Base Excision Repair Capacity Research Articles

Genotoxic effects of occupational exposure to glass fibres - A human biomonitoring study

As part of a large human biomonitoring study, we conducted occupational monitoring in a glass fibre factory in Slovakia. Shopfloor workers (n = 80), with a matched group of administrators in the same factory (n = 36), were monitored for exposure to glass fibres and to polycyclic aromatic hydrocarbons (PAHs). The impact of occupational exposure on chromosomal aberrations, DNA damage and DNA repair, immunomodulatory markers, and the role of nutritional and lifestyle factors, as well as the effect of polymorphisms in metabolic and DNA repair genes on genetic stability, were investigated.The (enzyme-modified) comet assay was employed to measure DNA strand breaks (SBs) and apurinic sites, oxidised and alkylated bases. Antioxidant status was estimated by resistance to H2O2-induced DNA damage. Base excision repair capacity was measured with an in vitro assay (based on the comet assay).Exposure of workers to fibres was low, but still was associated with higher levels of SBs, and SBs plus oxidised bases, and higher sensitivity to H2O2. Multivariate analysis showed that exposure increased the risk of high levels of SBs by 20%. DNA damage was influenced by antioxidant enzymes catalase and glutathione S-transferase (measured in blood). DNA repair capacity was inversely correlated with DNA damage and positively with antioxidant status. An inverse correlation was found between DNA base oxidation and the percentage of eosinophils (involved in the inflammatory response) in peripheral blood of both exposed and reference groups. Genotypes of XRCC1 variants rs3213245 and rs25487 significantly decreased the risk of high levels of base oxidation, to 0.50 (p = 0.001) and 0.59 (p = 0.001), respectively.Increases in DNA damage owing to glass fibre exposure were significant but modest, and no increases were seen in chromosome aberrations or micronuclei. However, it is of concern that even low levels of exposure to these fibres can cause significant genetic damage.

Acetylation of p62 regulates base excision repair through interaction with APE1.

p62, a well-known adaptor of autophagy, plays multiple functions in response to various stresses. Here, we report a function for p62 in base excision repair that is distinct from its known functions. Loss of p62 impairs base excision repair capacity and increases the sensitivity of cancer cells to alkylating and oxidizing agents. In response to alkylative and oxidative damage, p62 is accumulated in the nucleus,acetylated by hMOF,and deacetylated by SIRT7, and acetylated p62 is recruited to chromatin. The chromatin-enriched p62 directly interacts with APE1, a key enzyme of the BER pathway, and promotes its endonuclease activity, which facilitates BER and cell survival. Collectively, our findings demonstrate that p62 is a regulator of BER and provide further rationale for targeting p62 as a cancer therapeutic strategy.

The E3 Ubiquitin Ligase NEDD4L Targets OGG1 for Ubiquitylation and Modulates the Cellular DNA Damage Response.

8-Oxoguanine DNA glycosylase (OGG1) is the major cellular enzyme required for the excision of 8-oxoguanine DNA base lesions in DNA through the base excision repair (BER) pathway, and therefore plays a major role in suppressing mutagenesis and in controlling genome stability. However, the mechanism of regulation of cellular OGG1 protein, particularly in response to oxidative stress, is unclear. We have purified the major E3 ubiquitin ligase responsible for OGG1 ubiquitylation from human cell extracts, and identify this as E3 ubiquitin-protein ligase NEDD4-like (NEDD4L). We demonstrate that recombinant NEDD4L stimulates ubiquitylation of OGG1 in vitro, particularly on lysine 341, and that NEDD4L and OGG1 interact in U2OS cells. Depletion of NEDD4L in U2OS cells has no impact on the stability and steady-state protein levels of OGG1, however, OGG1 stability is enhanced in response to oxidative stress induced by ionizing radiation. Furthermore, ubiquitylation of OGG1 by NEDD4L in vitro inhibits its DNA glycosylase/lyase activity. As a consequence of prolonged OGG1 stability and increased excision activity in the absence of NEDD4L, cells display increased DNA repair capacity but conversely that this decreases cell survival post-irradiation. This effect can be reproduced following OGG1 overexpression, suggesting that dysregulation of OGG1 increases the formation of lethal intermediate DNA lesions. Our study therefore highlights the importance of balancing OGG1 protein levels and BER capacity in maintaining genome stability.

Non-muscle invasive bladder cancer tissues have increased base excision repair capacity

The molecular mechanisms underlying the development and progression of bladder cancer (BC) are complex and have not been fully elucidated. Alterations in base excision repair (BER) capacity, one of several DNA repair mechanisms assigned to preserving genome integrity, have been reported to influence cancer susceptibility, recurrence, and progression, as well as responses to chemotherapy and radiotherapy. We report herein that non-muscle invasive BC (NMIBC) tissues exhibit increased uracil incision, abasic endonuclease and gap-filling activities, as well as total BER capacity in comparison to normal bladder tissue from the same patient (p < 0.05). No significant difference was detected in 8-oxoG incision activity between cancer and normal tissues. NMIBC tissues have elevated protein levels of uracil DNA glycosylase, 8-oxoguanine DNA glycosylase, AP endonuclease 1 and DNA polymerase β protein. Moreover, the fold increase in total BER and the individual BER enzyme activities were greater in high-grade tissues than in low-grade NMIBC tissues. These findings suggest that enhanced BER activity may play a role in the etiology of NMIBC and that BER proteins could serve as biomarkers in disease prognosis, progression or response to genotoxic therapeutics, such as Bacillus Calmette–Guérin.

Androgen Receptor and Poly(ADP-ribose) Glycohydrolase Inhibition Increases Efficiency of Androgen Ablation in Prostate Cancer Cells

There is mounting evidence of androgen receptor signaling inducing genome instability and changing DNA repair capacity in prostate cancer cells. Expression of genes associated with base excision repair (BER) is increased with prostate cancer progression and correlates with poor prognosis. Poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) are key enzymes in BER that elongate and degrade PAR polymers on target proteins. While PARP inhibitors have been tested in clinical trials and are a promising therapy for prostate cancer patients with TMPRSS2-ERG fusions and mutations in DNA repair genes, PARG inhibitors have not been evaluated. We show that PARG is a direct androgen receptor (AR) target gene. AR is recruited to the PARG locus and induces PARG expression. Androgen ablation combined with PARG inhibition synergistically reduces BER capacity in independently derived LNCaP and LAPC4 prostate cancer cell lines. A combination of PARG inhibition with androgen ablation or with the DNA damaging drug, temozolomide, significantly reduces cellular proliferation and increases DNA damage. PARG inhibition alters AR transcriptional output without changing AR protein levels. Thus, AR and PARG are engaged in reciprocal regulation suggesting that the success of androgen ablation therapy can be enhanced by PARG inhibition in prostate cancer patients.

Base Excision Repair in Chromatin and the Requirement for Chromatin Remodelling.

Base excision repair (BER) is a co-ordinated DNA repair pathway that recognises and repairs chemically modified bases and DNA single strand breaks. It is essential for the maintenance of genome integrity and thus in the prevention of the development of human diseases, including premature ageing, neurodegenerative diseases and cancer. Within the cell, DNA is usually packaged with histone proteins to form chromatin which imposes major constraints on the capacity of cells to perform BER. Therefore chromatin remodelling, stimulated through histone post-translational modifications (PTMs) or ATP-dependent chromatin remodelling complexes (ACRs), are required to stimulate access to the DNA damage and therefore enhance the BER process. Despite this, the molecular mechanisms through which this is co-ordinated and the specific enzymes that promote chromatin remodelling required for BER remain elusive. In this review, we summarise the multitude of in vitro studies utilising mononucleosome substrates containing site-specific DNA base damage that demonstrate the requirement for chromatin remodelling to facilitate BER, particularly in occluded regions. We also highlight preliminary evidence to date for the identity of ACRs, their mechanisms and the role of histone PTMs in modulating the cellular capacity for BER.

Inhibition of base excision repair by natamycin suppresses prostate cancer cell proliferation

Prostate cancer (PCa) progression is characterized by increased expression and transcriptional activity of the androgen receptor (AR). In the advanced stages of prostate cancer, AR significantly upregulates the expression of genes involved in DNA repair. Upregulation of expression for base excision repair (BER) related genes is associated with poor patient survival. Thus, inhibition of the BER pathway may prove to be an effective therapy for prostate cancer. Using a high throughput BER capacity screening assay, we sought to identify BER inhibitors that can synergize with castration therapy. An FDA-approved drug library was screened to identify inhibitors of BER using a fluorescence-based assay suitable for HTS. A gel-based secondary assay confirmed the reduction of BER capacity by compounds identified in the primary screen. Five compounds were then selected for further testing in the independently derived, androgen-dependent prostate cancer cell lines, LNCaP and LAPC4, and in the nonmalignant prostate derived cell lines PNT1A and RWPE1. Further analysis led to the identification of a lead compound, natamycin, as an effective inhibitor of key BER enzymes DNA polymerase β (pol β) and DNA Ligase I (LIG I). Natamycin significantly inhibited proliferation of PCa cells in an androgen depleted environment at 1 μM concentration, however, growth inhibition did not occur with nonmalignant prostate cell lines, suggesting that BER inhibition may improve efficacy of the castration therapies.

Aberrant DNA Polymerase Beta Enhances H. pylori Infection Induced Genomic Instability and Gastric Carcinogenesis in Mice.

H. pylori is a significant risk factor of gastric cancer that induces chronic inflammation and oxidative DNA damage to promote gastric carcinoma. Base excision repair (BER) is required to maintain the genome integrity and prevent oxidative DNA damage. Mutation in DNA polymerase beta (Pol β) impacts BER efficiency and has been reported in approximately 30–40% of gastric carcinoma tumors. In this study, we examined whether reduced BER capacity associated with mutation in the POLB gene, along with increased DNA damage generated by H. pylori infection, accelerates gastric cancer development. By infecting a Pol β mutant mouse model that lacks dRP lyase with H. pylori, we show that reactive oxygen and nitrogen species (RONS) mediated DNA damage is accumulated in Pol β mutant mice (L22P). In addition, H. pylori infection in Leu22Pro (L22P) mice significantly increases inducible nitric oxide synthesis (iNOS) mediated chronic inflammation. Our data show that L22P mice exhibited accelerated H. pylori induced carcinogenesis and increased tumor incidence. This work shows that Pol β mediated DNA repair under chronic inflammation conditions is an important suppressor of H. pylori induced stomach carcinogenesis.

Base excision repair capacity as a determinant of prognosis and therapy response in colon cancer patients

The DNA-damaging agent 5-fluorouracil represents the most commonly used chemotherapeutic drug for colorectal cancer patients. DNA lesions associated with 5-fluorouracil therapy are primarily repaired by base excision repair (BER) and mismatch repair (MMR) pathways. Published evidence suggests that the individual DNA repair capacity (DRC) may affect a patient’s prognosis and response to chemotherapy.With this in mind, we designed a prospective study of which the main aim was to investigate BER-DRC in relation to 5-fluorouracil response as potential predictive and/or prognostic biomarker. BER-DRC was supplemented by a microsatellite instability (MSI) analysis which represents an indirect marker of MMR activity in the tumor. All parameters were measured in paired samples of tumor tissue and non-malignant adjacent mucosa of 123 incident colon cancer patients.Our results indicate that BER-DRC in non-malignant adjacent mucosa was positively associated with overall survival (P = 0.007) and relapse-free survival (P = 0.04). Additionally, in multivariate analysis, good therapy responders in TNM stage II and III with an elevated BER-DRC in mucosa exhibited better overall survival. Moreover, the overall survival of these patients was even better in the presence of a decreased BER-DRC in tumor tissue. The ratio of BER-DRC in tumor tissue over BER-DRC in mucosa positively correlated with advanced tumor stage (P = 0.003). With respect to MSI, we observed that MSI-high tumors were mostly localized in proximal colon; however, in our cohort, the MSI status affected neither patients’ prognosis nor survival.In summary, the results of the present study suggest that the level of BER-DRC is associated with patients’ survival. BER-DRC represents a potential prognostic biomarker, applicable for prediction of therapy response and useful for individual approach to patients.

Oxidatively-induced DNA damage and base excision repair in euthymic patients with bipolar disorder

Oxidatively-induced DNA damage has previously been associated with bipolar disorder. More recently, impairments in DNA repair mechanisms have also been reported. We aimed to investigate oxidatively-induced DNA lesions and expression of DNA glycosylases involved in base excision repair in euthymic patients with bipolar disorder compared to healthy individuals. DNA base lesions including both base and nucleoside modifications were measured using gas chromatography-tandem mass spectrometry and liquid chromatography-tandem mass spectrometry with isotope-dilution in DNA samples isolated from leukocytes of euthymic patients with bipolar disorder (n = 32) and healthy individuals (n = 51). The expression of DNA repair enzymes OGG1 and NEIL1 were measured using quantitative real-time polymerase chain reaction. The levels of malondialdehyde were measured using high performance liquid chromatography. Seven DNA base lesions in DNA of leukocytes of patients and healthy individuals were identified and quantified. Three of them had significantly elevated levels in bipolar patients when compared to healthy individuals. No elevation of lipid peroxidation marker malondialdehyde was observed. The level of OGG1 expression was significantly reduced in bipolar patients compared to healthy individuals, whereas the two groups exhibited similar levels of NEIL1 expression. Our results suggest that oxidatively-induced DNA damage occurs and base excision repair capacity may be decreased in bipolar patients when compared to healthy individuals. Measurement of oxidatively-induced DNA base lesions and the expression of DNA repair enzymes may be of great importance for large scale basic research and clinical studies of bipolar disorder.

DNA Base Excision Repair: The Achilles' Heel of Tumour Cells and their Microenvironment?

Our current understanding of cancer suggests that every tumour has individual features. Approaches to cancer treatment require thorough comprehension of the mechanisms triggering genomic instability and protecting cancer cells from therapeutic treatments. Base excision repair (BER) is a frontline DNA repair system that is responsible for maintaining genome integrity. The BER pathway prevents the occurrence of disease, including cancer, by constantly repairing DNA base lesions and DNA single strand breaks caused by endogenous and exogenous mutagens. BER is an important DNA repair system for cancer cell survival, as it can affect both chemoand radio-resistance of tumours. Variations in BER capacity are likely responsible for a number of cases of sporadic cancer and may also modulate cancer sensitivity and resistance to therapeutic treatments. For these reasons, it is broadly accepted that targeting BER enzymes might be a promising approach to personalised anti-cancer therapy. However, recent advances in both treatment strategies and the comprehension of cancer development call for a better understanding of the consequences of BER inhibition. Indeed, the impact on both the tumour microenvironment and healthy tissues is still unclear. This review will summarise the current status of the approaches exploiting BER targeting, describing the most promising small molecule inhibitors and synthetic lethality strategies, as well as potential limitations of these approaches.

In vivo measurements of interindividual differences in DNA glycosylases and APE1 activities

The integrity of our DNA is challenged with at least 100,000 lesions per cell on a daily basis. Failure to repair DNA damage efficiently can lead to cancer, immunodeficiency, and neurodegenerative disease. Base excision repair (BER) recognizes and repairs minimally helix-distorting DNA base lesions induced by both endogenous and exogenous DNA damaging agents. Levels of BER-initiating DNA glycosylases can vary between individuals, suggesting that quantitating and understanding interindividual differences in DNA repair capacity (DRC) may enable us to predict and prevent disease in a personalized manner. However, population studies of BER capacity have been limited because most methods used to measure BER activity are cumbersome, time consuming and, for the most part, only allow for the analysis of one DNA glycosylase at a time. We have developed a fluorescence-based multiplex flow-cytometric host cell reactivation assay wherein the activity of several enzymes [four BER-initiating DNA glycosylases and the downstream processing apurinic/apyrimidinic endonuclease 1 (APE1)] can be tested simultaneously, at single-cell resolution, in vivo. Taking advantage of the transcriptional properties of several DNA lesions, we have engineered specific fluorescent reporter plasmids for quantitative measurements of 8-oxoguanine DNA glycosylase, alkyl-adenine DNA glycosylase, MutY DNA glycosylase, uracil DNA glycosylase, and APE1 activity. We have used these reporters to measure differences in BER capacity across a panel of cell lines collected from healthy individuals, and to generate mathematical models that predict cellular sensitivity to methylmethane sulfonate, H2O2, and 5-FU from DRC. Moreover, we demonstrate the suitability of these reporters to measure differences in DRC in multiple pathways using primary lymphocytes from two individuals.

Development of a Cell-Based Assay for Measuring Base Excision Repair Responses

Base excision repair (BER) is the predominant pathway for coping with most forms of hydrolytic, oxidative or alkylative DNA damage. Measuring BER capacity in living cells is valuable for both basic science applications and epidemiological studies, since deficiencies in this pathway have been associated with cancer susceptibility and other adverse health outcomes. At present, there is an ongoing effort to develop methods to effectively quantify the rate of BER as a whole. We present a variation of a previously described “Oligonucleotide Retrieval Assay” designed to measure DNA excision repair that is capable of quantifying the rate of repair of thymine glycol in a variety of human cells with a high degree of sensitivity.

Genome-wide map of Apn1 binding sites under oxidative stress in Saccharomyces cerevisiae.

The DNA is cells is continuously exposed to reactive oxygen species resulting in toxic and mutagenic DNA damage. Although the repair of oxidative DNA damage occurs primarily through the base excision repair (BER) pathway, the nucleotide excision repair (NER) pathway processes some of the same lesions. In addition, damage tolerance mechanisms, such as recombination and translesion synthesis, enable cells to tolerate oxidative DNA damage, especially when BER and NER capacities are exceeded. Thus, disruption of BER alone or disruption of BER and NER in Saccharomyces cerevisiae leads to increased mutations as well as large-scale genomic rearrangements. Previous studies demonstrated that a particular region of chromosome II is susceptible to chronic oxidative stress-induced chromosomal rearrangements, suggesting the existence of DNA damage and/or DNA repair hotspots. Here we investigated the relationship between oxidative damage and genomic instability utilizing chromatin immunoprecipitation combined with DNA microarray technology to profile DNA repair sites along yeast chromosomes under different oxidative stress conditions. We targeted the major yeast AP endonuclease Apn1 as a representative BER protein. Our results indicate that Apn1 target sequences are enriched for cytosine and guanine nucleotides. We predict that BER protects these sites in the genome because guanines and cytosines are thought to be especially susceptible to oxidative attack, thereby preventing large-scale genome destabilization from chronic accumulation of DNA damage. Information from our studies should provide insight into how regional deployment of oxidative DNA damage management systems along chromosomes protects against large-scale rearrangements. Copyright © 2017 John Wiley & Sons, Ltd.

Modulation of proteostasis counteracts oxidative stress and affects DNA base excision repair capacity in ATM-deficient cells.

Ataxia telangiectasia (A-T) is a syndrome associated with loss of ATM protein function. Neurodegeneration and cancer predisposition, both hallmarks of A-T, are likely to emerge as a consequence of the persistent oxidative stress and DNA damage observed in this disease. Surprisingly however, despite these severe features, a lack of functional ATM is still compatible with early life, suggesting that adaptation mechanisms contributing to cell survival must be in place. Here we address this gap in our knowledge by analysing the process of human fibroblast adaptation to the lack of ATM. We identify profound rearrangement in cellular proteostasis occurring very early on after loss of ATM in order to counter protein damage originating from oxidative stress. Change in proteostasis, however, is not without repercussions. Modulating protein turnover in ATM-depleted cells also has an adverse effect on the DNA base excision repair pathway, the major DNA repair system that deals with oxidative DNA damage. As a consequence, the burden of unrepaired endogenous DNA lesions intensifies, progressively leading to genomic instability. Our study provides a glimpse at the cellular consequences of loss of ATM and highlights a previously overlooked role for proteostasis in maintaining cell survival in the absence of ATM function.

Oxidative Stress, DNA Damage and DNA Repair in Female Patients with Diabetes Mellitus Type 2.

BackgroundDiabetes mellitus type 2 (T2DM) is associated with oxidative stress which in turn can lead to DNA damage. The aim of the present study was to analyze oxidative stress, DNA damage and DNA repair in regard to hyperglycemic state and diabetes duration.MethodsFemale T2DM patients (n = 146) were enrolled in the MIKRODIAB study and allocated in two groups regarding their glycated hemoglobin (HbA1c) level (HbA1c≤7.5%, n = 74; HbA1c>7.5%, n = 72). In addition, tertiles according to diabetes duration (DD) were created (DDI = 6.94±3.1 y, n = 49; DDII = 13.35±1.1 y, n = 48; DDIII = 22.90±7.3 y, n = 49). Oxidative stress parameters, including ferric reducing ability potential, malondialdehyde, oxidized and reduced glutathione, reduced thiols, oxidized LDL and F2-Isoprostane as well as the activity of antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase were measured. Damage to DNA was analyzed in peripheral blood mononuclear cells and whole blood with single cell gel electrophoresis. DNA base excision repair capacity was tested with the modified comet repair assay. Additionally, mRNA expressions of nine genes related to base excision repair were analyzed in a subset of 46 matched individuals.ResultsNo significant differences in oxidative stress parameters, antioxidant enzyme activities, damage to DNA and base excision repair capacity, neither between a HbA1c cut off />7.5%, nor between diabetes duration was found. A significant up-regulation in mRNA expression was found for APEX1, LIG3 and XRCC1 in patients with >7.5% HbA1c. Additionally, we observed higher total cholesterol, LDL-cholesterol, LDL/HDL-cholesterol, triglycerides, Framingham risk score, systolic blood pressure, BMI and lower HDL-cholesterol in the hyperglycemic group.ConclusionBMI, blood pressure and blood lipid status were worse in hyperglycemic individuals. However, no major disparities regarding oxidative stress, damage to DNA and DNA repair were present which might be due to good medical treatment with regular health checks in T2DM patients in Austria.

Enhanced base excision repair capacity in carotid atherosclerosis may protect nuclear DNA but not mitochondrial DNA

BackgroundLesional and systemic oxidative stress has been implicated in the pathogenesis of atherosclerosis, potentially leading to accumulation of DNA base lesions within atherosclerotic plaques. Although base excision repair (BER) is a major pathway counteracting oxidative DNA damage, our knowledge on BER and accumulation of DNA base lesions in clinical atherosclerosis is scarce. Here, we evaluated the transcriptional profile of a wide spectrum of BER components as well as DNA damage accumulation in atherosclerotic and non-atherosclerotic arteries. MethodsBER gene expression levels were analyzed in 162 carotid plaques, 8 disease-free carotid specimens from patients with carotid plaques and 10 non-atherosclerotic control arteries. Genomic integrity, mitochondrial (mt) DNA copy number, oxidative DNA damage and BER proteins were evaluated in a subgroup of plaques and controls. ResultsOur major findings were: (i) The BER pathway showed a global increased transcriptional response in plaques as compared to control arteries, accompanied by increased expression of several BER proteins. (ii) Whereas nuclear DNA stability was maintained within carotid plaques, mtDNA integrity and copy number were decreased. (iii) Within carotid plaques, mRNA levels of several BER genes correlated with macrophage markers. (iv) In vitro, some of the BER genes were highly expressed in the anti-inflammatory and pro-resolving M2 macrophages, showing increased expression upon exposure to modified lipids. ConclusionsThe increased transcriptional response of BER genes in atherosclerosis may contribute to lesional nuclear DNA stability but appears insufficient to maintain mtDNA integrity, potentially influencing mitochondrial function in cells within the atherosclerotic lesion.

Chk1 inhibitor synergizes quinacrine mediated apoptosis in breast cancer cells by compromising the base excision repair cascade

Quinacrine (QC) causes apoptosis in breast cancer cells by induction of DNA damage, arrest of cells in S-phase, and by topoisomerase inhibition. Here, we show that QC-mediated apoptosis is not only due to increased DNA damage but also by compromising cell cycle checkpoints and base excision repair (BER) capacity in breast cancer cells. QC decreased CHK1, CDKs (CDC2, MDM2, CDC6), cyclins (B1, E1) and CDC25-A in a dose dependent manner. The expression of basal ATR remains unaltered but pATR (Ser-428) increased after QC treatment. A CHK1 inhibitor, SB218078, was also tested alone and in combination with QC. Like QC, SB218078 caused apoptosis by DNA damage and S-phase arrest. The combination of QC and SB218078 increased apoptosis by blocking the cell cycle in G2/M, which caused a mitotic catastrophe, and induced DNA damage at a higher level in comparison to individual compound treatments. Both drugs individually or in combination decreased the levels of replication protein A (RPA). Measurement of the expression of BER (SP- and LP-BER) proteins and direct in vivo BER activity revealed that the QC/SB218078 combination caused apoptosis in cancer cells by disrupting the induction of BER, which represents a novel means of potentially treating breast cancer.

Gut Microbiota Imbalance and Base Excision Repair Dynamics in Colon Cancer.

Gut microbiota are required for host nutrition, energy balance, and regulating immune homeostasis, however, in some cases, this mutually beneficial relationship becomes twisted (dysbiosis), and the gut flora can incite pathological disorders including colon cancer. Microbial dysbiosis promotes the release of bacterial genotoxins, metabolites, and causes chronic inflammation, which promote oxidative DNA damage. Oxidized DNA base lesions are removed by base excision repair (BER), however, the role of this altered function of BER, as well as microbiota-mediated genomic instability and colon cancer development, is still poorly understood. In this review article, we will discuss how dysbiotic microbiota induce DNA damage, its impact on base excision repair capacity, the potential link of host BER gene polymorphism, and the risk of dysbiotic microbiota mediated genomic instability and colon cancer.

The efficiency of the translesion synthesis across abasic sites by mitochondrial DNA polymerase is low in mitochondria of 3T3 cells

Translesion synthesis by specialized DNA polymerases is an important strategy for mitigating DNA damage that cannot be otherwise repaired either due to the chemical nature of the lesion. Apurinic/Apyrimidinic (abasic, AP) sites represent a block to both transcription and replication, and are normally repaired by the base excision repair (BER) pathway. However, when the number of abasic sites exceeds BER capacity, mitochondrial DNA is targeted for degradation. Here, we used two uracil-N-glycosylase (UNG1) mutants, Y147A or N204D, to generate AP sites directly in the mtDNA of NIH3T3 cells in vivo at sites normally occupied by T or C residues, respectively, and to study repair of these lesions in their native context. We conclude that mitochondrial DNA polymerase γ (Pol γ) is capable of translesion synthesis across AP sites in mitochondria of the NIH3T3 cells, and obeys the A-rule. However, in our system, base excision repair (BER) and mtDNA degradation occur more frequently than translesion bypass of AP sites.