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Oxidative Stress Dna Damage

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Last Updated: 02 July 2021

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General | Latest Info

Numerous studies have linked excess generation of reactive oxygen species with cellular damage and atherogenesis. 1 2 Although this notion is widely hold, thorough factual evidence is lacking. Ros has been implicated in a variety of distinct cellular processes, including initiation of gene expression and promotion of cell proliferation, hypertrophy, growth arrest, and / or apoptosis. 1 2, on the other hand, ROS is involved in oxidation of LDL, which is considered a fundamental step in initiation and progression of atherosclerosis. It is also tempting to speculate that ROS may have some deleterious effects on DNA. Indeed, ROS CAN provoke extensive oxidative DNA damage, DNA strand breaks, and chromosomal aberrations. 3 Significant damage to DNA resulting from endogenous free radical attack has already been suggested to contribute to the pathology of cancer 4 and several neurodegenerative diseases. 5 6 The growing body of evidence indicates that oxidative DNA damage is also a prominent feature of atherosclerotic plaques. 7 - 9 in light of this, we have recently described elevated levels of oxidative DNA damage and repair in thoracic aorta of cholesterol - fed rabbits. The aim of the present study was to investigate whether oxidative DNA damage occurs in human atherosclerotic plaques and whether DNA repair mechanisms are upregulated in response to DNA damage.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

Introduction

Identification of oxidatively damage in cellular DNA has been until recently hampered by lack of accurate and sensitive detection methods. As exception, HPLC coupled with electrochemical Detection that was applied almost 30 years ago has allowed to monitor formation in cellular DNA of 8 - oxo - 7 8 - dihydroguanine, ubiquitous guanine oxidation product. However, 15 years have been necessary to extend measurement of 8 - oxoGua to about 25% of other 80 oxidized nucleobases that were characterized in model studies. This has been made possible by the advent of the HPLC - MS / MS versatile method that provides unambiguous assignment of damage and quantitative measurement through use of isotope dilution technique. Thus, 16 single Base lesions together with more complex damage such as intra - and inter - strand cross - links have been accurately detected in DNA of cultured cells exposed to biologically relevant oxidizing agents including hydroxyl Radical, singlet Oxygen, one - electron oxidants and Ten - Eleven Translocation enzymes. In addition, several modifications whose generation is triggered by activation of myeloperoxidases and release of reactive aldehydes from breakdown of lipid peroxides have been measured in cellular DNA. In contrast, formation of secondary oxidation products of 8 - oxoGua that has received strong attention from chemists and biochemists is at best a very minor process in cells, This remark applies as well to questionable formation of purine 5 8 - cyclo - 2deoxyribonucleosides that have been shown to be repair in model studies by Nucleotide Excision Repair pathway and not by Base Excision Repair that operate on single oxidize nucleobases. Recently collected data on cytosine and 5 - methylcytosine confirm that steady - state levels of oxidatively induced Base lesions are rather low being comprise in most cases within the range of 1 modification PAR 106 to 107 nucleobases. It may also be noted that there is still a paucity of information on kinetics of Base damage removal in cellular DNA. However, as one of the main exceptions, it was shown that 8 - oxoGua is removed from DNA in cells and animal organs, typically half of the lesion being repaired in 2h - 4h time period. Further work should focus on repair of other single oxidized bases as well as identification of tandem modifications whose measurement in cellular DNA remains a challenging analytical issue. Base Excision Repair in humans utilizes DNA glycosylase to initiate lesion removal. Three critical human DNA glycosylases include NEIL1, NEIL2, and NEIL3. Neils possess superior kinetic preference for oxidatively modified guanine lesions spiroiminodihydantoin, 5 - guanidinohydantoin, and 5 - carboxamido - 5formamido - 2iminohydantoin; in contrast, NEILs operate poorly on 8 - oxo - 7 8 - dihydroguanine and 2 6 - diamino - 4hydroxy - 5formamidopyrimidine. The unique feature of NEIL glycosylases resides in their ability to remove hydantoins from single - strand and double - strand DNA, as well as G - quadruplex DNA. More specifically, NEIL1 initiates hydantoin removal best in ssDNA and dsDNA, while NEIL2 removes hydantoins from ssDNA, and NEIL3 liberates hydantoins from ssDNA and G4 DNA, albeit very slowly.

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Discussion

Diet, and nutrition have been considered as effective strategies in prevention of Breast cancer 41. Moreover, numerous studies indicate that quite diverse category of dietary natural components can slow down breast cancer progression at different stages through induction of apoptosis, proliferation, angiogenesis, and metastasis inhibition as well as sensitization of breast cancer cells to chemotherapy. Hence, natural dietary ingredients can serve not only in prevention of breast cancer, but also their bioactive components can be considered as possible antitumor drugs 42. One of major obstacles in cancer therapy is multidrug resistance which causes repeated reoccurrence of tumor at the primary site of incidence. To tackle this problem, various studies have reported anticancer potential of herbal extracts with minimal toxicity to normal cells. Taking these facts into consideration, two plant products were selected based on their therapeutic potential. Pectin is heteropolysaccharide found naturally in the cell wall of fruits, and vegetables with beneficial effects on human health 43. On industrial scale, main sources of Pectin are Apple and Citrus fruits. Pectin biological properties depend largely on its molecular weight, degree of esterification, and viscosity 20 44. Pectins function as anticancer agents depending on their ability to induce apoptosis in cancer cells, and in different studies, CP, and AP have been modified in different conditions to improve this ability. Previous studies have shown that CP, and AP can induce apoptosis in cancer cells,. However, their mechanism of action has not been understood very well. In this study, we have investigated whether CP and AP in their intact forms are able to induce apoptosis in MDA - MB - 231 cells, blocking Galectin - 3, and interacting with genomic DNA. To examine this, cytotoxicity of CP, and AP at different concentrations was assessed on T47D, MDA - MB - 231, and MCF - 7 cancer cells. As MTT Assay results indicate, both compounds were capable of increasing cell death in breast cancer cells in dose, and in a time - dependent manner. Higher cytotoxicity was obtained when MDA - MB - 231 cells were treated with AP, and T47D cells with CP For 72 H, which also caused dose - dependent reduction in cell viability. Furthermore, apoptosis specific morphological alterations, and DNA fragmentation, were observed in treated cells as light and fluorescent microscopy, and tunnel Assay indicate, which support previous studies showing antiproliferative, and apoptotic effects of Pectin in Breast cancer cells 22. It is worth noting that, treating L929 normal cells with CP, and AP does not induce any cytotoxicity. The Anti - proliferative effect of both pectins on malignant cells has been attributed to various mechanisms. Delphi et al. Have reported that Apple Pectic - oligosaccharides cause sub - G1 cell cycle arrest, and induce caspase dependent apoptosis in MDA - MB - 231 cells 45. Moreover, Sun et al. Have shown that, Apple extract arrest MCF - 7 cells at G1 phase of the cell cycle by reducing cyclin D1, and Cdk4 proteins expression 46.

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Methods

Indirect or biochemical approaches have been used to detect DNA lesions in cells. First attempts have been made to use antibodies raised against DNA lesions. Thus, specific antibodies raised against pyrimidine dimers were developed and were found to be specific enough to detect formation of these UV - induced lesions. For Oxidative DNA lesions, and mostly 8 - oxodGuo, developed antibodies were found to be not specific enough and thus cross - reaction with guanine Base was found to give overestimated results. Other indirect methods are based on detection of DNA strand breaks. Among them, alkaline elution or the more recently developed Comet assay are well suit. These methods enable measurement of strand breaks Base on FACT that alkaline elution of DNA through filter is faster IF it contains breaks, or that electrophoresis of DNA embed in agarose gel is increased in the presence of SSB. To increase the versatility of assay, approach could be combined with DNA repair enzymes like human or bacterial glycosylases that excise oxidative DNA lesions and thus induce additional breaks. Thus, prior to electrophoresis, DNA can be treated by these glycosylases and thus additional strand breaks are interpreted as Base modifications that have been recognized by DNA Repair enzymes. By running electrophoresis under alkaline conditions, total lesions are measure, while under neutral conditions, bistranded DNA lesions, ie, two lesions located on opposing strands, are quantify. An Adaptation of this approach for detection of bistranded clustered DNA lesions is present in the next section. Coming back to the idea of using DNA Repair enzymes as damage probes, for example, it is accepted that Fpg - Sensitive sites are mostly due to the presence of oxidized purine bases, including mostly 8 - oxodGuo. Using such approach, relative proportion of direct strand breaks, oxidized purine and pyrimidine bases has been determined in cells exposed to ionizing radiation. Formation of these lesions was found to increase linearly with radiation dose. The amount of Fpg - Sensitive sites was found to be similar to that of EndoIII Sensitive sites, suggesting that almost similar amount of oxidized pyrimidine and purine bases is produce. In addition, number of direct strand breaks was found to be similar to the number of modified bases. As already mention, increasing LET of particle was found to lower the yield of formation of individual lesions. For comparison, it has been demonstrated that singlet Oxygen only produces 8 - oxodGuo in cellular DNA, in the absence of significant formation of strand breaks. Using specific measurement of several DNA lesions, attempts have been made to determine the relative importance of direct vs. Indirect effect. This remains to determine the relative proportion of lesions produced by one - electron oxidation mechanism, compared to lesions produced by HO. As mentioned above, one - electron oxidation of DNA produces mostly 8 - oxodGuo, and this has been demonstrated experimentally using two photon ionization system. Ho produces several lesions, including 8 - oxodGuo.

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Acknowledgements

Human samples were obtained from the Clinical Institute of Pathology at Medical University of Vienna. Ihc of formalin - fix paraffin - embed slides was performed as described previously with primary antibodies against 8 - oxoG, H2AX, MRE11, KU70, BRCA1, Nrf2, and p53. Briefly, slides were deparaffinized with 2 cycles of xylol and concomitant rehydration with descendent alcohol series. Heat - induced antigen retrieval was carried out either with citrate buffer or with Tris EDTA for nuclear proteins. Normal goat serum buffer was used as a blocking solution, followed by optional MOM blocking and subsequent primary antibody incubation. Staining was performed using biotinylated antibody, Avidin - Biotin - HRP Complex, and diaminobenzidine. Slides were counterstained with hematoxylin. Loss of heterozygosity was examined for Trp53 and Apc with 3 loci - aligning dinucleotide PCR amplifications, which were selected from ABI prism mouse mapping primer list. For Trp53, primer pairs were specific for segments D11mit86, d11mit4, and D11mit285. Apc markers were specific for segments D18mit12, d18mit177, and D18mit91. For amplification, Multiplex PCR Kit was used. All primers were labelled with fluorescent dye. Pcr reaction consists of 7. 5 L master mix, 1. 5 L Solution Q, 0. 5 L per primer, and DNA amount of 5 - 10 ng. For MSI Analysis, reagents were combined prior to Analysis: 8. 6 L formamide, 0. 4 L GeneScan 500 LIZ size standard, and 0. 4 L PCR product is followed by denaturation step at 95C and immediately cooled on ice. Carry out under the following PCR conditions: 15 minutes at 95C, 35 cycles at 94C for 30 seconds; 60C for 30 seconds, and 72C for 30 seconds; followed by 72C for 10 minutes. Analysis was done by comparing noninflamed, inflame, and dysplastic tissue using GeneMapper Software 4. 0 To additionally analyze microsatellite status, 4 dinucleotide and 2 polyA repeat markers were used and handled and analyzed as described previously. All experiments were carried out on Genetic Analyzer 3500. Small intestinal and large bowel organoids were prepared from IL10 KO and wild - type mice. Organoids were cultured in Matrigel with passage every 3 to 5 days in a 1: 2 ratio. Treatment of organoids was carried out with TNF, IL6, recombinant murine IL10, rmIL22, and sulforaphane for 24 hours or with H 2 O 2 for 1 hour. For Western blotting, organoids were washed and resuspend in 1 mL PBS and transferred into a precooled Eppendorf tube, followed by centrifugation at 400 G for 5 minutes at 4C. Supernatant was discarded and the washing step was repeat. The Cell pellet was lysed in RIPA buffer, followed by short sonication and subsequent cooling on ice for 30 minutes. Using Bradford assay, protein concentration was measured and 20 G of protein was used for SDS - PAGE. Immunoblotting was performed on PVDF membrane. Protein bands were visualized with IRDye couple anti - rabbit or anti - mouse antibodies and scanned on Odyssey imager.


INTRODUCTION

The brain is exquisitely sensitive to oxidative stress 1, never more so than in developing embryo 2 3. Oxidative stress can arise internally, since all cells produce oxygen free radicals in the form of reactive oxygen species as byproducts of ATP synthesis during oxidative phosphorylation. Oxidative stress also originates from external sources, such as nitric oxide or peroxide. Ros damages protein, lipid, carbohydrate,s and RNA, all of which can be resynthesized. However, unless oxidatively damaged DNA is repair, genetic information could be lose, cells might die, or they might become transformed. Although large amounts of ROS are deleterious, small amounts are particularly important during early embryogenesis 4 and are required for microglial activation and self - renewal 5 6. Therefore, generation of ROS and repair of oxidatively damaged DNA are tightly regulated processes. Most DNA damage arising from oxidative damage is repaired by base excision repair pathway 7 8. Ber also repairs small lesions arising from alkylation, deamination, depurination / depyrimidination, and presence of uracil 9 10. Several proteins that participate in the BER pathway are embryonic or peri - embryonic lethals in mice, most notably AP endonuclease 1, DNA polymerase, XRCC1, and flap endonuclease 1 11 12 13 14. Furthermore, uracil DNA glycosylase, which recognizes uracil and serves as an entry point to BER, is embryonic lethal in zebrafish 15. Zebrafish embryos in which Apex1 has been knocked down completely arrested at midblastula transition, when zygotic transcription is initiated and differentiation begins. Embryos in which Apex1 or Ogg1 protein has been partially knocked down show defective brain and heart development 16 17. Recent evidence from this laboratory 18 indicates that Apex1 is responsible for maintaining transcript and protein levels of transcription factor Creb1 and its binding partners Crem, torc1 and 3, and CBP. Furthermore, damage to Creb consensus sequence that is repaired by BER can affect Creb1 binding both positively and negatively 19 20. Creb1 has a close relationship with normal brain development and neuronal function 21 22 23 24 25, while CBP is known to regulate differentiation and survival of interneurons 26. Apex1 regulates levels of DNA polymerase protein, next participant in BER pathway 18, via Creb1. Indeed, Creb1 activity is also associated with modulating neural cell proliferation, midbrain - hindbrain organization, and patterning 27. Apex1 is an excellent marker for rapid proliferation in cancer cells including glioma, prostate, head and neck, pancreas, colon and breast 28 29 30 31 32 33. Consequently, it has frequently been marked as a potential target for chemotherapy 34. Not surprisingly, most, if not all, of the transcription factors with which Apex1 is known to interact by various methods, including AP - 1 35, Jag1 32, Egr1 32, Mdm2 36, p53 37, HIF - 1 38 and NF - kB 39 among others, are directly or indirectly dependent on Creb1 for regulation of expression and their upregulation has been associate with poor outcomes for cancer chemotherapy. Creb1 plays a vital role in the central nervous system, and genetic disruption of Creb1 leads to neurodegeneration in the brain at 40.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

BACKGROUND

Relative Cell Type Abundance

Cell TypeDescription
B CellsB cells are the primary mediators of the humoral immune response, bearing antigen-specific B cell receptors and producing antibodies that can enable the immune system to respond to a broad variety of antigens. B cells can also function as MHC class II antigen presenting cells to stimulate T cell immunity.
T CellsT-cells mediate cell-based immunity by recognizing primarily peptide antigens displayed on MHC class I or class II and either producing cytokines or directly killing the presenting cell.
TH1CD4+ T cell subset that produces IL2 and Interferon-gamma to promote cellular immunity by acting on CD8+ T Cells, NK Cells and Macrophages
Regulatory T Cells (Tregs)CD4+ T Cells that suppress effector B and T Cells and play a central role in suppression of the immune response and tolerance to self-antigens
CD8+ T CellsA subset of T cells that are capable of binding cognate-antigen expressing cells via class I MHC and directly lysing them via perforin and granzymes.
Exhausted CD8+ T CellsT-cells overstimulated by antigen can develop an "exhausted" phenotype, in which they are no longer effective in targeting antigen-bearing cells.
Cytotoxic CellsAll cells capable of cytotoxic activity, which can include T, NKT, and NK-cells.
Dendritic CellsProfessional antigen presenting cells that internalize, process, and present antigens to lymphocytes via MHC class I and class II along with costimulatory signals to initiate cellular immune responses.
MacrophagesPluripotent cells with critical roles in initiating innate and adaptive immune responses, phagocytosing abnormal cells, and regulating wound healing and tissue repair.
Mast CellsMast cells release histamine containing granules and other signals in order to promote inflammation and regulate allergic responses.
NeutrophilsNeutrophils are highly abundant cells that respond early to sites of infection or inflammation, phagocytose cellular debris, and promote downstream immunity.
Natural Killer (NK) CellsCytotoxic cells of the innate immune system that are a significant source of interferon-gamma and are capable of directly killing targeted cells via detection of a loss in MHC surface expression.
NK CD56dim cellsThe amount of CD56 present on an NK cell is indicative of its age and differentiation state; CD56dim cells are mature NK cells, more commonly found in peripheral blood than secondary lymphoid tissues, and have the greatest cytolytic activity.

Several techniques have been developed in recent years to measure oxidative DNA damage and repair intermediates. Each method comes with unique benefits and pitfalls. They are all Base on Enrichment of damaged DNA through arrays or sequencing, either by pull - down of damaged DNA, or analogous strategies. 8 - oxoG location in Genome was first addressed using antibodies, initially at large - scale resolution. Amente et al. Adapt this approach for next generation sequencing As OxiDIP - Seq. In short, DNA is fragmented into several hundred base pairs long fragments, bind by 8 - oxoG specific antibody, and enriched through protein - Gcoated magnetic beads. Enrich DNA, as well as non - enrich input DNA is then prepared for next generation sequencing and resulting fragments align back to respective genome. Increase sequencing read coverage is used to quantify damage levels Genome - wide. Using this method, Amente et al. It could detect 8 - oxoG with resolution of several hundred Base pairs. In addition to antibody - mediate enrichment, several techniques were developed that enrich oxidatively damaged DNA using chemical biology. Og - Seq, method developed by Ding et al. Is taking advantage of the propensity of 8 - oxoG to become hyperoxidated in response to mild oxidants. Using this chemical property, damage Base is converted into acceptor for biotin tag. After this chemical reaction, covalently tagged DNA is enriched through biotin - streptavidine pull - down and subsequently sequence. The resolution of this method is similar to OxiDIP - Seq limited only by size of DNA fragmentation. In Click - code - Seq, developed by Wu et al., Specificity for 8 - oxoG is achieved through utilizing specific glycosylase formamidopyrimidine DNA glycosylase to remove damaged Base and APE1 to create strand break. The Single nucleotide gap is then refilled with Click - tag guanine. Using this nucleotide as acceptor for Click reaction, code sequence is added to DNA at position where 8 - oxoG has been locate. This oligonucleotide then serves as an adapter for sequencing. Click - code - Seq is so far the only method achieving single - nucleotide resolution. While used to measure 8 - oxoG, this method would in principle be applicable to measureing other Base adducts through use of different glycosylases, as well as AP sites. In addition, our method, so called AP - Seq, can be used to measure AP sites as first Repair intermediate, but indiscriminate of source of AP site. To prevent other sources of AP sites, such as spontaneous depurination from confounding measurements, careful experimental design that focuses on specific introduction of oxidative DNA damage allows interrogation of early steps of damage processing when compared to preexisting background AP site levels. For AP - Seq, AP sites are tagged with biotin using Aldehyde Reactive Probe. This probe was developed by Kubo et al. In 1992 and used for a variety of assays over decades since,. Arp reacts under recommended conditions, specifically with the Aldehyde group of AP site and consequently introduces covalent biotin tag into DNA at the damage site.


Introduction

The integrity of DNA is constantly challenged by damaging agents and chemical modifications. Base oxidation is a frequent insult that can arise from endogenous metabolic processes as well as from exogenous sources such as ionizing radiation. At background levels, human cell is estimated to undergo 100 to 500 such modifications per day, most commonly resulting in 8 - oxo - 7 8 - dihydroguanine and related products, which are then processed into repair intermediates. At steady state, up to 2400 8 - oxoG sit per cell is report. However, estimates differ widely due to differences in methodology. Oxidative damage is processed in a two - step process through base excision repair pathway. The damage base is first recognized and excised by 8 - oxoguanine DNA glycosylase 1, leaving the apurinic site. Glycohydrolysis is highly efficient, with an 8 - oxoG half - life of 11 min. Ap - sites are removed through backbone incision by AP - lyase, and end processing through flap - endonuclease 1, and base is subsequently replaced with undamaged nucleotide. Alternatively, in short - patch base excision repair, replacement is dependent on polymerase beta. Other sources of AP - sites include spontaneous depurination and excision of non - oxidative base modifications, such as uracil. Cells are reported to typically present with a steady state of ~15 000 to ~30 000 AP - sites per cell, which include associate beta - elimination product. Left unrepaired, 8 - oxoG can compromise transcription, DNA replication, and telomere maintenance. Also, AP - sites can lead to genomic instability and compromise genomic processes. Moreover, damage sites provide direct and indirect routes to C - to - mutagenesis. Ionizing radiation is one of the most relevant exogenous sources of high - level oxidative DNA damage and DNA strand breaks. Each gray is estimated to lead to ~10 6 ionization events in the nucleus, only ~2000 of which are supposed to target DNA directly. Most DNA damage from ionizing radiation occurs indirectly from radiolysed water and 60 - 70% can be prevented through radical scavenging. While absolute numbers differ throughout literature, Lehnert estimates 1000 - 2000 base modifications per gray, 250 alkali labile sites, 1000 single - strand breaks, and 40 double - strand breaks. Others report base modifications to be threefold more prevalent than SSBs or even several orders of magnitude increase. Interestingly, direct formation of AP - sites, however, has been shown not to increase more than 5% from background levels. Therefore, after ionizing radiation, most AP - sites likely arise from excision of oxidized bases, which comprise mostly of 8 - oxoG and related modification FaPy - guanine. Though originally controversial, there is now broad acceptance that mutation rates vary across different genomic regions. Background mutation rates in Escherichia coli were shown to vary non - randomly between genes by order of magnitude, with highly expressed genes displaying lower mutation rates. In cancer genomes, single nucleotide variants tend to accumulate preferentially in heterochromatin. More recently, it was reported that SNV densities in cancers are lower in regions surrounding transcription factor binding but are elevated at binding sit themselves and at sites with high nucleosome occupancy. These variabilities likely arise through combination of regional differences in damage sensitivity and accessibility to DNA repair machinery.

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Table

ConditionDescription
Rheumatoid ArthritisChronic inflammatory disorder that impacts the lining of joints.
Inflammatory Bowel DiseaseChronic inflammation of the digestive tract.
Type 1 DiabetesAutoimmune disease where the immune system mistakenly attacks the insulin-producing cells of the pancreas.
Multiple SclerosisDemyelinating disease that disrupts communication within the nervous system.
Systemic Lupus ErythematosusChronic inflammatory disease that manifests systemically throughout the body.
PsoriasisImmune-mediated disease that causes skin cells to rapidly build up on the surface of the skin.
Induced Adverse Immune EventsCertain treatments and infections have been reported to interfere with the immune system and induce a series of autoimmune disease or adverse immune-related events.
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Sequence context

The conventional view of oxidatively inducing DNA damage, such as 8 - oxo - 7 8 - dihydroguanine, is that it is detrimental to cellular processes. For example, when OG is located in recognition elements of nuclear factor kappa - light - chain enhancer of activated B cells, specificity protein 1, or CAMP responsive element binding protein 1 transcription factors, protein - binding affinity was significantly reduce. When OG was present in template strand of protein - coding regions, modest stalling of RNA Pol II occur, whereas initiation of DNA repair at OG to yield abasic site stop RNA Pol II, leading to truncated transcripts. These observations support the hypothesis of OG decreasing gene transcription. In contrast, livers of mice with infection - induced colitis exhibit increased levels of genomic OG in tandem with enhanced expression of many DNA repair, cell cycle, and stress response genes. Another notable example appears when rat pulmonary artery endothelial cells are subject to hypoxia; strong positive correlation between OG in Promoter regions and elevated expression of > 100 genes was observe. One gene in particular is vascular endothelial growth factor, for which OG was found in G - rich potential G - quadruplex sequence demonstrated to be responsible for transcriptional regulation of gene. Strong cellular evidence for enhancement of transcription via folding of Promoter G - quadruplexes was recently demonstrated by Balasubramanian laboratory. Therefore, we hypothesize the formation of OG in VEGF PQS under oxidative stress conditions functions as a signaling mark to unmask G - quadruplex fold, thus leading to transcriptional activation. Experiments supporting this hypothesis are described herein. Vegf PQS possess five G - tracks in which four tracks required for folding to parallel - strand G - quadruplex are four 5 - most. Our previous work mapped most Reactive guanine bases in VEGF toward oxidation, leading to OG and other secondary oxidation products. When DNA damage resides in four G - tracks of VEGF G - quadruplex, DNA repair was not observe; To our surprise, addition of fifth G - track allowed structural transition to competent G - quadruplex fold by extruding damage G run into long Loop, folding in fifth track to reform G4, and allowing faithful DNA repair of Loop out, damage G. In present work, mechanism for gene induction drive by G oxidation to OG that induce structural switch in VEGF PQS Promoter element is propose and experimentally validate in human and mouse cells. Induction of transcription was found to require 8 - oxoguanine DNA glycosylase and apurinic / apyrimidinic endoDNase 1, also known as redox effector factor 1, in base excision repair pathway. The Coupling of BER of OG and transcriptional activation observed in the present study leads to the hypothesis that G modification to OG may be epigenetic modification that regulates gene expression. Oxidation of G in VEGF PQS induces transcription. G oxidation to OG. Vegf G - quadruplex, label with positions study. Vegf sequence with Gs in core underline, reporter System design, and method for site - specific incorporation of DNA modifications.


Results and Discussion

Demonstration that OG drives VEGF PQS promoter element to induce transcription was accomplished using luciferase reporter plasmid. Key features of the reporter system include VEGF PQS promoter element with all five G runs regulating Renilla luciferase gene. The Regulatory sequence has flanking nicking endonuclease sequences allowing replacement of G - rich sequence with synthetic oligomer containing single, site - specific OG. Additionally, plasmid possess firefly luciferase gene regulated by unmodified promoter used as an internal standard. Og positions selected are based on the VEGF G - quadruplex structure solved by nuclear magnetic resonance. Changes in gene expression as function of OG position focus on oxidation - prone VEGF PQS sit 7, 12 14, and 18. Position 12 is in the loop, whereas 7, 14, and 18 occupy core positions in G4, providing contrasting views on DNA damage and structure. Also study was position 29, residing on fifth G - track 3 to principal G4 structure. First, time - dependent expression of Rluc with OG incorporated at position 12 was evaluated upon transfection of plasmid into glioblastoma cells, and report expression was normalized against expression of internal standard luc. From 12 to 48 H posttransfection, expression of Rluc significantly increased to nearly threefold when OG was present compared with WT plasmid. Next, when OG was analyzed at other sites, measurements make 48 H posttransfection found Rluc expression was enhanced by 2. 2 - to 3. 0 - fold. These results demonstrate that the presence of OG in VEGF promoter PQS enhances transcriptional output of reporter gene; significantly, OG was not detrimental, but rather increased transcription. This observation is consistent with previous studies monitoring VEGF expression under hypoxic conditions that identified approximately threefold greater expression in tandem with presence of OG in PQS promoter element. Experiments to reveal molecular details by which OG induces gene expression were conduct. In the mammalian genome, OG is bind and cloven by OGG1 in the first step of BER. Whether OGG1 cleaves phosphodiester backbone or APE1 catalyzes this step remains unanswered. To establish whether OGG1 was involved in gene induction, we conducted comparative studies in WT and OGG1 / mouse embryonic fibroblasts transfected with OG - containing plasmids. In WT MEFs, depending on the position of OG, Rluc expressions increase by 2. 5 - to 3. 9 - fold, whereas OG - containing plasmids in OGG1 / MEFs yield essentially no change in amounts of Rluc expression compared with WT plasmid. Because OG was in the coding strand of promoter, it did not interfere with advancement of RNA Pol II on promoter to transcription start site in OGG1 / MEFs, and, therefore, same gene output was observed with and without OG. The observation that OG does not increase transcription in OGG1 / MEFs confirms OGG1 was critical to expression enhancement; However, more questions remain.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

Alternatives to mutation

Loss of FUS induces SSB accumulation and ROS sensitization., B Immunoblot showing FUS knockdown by shRNAs. Total lysate was extracted from SH - SY5Y cells 48 H after transfection with two individual FUS shRNAs. - Actin was probe AS loading Control. Quantitation of relative band intensity of FUS shRNA vs Control shRNA shows in B. Error bars are standard deviation of experiments performed in triplicate. C Alkaline and neutral comet assay of Control vs FUS KD SH - SY5Y cells. Quantitation of mean tail moment from 50 randomly selected nuclei is shown in histogram. Error bars are standard deviation. D, e MTT - base viability analysis of FUS KD SH - SY5Y cells, treated with increasing doses of GO or H 2 O 2. Cells were incubated with GO for 1 H or H 2 O 2 AS indicate dose of 3 H, 48 H after FUS shRNA transfection. Mtt assay was performed 24 H after treatment. Error bars are standard deviation of experiment performed in triplicate. F Clonogenic survival analysis of FUS KD HEK293 cells after GO treatment, AS in Fig. 1c. Error bars are standard deviation of experiment performed in triplicate. G, H Repair kinetics of induced oxidative genome damage after FUS KD. Alkaline comet assay of Control or FUS knockdown SH - SY5Y cells at 0 30, and 150 min post GO treatment. The Histogram represents quantitation of mean tail moment from 50 randomly selected nuclei. Error bars are standard deviation FUS forms complex with PARP1, XRCC1, and LigIII. Ib of FLAG - FUS co - Immunoprecipitation from HEK293 cells for oxidative DNA damage Repair proteins with or without GO treatment. Ip was performed with anti - FLAG antibody. B IB of endogenous FUS co - IP from SH - SY5Y cells for PARP1, XRCC1, and LigIII. Ip was performed with anti - FUS antibody. C Scheme of human iPSC line differentiation to motor neurons. Representative phase - contrast images of iPSC and differentiated motor neurons are show. Scale bar = 50 M. D Immunofluorescence staining of motor neuron markers. Representative images of motor neurons that stain Isl - 1, MAP2, or III - tubulin indicate ~80% differentiation efficiency. Scale bar = 50 M. E IB of endogenous FUS co - IP from differentiated motor neurons for PARP1, XRCC1, and LigIII. F Quantitation of IB band intensity in Fig. 2b, e to show level change of PARP - 1, XRCC1, and LigIII in FUS IP after GO treatment. Error bars are standard deviation of experiments performed in triplicate. G PLA of FUS vs PARP1, XRCC1, or LigIII in SH - SY5Y and iPSC - derive motor neurons with or without GO treatment. Nuclei stain with DAPI. Scale bar = 5 M. H Quantitation of PLA foci from 25 motor neuronal cells. Error bars are standard deviation FUS activate LigIII for DNA ligation via direct interaction. In vitro affinity co - elution of purified GST - FUS with XRCC1 / LigIII complex, or XRCC1, LigIII, and PARP1 separately. Gst or GST - FUS was detected by coomassie staining or IB. Xrcc1, LigIII and PARP1 were detected by IB. B and C. In vitro DNA nick ligation activity assay.


INTRODUCTION

Oxidative DNA damage is a constant challenge to the genome, arising from exposure to reactive oxygen species. These can come from multiple external and internal sources, including but not limited to endogenous chemical processes through cells ' own metabolism and enzymatic activity, inflammatory processes, toxins, or ionizing radiation,. When persistent, damage harbors the risk of disrupting cellular function and causing mutation. To cope with this, living organisms have evolved very efficient repair mechanisms. However, these pathways operate without risks. This is because repair intermediates may increase genome instability and provide indirect routes to disruption of genome function and mutation. Therefore, it is a delicate decision for cells to balance out whether to start the repair process of a particular oxidative DNA damage site or to leave it unrepaired. Oxidative DNA damage is among the main mutagenic processes in germline and accompanies early development,. Therefore, harmful mutations can potentially be passed on to the majority of cells in the body and damage sites can also impact embryonic development. Throughout life, oxidative DNA damage and associated mutations contribute to the ageing process and development of age - related diseases such as neurodegeneration and cancer,. Most cancer treatments cause oxidative DNA damage and DNA strand breaks and thus oxidative DNA damage contributes to long - term side effects in cancer survivors. Therefore, unsurprisingly, oxidative DNA damage and its repair have been of high interest for decades, leading to profound knowledge about its biochemistry as a whole. However, thanks to novel approaches using sequencing - base techniques, it has become apparent that oxidative DNA damage, repair intermediates, and related mutagenesis, are distributed heterogeneously over genome at multiple levels of resolution. Heterogenous distribution adds to above - mentioned processes and mechanisms an additional dimension, because it suggests that functional genomic processes impact on oxidative DNA damage distribution and repair specificity. In addition, oxidative DNA damage itself has been identified to have an active role as site - specific gene regulator and impacts on functional genomics dependent on its genomic location,. This functional role may come as a double - edge sword due to its side effects on mutagenesis and additional genome instability. There are now several datasets published that address oxidative DNA damage and repair genome - wide in human, mouse, and yeast, using various conditions and methodology. Increasing amounts of re - sequencing data from tumors, and healthy tissues, as well as novel computational methods to extract biological information from mutation data, contribute to understanding of how oxidative DNA damage affects mutagenesis. Due to distinct methodological approaches and perspectives, these studies come to diverging conclusions. This review aims at discussing different methods and attempts to unite existing viewpoints regarding mechanistic insight we have achieved on genomic distribution of oxidative DNA damage, its repair, and consequences for genome function and distribution of mutations.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

Noncancerous disease

The association between inflammation and Oxidative Stress is well document, with studies of inflammatory conditions or infections reporting elevated levels of 8OHdG: hepatitis, hepatitis C infection, and atopic dermatitis. Bactericidal species, generated from respiratory burst of invading neutrophils, macrophages, and eosinophils damage surrounding tissue, initiating further radical reactions and potentially Oxidative Stress. Chronic inflammation and hence Oxidative Stress have been closely linked to pathogenesis of such autoimmune diseases as rheumatoid arthritis and systemic lupus erythematosus, with radical production resulting not only in connective tissue damage, but also modified biomolecules being exposed to systemic circulation, postulated to be antigen driving autoantibody production. Mechanistically, chronic inflammation can be closely linked to carcinogenesis, although there is little evidence to suggest that patients with chronic inflammatory diseases such as systemic lupus erythematosus have an increased rate of cancer development. Nevertheless, DNA damage does occur in cells cocultured with activated phagocytes, with lymphocyte DNA from patients with RA, SLE, vasculitis, or Behcet's disease containing elevated levels of 8OHdG; furthermore, lymphocytes from RA and SLE patients are more sensitive to cytotoxic effects of hydrogen peroxide. Such damage may fulfill initiation; tumor promotors have been reported to recruit inflammatory cells that, with their potential to generate ROS, may provide appropriate stimuli to lead to promotion.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

CONCLUSIONS

Demonstrating the link between defects in repair of oxidative DNA damage and propensity for disease has not been easy. Experiments with singlegene knockout mice have been rather disappointing, with mice thus far displaying few ill effects. Combine gene knockouts such as OGG1 and CSB have been more promising, with elevated tissue levels of 8OHdG, although pathological consequences of this have yet to be report. It is worth noting that whereas NER might be thought of as backup for glycosylases in repair of oxidative DNA damage, relative contribution of each repair process may vary from tissue to tissue. Such hypothesis might support findings that suggest that defect in NER of oxidative lesions in xeroderma pigmentosum account for accumulation of damage and increased frequency of internal cancers and, in certain cases, neurological degeneration characteristic of this disease. Nevertheless, it is not unreasonable to speculate that given multiple pathways for its repair, oxidative DNA damage is likely to play an important role in disease. Indeed, it seems that ROS and oxidative DNA damage are omnipresent in disease; for researchers, this means there is NO limit to conditions in which oxidative stress may be study. However, mere presence of damage is not proof of causative link, although give close link between ROS formation and oxidative DNA damage and the importance of DNA damage and mutation in carcinogenesis, it is not large leap of intuition to link oxidative DNA lesions and cancer. With this accept, it is nevertheless difficult to account for why elevated ROS / DNA damage in other diseases does not in itself lead to malignancy. The basis of this apparent contradiction and failure of current studies to definitively establish the significance of oxidative DNA damage in disease may lie with numerous factors operating simultaneously in pathogenesis. It would be unrealistic for a single experiment to be expected to consider all these factors, particularly as new factors are continually being identified and the importance of existing factors reevaluate. Clearly, a great deal of work remains to be done in defining the exact roles of oxidative DNA damage in pathogenesis of disease; with this establish, it might be possible to determine how modulation of repair might be useful in disease prevention and therapy.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

Sources

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

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