the adverse effects of ethanol on the brain.     

Beyond base excision repair: an evolving picture of mitochondrial DNA repair

Mitochondria are highly specialised organelles required for key cellular processes including ATP production through cellular respiration and controlling cell death via apoptosis. Unlike other organelles, mitochondria contain their own DNA genome which encodes both protein and RNA required for cellular respiration. Each cell may contain hundreds to thousands of copies of the mitochondrial genome, which is essential for normal cellular function – deviation of mitochondrial DNA (mtDNA) copy number is associated with cellular ageing and disease. Furthermore, mtDNA lesions can arise from both endogenous or exogenous sources and must either be tolerated or corrected to preserve mitochondrial function. Importantly, replication of damaged mtDNA can lead to stalling and introduction of mutations or genetic loss, mitochondria have adapted mechanisms to repair damaged DNA. These mechanisms rely on nuclear-encoded DNA repair proteins that are translocated into the mitochondria.Despite the presence of many known nuclear DNA repair proteins being found in the mitochondrial proteome, it remains to be established which DNA repair mechanisms are functional in mammalian mitochondria. Here, we summarise the existing and emerging research, alongside examining proteomic evidence, demonstrating that mtDNA damage can be repaired using Base Excision Repair (BER), Homologous Recombination (HR) and Microhomology-mediated End Joining (MMEJ). Critically, these repair mechanisms do not operate in isolation and evidence for interplay between pathways and repair associated with replication is discussed. Importantly, characterising non-canonical functions of key proteins and understanding the bespoke pathways used to tolerate, repair or bypass DNA damage will be fundamental in fully understanding the causes of mitochondrial genome mutations and mitochondrial dysfunction.     

New tricks for old drugs: the anticarcinogenic potential of DNA repair inhibitors

Defective or abortive repair of DNA lesions has been associated with carcinogenesis. Therefore it is imperative for a cell to accurately repair its DNA after damage if it is to return to a normal cellular phenotype. In certain circumstances, if DNA damage cannot be repaired completely and with high fidelity, it is more advantageous for an organism to have some of its more severely damaged cells die rather than survive as neoplastic transformants. A number of DNA repair inhibitors have the potential to act as anticarcinogenic compounds. These drugs are capable of modulating DNA repair, thus promoting cell death rather than repair of potentially carcinogenic DNA damage mediated by error-prone DNA repair processes. In theory, exposure to a DNA repair inhibitor during, or immediately after, carcinogenic exposure should decrease or prevent tumorigenesis. However, the ability of DNA repair inhibitors to prevent cancer development is difficult to interpret depending upon the system used and the type of genotoxic stress. Inhibitors may act on multiple aspects of DNA repair as well as the cellular signaling pathways activated in response to the initial damage. In this review, we summarize basic DNA repair mechanisms and explore the effects of a number of DNA repair inhibitors that not only potentiate DNA-damaging agents but also decrease carcinogenicity. In particular, we focus on a novel anti-tumor agent, β-lapachone, and its potential to block transformation by modulating poly(ADP-ribose) polymerase-1.     

DNA damage and repair in Helicobacter pylori-infected gastric mucosa cells

Helicobacter pylori is a common human pathogen and its infection is believed to contribute to gastric cancer. Impaired DNA repair may fuel up cancer transformation by the accumulation of mutation and increased susceptibility to exogenous carcinogens. To evaluate the role of infection of H. pylori in DNA damage and repair we determined: (1) the level of endogenous basal, oxidative and alkylative DNA damage, and (2) the efficacy of removal of DNA damage induced by hydrogen peroxide and the antibiotic amoxicillin in the H. pylori-infected and non-infected GMCs. DNA damage and the efficacy of DNA repair were evaluated by the alkaline single cell gel electrophoresis (comet assay). Specific damage to the DNA bases were assayed with the DNA repair enzymes formamidopyrimidine-DNA glycosylase (Fpg) recognizing oxidized DNA bases and 3-methyladenine-DNA glycosylase II (AlkA) recognizing alkylated bases. The level of basal and oxidative DNA in the infected GMCs was higher than non-infected cells. H. pylori-infected GMCs displayed enhanced susceptibility to hydrogen peroxide than control cells. There was no difference between the efficacy of DNA repair in the infected and non-infected cells after treatment with hydrogen peroxide and amoxicillin. Our results indicate that H. pylori infection may be correlated with oxidative DNA damage in GMCs. Therefore, these features can be considered as a risk marker for gastric cancer associated with H. pylori infection and the comet assay may be applied to evaluate this marker.     

Critical roles of ring finger protein RNF8 in replication stress responses

Histone ubiquitylation is emerging as an important protective component in cellular responses to DNA damage. The ubiquitin ligases RNF8 and RNF168 assemble ubiquitin chains onto histone molecules surrounding DNA breaks and facilitate retention of DNA repair proteins. Although RNF8 and RNF168 play important roles in repair of DNA double strand breaks, their requirement for cell protection from replication stress is largely unknown. In this study, we uncovered RNF168-independent roles of RNF8 in repair of replication inhibition-induced DNA damage. We showed that RNF8 depletion, but not RNF168 depletion, hyper-sensitized cells to hydroxyurea and aphidicolin treatment. Consistently, hydroxyurea induced persistent single strand DNA lesions and sustained CHK1 activation in RNF8-depleted cells. In line with strict requirement for RAD51-dependent repair of hydroxyurea-stalled replication forks, RNF8 depletion compromised RAD51 accumulation onto single strand DNA lesions, suggesting that impaired replication fork repair may underlie the enhanced cellular sensitivity to replication arrest observed in RNF8-depleted cells. In total, our study highlights the differential requirement for the ubiquitin ligase RNF8 in facilitating repair of replication stress-associated DNA damage.     

Further in vivo evidence implying DNA apurinic/apyrimidinic endonuclease activity in Trypanosoma cruzi oxidative stress survival

Trypanosoma cruzi is under the attack of reactive species produced by its mammalian and insect hosts. To survive, it must repair its damaged DNA. We have shown that a base excision DNA repair (BER)-specific parasite TcAP1 endonuclease is involved in the resistance to H₂O₂. However, a putative TcAP1 negative dominant form impairing TcAP1 activity in vitro did not show any in vivo effect. Here, we show that a negative dominant form of the human APE1 apurinic/apyrimidinic (AP) endonuclease (hAPE1DN) induces a decrease in epimastigote and metacyclic trypomastigote viability when parasites were exposed to H₂O₂. Those results confirm that TcAP1 AP endonuclease activity plays an important role in epimastigote and in infective metacyclic trypomastigote oxidative DNA damage resistance leading to parasite persistence in the insect and mammalian hosts. All along its biological cycle and in its different cellular forms, T. cruzi, the etiological parasite agent of Chagas’ disease, is under the attack of reactive species produced by its mammalian and insect hosts. To survive, T. cruzi must repair their oxidative damaged DNA. We have previously shown that a specific parasite TcAP1 AP endonuclease of the BER is involved in the T. cruzi resistance to oxidative DNA damage. We have also demonstrated that epimastigotes and cell-derived trypomastigotes parasite forms expressing a putative TcAP1 negative dominant form (that impairs the TcAP1 activity in vitro), did not show any in vivo effect in parasite viability when exposed to oxidative stress. In this work, we show the expression of a negative dominant form of the human APE1 AP endonuclease fused to a green fluorescent protein (GFP; hAPE1DN-GFP) in T. cruzi epimastigotes. The fusion protein is found both in the nucleus and cytoplasm of noninfective epimastigotes but only in the nucleus in metacyclic and cell-derived trypomastigote infective forms. Contrarily to the TcAP1 negative dominant form, the ectopic expression of hAPE1DN-GFP induces a decrease in epimastigote and metacyclic trypomastigote viability when parasites were exposed to increasing H₂O₂ concentrations. No such effect was evident in expressing hAPE1DN-GFP cell-derived trypomastigotes. Although the viability of both wild-type infective trypomastigote forms diminishes when parasites are submitted to acute oxidative stress, the metacyclic forms are more resistant to H₂O₂ exposure than cell-derived trypomastigotes.Those results confirm that the BER pathway and particularly the AP endonuclease activity play an important role in epimastigote and metacyclic trypomastigote oxidative DNA damage resistance leading to parasite survival and persistence inside the mammalian and insect host cells.     

Degradation of p12 Subunit by CRL4Cdt2 E3 Ligase Inhibits Fork Progression after DNA Damage

After acute DNA damage, the cell arrests S-phase progression by inhibiting origin initiation and fork progression to repair damaged DNA. The intra-S-phase checkpoint kinase Chk1 phosphorylates Cdc25A to target the latter for degradation by CRL1^β-TrCP and so inhibit origin firing. The mechanism for inhibiting fork progression, however, has not been identified. Here, we show that degradation of p12, the fourth subunit of DNA polymerase δ, is critical for inhibiting fork progression. CRL4^Cdt2 is an E3 ligase that ubiquitinates and degrades p12 after UV treatment. Cells expressing a stable form of p12 exhibit UV-resistant DNA synthesis. DNA fiber assay and alkaline-sucrose gradient assay demonstrate that the impairment of fork progression after DNA damage requires p12 degradation. These results suggest that ubiquitination of p12 through CRL4^Cdt2 and subsequent degradation form one mechanism by which a cell responds to DNA damage to inhibit fork progression.     

RNF8/RNF168信号通路在DNA损伤修复中的作用

细胞内DNA会受部分外界因素(如紫外辐射,电离辐射和化学毒素)和内部因素(如复制错误)的影响而发生损伤,包括DNA双链断裂、DNA错配和DNA交链等。DNA损伤发生后,损伤部位会被一些蛋白识别,进而招募一系列蛋白至损伤部位,形成一个修复系统。DNA双链断裂是最严重的一种DNA损伤,错误修复往往导致疾病的发生。DNA双链断裂(double strand break, DSB)后,细胞启动RNF8/RNF168信号通路进行修复。RNF8和RNF168是这条通路的枢纽蛋白;53BP和BRCA1是关键的效应蛋白,决定着DSB修复的方式;组蛋白泛素化、磷酸化和甲基化等翻译后修饰是这条通路顺利进行的基本条件;染色质重塑、泛素化酶/去泛素化酶平衡和蛋白稳定性是这条通路的主要调节方式。本综述对RNF8/RNF168信号通路进行了梳理总结,希望其能对相关研究者起到参考作用。     

Evidence that hydrogen sulfide is a genotoxic agent

Hydrogen sulfide (H2S) produced by commensal sulfate-reducing bacteria, which are often members of normal colonic microbiota, represents an environmental insult to the intestinal epithelium potentially contributing to chronic intestinal disorders that are dependent on gene-environment interactions. For example, epidemiologic studies reveal either persistent sulfate-reducing bacteria colonization or H2S in the gut or feces of patients suffering from ulcerative colitis and colorectal cancer. However, a mechanistic model that explains the connection between H2S and ulcerative colitis or colorectal cancer development has not been completely formulated. In this study, we examined the chronic cytotoxicity of sulfide using a microplate assay and genotoxicity using the single-cell gel electrophoresis (SCGE; comet assay) in Chinese hamster ovary (CHO) and HT29-Cl.16E cells. Sulfide showed chronic cytotoxicity in CHO cells with a %C1/2 of 368.57 micromol/L. Sulfide was not genotoxic in the standard SCGE assay. However, in a modified SCGE assay in which DNA repair was inhibited, a marked genotoxic effect was observed. A sulfide concentration as low as 250 micromol/L (similar to that found in human colon) caused significant genomic DNA damage. The HT29-Cl.16E colonocyte cell line also exhibited increased genomic DNA damage as a function of Na2S concentration when DNA repair was inhibited, although these cells were less sensitive to sulfide than CHO cells. These data indicate that given a predisposing genetic background that compromises DNA repair, H2S may lead to genomic instability or the cumulative mutations found in adenomatous polyps leading to colorectal cancer.     

Genetic controls of DNA damage avoidance in response to acetaldehyde in fission yeast

Acetaldehyde, a primary metabolite of alcohol, forms DNA adducts and disrupts the DNA replication process, causing genomic instability, a hallmark of cancer. Indeed, chronic alcohol consumption accounts for approximately 3.6% of all cancers worldwide. However, how the adducts are prevented and repaired after acetaldehyde exposure is not well understood. In this report, we used the fission yeast Schizosaccharomyces pombe as a model organism to comprehensively understand the genetic controls of DNA damage avoidance in response to acetaldehyde. We demonstrate that Atd1 functions as a major acetaldehyde detoxification enzyme that prevents accumulation of Rad52-DNA repair foci, while Atd2 and Atd3 have minor roles in acetaldehyde detoxification. We found that acetaldehyde causes DNA damage at the replication fork and activates the cell cycle checkpoint to coordinate cell cycle arrest with DNA repair. Our investigation suggests that acetaldehyde-mediated DNA adducts include interstrand-crosslinks and DNA-protein crosslinks. We also demonstrate that acetaldehyde activates multiple DNA repair pathways. Nucleotide excision repair and homologous recombination, which are both epistatically linked to the Fanconi anemia pathway, have major roles in acetaldehyde tolerance, while base excision repair and translesion synthesis also contribute to the prevention of acetaldehyde-dependent genomic instability. We also show the involvement of Wss1-related metalloproteases, Wss1 and Wss2, in acetaldehyde tolerance. These results indicate that acetaldehyde causes cellular stresses that require cells to coordinate multiple cellular processes in order to prevent genomic instability. Considering that acetaldehyde is a human carcinogen, our genetic studies serve as a guiding investigation into the mechanisms of acetaldehyde-dependent genomic instability and carcinogenesis.     

Protein phosphatase PP4 is involved in NHEJ-mediated repair of DNA double-strand breaks

Reversible phosphorylation is an essential posttranslational modification to turn on/off a protein function and to regulate many cellular activities, including DNA repair. A DNA double-strand break (DSB) is the most lethal form of DNA damage and is mainly fixed by the error-prone nonhomologous end joining (NHEJ)-mediated repair and by the high-fidelity homology recombination (HR)-mediated repair. We found previously that protein phosphatase PP4 is required for HR-mediated DSB repair. In this report, we showed that depletion of PP4C by siRNA compromised NHEJ-mediated repair of DSBs induced by the nuclease I-SceI. Both PP4C and its regulatory subunit PP4R2 physically interacted with the chromatin condensation factor KAP1 (KRAB-associated protein 1). Depletion of PP4C led to sustained phosphorylation of KAP1 at Ser824. Conversely, overexpression of PP4C resulted in a decrease of KAP1 phosphorylation. PP4 dephosphorylated pKAP1 in vitro. Inhibition of KAP1 expression resulted in a defect on NHEJ-mediated DSB repair, and co-depletion of PP4c and KAP1 did not have significant synergistic effect on NHEJ-mediated DSB repair. Taken together, our results suggest that PP4C and KAP1 are in the same epistasis group, and PP4 is involved in NHEJ-mediated DSB repair, possibly through regulating the phosphorylation status of KAP1.     

The role of NBS1 in DNA double strand break repair, telomere stability, and cell cycle checkpoint control

The genomes of eukaryotic cells are under continuous assault by environmental agents and endogenous metabolic byproducts. Damage induced in DNA usually leads to a cascade of cellular events, the DNA damage response. Failure of the DNA damage response can lead to development of malignancy by reducing the efficiency and fidelity of DNA repair. The NBS1 protein is a component of the MRE11/RAD50/NBS 1 complex （MRN） that plays a critical role in the cellular response to DNA damage and the maintenance of chromosomal integrity. Mutations in the NBS1 gene are responsible for Nijmegen breakage syndrome （NBS）, a hereditary disorder that imparts an increased predisposition to development of malignancy. The phenotypic characteristics of cells isolated from NBS patients point to a deficiency in the repair of DNA double strand breaks. Here, we review the current knowledge of the role of NBS1 in the DNA damage response. Emphasis is placed on the role of NBS1 in the DNA double strand repair, modulation of the DNA damage sensing and signaling, cell cycle checkpoint control and maintenance oftelomere stability.     

The interplay of DNA polymerase λ in diverse DNA damage repair pathways in higher plant genome in response to environmental and genotoxic stress factors

DNA repair mechanisms are essential for the maintenance of genomic stability, proper cellular function and survival for all organisms. Plants, with their intrinsic immobility, are vastly exposed to a wide range of environmental agents and also endogenous processes which frequently cause damage to DNA and impose genotoxic stress. Therefore, in order to survive under frequent and extreme environmental stress conditions, plants have developed a vast array of efficient and powerful DNA damage repair mechanisms to ensure rapid and precise repair of genetic material for maintaining genome stability and faithful transfer of genetic information over generations.¹ Recently, we have defined the role of DNA polymerase λ in repair of UV-B-induced photoproducts in Arabidopsis thaliana via nucleotide excision repair pathway.² Here, we have further discussed potential function of DNA polymerase λ in various DNA repair pathways in higher plant genome in response to environmental and genotoxic stress factors.     

Chromodomain helicase DNA-binding protein 4 (CHD4) regulates homologous recombination DNA repair, and its deficiency sensitizes cells to poly(ADP-ribose) polymerase (PARP) inhibitor treatment

To ensure genome stability, cells have evolved a robust defense mechanism to detect, signal, and repair damaged DNA that is generated by exogenous stressors such as ionizing radiation, endogenous stressors such as free radicals, or normal physiological processes such as DNA replication. Homologous recombination (HR) repair is a critical pathway of repairing DNA double strand breaks, and it plays an essential role in maintaining genomic integrity. Previous studies have shown that BRIT1, also known as MCPH1, is a key regulator of HR repair. Here, we report that chromodomain helicase DNA-binding protein 4 (CHD4) is a novel BRIT1 binding partner that regulates the HR repair process. The BRCA1 C-terminal domains of BRIT1 are required for its interaction with CHD4. Depletion of CHD4 and overexpression of the ATPase-dead form of CHD4 impairs the recruitment of BRIT1 to the DNA damage lesions. As a functional consequence, CHD4 deficiency sensitizes cells to double strand break-inducing agents, reduces the recruitment of HR repair factor BRCA1, and impairs HR repair efficiency. We further demonstrate that CHD4-depleted cells are more sensitive to poly(ADP-ribose) polymerase inhibitor treatment. In response to DNA damage induced by poly(ADP-ribose) polymerase inhibitors, CHD4 deficiency impairs the recruitment of DNA repair proteins BRIT1, BRCA1, and replication protein A at early steps of HR repair. Taken together, our findings identify an important role of CHD4 in controlling HR repair to maintain genome stability and establish the potential therapeutic implications of targeting CHD4 deficiency in tumors.     

Crosstalk between autophagy and DNA repair systems

Autophagy and DNA repair are two essential biological mechanisms that maintain cellular homeostasis. Impairment of these mechanisms was associated with several pathologies such as premature aging, neurodegenerative diseases, and cancer. Intrinsic or extrinsic stress stimuli (e.g., reactive oxygen species or ionizing radiation) cause DNA damage. As a biological stress response, autophagy is activated following insults that threaten DNA integrity. Hence, in collaboration with DNA damage repair and response mechanisms, autophagy contributes to the maintenance of genomic stability and integrity. Yet, connections and interactions between these two systems are not fully understood. In this review article, current status of the associations and crosstalk between autophagy and DNA repair systems is documented and discussed.     

Oxidative and nitrative DNA damage: key events in opisthorchiasis-induced carcinogenesis

Chronic inflammation induced by liver fluke (Opisthorchis viverrini) infection is the major risk factor for cholangiocarcinoma (CCA) in Northeastern Thailand. Increased levels of proinflammatory cytokines and nuclear factor kappa B that control cyclooxygenase-2 and inducible nitric oxide activities, disturb the homeostasis of oxidants/anti-oxidants and DNA repair enzymes, all of which appear to be involved in O. viverrini-associated inflammatory processes and CCA. Consequently oxidative and nitrative stress-related cellular damage occurs due to the over production of reactive oxygen and nitrogen species in inflamed target cells. This is supported by the detection of high levels of oxidized DNA and DNA bases modified by lipid peroxidation products in both animal and human tissues affected by O. viverrini-infection. Treatment of opisthorchiasis patients with praziquantel, an anti- trematode drug was shown to reduce inflammation-mediated tissue damage and carcinogenesis. The principal mechanisms that govern the effects of inflammation and immunity in liver fluke-associated cholangiocarcinogenesis are reviewed. The validity of inflammation-related biomolecules and DNA damage products to serve as predictive biomarkers for disease risk evaluation and intervention is discussed.