Endogenous oxidative DNA damage is caused by multiple endogenous and exogenous factors. It is not completely known whether coronary angiography has an effect on DNA damage. The aim of this study was to investigate whether coronary angiography causes oxidative DNA damage. Fifty-four patients who underwent elective coronary angiography for diagnostic purpose were enrolled to the study. For each subject, the frequency of oxidative DNA damage was analyzed by using the comet assay, which is a sensitive biomarker of DNA damage, before and after diagnostic procedures. A highly significant increase of DNA damage mean score was observed in all patients after the coronary angiography procedure (p < 0.001). No significant associations were found between the change in oxidative DNA damage and dose of contrast media and radiation exposure time. A significant correlation was observed between the change of DNA damage and age, hyperlipidemia, hypertension, smoking, Gensini score index, and vitamin B,2 (r = ...
The cellular response to DNA damage is critical for maintenance of genomic integrity and inhibition of tumorigenesis. Mutations or aberrant expression of the E3 ubiquitin ligase EDD have been observed in a number of carcinomas and we recently reported that EDD modulates activity of the DNA damage checkpoint kinase, CHK2. Here, we demonstrate that EDD is necessary for G(1)/S and intra S phase DNA damage checkpoint activation and for the maintenance of G(2)/M arrest after double strand DNA breaks. Defective checkpoint activation in EDD-depleted cells led to radio-resistant DNA synthesis, premature entry into mitosis, accumulation of polyploid cells, and cell death via mitotic catastrophe. In addition to decreased CHK2 activation in EDD-depleted cells, the expression of several key cell cycle mediators including Cdc25A/C and E2F1 was altered, suggesting that these checkpoint defects may be both CHK2-dependent and -independent. These data support a role for EDD in the maintenance of genomic stability,
DNA damage plays a causal role in numerous human pathologies including cancer, premature aging and chronic inflammatory conditions. In response to genotoxic insults, the DNA damage response (DDR) orchestrates DNA damage checkpoint activation and facilitates the removal of DNA lesions. The DDR can also arouse the immune system by for example inducing the expression of antimicrobial peptides as well as ligands for receptors found on immune cells. The activation of immune signalling is triggered by different components of the DDR including DNA damage sensors, transducer kinases, and effectors. In this review, we describe recent advances on the understanding of the role of DDR in activating immune signalling. We highlight evidence gained into (i) which molecular and cellular pathways of DDR activate immune signalling, (ii) how DNA damage drives chronic inflammation, and (iii) how chronic inflammation causes DNA damage and pathology in humans.
Protein synthesis and degradation are posttranscriptional pathways used by cells to regulate protein levels. We have developed a systems biology approach to identify targets of posttranscriptional regulation and we have employed this system in Saccharomyces cerevisiae to study the DNA damage response. We present evidence that 50% to 75% of the transcripts induced by alkylation damage are regulated posttranscriptionally. Significantly, we demonstrate that two transcriptionally-induced DNA damage response genes, RNR1 and RNR4, fail to show soluble protein level increases after DNA damage. To determine one of the associated mechanisms of posttranscriptional regulation, we tracked ribonucleotide reductase 1 (Rnr1) protein levels during the DNA damage response. We show that RNR1 is actively translated after damage and that a large fraction of the corresponding Rnr1 protein is packaged into a membrane-bound structure and transported to the vacuole for degradation, with these last two steps dependent ...
One function of cell cycle checkpoints is to integrate cell cycle progression with DNA replication and repair. Therefore, the integrity of these checkpoints is considered essential in maintaining genetic stability. Mutations in checkpoint components may lead to aberrant cell cycle progression and, in the presence of DNA damage, may lead to subsequent genetic instability. DNA damage triggers a variety of cellular responses, including activation of DNA damage response pathways. In fission yeast, genetic evidence pointed to a model in which five checkpoint Rad proteins, Rad1, Rad9, Rad17, Rad26, and Hus1, sense DNA alterations and then cooperate to send a signal through Rad3 (1) . Rad3 can also function in the absence of several of these Rad genes, suggesting that Rad3 may interact with other proteins involved in the DNA damage response (4) .. In S. pombe the cell cycle checkpoint gene Rad1 is required to ensure that mitosis does not occur in the presence of DNA damage (8 , 9) . X-Spy1 was ...
Lans H, Lindvall JM, Thijssen K, Karambelas AE, Cupac D, Fensgård O, Jansen G, Hoeijmakers JH, Nilsen H, Vermeulen W Cell Death Differ. 20 (12) 1709-1718 [2013-12-00; online 2013-09-10] Human-nucleotide-excision repair (NER) deficiency leads to different developmental and segmental progeroid symptoms of which the pathogenesis is only partially understood. To understand the biological impact of accumulating spontaneous DNA damage, we studied the phenotypic consequences of DNA-repair deficiency in Caenorhabditis elegans. We find that DNA damage accumulation does not decrease the adult life span of post-mitotic tissue. Surprisingly, loss of functional ERCC-1/XPF even further extends the life span of long-lived daf-2 mutants, likely through an adaptive activation of stress signaling. Contrariwise, NER deficiency leads to a striking transgenerational decline in replicative capacity and viability of proliferating cells. DNA damage accumulation induces severe, stochastic impairment of development and ...
Proteins involved in the DNA damage response accumulate as microscopically-visible nuclear foci on the chromatin flanking DNA double-strand breaks (DSBs). As growth of ionizing radiation (IR)-induced foci amplifies the ATM-dependent DNA damage signal, the formation of discrete foci plays a crucial role in cell cycle checkpoint activation, especially in cells exposed to lower doses of IR. However, there is no quantitative parameter for the foci which considers both the number and their size. Therefore, we have developed a novel parameter for DNA damage signal based on the image analysis of the foci and quantified the amount of the signal sufficient for G2 arrest. The parameter that we have developed here was designated as SOID. SOID is an abbreviation of Sum Of Integrated Density, which represents the sum of fluorescence of each focus within one nucleus. The SOID was calculated for individual nucleus as the sum of (area (total pixel numbers) of each focus) x (mean fluorescence intensity per pixel of each
Daily exposure to environmental agents (reactive oxygen species, methylating agents, UV light, and other ionizing radiation) and normal physiological processes (replication and recombination) all damage DNA. Cells must repair DNA damage to prevent mutations from propagating and accumulating, and to maintain genome integrity and stability. The ATM and ATR genes often initiate the DNA damage response, activating signal transduction pathways that arrest the cell cycle and increase the expression of DNA repair genes. Enzymes involved in base-excision, nucleotide excision, mismatch, double-strand break, and other DNA repair processes all respond in a pre- and post-transcriptionally regulated fashion to DNA damage. Incomplete DNA repair normally activates cell death pathways such as apoptosis. Cells unable to sense and repair DNA damage may continue to grow and divide, eventually causing cellular dysfunction and death, a hallmark of diseases such as neurological defects and infertility. However, ...
DNA damage (alkaline filter elution) and sister chromatid exchange (SCE) frequencies were measured in lymphocytes of 39 welders and 39 controls. The welders showed a significantly higher rate of DNA single-strand breakages and significantly elevated SCE values. These results are not in accordance with those of a former study in which only DNA-protein cross-links were measured. The different results may be explained on the basis of different exposure levels for chromium(VI) and nickel. Both methods are not specific but sensitive enough to measure genotoxic damage after occupational exposure to chromium(VI) and nickel in the range of threshold values for the workplace on a collective basis. Additionally, the results indicate that DNA single-strand breakage and DNA-protein cross-links show different increases depending on the exposure levels for chromium and nickel. ...
TY - JOUR. T1 - Attenuating the DNA damage response to double-strand breaks restores function in models of CNS neurodegeneration. AU - Tuxworth , Richard AU - Taylor, Matthew AU - Anduaga, Ane Martin AU - Hussien-Ali, Al. AU - Chatzimatthaiou, Sotiroula AU - Longland, Joanne AU - Thompson, Adam. AU - Almutiri, Sharif AU - Alifragis, Pavlos. AU - Kyriacou, Charalambos AU - Kysela, Boris AU - Ahmed, Zubair PY - 2019/7/2. Y1 - 2019/7/2. N2 - DNA double-strand breaks are a feature of many acute and long-term neurological disorders, including neurodegeneration, following neurotrauma and after stroke. Persistent activation of the DNA damage response in response to double-strand breaks contributes to neural dysfunction and pathology as it can force post-mitotic neurons to re-enter the cell cycle leading to senescence or apoptosis. Mature, non-dividing neurons may tolerate low levels of DNA damage, in which case muting the DNA damage response might be neuroprotective. Here, we show that attenuating the ...
Faithful duplication and segregation of undamaged DNA is critical to the survival of all organisms and prevention of oncogenesis in multicellular organisms. To ensure inheritance of intact DNA, cells rely on checkpoints. Checkpoints alter cellular processes in the presence of DNA damage preventing cell cycle transitions until replication is completed or DNA damage is repaired. Several checkpoints are specific to S-phase. The S-M replication checkpoint prevents mitosis in the presence of unreplicated DNA. Rather than outright halting replication, the S-phase DNA damage checkpoint slows replication in response to DNA damage. This checkpoint utilizes two general mechanisms to slow replication. First, this checkpoint prevents origin firing thus limiting the number of replication forks traversing the genome in the presence of damaged DNA. Second, this checkpoint slows the progression of the replication forks. Inhibition of origin firing in response to DNA damage is well established, however when this thesis
DNA houses the blueprint that dictates how an organism will develop. However, DNA features numerous reactive sites that can be attacked by chemicals and radiation, resulting in DNA damage and possibly mutations. Chemical products and other environmental toxins must be tested for genetic abnormalities due to exposure. Traditional DNA damage detection can be tedious, time-consuming, and cost prohibitive. Electrochemical methods to detect DNA damage offer remedies to these drawbacks. Simple and sensitive DNA hybridization sensors are widely used for DNA detection and studying biochemical processes at specific DNA sequences. An electrochemical DNA hybridization sensor designed to detect DNA damage at hotspot TP53 gene sequences resulting from bioactivated benzo[a]pyrene (BP) will be discussed. TP53 codes for the p53 protein, and mutations at the studied genetic sequence have been shown to be prevalent in many different cancers. Double stranded DNA 21-mers were absorbed on gold electrodes followed by ...
In light of the studies discussed above (and below), we suggest a revised model of the DNA damage response (Fig. 2). In this model, DNA damage is initially detected by specific repair factor(s) that have an affinity for specific types of primary DNA lesion. In some cases, the lesion may be relatively easy to repair so that the DNA damage becomes rapidly reversed after initial detection. Under these circumstances, repair would occur sufficiently quickly to prevent recognition by components of the Mec1p/Tel1p signaling network and initiation of the DNA damage response. Consistent with this, it has been reported that HO-induced DSBs only trigger Rad53p activation when repair of these lesions is prevented by inactivation of HR (50). Similarly, even though hydrogen peroxide-induced DNA base damage does not trigger Rad53p activation during G1 or G2 in wild-type yeast cells, decreasing the efficiency of BER allows Rad53p activation in response to this type of DNA damage (51). Furthermore, when ...
As part of the heterotrimeric replication protein A complex (RPA/RP-A), binds and stabilizes single-stranded DNA intermediates, that form during DNA replication or upon DNA stress. It prevents their reannealing and in parallel, recruits and activates different proteins and complexes involved in DNA metabolism. Thereby, it plays an essential role both in DNA replication and the cellular response to DNA damage. In the cellular response to DNA damage, the RPA complex controls DNA repair and DNA damage checkpoint activation. Through recruitment of ATRIP activates the ATR kinase a master regulator of the DNA damage response. It is required for the recruitment of the DNA double-strand break repair factors RAD51 and RAD52 to chromatin in response to DNA damage. Also recruits to sites of DNA damage proteins like XPA and XPG that are involved in nucleotide excision repair and is required for this mechanism of DNA repair. Plays also a role in base excision repair (BER) probably through interaction with ...
The vast majority of DNA damage affects the primary structure of the double helix in which the bases are chemically modified. These modifications can in turn disrupt the molecules regular helical structure by introducing non-native chemical bonds or bulky adducts that do not fit in the standard double helix. DNA damages that are naturally occurring due to metabolism and its byproducts occur at a high rate in the body. Most of these damages to DNA from naturally occurring metabolism are repaired. However, there may remain some DNA damage despite the action of repair processes. These remaining DNA damages accumulate in the tissues.. There are a number of sources that contribute to DNA damage. DNA damage can be subdivided into two main types, either endogenous damage from naturally occurring metabolic processes and/or exogenous damage caused by external agents.. Endogenous damage caused by metabolic byproducts (naturally occurring):. ...
The cyclin-dependent kinase 12 (CDK12) modulates transcription elongation by phosphorylating the carboxy-terminal domain of RNA polymerase II and appears to selectively affect the expression of DNA damage response (DDR) and mRNA processing genes. Yet, the mechanism(s) by which it achieves this selectivity remains unclear. Using a highly selective CDK12/13 inhibitor, THZ531, and nascent RNA sequencing, we show that CDK12 inhibition results in gene length-dependent elongation defects, leading to premature cleavage and polyadenylation (PCPA) as well as loss of expression of long (,45 kb) genes, a substantial proportion of which participate in the DDR. This early termination phenotype correlated with an increased proportion of intronic polyadenylation sites, a feature that was especially prominent among DDR genes. Finally, phosphoproteomic analysis indicated that pre-mRNA processing factors, including those involved in PCPA, are direct phosphotargets of CDKs 12 and 13. These results support a model ...
Tumors exhibit genomic instability that arises from environmental and endogenous sources of DNA damage. To prevent the propagation of unstable genomes, the DNA damage response DDR pathway is activated during each cell cycle to ensure accurate DNA replication, repair of damaged DNA and apoptosis of heavily damaged cells. Thus, the DDR pathway functions as a barrier to cancer. DDR activation has been observed in precancerous lesions and has led to the idea that unresolved problems accumulated during DNA replication promote tumorigenesis. Such damaged sites halt the progression of DNA polymerases and cause replication stress that activates the DDR. Repair and restart of damaged replication forks requires the concerted effort of several DNA repair proteins, including the tumor suppressor BRCA1 breast cancer 1 and other DDR proteins that have yet to be characterized. I hypothesized that multiple DDR pathways prevent the accumulation of replication stress observed in precancerous and cancerous lesions of the
The COMET test measures sperm DNA damage (fragmentation). Sperm DNA can be damaged when sperm are made, breaking the DNA into smaller fragments. Men with high levels of sperm DNA damage are less likely to get their partner pregnant and have increased risk of miscarriage (1-5). Even if your sperm count is normal, the sperm may not be of good quality, and therefore sperm DNA damage can reduce the chance of you/partner having a baby (1-5).. Why should I get tested?. Knowing whether you have sperm DNA damage can help you make informed decisions about the type of treatment and/or lifestyle changes to improve your sperm DNA and fertility.. Can I improve my sperm DNA?. DNA damage is usually caused by oxidative stress. Oxidative stress produces free radicals which attack the DNA molecule causing breaks in the DNA strands. Sperm DNA damage is often associated with underlying medical conditions (such as varicocoele, infection or fever) or certain lifestyle choices (such as smoking or heat).. Your ...
TY - JOUR. T1 - Undamaged DNA transmits and enhances DNA damage checkpoint signals in early embryos. AU - Peng, Aimin. AU - Lewellyn, Andrea L.. AU - Maller, James L.. PY - 2007/10/1. Y1 - 2007/10/1. N2 - In Xenopus laevis embryos, the midblastula transition (MBT) at the 12th cell division marks initiation of critical developmental events, including zygotic transcription and the abrupt inclusion of gap phases into the cell cycle. Interestingly, although an ionizing radiation-induced checkpoint response is absent in pre-MBT embryos, introduction of a threshold amount of undamaged plasmid or sperm DNA allows a DNA damage checkpoint response to be activated. We show here that undamaged threshold DNA directly participates in checkpoint signaling, as judged by several dynamic changes, including H2AX phosphorylation, ATM phosphorylation and loading onto chromatin, and Chk1, Chk2 phosphorylation and release from nuclear DNA. These responses on physically separate threshold DNA require γ-H2AX and are ...
Background Dot1L, a histone methyltransferase that targets histone H3 lysine 79 (H3K79), has been implicated in gene regulation and the DNA damage response although its functions in these processes remain poorly defined. Methodology/Principal Findings Using the chicken DT40 model system, we generated cells in which the Dot1L gene is disrupted to examine the function and focal recruitment of the 53Bp1 DNA damage response protein. Detailed kinetic and dose response assays demonstrate that, despite the absence of H3K79 methylation demonstrated by mass spectrometry, 53Bp1 focal recruitment is not compromised in these cells. We also describe, for the first time, the phenotypes of a cell line lacking both Dot1L and 53Bp1. Dot1L¿/¿ and wild type cells are equally resistant to ionising radiation, whereas 53Bp1¿/¿/Dot1L¿/¿ cells display a striking DNA damage resistance phenotype. Dot1L and 53Bp1 also affect the expression of many genes. Loss of Dot1L activity dramatically alters the mRNA levels of ...
The DNA damage response (DDR) is a signal transduction pathway that decides the cells fate either to repair DNA damage or to undergo apoptosis if there is too much damage. Post-translational modifications modulate the assembly and activity of protein complexes during the DDR pathways. MicroRNAs (miRNAs) are emerging as a class of endogenous gene modulators that control protein levels, thereby adding a new layer of regulation to the DDR. In this review, we describe a new role for miRNAs in regulating the cellular response to DNA damage with a focus on DNA double-strand break damage. We also discuss the implications of miRNAs role in the DDR to stem cells, including embryonic stem cells and cancer stem cells, stressing the potential applications for miRNAs to be used as sensitizers for cancer radiotherapy and chemotherapy.
The excision of mutagenic DNA adducts by the nucleotide excision repair (NER) pathway is essential for genome stability, which is key to avoiding genetic diseases, premature aging, cancer and neurologic disorders. Due to the need to process an extraordinarily high damage density embedded in the nucleosome landscape of chromatin, NER activity provides a unique functional caliper to understand how histone modifiers modulate DNA damage responses. At least three distinct lysine methyltransferases (KMTs) targeting histones have been shown to facilitate the detection of ultraviolet (UV) light-induced DNA lesions in the difficult to access DNA wrapped around histones in nucleosomes. By methylating core histones, these KMTs generate docking sites for DNA damage recognition factors before the chromatin structure is ultimately relaxed and the offending lesions are effectively excised. In view of their function in priming nucleosomes for DNA repair, mutations of genes coding for these KMTs are expected to cause
The circadian control of an organisms response to DNA damage response rests upon circadian proteins which play important roles in the processes of cell proliferation and control of response to genotoxic stress both at the cellular and organismal levels [83]. DNA damage triggers cellular stress response pathways which may result in checkpoint cell cycle arrest, apoptosis or DNA repair. DNA damage leads to activation of critical components of cellular stress response pathways including ATM/ATR (ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related) and CHK1/2 (checkpoint kinase1/2) which in turn activates tumour suppressor protein p53 and subsequently causes cell cycle arrest or apoptosis [84]. It has been shown that Bmal1-deficient human cells are unable to undergo growth arrest on p53 activation by DNA damage. Contrary to in vivo mouse data connecting BMAL1-dependent delay in G1 progression to upregulation of p21 [82], radiation induced growth arrest in Bmal1-deficient human ...
One function of cell cycle checkpoints is to integrate cell cycle progression with DNA replication and repair. Therefore, the integrity of these checkpoints is considered essential in maintaining genetic stability. Mutations in checkpoint components may lead to aberrant cell cycle progression and, in the presence of DNA damage, may lead to subsequent genetic instability. DNA damage triggers a variety of cellular responses, including activation of DNA damage response pathways. In fission yeast, genetic evidence pointed to a model in which five checkpoint Rad proteins, Rad1, Rad9, Rad17, Rad26, and Hus1, sense DNA alterations and then cooperate to send a signal through Rad3 (1) . Rad3 can also function in the absence of several of these Rad genes, suggesting that Rad3 may interact with other proteins involved in the DNA damage response (4) .. In S. pombe the cell cycle checkpoint gene Rad1 is required to ensure that mitosis does not occur in the presence of DNA damage (8 , 9) . X-Spy1 was ...
Strand breaks in cellular DNA occur continuously as a consequence of normal processes such as recombination or the infliction of DNA damage. DNA damage triggers several signal transduction pathways that lead either to damage repair coupled with attenuation of cell cycle progression, or to programmed cell death (apoptosis). A junction of such pathways is controlled by the transcription factor p53. After DNA damage, the amount of p53 in cells is increased through posttranscriptional mechanisms and its transactivation activity is enhanced, leading to the activation of downstream genes (1).. The genetic disorder A-T results in genome instability, cerebellar and thymic degeneration, immunodeficiency, gonadal dysgenesis, radiation sensitivity, and predisposition to cancer. A-T cells exhibit acute sensitivity to ionizing radiation and radiomimetic chemicals, and their cell cycle checkpoints fail to be activated after treatment with these agents (2). The responsible gene, ATM, encodes a 370-kD protein ...
PubMedID: 23220418 | Dimer exchange and cleavage specificity of the DNA damage response protein UmuD. | Biochimica et biophysica acta | 2/1/2013
This is the first out of two pathways which deals with the DNA damage response. It is comprised of two central gene products (ATM and ATR) influenced by different sources of DNA damage (in blue). The two central genes can both be divides into their most important genes. For the ATM pathway these are TP53 and CHEK2, while CHEK1 is most important for the ATR pathway. The goal of this first pathway is to provide an overview of the most important gene products, processes and changes in cell condition elicited by the DNA damage response while keeping it clear and understandable. Also some microRNAs are implemented to visualize the possible effects they can induce. By doing so a better understanding of the role microRNA play in the DNA damage response might arise. All processes take place in the cytoplasm, except when mentioned differently. ...
DNA replication forks that are stalled by DNA damage activate an S-phase checkpoint that prevents irreversible fork arrest and cell death. The increased cell death caused by DNA damage in budding yeast cells lacking the Rad53 checkpoint protein kinase is partially suppressed by deletion of the gene. Using a whole-genome sequencing approach, we identified two additional genes, and , whose mutation can also partially suppress this DNA damage sensitivity. We provide evidence that and act in a common pathway, which is distinct from the pathway. Analysis of additional mutants indicates that suppression works through the loss of the Rpd3L histone deacetylase complex. Our results suggest that the loss or absence of histone acetylation, perhaps at stalled forks, may contribute to cell death in the absence of a functional checkpoint. ...
TY - JOUR. T1 - Maintenance of the DNA-damage checkpoint requires DNA-damage-induced mediator protein oligomerization. AU - Usui, Takehiko AU - Foster, Steven. AU - Petrini, John H. J.. N1 - Open Archive. PY - 2009/1/30. Y1 - 2009/1/30. N2 - Oligomeric assembly of Brca1 C-terminal (BRCT) domain-containing mediator proteins occurs at sites of DNA damage. However, the functional significance and regulation of such assemblies are not well understood. In this study, we defined the molecular mechanism of DNA-damage-induced oligomerization of the S. cerevisiae BRCT protein Rad9. Our data suggest that Rad9s tandem BRCT domain mediates Rad9 oligomerization via its interaction with its own Mec1/Tel1-phosphorylated SQ/TQ cluster domain (SCD). Rad53 activation is unaffected by mutations that impair Rad9 oligomerization, but checkpoint maintenance is lost, indicating that oligomerization is required to sustain checkpoint signaling. Once activated, Rad53 phosphorylates the Rad9 BRCT domain, which attenuates ...
CRISPR/Cas9 induces DNA double-strand breaks that are repaired by cell-autonomous repair pathways, namely, non-homologous end-joining (NHEJ), or homology-directed repair (HDR). While HDR is absent in G1, NHEJ is active throughout the cell cycle and, thus, is largely favored over HDR. We devised a strategy to increase HDR by directly synchronizing the expression of Cas9 with cell-cycle progression. Fusion of Cas9 to the N-terminal region of human Geminin converted this gene-editing protein into a substrate for the E3 ubiquitin ligase complex APC/Cdh1, resulting in a cell-cycle-tailored expression with low levels in G1 but high expression in S/G2/M. Importantly, Cas9-hGem(1/110) increased the rate of HDR by up to 87% compared to wild-type Cas9. Future developments may enable high-resolution expression of genome engineering proteins, which might increase HDR rates further, and may contribute to a better understanding of DNA repair pathways due to spatiotemporal control of DNA damage induction ...
The kinase ATM is classically known for its role in coordinating the response to DNA damage. DNA damage is caused by various intracellular and extracellular stimuli, including oxidative stress and free radicals. Lee et al. found critical amino acid residues that enable ATM to coordinate a response to DNA damage that is independent of its response to oxidative stress. Activation of ATM by either pathway promoted mitochondrial function and autophagy, thus mediating cell survival through metabolic changes. ATM activation via oxidative stress additionally promoted the clearance of toxic protein aggregates. These findings expand the roles of ATM and suggest that the loss of ATM function, such as in the neurodegenerative disease ataxia telangiectasia (A-T), causes broader cellular stress than that limited to a defective DNA damage response. ...
DNA damage (or RNA damage in the case of some virus genomes) appears to be a fundamental problem for life. As noted by Haynes,[14] the subunits of DNA are not endowed with any peculiar kind of quantum mechanical stability, and thus DNA is vulnerable to all the chemical horrors that might befall any such molecule in a warm aqueous medium. These chemical horrors are DNA damages that include various types of modification of the DNA bases, single- and double-strand breaks, and inter-strand cross-links (see DNA damage (naturally occurring). DNA damages are distinct from mutations although both are errors in the DNA. Whereas DNA damages are abnormal chemical and structural alterations, mutations ordinarily involve the normal four bases in new arrangements. Mutations can be replicated, and thus inherited when the DNA replicates. In contrast, DNA damages are altered structures that cannot, themselves, be replicated. Several different repair processes can remove DNA damages (see chart in DNA repair). ...
Time resolved data of DNA damage and repair after radiotherapy elucidates the relation between damage, repair, and cell survival. While well characterized in vitro, little is known about the time-course of DNA damage response in tumors sampled from individual patients. Kinetics of DNA damage after radiotherapy was assessed in eight dogs using repeated in vivo samples of tumor and co-irradiated normal tissue analyzed with comet assay and phosphorylated H2AX (γH2AX) immunohistochemistry. In vivo results were then compared (in silico) with a dynamic mathematical model for DNA damage formation and repair. Maximum %DNA in tail was observed at 15-60 min after irradiation, with a rapid decrease. Time-courses of γH2AX-foci paralleled these findings with a small time delay and were not influenced by covariates. The evolutionary parameter search based on %DNA in tail revealed a good fit of the DNA repair model to in vivo data for pooled sarcoma time-courses, but fits for individual sarcoma time-courses suffer
By Kristian Moss Bendtsen, Jeppe Juul, Ala Trusina. DNA damages, as well as mutations, increase with age. It is believed that these result from increased genotoxic stress and decreased capacity for DNA repair. The two causes are not independent, DNA damage can, for example, through mutations, compromise the capacity for DNA repair, which in turn increases the amount of unrepaired DNA damage. Despite this vicious circle, we ask, can cells maintain a high DNA repair capacity for some time or is repair capacity bound to continuously decline with age? We here present a simple mathematical model for ageing in multicellular systems where cells subjected to DNA damage can undergo full repair, go apoptotic, or accumulate mutations thus reducing DNA repair capacity. Our model predicts that at the tissue level repair rate does not continuously decline with age, but instead has a characteristic extended period of high and non-declining DNA repair capacity, followed by a rapid decline. Furthermore, the time ...
Upon viral infection, the major defense mounted by the host innate immune system is activation of the IFN- and apoptosis-mediated antiviral pathway. In order to complete their life cycle, viruses that are obligatory intracellular parasites must modulate these host immune responses. We have previously shown that the γHV68 latency-associated M2 protein effectively downregulates STAT1 and STAT2, resulting in the inhibition of type I and II IFN-mediated transcriptional activation. Here, we demonstrate that M2 interacts with ATM, a DNA damage signal transducer, and the DDB1/COP9/cullin DNA damage effector complex. This interaction blocked DNA damage-sensing activity as well as DNA damage repair activity, thereby rendering cells resistant to DNA damage-induced apoptosis. These results indicate that γHV68 encodes M2, a latency-associated gene, to antagonize both IFN- and apoptosis-mediated host innate immunities and thus is important in establishing and maintaining viral latency in infected ...
The endoribonuclease Dicer is a key component of the human RNA interference pathway and is known for its role in cytoplasmic microRNA production. Recent findings suggest that noncanonical Dicer generates small noncoding RNA to mediate the DNA damage response (DDR). Here, we show that human Dicer is phosphorylated in the platform-Piwi/Argonaute/Zwille-connector helix cassette (S1016) upon induction of DNA damage. Phosphorylated Dicer (p-Dicer) accumulates in the nucleus and is recruited to DNA double-strand breaks. We further demonstrate that turnover of damage-induced nuclear, double-stranded (ds) RNA requires additional phosphorylation of carboxy-terminal Dicer residues (S1728 and S1852). DNA damage-induced nuclear Dicer accumulation is conserved in mammals. Dicer depletion causes endogenous DNA damage and delays the DDR by impaired recruitment of repair factors MDC1 and 53BP1. Collectively, we place Dicer within the context of the DDR by demonstrating a DNA damage-inducible phosphoswitch that causes
This is the first pathway out of two pathways which deals with DNA damage response. It has two central gene products (ATM and ATR) which are connected to the sources of DNA damage (in blue). The two central genes can be divides furthermore into their most important genes. In the ATM pathway are the most important genes TP53 and CHEK2 and on the other hand in the ATR pathway is this CHEK1. If it is not mentioned different, the processes take place in the cell cytoplasm. The goal of this first pathway is to give an overview of the most important gene products, processes and changes in the cell condition through the DNA damage response pathway and at the same time to keep it clearly ...
MCC is a potential tumor suppressor gene, which is silenced by promoter hypermethylation in a subset of colorectal cancers. However, its functions have remained poorly understood. In the present study, we describe a novel function of MCC in the DNA damage response. Several novel phosphorylation sites were identified by mass spectrometry, including 2 highly conserved ATM/ATR consensus sites at serine 118 and serine 120. In addition, exposure to ultraviolet radiation (UV), but not phleomycin, caused PI3K-dependent phosphorylation of MCC and its nuclear localization. Re-expression of MCC in HCT15 colorectal cancer cells led to a G2/M arrest, and MCC knockdown impaired the induction of a G2/M arrest following UV radiation. Finally, mutation of S118/120 to alanine did not affect MCC nuclear shuttling following UV but did impair MCC G2/M checkpoint activity. Thus, these results suggest that MCC is a novel target of the DNA damage checkpoint and that MCC is required for the complete cell cycle arrest in the G2
The 25 gene deletions that conferred increased Dna2 foci were strongly enriched for genes involved in DNA repair and DNA damage response (Figure 4B) (P = 2×10−17 and P = 5×10−16). We compared these genes to those identified in a recent constitutive RNR3 expression screen (Hendry et al. 2015) and found significant overlap (16 genes, hypergeometric P = 4×10−21), suggesting the presence of increased spontaneous DNA damage in these mutants, as expression of RNR3 responds specifically to DNA damage (Elledge and Davis 1990). We compared the genes that, when deleted, caused increased Dna2 foci to those that cause increased Rad52 foci (Alvaro et al. 2007), again finding significant overlap (10 genes, hypergeometric P = 2×10−11). Finally, we compared the set of genes with negative genetic interactions with dna2-1 or dna2-2 (Budd et al. 2005), which could indicate spontaneous damage that requires Dna2 for its repair. We noted a significant overlap (10 genes, hypergeometric P = 2×10−14). ...
Stem cell dysfunction is closely linked to tissue and organismal aging and age-related diseases, and heavily influenced by the niche cells environment. The DNA damage response (DDR) is a key pathway for tissue degeneration and organismal aging; however, the precise protective role of DDR in stem cell/niche aging is unclear. The Drosophila midgut is an excellent model to study the biology of stem cell/niche aging because of its easy genetic manipulation and its short lifespan. Here, we showed that deficiency of DDR in Drosophila enterocytes (ECs) accelerates intestinal stem cell (ISC) aging. We generated flies with knockdown of Mre11, Rad50, Nbs1, ATM, ATR, Chk1, and Chk2, which decrease the DDR system in ECs. EC-specific DDR depletion induced EC death, accelerated the aging of ISCs, as evidenced by ISC hyperproliferation, DNA damage accumulation, and increased centrosome amplification, and affected the adult flys survival. Our data
Stem cell dysfunction is closely linked to tissue and organismal aging and age-related diseases, and heavily influenced by the niche cells environment. The DNA damage response (DDR) is a key pathway for tissue degeneration and organismal aging; however, the precise protective role of DDR in stem cell/niche aging is unclear. The Drosophila midgut is an excellent model to study the biology of stem cell/niche aging because of its easy genetic manipulation and its short lifespan. Here, we showed that deficiency of DDR in Drosophila enterocytes (ECs) accelerates intestinal stem cell (ISC) aging. We generated flies with knockdown of Mre11, Rad50, Nbs1, ATM, ATR, Chk1, and Chk2, which decrease the DDR system in ECs. EC-specific DDR depletion induced EC death, accelerated the aging of ISCs, as evidenced by ISC hyperproliferation, DNA damage accumulation, and increased centrosome amplification, and affected the adult flys survival. Our data
In mammalian cells, RB/E2F and p53 are intimately connected, and crosstalk between these pathways is critical for the induction of cell cycle arrest or cell death in response to cellular stresses. Here we have investigated the genetic interactions between RBF/E2F and p53 pathways during Drosophila development. Unexpectedly, we find that the pro-apoptotic activities of E2F and p53 are independent of one another when examined in the context of Drosophila development: apoptosis induced by the deregulation of dE2F1, or by the overexpression of dE2F1, is unaffected by the elimination of dp53; conversely, dp53-induced phenotypes are unaffected by the elimination of dE2F activity. However, dE2F and dp53 converge in the context of a DNA damage response. Both dE2F1/dDP and dp53 are required for DNA damage-induced cell death, and the analysis of rbf1 mutant eye discs indicates that dE2F1/dDP and dp53 cooperatively promote cell death in irradiated discs. In this context, the further deregulation in the expression
Radiotherapy resistance is one of the major factors limiting the efficacy of radiotherapy in lung cancer patients. The extensive investigations indicate the diversity in the mechanisms underlying radioresistance. Here, we revealed that DNA damage binding protein 2 (DDB2) is a potential regulator in the radiosensitivity of non-small cell lung cancer (NSCLC) cells. DDB2, originally identified as a DNA damage recognition factor in the nucleotide excision repair, promotes the survival and inhibits the apoptosis of NSCLC cell lines upon ionizing radiation (IR). Mechanistic investigations demonstrated that DDB2 is able to facilitate IR-induced phosphorylation of Chk1, which plays a critical role in the cell cycle arrest and DNA repair in response to IR-induced DNA double-strand breaks (DSBs). Indeed, knockdown of DDB2 compromised the G2 arrest in the p53-proficient A549 cell line and reduced the efficiency of homologous recombination (HR) repair. Taken together, our data indicate that the expression of DDB2
Stress overload of the DDR pathway should also show efficacy in cancer treatment. Although it is not clear why DNA damaging agents, such as IR and chemotherapy, are effective cancer therapies, it is possible that these are examples of stress overload, where cancer cells with already elevated levels of DNA damage and replication stress cannot repair the additional damage inflicted by these agents. An alternative explanation is that during tumorigenesis, the persistence of DNA damage selects for cells with mutations that abrogate part of the DDR pathway and therefore cannot properly sense and respond to DNA damage. These cells with a partially defective DDR might therefore be more vulnerable to the extensive DNA damage resulting from radiation or chemotherapy that is lethal without a normal DDR pathway. In this context, DNA damage exploits a stress phenotype of tumors that is analogous to non-oncogene addition.. Given the sensitivity of many cancers to DNA damaging agents, there should exist genes ...
Research has indicated that oxidative stress is the cause of many serious diseases such as cancer, Alzheimers, arteriosclerosis and diabetes.
Emerging data strongly suggest that the oxidation of DNA bases can contribute to genomic instability. Structural changes to DNA, induced by base oxidation, may reduce the fidelity of DNA replication and interfere with sequence-specific DNA−protein interactions. We have examined the structures of a series of pyrimidine deoxynucleoside oxidation damage products in aqueous solution. The modified nucleosides studied include the deoxynucleoside derivatives of 5-hydroxyuracil, 5-hydroxycytosine, 5-(hydroxymethyl)uracil, 5-(hydroxymethyl)cytosine, 5-formyluracil, and 5-formylcytosine. The influence of base oxidation on ionization constants, sugar conformation, and tautomeric configuration has been determined on the basis of UV, proton, and nitrogen NMR spectra of the ^(15)N-enriched derivatives. The potential biological consequences of the structural perturbations resulting from base oxidation are discussed. ...
DNA damage and repair and their role in carcinogenesis A DNA sequence can be changed by copying errors introduced by DNA polymerase during replication and by environmental agents such as chemical mutagens or radiation If uncorrected, such changes may interfere with the ability of the cell to function DNA damage can be repaired by several mechanisms All carcinogens cause changes in the DNA sequence and thus DNA damage and repair are important aspects in the development of cancer Prokaryotic and eukaryotic DNA-repair systems are analogous
Cells of metazoan organisms respond to DNA damage by arresting their cell cycle to repair DNA, or they undergo apoptosis. Two protein kinases, ataxia-telangiectasia mutated (ATM) and ATM and Rad-3 related (ATR), are sensors for DNA damage. In humans, ATM is mutated in patients with ataxia-telangiectasia (A-T), resulting in hypersensitivity to ionizing radiation (IR) and increased cancer susceptibility. Cells from A-T patients exhibit chromosome aberrations and excessive spontaneous apoptosis. We used Drosophila as a model system to study ATM function. Previous studies suggest that mei-41 corresponds to ATM in Drosophila; however, it appears that mei-41 is probably the ATR ortholog. Unlike mei-41 mutants, flies deficient for the true ATM ortholog, dATM, die as pupae or eclose with eye and wing abnormalities. Developing larval discs exhibit substantially increased spontaneous chromosomal telomere fusions and p53-dependent apoptosis. These developmental phenotypes are unique to dATM, and both dATM ...
Glioblastoma multiforme (GBM) is the most common form of brain tumor with a poor prognosis and resistance to radiotherapy. Recent evidence suggests that glioma-initiating cells play a central role in radioresistance through DNA damage checkpoint activation and enhanced DNA repair. To investigate this in more detail, we compared the DNA damage response in nontumor forming neural progenitor cells (NPC) and glioma-initiating cells isolated from GBM patient specimens. As observed for GBM tumors, initial characterization showed that glioma-initiating cells have long-term self-renewal capacity. They express markers identical to NPCs and have the ability to form tumors in an animal model. In addition, these cells are radioresistant to varying degrees, which could not be explained by enhanced nonhomologous end joining (NHEJ). Indeed, NHEJ in glioma-initiating cells was equivalent, or in some cases reduced, as compared with NPCs. However, there was evidence for more efficient homologous recombination ...
TY - JOUR. T1 - The novel histone deacetylase inhibitor, LBH589, induces expression of DNA damage response genes and apoptosis in Ph - acute lymphoblastic leukemia cells. AU - Scuto, Anna. AU - Kirschbaum, Mark. AU - Kowolik, Claudia. AU - Kretzner, Leo. AU - Juhasz, Agnes. AU - Atadja, Peter. AU - Pullarkat, Vinod. AU - Bhatia, Ravi. AU - Forman, Stephen. AU - Yen, Yun. AU - Jove, Richard. PY - 2008/5/15. Y1 - 2008/5/15. N2 - We investigated the mechanism of action of LBH589, a novel broad-spectrum HDAC inhibitor belonging to the hydroxamate class, in Philadelphia chromosome-negative (Ph -) acute lymphoblastic leukemia (ALL). Two model human Ph - ALL cell lines (T-cell MOLT-4 and pre-B-cell Reh) were treated with LBH589 and evaluated for biologic and gene expression responses. Low nanomolar concentrations (IC 50:5-20 nM) of LBH589 induced cell-cycle arrest, apoptosis, and histone (H3K9 and H4K8) hyperacetylation. LBH589 treatment increased mRNA levels of proapoptosis, growth arrest, and DNA ...
TY - JOUR. T1 - Suppression of replication fork progression in low-dose-specific p53-dependent S-phase DNA damage checkpoint. AU - Shimura, T.. AU - Toyoshima, M.. AU - Adiga, S. K.. AU - Kunoh, T.. AU - Nagai, H.. AU - Shimizu, N.. AU - Inoue, M.. AU - Niwa, O.. PY - 2006/9/28. Y1 - 2006/9/28. N2 - The S-phase DNA damage checkpoint is activated by DNA damage to delay DNA synthesis allowing time to resolve the replication block. We previously discovered the p53-dependent S-phase DNA damage checkpoint in mouse zygotes fertilized with irradiated sperm. Here, we report that the same p53 dependency holds in mouse embryonic fibroblasts (MEFs) at low doses of irradiation. DNA synthesis in p53 wild-type (WT) MEFs was suppressed in a biphasic manner in which a sharp decrease below 2.5 Gy was followed by a more moderate decrease up to 10 Gy. In contrast, p53-/- MEFs exhibited radioresistant DNA synthesis below 2.5 Gy whereas the cells retained the moderate suppression above 5 Gy. DNA fiber analysis ...
TY - JOUR. T1 - DNA damage and breast cancer risk. AU - Smith, Tasha R.. AU - Miller, Mark S.. AU - Lohman, Kurt K.. AU - Case, L. Douglas. AU - Hu, Jennifer H.. PY - 2003/5/1. Y1 - 2003/5/1. N2 - To evaluate whether deficient DNA repair contributes to elevated DNA damage and breast carcinogenesis, we used the comet assay (single-cell alkaline gel electrophoresis) to measure the levels of DNA damage in peripheral lymphocytes from 70 breast cancer cases and 70 controls. DNA damage, measured as the comet tail moment, was not influenced by age, family history (FH), age at menarche, age at first birth or parity. The results showed that cancer cases had significantly higher DNA damage compared with controls; the comet tail moments (mean ± SD) for cases and controls were: 10.78 ± 3.63 and 6.86 ± 2.76 (P , 0.001) for DNA damage at baseline (DB), 21.24 ± 4.88 and 14.97 ± 4.18 (P , 0.001) for DNA damage after exposure to 6 Gy of ionizing radiation (DIR), and 14.76 ± 5.35 and 9.75 ± 3.35 (P , ...
Compounds of nickel(II) and cadmium(II) are carcinogenic to humans and to experimental animals. One frequently discussed mechanism involved in tumor formation is an increase in reactive oxygen species by both metals with the subsequent generation of oxidative DNA damage. In the present study we used human HeLa cells to investigate the potential of nickel(II) and cadmium(II) to induce DNA lesions typical for oxygen free radicals in intact cells and the effect on their repair. As indicators of oxidative DNA damage, we determined the frequencies of DNA strand breaks and of lesions recognized by the bacterial formamidopyrimidine-DNA glycosylase (Fpg protein), including 7,8-dihydro-8-oxoguanine (8-hydroxyguanine), a pre-mutagenic DNA base modification. Nickel(II) caused a slight increase in DNA strand breaks at 250 microM and higher, while the frequency of Fpg-sensitive sites was enhanced only at the cytotoxic concentration of 750 microM. The repair of oxidative DNA lesions induced by visible light ...
To investigate how the nucleotide excision repair initiator XPC locates DNA damage in mammalian cell nuclei we analyzed the dynamics of GFP-tagged XPC. Photobleaching experiments showed that XPC constantly associates with and dissociates from chromatin in the absence of DNA damage. DNA-damaging agents retard the mobility of XPC, and UV damage has the most pronounced effect on the mobility of XPC-GFP. XPC exhibited a surprising distinct dynamic behavior and subnuclear distribution compared with other NER factors. Moreover, we uncovered a novel regulatory mechanism for XPC. Under unchallenged conditions, XPC is continuously exported from and imported into the nucleus, which is impeded when NER lesions are present. XPC is omnipresent in the nucleus, allowing a quick response to genotoxic stress. To avoid excessive DNA probing by the low specificity of the protein, the steady-state level in the nucleus is controlled by nucleus-cytoplasm shuttling, allowing temporally higher concentrations of XPC in ...
Cancer is one of the most unwanted menaces in the human body. Cancers of lower abdomen are not only life threatening but also painful and survival rate is low. Cervical cancer is second most common worldwide and fifth deadliest in women. It affects about 16 per 100,000 women per year and kills about 9 per 100,000 in a year. In developing countries occurrence rate is 80%. It is possible that there may be no symptom until an advance stage of the cancer is progressed. The single cell gel electrophoresis assay also called comet assay, which is versatile, reliable, powerful, uncomplicated and sensitive technique for detection of DNA damage at the level of individual eukaryotic cell. Understanding the extent of DNA damage in neoplastic cells discarded in the urine of cancer patients through comet assay. Usually normal urine sample have very rare cells. In case of cancer patient the number of cells increases drastically. The type of cells passed in the urine of cancer patient contains mainly ...
Many cancers display both structural (s-CIN) and numerical (w-CIN) chromosomal instabilities. Defective chromosome segregation during mitosis has been shown to cause DNA damage that induces structural rearrangements of chromosomes (s-CIN). In contrast, whether DNA damage can disrupt mitotic processes to generate whole chromosomal instability (w-CIN) is unknown. Here we show that activation of the DNA damage response (DDR) during mitosis selectively stabilizes kinetochore-microtubule (k-MT) attachments to chromosomes through Aurora-A and Plk1 kinases, thereby increasing the frequency of lagging chromosomes during anaphase. Inhibition of DDR proteins, ATM or Chk2, abolishes the effect of DNA damage on k-MTs and chromosome segregation, whereas activation of the DDR in the absence of DNA damage is sufficient to induce chromosome segregation errors. Finally, inhibiting the DDR during mitosis in cancer cells with persistent DNA damage suppresses inherent chromosome segregation defects. Thus, DDR ...
Coordination of the multiple processes underlying DNA replication is key for maintaining genome stability and preventing tumorigenesis. CLASPIN, a critical player in replication fork stabilization and checkpoint responses, must be tightly regulated during the cell cycle to prevent the accumulation of DNA damage. In this study, we used a quantitative proteomics approach and identified USP9X as a novel CLASPIN-interacting protein. USP9X is a deubiquitinase involved in multiple signaling and survival pathways whose tumor suppressor or oncogenic activity is highly context dependent. We found that USP9X regulated the expression and stability of CLASPIN in an S-phase-specific manner. USP9X depletion profoundly impairs the progression of DNA replication forks, causing unscheduled termination events with a frequency similar to CLASPIN depletion, resulting in excessive endogenous DNA damage. Importantly, restoration of CLASPIN expression in USP9X-depleted cells partially suppressed the accumulation of ...
TY - JOUR. T1 - Comparison of UVB and UVC effects on the DNA damage-response protein 53BP1 in human pancreatic cancer. AU - Uehara, Fuminari. AU - Miwa, Shinji. AU - Tome, Yasunori. AU - Hiroshima, Yukihiko. AU - Yano, Shuya. AU - Yamamoto, Mako. AU - Efimova, Elena. AU - Matsumoto, Yasunori. AU - Maehara, Hiroki. AU - Bouvet, Michael. AU - Kanaya, Fuminori. AU - Hoffman, Robert M.. N1 - Copyright: Copyright 2016 Elsevier B.V., All rights reserved.. PY - 2014/10. Y1 - 2014/10. N2 - We have previously demonstrated that ultraviolet (UV) light is effective against a variety of cancer cells expressing fluorescent proteins in vivo as well as in vitro. In the present report, we compared the DNA damage repair (DDR) response of pancreatic cancer cells after UVB or UVC irradiation. The UV-induced DNA damage repair was imaged with green fluorescent protein (GFP) fused to the DDR-related chromatin-binding protein 53BP1 in MiaPaCa-2 human pancreatic cancer cells growing in 3D Gelfoam® histoculture and in ...
TY - JOUR. T1 - Yeast Rap1 contributes to genomic integrity by activating DNA damage repair genes. AU - Tomar, Raghuvir S.. AU - Zheng, Suting. AU - Brunke-Reese, Deborah. AU - Wolcott, Holly N.. AU - Reese, Joseph C.. N1 - Copyright: Copyright 2008 Elsevier B.V., All rights reserved.. PY - 2008/6/4. Y1 - 2008/6/4. N2 - Rap1 (repressor-activator protein 1) is a multifunctional protein that controls telomere function, silencing and the activation of glycolytic and ribosomal protein genes. We have identified a novel function for Rap1, regulating the ribonucleotide reductase (RNR) genes that are required for DNA repair and telomere expansion. Both the C terminus and DNA-binding domain of Rap1 are required for the activation of the RNR genes, and the phenotypes of different Rap1 mutants suggest that it utilizes both regions to carry out distinct steps in the activation process. Recruitment of Rap1 to the RNR3 gene is dependent on activation of the DNA damage checkpoint and chromatin remodelling by ...
Here, we report that genome editing by CRISPR-Cas9 induces a p53-mediated DNA damage response and cell cycle arrest in immortalized human retinal pigment epithelial cells, leading to a selection against cells with a functional p53 pathway. Inhibition of p53 prevents the damage response and increases the rate of homologous recombination from a donor template. These results suggest that p53 inhibition may improve the efficiency of genome editing of untransformed cells and that p53 function should be monitored when developing cell-based therapies utilizing CRISPR-Cas9. CRISPR-Cas9-induced DNA damage triggers p53 to limit the efficiency of gene editing in immortalized human retinal pigment epithelial cells.
The Comet Assay, also called single cell gel electrophoresis (SCGE), is a sensitive and rapid technique for quantifying and analyzing DNA damage in individual cells.
BACKGROUND. Chronic obstructive pulmonary disease (COPD) is characterised by oxidative stress and increased risk of lung carcinoma. Oxidative stress causes DNA damage which can be repaired by DNA-dependent protein kinase complex.. OBJECTIVES. To investigate DNA damage/repair balance and DNA-dependent protein kinase complex in COPD lung and in an animal model of smoking-induced lung damage and to evaluate the effects of oxidative stress on Ku expression and function in human bronchial epithelial cells.. METHODS. Protein expression was quantified using immunohistochemistry and/or western blotting. DNA damage/repair was measured using colorimetric assays.. RESULTS. 8-OH-dG, a marker of oxidant-induced DNA damage, was statistically significantly increased in the peripheral lung of smokers (with and without COPD) compared with non-smokers, while the number of apurinic/apyrimidinic (AP) sites (DNA damage and repair) was increased in smokers compared with non-smokers (p = 0.0012) and patients with COPD ...
The recent observations that both DNA adducts and oxidative base damage are increased in the brains from Alzheimer s and Parkinson s patients and in spinal cord tissue of patients with amyotrophic lateral sclerosis (ALS) support the idea that oxidative DNA damage may contribute to the observed loss of neurons in these neurological disorders (8,9). Therefore, understanding how oxidative DNA damage is repaired (or not) is critical for understanding the pathology underlying these diseases. The overall hypothesis of this proposal is that oxidative stress induced DNA damage in neuronal cell lines leading to cellular malfunction and/or death and DNA repair enzymes are critical for the protection of neurons against oxidative stress. The following specific Aims will address this hypothesis. Specific Aim 1: Functional and biochemical characterization of the repair enzymes Ogg1, NTH, MTH, and MYH in the neuroblastoma cell lines SHSY5Y and Neuro-2A and in primary cultures of hippocampal neurons. We will ...
UV-induced damage can induce apoptosis or trigger DNA repair mechanisms. Minor DNA damage is thought to halt the cell cycle to allow effective repair, while more severe damage can induce an apoptotic program. Of the two major types of UV-induced DNA lesions, it has been reported that repair of CPD, but not 6-4PP, abrogates mutation. To address whether the two major forms of UV-induced DNA damage, can induce differential biological effects, NER-deficient cells containing either CPD photolyase or 6-4 PP photolyase were exposed to UV and examined for alterations in cell cycle and apoptosis. In addition, pTpT, a molecular mimic of CPD was tested in vitro and in vivo for the ability to induce cell death and cell cycle alterations. NER-deficient XPA cells were stably transfected with CPD-photolyase or 6-4PP photolyase to specifically repair only CPD or only 6-4PP. After 300 J/m2 UVB exposure photoreactivation light (PR, UVA 60 kJ/m2) was provided for photolyase activation and DNA repair. Apoptosis was
BioTek Note applicative, 07-Jul-16, Automated Comet Assay Imaging and Dual-Mask Analysis to Determine DNA Damage on an Individual Comet Basis
The underlying cause of cancer is spontaneous mutations introduced to genomic DNA. Reactive products of cellular metabolism and external genotoxic agents cause persistent DNA damage, which is constantly removed through various DNA repair mechanisms. It is unavoidable, however, that some DNA modifications (lesions) persist into S-phase, creating blocks for progression of the DNA replication machinery. To circumvent this problem organisms in all kingdoms of life have evolved DNA damage tolerance pathways, employing specialized enzymes that bypass DNA lesions while temporarily leaving DNA damage unrepaired. The vast majority of mutations are introduced in the genome by enzymes of error-prone branch of DNA damage tolerance - translesion DNA synthesis (TLS). Genetic changes that ensue as a result of TLS are at the root of the onset of cancer and the development of various resistance mechanisms displayed by relapsed tumors, which represents a major problem for treatment of some types of cancer, ...
Here, we demonstrate that OTUD4 may serve as a master regulator of alkylation damage resistance through stabilization of the human AlkB homologues. A number of distinct lines of evidence support this role for OTUD4. First, OTUD4 interacts specifically with ALKBH2 and ALKBH3 and encodes a K48‐specific DUB (Fig 1). Consistently, ALKBH3 is subjected to K48‐linked ubiquitination and proteasomal degradation (Fig 2A-D). OTUD4 antagonizes ALKBH3 ubiquitination and stabilizes both ALKBH2 and ALKBH3 in vivo (Fig 2E-H). ALKBH3 protein levels do not correlate well with ALKBH3 mRNA levels in various tumor cell lines but do correlate with OTUD4 levels (Supplementary Fig S2). Finally, overexpression of ALKBH3 in PC‐3 cells, which depend primarily on ALKBH3 instead of ALKBH2 for alkylation damage resistance, is sufficient to rescue alkylation damage sensitivity upon loss of OTUD4 (Fig 7G).. What is most striking is that we find OTUD4 catalytic activity to be apparently dispensable for its stabilization ...
The Mre11-Rad50-Nbs1 (MRN) complex has many biological functions: processing of double-strand breaks in meiosis, homologous recombination, telomere maintenance, S-phase checkpoint, and genome stability during replication. In the S-phase DNA damage checkpoint, MRN acts both in activation of checkpoint signaling and downstream of the checkpoint kinases to slow DNA replication. Mechanistically, MRN, along with its cofactor Ctp1, is involved in 5 resection to create single-stranded DNA that is required for both signaling and homologous recombination. However, it is unclear whether resection is essential for all of the cellular functions of MRN. To dissect the various roles of MRN, we performed a structure-function analysis of nuclease dead alleles and potential separation-of-function alleles analogous to those found in the human disease ataxia telangiectasia-like disorder, which is caused by mutations in Mre11. We find that several alleles of rad32 (the fission yeast homologue of mre11), along with
Nitric oxide (NO) as well as its donors has been shown to generate mutation and DNA damage in in vitro assays. The objective of this study was to identify that DNA single-strand breaks (SSBs) could be elicited by NO, not only in vitro but also in vivo. The alkaline single-cell gel electrophoresis (SCGE) was performed to examine the DNA damage in g12 cells and the cells isolated from the organs of mice exposed to sodium nitroprusside (SNP). A modified method, in which neither collagenase nor trypsin was necessary, was used to prepare the single-cell suspension isolated from organs of mice. Results showed that the exposure of g12 cells to 0.13-0.5 mu mol/ml SNP with S9 for 1 h induced a concentration-dependent increase in DNA SSBs in g12 cells. The significant increase in DNA migration and comet frequency has appeared in the cells isolated from the spleen, thymus, and peritoneal macrophages of mice after injecting i.p. SNP in the dosage range of 0.67-6.0 mg/kg b.wt for 1 h. However, no obvious ...
Article Effects of proton beams from the folded tandem ion accelerator on DNA damage in mouse leukocytes using the comet assay. Single cell gel electrophoresis or the comet assay is a sensitive technique to quantify different types of DNA damage, e.g...
This unit describes immunocytochemical detection of phosphorylated histone H2AX for revealing the presence of DNA double-strand breaks. Double-strand breaks indicate DNA damage induced by ionizing radiation or by treatment with antitumor drugs such as DNA topoisomerase inhibitors. However, double-strand breaks can also be intrinsic, occurring in healthy, nontreated cells for a variety of reasons, and are generated in the course of DNA fragmentation in apoptotic cells. The unit presents strategies to distinguish radiation- or drug-induced breaks from those intrinsically formed in untreated cells or associated with apoptosis. The protocol describes the immunocytochemical detection of histone H2AX phosphorylated on Ser-139 combined with measurement of DNA content to identify cells that have DNA double-strand breaks and to concurrently assess their cell cycle phase. The detection is based on indirect immunofluorescence using a FITC-labeled secondary antibody, and DNA is counterstained with propidium ...
Time-lapse imaging can make complicated processes easier to grasp-think of a stitched-together sequence of photos that chronicles the construction of a building. Now, scientists from the Department of Energys Lawrence Berkeley National Laboratory (Berkeley Lab) are using a similar approach to study how cells repair DNA damage.
TY - JOUR. T1 - Cytogenetic analysis of human cells reveals specific patterns of DNA damage in replicative and oncogene-induced senescence. AU - Falcone, Germana. AU - Mazzola, Alessia. AU - Michelini, Flavia. AU - Bossi, Gianluca. AU - Censi, Federica. AU - Biferi, Maria G.. AU - Minghetti, Luisa. AU - Floridia, Giovanna. AU - Federico, Maurizio. AU - Musio, Antonio. AU - Crescenzi, Marco. PY - 2013. Y1 - 2013. N2 - Senescence is thought to be triggered by DNA damage, usually indirectly assessed as activation of the DNA damage response (DDR), but direct surveys of genetic damage are lacking. Here, we mitotically reactivate senescent human fibroblasts to evaluate their cytogenetic damage. We show that replicative senescence is generally characterized by telomeric fusions. However, both telomeric and extratelomeric aberrations are prevented by hTERT, indicating that even non-telomeric damage descends from the lack of telomerase. Compared with replicative senescent cells, oncogene-induced ...
TY - JOUR. T1 - Defense mechanism to oxidative DNA damage in glial cells. AU - Iida, Takashi. AU - Furuta, Akiko. AU - Nakabeppu, Yusaku. AU - Iwaki, Toru. PY - 2004/6/1. Y1 - 2004/6/1. N2 - Astrocytosis is a sequential morphological change of astrocytic reaction to tissue damage, and is associated with regulation of antioxidant defense mechanisms to reduce oxidative damage. The repair enzymes to oxidative DNA damage, oxidized purine-nucleoside triphosphatase (hMTH1) and a mitochondrial type of 8-oxoguanine DNA glycosylase (hOGG1-2a) in brain tumors and neurons of Alzheimers disease, were previously reported. In the present study, glial expression of these repair enzymes under such pathological conditions as cerebrovascular diseases and metastatic brain tumors, were investigated. Furthermore, an in-vitro experiment using a glioma cell-line under oxidative stress was performed to verify the immunohistochemical results of post-mortem materials. As a result, hOGG1-2a immunoreactivities in reactive ...
L to R) David Jeruzalmi, Danaya Pakotiprapha, and Martin Samuels. DNA damage is a fact of life for all living organisms. Some have estimated that each of our human cells absorbs thousands of damaging events each and every day. Left uncorrected, such damage can lead to permanent, disruptive, changes in the genome, and cause various diseases including cancer. Without an active method of patrolling the genome for damage, and repairing it when found, life on earth would not last very long. Thus, all organisms deploy a panoply of DNA repair pathways. One of these, the nucleotide excision repair (NER) pathway, is unique for its ability to repair a chemically diverse set of lesions that each alters the structure of DNA in profoundly different ways. Conserved throughout evolution, NER involves several major steps: genome scanning, damage recognition, incision, and repair synthesis. Mutations in this pathway give rise to many human diseases including xeroderma pigmentosum, Cockayne syndrome, and ...
Genome instability (also genetic instability or genomic instability) refers to a high frequency of mutations within the genome of a cellular lineage. These mutations can include changes in nucleic acid sequences, chromosomal rearrangements or aneuploidy. Genome instability does occur in bacteria. In multicellular organisms genome instability is central to carcinogenesis, and in humans it is also a factor in some neurodegenerative diseases such as amyotrophic lateral sclerosis or the neuromuscular disease myotonic dystrophy. The sources of genome instability have only recently begun to be elucidated. A high frequency of externally caused DNA damage can be one source of genome instability since DNA damages can cause inaccurate translesion synthesis past the damages or errors in repair, leading to mutation. Another source of genome instability may be epigenetic or mutational reductions in expression of DNA repair genes. Because endogenous (metabolically-caused) DNA damage is very frequent, ...
Pro-carcinogens, such as benzoapyrene (BaP), that are exogenous ligands of the aromatic hydrocarbon receptor may influence the susceptibility of target-cell populations through the up-regulation of cytochrome P450 (CYP) mixed function oxidases. We examined whether the growth kinetics of MCF-7 cells might determine the level of up-regulation of CYP1A1, CYP1A2 or CYP1B1 by BaP, and whether this could then influence subsequent levels of DNA damage. Cell cultures manipulated to be G0/G1-phase concentrated, S-phase concentrated or G2/M-phase concentrated were treated with BaP and the expression levels of CYP1A1, CYP1A2, CYP1B1, cyclin-dependent kinase inhibitor 1A CDKN1A (P21WAF1/CIP1), B-cell leukaemia/lymphoma-2 (BCL-2), and Bcl-2-associated X levels were determined. Levels of DNA damage were measured as DNA single-strand breaks (SSBs) by the alkaline single-cell gel electrophoresis (comet) assay or as DNA adducts by 32P-postlabelling analysis. BaP-induced up-regulation of CYP1A1 was ...
Every day we are exposed to carcinogens, including ultraviolet radiation (UVR). High levels of ultraviolet radiation are known to cause DNA damage. One of the most common forms of UVR-induced damage, the pyrimidine dimer, is repaired by an enzymatic reaction powered by visible light. We wanted to find out if there is variation in the level of UVR-induced DNA damage induced in two different clones of Daphnia magna, a model organism for ecotoxicology. One clone was derived from a mid-latitude deep reservoir, where escape from UVR is possible via vertical migration. The second clone was from a high-latitude shallow rock pool, where D. magna are exposed to high levels of UVR. Pregnant mothers from each clone line were subjected to ecologically relevant levels of UVR in the lab. Immediately afterwards, we extracted the embryos, suspended the cells in agarose, and performed a comet assay, which allows for quantification of DNA damage within individual cells. The slides were viewed under a fluorescent
Morphological transformation of C3H/M2 mouse fibroblasts by, and genotoxicity of, extracts of human milk. Breast cancer may be initiated by environmental/dietary agents and human milk may act as an ex vivo indicator of in vivo exposure of mammary epithelial cells to genotoxins. Extracts of human milk from UK-resident women (n = 7) were tested for their abilities to morphologically transform C3H/M2 mouse fibroblasts. Genotoxicities were assessed in the Salmonella typhimurium reverse-mutation assay in the presence of S9 using strains TA1538 and YG1019, and in metabolically-competent human MCL-5 cells with the micronucleus and with the alkaline single cell gel electrophoresis (comet) assays. Two of the seven extracts were inactive in the transformation assay both in the presence or absence of S9, two appeared to be equally transforming either in the presence or absence of S9, and two other extracts induced increased transformation frequencies in the presence of S9. A seventh extract, tested only in ...
Autophagy and the DNA damage response (DDR) are biological processes essential for cellular and organismal homeostasis. Herein we summarize and discuss emerging evidence linking DDR to autophagy. We highlight published data suggesting that autophagy is activated by DNA damage and is required for several functional outcomes of DDR signaling, including repair of DNA lesions, senescence, cell death, and cytokine secretion. Uncovering the mechanisms by which autophagy and DDR are intertwined provides novel insight into the pathobiology of conditions associated with accumulation of DNA damage, including cancer and aging, and novel concepts for the development of improved therapeutic strategies against these pathologies.
Among numerous types of oxidative DNA damage, 8-hydroxydeoxyguanosine (8-OHdG) is a ubiquitous marker of oxidative stress. 8-OHdG, one of the byproducts of oxidative DNA damage, is physiologically formed and enhanced by chemical carcinogens. Our OxiSelect™ Oxidative DNA Damage ELISA Kit (8-hydroxydeoxyguanosine assay) provides a powerful method for rapid, sensitive quantitation of 8-OHdG in DNA samples.
The promoter of nrdA gene which is related with DNA synthesis was used to construct a DNA damage sensitive biosensor. A recombinant bioluminescent E. coli strain, BBTNrdA, harboring a plasmid with the nrdA promoter fused to the luxCDABE operon, was successfully constructed. Its response to various chemicals including genotoxic chemicals substantiates it as a DNA damage biosensor. In characterization, three different classes of toxicants were used: DNA damaging chemicals, oxidative stress chemicals, and phenolics. BBTNrdA only responded strongly to DNA damaging chemicals, such as nalidixic acid (NDA), mitomycin C (MMC), 1-methyl-1-nitroso-N-methylguanidine (MNNG), and 4-nitroquinoline N-oxide (4-NQO). In contrast, there were no responses from the oxidative stress chemicals and phenolics, except from hydrogen peroxide (H2O2) which is known to cause DNA damage indirectly. Therefore, the results of the study demonstrate that BBTNrdA can be used as a DNA damage biosensor.
The Comet Assay for detection of DNA repair and DNA damage by tail intensity. In Comet Assays the damage is detected with the single-stranded or double-stranded DNA breaks. DNA damage can be induced by chemicals or UV light radiation. A single cell is paced in low melting agarose on a microscope slide. The cell membrane will be lysed with detergent and salt concentration. Nucleotides will be released from the nucleus and electrophoresis will migrate the damaged DNA to the + anode electric field. The more the DNA is damaged the more there will appear a migration tail. Endonucleases damage increases the DNA migration, whereas DNA-DNA and DNA-protein cross-links result in retarded DNA migration, Tice, 2000. The comet assay is used in DNA repair studies, in animal and clinical studies for base excision repair (BER), nucleotide excision repair (NER) like the Vitotox L. Gevaert, 2009 measures the cytotoxicity in Salmonella lux promotor cloned cells. They show that the Comet Assay or single cell gel ...
Highlights: • Aging process increases ROS accumulation. • Aging process increases DNA damage levels. • Absence of SOD activity does not cause DNA damage in young cells. • Absence of SOD activity accelerate aging and increase oxidative DNA damages during the aging process. - Abstract: Superoxide dismutases (SOD) serve as an important antioxidant defense mechanism in aerobic organisms, and deletion of these genes shortens the replicative life span in the budding yeast Saccharomyces cerevisiae. Even though involvement of superoxide dismutase enzymes in ROS scavenging and the aging process has been studied extensively in different organisms, analyses of DNA damages has not been performed for replicatively old superoxide dismutase deficient cells. In this study, we investigated the roles of SOD1, SOD2 and CCS1 genes in preserving genomic integrity in replicatively old yeast cells using the single cell comet assay. We observed that extend of DNA damage was not significantly different among the ...
TY - JOUR. T1 - DNA damage repair and telomere length in normal breast, preneoplastic lesions, and invasive cancer. AU - Raynaud, Christophe M.. AU - Hernandez, Juana. AU - Llorca, Frédérique P.. AU - Nuciforo, Paolo. AU - Mathieu, Marie Christine. AU - Commo, Frederic. AU - Delaloge, Suzette. AU - Sabatier, Laure. AU - André, Fabrice. AU - Soria, Jean Charles. PY - 2010/8/1. Y1 - 2010/8/1. N2 - Objectives: Carcinogenesis is a multistep process involving the accumulation of genetic and molecular abnormalities. It has been suggested that there is a relationship between telomere attrition in the early stages of carcinogenesis and activation of the DNA damage response machinery. We explored telomere length modification and damage response pathway activation at 3 steps of breast carcinogenesis. Methods: We carried out a retrospective immunohistochemical analysis of pathway ataxia telangiectasia mutated (p-ATM) (series 1981) and +-H2AX (series 139) levels in normal breast, preneoplastic lesions, ...
In every cell cycle, DNA accumulates lesions that impair the advance of the replication forks. Eventually, this leads to an accumulation of single‐stranded DNA (ssDNA), which activates a number of mechanisms aimed at ensuring that DNA replication passes through DNA lesions and repairing the gaps. Defects in this response cause replication fork stalling and genetic instability in yeast (Vázquez et al, 2008; Putnam et al, 2010) and are associated with cancer in humans (Moynahan and Jasin, 2010). This DNA damage tolerance (DDT) response relies in error‐prone translesion synthesis (TLS) and error‐free template switch (TS) mechanisms. TLS fills the gap by extending the 3′‐end past the damaged template, using specialized DNA polymerases that are able to incorporate a nucleotide opposite the lesion, while TS uses the information of the sister chromatid to bypass damage (Friedberg, 2005). A crucial protein in this decision is the DNA polymerase processivity factor PCNA, which functions as a ...
The rates at which lesions are removed by DNA repair can vary widely throughout the genome, with important implications for genomic stability. To study this, we measured the distribution of nucleotide excision repair (NER) rates for UV-induced lesions throughout the budding yeast genome. By plotting these repair rates in relation to genes and their associated flanking sequences, we reveal that, in normal cells, genomic repair rates display a distinctive pattern, suggesting that DNA repair is highly organized within the genome. Furthermore, by comparing genome-wide DNA repair rates in wild-type cells and cells defective in the global genome-NER (GG-NER) subpathway, we establish how this alters the distribution of NER rates throughout the genome. We also examined the genomic locations of GG-NER factor binding to chromatin before and after UV irradiation, revealing that GG-NER is organized and initiated from specific genomic locations. At these sites, chromatin occupancy of the histone ...
Following DNA damage, mRNA levels decrease, reflecting a coordinated interaction of the DNA repair, transcription and RNA processing machineries. In this study, we provide evidence that transcription and polyadenylation of mRNA precursors are both affected in vivo by UV treatment. We next show that the polyadenylation factor CstF, plays a direct role in the DNA damage response. Cells with reduced levels of CstF display decreased viability following UV treatment, reduced ability to ubiquitinate RNA polymerase II (RNAP II), and defects in repair of DNA damage. Furthermore, we show that CstF, RNAP II and BARD1 are all found at sites of repaired DNA. Our results indicate that CstF plays an active role in the response to DNA damage, providing a link between transcription-coupled RNA processing and DNA repair.
Investigating the cellular and molecular signatures in eukaryotic cells following exposure to nanoparticles will further our understanding on the mechanisms mediating nanoparticle induced effects. This study illustrates the molecular effects of silver nanoparticles (Ag-np) in normal human lung cells, IMR-90 and human brain cancer cells, U251 with emphasis on gene expression, induction of inflammatory mediators and the interaction of Ag-np with cytosolic proteins. We report that silver nanoparticles are capable of adsorbing cytosolic proteins on their surface that may influence the function of intracellular factors. Gene and protein expression profiles of Ag-np exposed cells revealed up regulation of many DNA damage response genes such as Gadd 45 in both the cell types and ATR in cancer cells. Moreover, down regulation of genes necessary for cell cycle progression (cyclin B and cyclin E) and DNA damage response/repair (XRCC1 and 3, FEN1, RAD51C, RPA1) was observed in both the cell lines. Double strand
Cell proliferation demands that the cells go through the mitotic cell cycle, involving duplication of their DNA before chromosome segregation and cell division. During G1 phase the decision is made whether to start a new round of the cell cycle, go into a quiescent state or enter into meiosis. It is critical for the cells to carefully regulate the progression through G1 phase, where the cells become committed to a new round in the cell cycle. Cell cycle progression is negatively regulated by checkpoint mechanisms to make sure that the events of one cell cycle phase have been completed before continuing to the next phase. Checkpoints delay the cell cycle in response to several forms of stress. DNA damage checkpoints are thought to allow additional time for DNA repair before DNA replication (S phase) and before chromosome segregation (mitosis). These responses are crucial for the cells to maintain their genetic integrity. The fact that the majority of cancer cells have defects in G1-S checkpoints ...
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Activating transcription factor 2 (ATF2) is regulated by JNK/p38 in response to stress. Here, we demonstrate that the protein kinase ATM phosphorylates ATF2 on serines 490 and 498 following ionizing radiation (IR). Phosphoantibodies to ATF2(490/8) reveal dose- and time-dependent phosphorylation of ATF2 by ATM that results in its rapid colocalization with gamma-H2AX and MRN components into IR-induced foci (IRIF). Inhibition of ATF2 expression decreased recruitment of Mre11 to IRIF, abrogated S phase checkpoint, reduced activation of ATM, Chk1, and Chk2, and impaired radioresistance. ATF2 requires neither JNK/p38 nor its DNA binding domain for recruitment to IRIF and the S phase checkpoint. Our findings identify a role for ATF2 in the DNA damage response that is uncoupled from its transcriptional activity. ...
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The nucleotide excision repair (NER) pathway operates through two sub-pathways: global genome repair (ggNER) and transcription-coupled repair (TCR) or gene- and strand-specific DNA repair [1, 2, 4]. The ggNER is a repair mechanism which has the ability to repair DNA damage to the overall genome with equivalent efficiency. In contrast, TCR is a kind of heterogeneous DNA repair, where repair to the damaged DNA in the status of transcription activity is superior to the silenced genes and the repair of the transcribed strand is superior to the untranscribed strand. Some DNA repair proteins and transcription factors have been identified to be involved in TCR such as CSA, CSB, XPG, XAB2, RNA polymerase II, and TFIIH [1, 7, 8, 24]. Blockage of RNA polymerase □ at the DNA damage site is believed to create a conducive environment for DNA repair [7, 9]. In this report, we provide evidence to demonstrate that DNA-PKcs, a known critical component in the NHEJ pathway of DNA double-strand breaks, is also ...