We are not surprised by the fact that almost all mutations tested in our study lead to increases in the recovery of events associated with de novo telomere formation. It is consistent with the idea that telomere formation might be a backup mechanism to all modes of damage repair and response in germ cells. Although our candidate approach in identifying factors essential for telomere establishment is far from com- prehensive, a picture has emerged in which factors responsible for the recruitment and execution of damage repair and re- sponse activities are also responsible for inhibiting telomere formation. In the absence of these activities, the DNA and chromatin structures at the ends might be sufﬁcient for the recruitment of the protective cap. If this were true, only de- fects in the protective cap itself would have a negative effect on telomere establishment on DSBs. We did not test this hy- pothesis due to the lack of hypomorphic mutations in capping components.
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We first sought to assess comprehensively the link between DSBs and epigenome marks or DNA motifs. As previously shown [6, 25], several epigenomic and chro- matin marks colocalized at DSBs (Fig. 2a). Among the most enriched marks were DNase I hypersensitive sites, H3H4 methylation, and CTCF (Fig. 2b). For instance, 91% of DSBs colocalized to a DNase site, whereas this percentage dropped to 11% for non-DSB regions. This corresponded to an odds ratio (OR) of 89.3. Similarly, high enrichment was found for H3K4me2 (74% versus 11%; OR = 22.4) and for the insulator protein CTCF (25% ver- sus 2%; OR = 19), which may involve its interactions with the insulator-related cofactor cohesin, which has been shown to protect genes from DSBs . As such, DSBs mostly localized within open and active regions that were often implicated in long-range contacts . Interestingly, DSBs also colocalized with tumor protein p63 binding (19.4% versus 1%; OR = 23.8), a member of the p53 gene family [28, 29]. In addition, we could distinguish DNase and CTCF sites that were enriched at the center of DSBs from histone marks that were found at the edges of DSB sites (Fig. 2c). Therefore, the strong enrichment of epige- nomic and chromatin marks at DSB sites suggests that DSB regions could be accurately predicted using avail- able ChIP-seq and DNase-seq data from public databases, including ENCODE and Roadmap Epigenomics.
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explore the possibility of ISWI being a substrate of ATR. Xenopus ISWI contains three potential ATR phosphorylation sites distributed throughout the protein. The first potential site lies within a highly conserved nucleotide-binding region found in a wide variety of helicases and helicase-related proteins. This site S499, conserved in all the observed ISWI homologs from yeast to humans, was not phosphorylated by ATR. A second site, T721, lying in an otherwise uncharacterized region of the protein that displays a lower degree of conservation was marginally phosphorylated by ATR and ATM. Finally, T832, which is highly conserved in eukaryotic ISWI homologs, was robustly phosphorylated by both ATM and ATR. This site lies directly upstream of the SANT DNA-binding domain, which in conjunction with the adjacent SLIDE domain is required to mediate the substrate recognition and chromatin remodeling activities of ISWI 161,162 . Mutation of this residue to a non-phosphorylatable alanine did not compromise the ability of ISWI to interact with ATR (data not shown). Conversely, albeit with some level of uncertainty, it diminishes the binding of ISWI onto chromatin that contains DSBs. The regulation of ISWI activity by post-translational modifications was recently observed by work done in Drosophila. Corona and colleagues observed the previously uncharacterized poly-ADP-ribosylation of ISWI by PARP. Poly-ADP-ribosylation inhibited the DNA and nucleosome binding abilities of ISWI as well as its ATPase activity in vitro 163 . It is tempting to speculate that
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Neuronal and astroglial cells were prepared from whole brain of three month and 30-month- old rats for study of alterations in the nuclear poly ADP-ribosylation and DNA breaks with age. The relative purity of the cell preparations was confirmed by the determination of the neurofilament (low molecular weight) and glutamine synthetase content of the cells using ELISA. An increase (75%) in the poly ADP-ribosylation was observed in the whole brain cell suspension of aged rats, whereas the increase was markedly pronounced (460%) when the reaction was measured in the purified neuronal preparations. The rate of poly ADP-ribosylation in the astroglial fractions prepared from aged rat brain was higher than that of adult levels (67%). An unexpectedly high increase of ADP-ribosylation in the neurons and a much lower rate in the astroglial cells was thus recorded. The amount of DNA breaks was also higher in the neuronal preparation in aged brain as compared to that of adult levels. The amount of DNA breaks was much lower in the astroglial cells and aging had no effect on DNA breaks of these cells. The close relationship between DNA breaks and poly ADP ribosylation in the different cell types suggest that neurons are more susceptible to the metabolic alterations of the aging process.
Alzheimer’s disease (AD) is characterized by neuronal death with an accumulation of intra-cellular neurofibrillary tangles (NFT) and extracellular amyloid plaques. Reduced DNA repair ability has been reported in AD brains. In neurons, the predominant mechanism to repair double-strand DNA breaks (DSB) is non-homologous end joining (NHEJ) that requires DNA-dependent protein kinase (DNA-PK) activity. DNA-PK is a holoenzyme comprising the p460 kD DNA-PK catalytic subunit (DNA-PKcs) and its activator Ku, a heterodimer of p86 (Ku80) and p70 (Ku70) subunits. Upon binding to double-stranded DNA ends, Ku recruits DNA-PKcs to process NHEJ. In AD brains, reduced NHEJ activity as well as DNA-PKcs and Ku protein levels have been shown. Normal aging brains also show a reduction in both DNA-PKcs and Ku levels questioning a direct link between NHEJ ability and AD, and suggesting additional players/ events in AD pathogenesis. Deficiency of Ku80, a somatostatin receptor, can disrupt somatostatin signaling thus inducing amyloid beta (Aβ) generation, which in turn can potentiate DNA-PKcs degradation and consequently loss of NHEJ activity, an additional step negatively affecting DSB repair. Trigger of these two different pathways culminating in genome instability may differentiate the outcomes between AD and normal aging.
infected with HCMV [30,31]. Taken together, these results suggest that UL76 is not only an essential gene for lytic replication but also implicate UL76 in viral latency. During the course of this study, Knizewski and colleagues proposed that the UL76 protein family contains a poten- tial endonuclease motif (Pfam accession number: PF01646) using computational analysis . We show here that in UL76-expressing cell lines chromosome aber- rations, micronuclei and chromosomal misalignments (laggings and bridgings) were significantly increased. Fur- ther, an increased number of these cells exhibited enhanced nuclear foci containing phosphorylated histone γ-H2AX. We also show that UL76 induces DNA breaks in proportion to its protein levels, and marginally subverts mitotic fidelity by inducing aberrant spindles and super- numerary centrosomes relative to control cells. Our results therefore suggest that HCMV UL76 may be a source of chromosomal abnormalities, and the fundamental alteration of the cellular biochemical environment may modulate viral production.
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In this issue, Butin-Israeli and col- leagues advance this knowledge gap by identifying a potentially novel and unex- pected mechanism, whereby transmigrat- ing neutrophils promote the accumula- tion of double-strand DNA breaks (DSBs) in intestinal epithelial cells by shuttling microRNA-containing microparticles (12). Patients with active inflammatory bowel disease (IBD) exhibited a marked accumu- lation of DSBs in intestinal epithelial cells, with a concomitant decrease in nuclear integrity. Moreover, these alterations were accompanied by increased expression of MPO, elastase, and inflammatory cyto- kines, such as TNF, IL-6, and IFN-γ. Using a series of reductionist in vitro culture sys- tems, including human colonic enteroids and in vivo models of biopsy-induced ster- ile injury, Butin-Israeli et al. demonstrate that neutrophil-derived microparticles can selectively induce downstream effects on DNA damage. Neutrophil-derived micro- particles alone induced DNA damage response in proliferating epithelial cells, a response that was blocked following inhi- bition of microparticle uptake. A decrease in epithelial cell proliferation did not Related Article: p. 712
pendent experiments are plotted. Figures 4–6 compare, respectively, chromosomal bands, PFGE migration profiles, and estimates of the numbers of DSBs produced in chromosomes from RAD52/ the spatial separation of individual chromosomes into RAD52 and rad52/rad52 cells. The fragmentation of distinct bands can be obtained according to molecular chromosomes from RAD52/RAD52 cells illustrated in weight and electric field interaction. Radiation-induced Figure 4 (lanes 1–7) was characteristic and reproducibly DSBs cause the distinct chromosomal bands to lessen obtained for several RAD52/RAD52 strains, but was very in intensity or disappear in a dose-dependent manner different from the fragmentation and apparent recon- (Contopoulou et al. 1987; Game et al. 1989; Geigl struction of chromosomes from rad52/rad52 cells (Fig- and Eckardt-Schupp 1990; Dardalhon et al. 1994). ure 4, lanes 8–14). The dose-dependent chromosomal Chromosomal bands reappear when the integrity of in- fragmentation caused by 1 and 2.5 g/ml of bleomycin dividual chromosomes is restored. The detection of is apparent in the PFGE migration profiles for the DSBs by PFGE is quite sensitive, and doses as low as 20 RAD52/RAD52 and rad52/rad52 strains, since the pro- Gy of ␥ rays or 0.01 g/ml bleomycin can be used files for each strain were highest for untreated cells and for mutant strains. DNA breaks increase approximately lowest after 2.5-g/ml treatments (Figure 5). Thus, the linearly with increasing doses of ␥ rays (Moore 1982b) fragmentation observed after PFGE (Figure 4) corre- or bleomycin (e.g., bleomycin A 2 or B 2 ; Moore 1990). sponds well to the changes in PFGE profiles (Figure 5).
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ratios of DNA breaks in comet assay indicates either elevated damage rate or repair approach (Collins et al., 1997; Abdel- Gawad et al., 2015). It can be seen that DNA damage reached the highest percentages in El Max site followed by Bahary area and the least were detected in El-Shatby. When comparing the DNA damage among sampled fish for the same area it’s clear that; white Sea bream showed DNA damage levels more than Sardine fish. The different answer to xenobiotics is related to the species-specificity and this is the reason why it is important the samples discrimination (Di Finizio et al., 2007; Mazzeo 2008; Guerriero et al., 2017c) Previous studies also indicated that El-Max bay is considered from the highly polluted sites when compared to other sites in Alexandria coast (Saad et al., 2017). El-Max bay suffered from continuous major drastic changes resulting from human activities; untreated industrial waste, domestic sewage, shipping industry and agricultural runoff which are being released into the bay (Abo-Taleb et al., 2015). Such activities have pronounced harmful impacts on marine fish filet sampled in our study. Referring to fish tissues and organs, it can be realized that gills followed by liver show more rates of DNA damage than filet in all sampled fish from different localities. The comet assay is an important tool used in environmental biomonitoring using fish as a model as it can presents rapid screening system to be adopted in biomonitoring food programs for detecting genotoxic potential as in environmental studies (Sharma et al., 2007; Nagpure et al., 2008; Abdel-Gawad et al., 2014; Abdel-Gawad et al., 2018).
H. pylori infection induces DNA damage on gastric epithelial cells .Contact dependent interactions between H. pylori bacteria and gastric epithelial cells activate intracellular signaling events that have further downstream effects via activation of the transcription factor NF-κB . In gastrointestinal epithelial cells, NF-κB has a central role in regulating genes that govern the onset of mucosal inflammatory responses following microbial infections [65,66]. NF-κB activation is effected through a series of phosphorylation and transactivation events triggering a downstream signaling pathway that contributes to gastric inflammation in H. pylori-infected individuals [67,68].
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There is much investigation regarding how organisms regulate the DSB repair pathway choice and increasing evidence suggests that Ku has an inhibitory effect on the other DSB pathways. Ku is one of the first proteins found at DSB regardless of cell cycle stage and cells will first attempt to repair DSBs by C-NHEJ if the ends are compatible (101, 107-109). HR is the preferred pathway in the S and G2 phases, and the initial end binding factors of HR, for example Mre11, antagonize Ku for DNA end binding. The binding of HR factors initiates DNA end resection to produce single-stranded DNA, which Ku does not have a strong affinity for, and promotes the completion of DSB repair by HR (61). In yeast, Ku appears to outcompete HR factors in G1 phase, as the loss of Ku results in increased Mre11 recruitment and Exo1 mediated resection (72, 110-113). Overexpression of Ku is even able to reduce recruitment of Mre11 in G2, when HR is the preferred DSB repair pathway (110). The HR inhibitory effect appears to be specifically dependent upon Ku’s DNA end binding function, as deletion of other NHEJ factors, such as ligase IV, were not able to increase HR activity to the same extent (110). Another study has implicated, not only Ku binding, but the kinase activity of DNA-PK CS in the
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Regulation of the cell cycle involves processes crucial to cell survival, including the detection and repair of genetic damage to ensure that damaged or incomplete DNA is not passed on to daughter cells. The prevention of gaps in replication is very important, because ensures that every portion of the cell genome will be replicated once and only once: daughter cells that are missing all or part of crucial genes will die. Two key classes of regulatory molecules determine the cell’s progress into the cycle: cyclins and cyclin-dependent kinases (CDKs) [24,25]. The close interaction of CDKs with cyclins and Cdk inhibitors (CKIs) is necessary for ensuring orderly progression through the cell cycle. These molecules act in concert in a cascade.
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abnormal metabolic milieu leads to irreversible changes in some cell populations. Since we have previously observed that high glucose concentrations induce an increase in single strand breaks in the DNA of cultured human endothelial cells, we have investigated whether the same abnormality occurs in cells derived from the in vivo diabetic environment.
Results: In this study, the genotoxic effect of aromatic hydrocarbons was assessed in peripheral blood lymphocytes of 24 workers occupationally exposed and 24 unexposed donors, by using Cytogenetic analysis and comet assay. A high frequency of Chromosomal alterations was found in the exposed group in comparison with those observed in the unexposed group. Among the total of CAs observed in the exposed group, fragilities were most frequently found (100 %), followed by chromosomal breaks (58 %), structural (41.2 %) and numerical chromosomal alterations (21 %). Numerical chromosomal alterations, fragilities and chromosomal breaks showed significant differences between exposed and unexposed groups. Among the fragilities, fra(9)(q12) was the most frequently observed. DNA damage index was also significantly higher in the exposed group compared to the unexposed group ( p < 0.000). Conclusions: Our results revealed that occupational exposure to aromatic hydrocarbons is significantly associated with Chromosomal and DNA damage in car paint shops workers and are also indicative of high chromosomal instability. The high frequency of both Chromosomal Alterations and DNA Damage Index observed in this study indicates an urgent need of intervention not only to prevent the increased risk of developing cancer but also to the application of strict health control and motivation to the use of appropriate protecting devices during work.
Classically, cancer has been viewed as a set of diseases that are driven by progressive genetic abnormalities that include mutations in tumour-suppressor genes and oncogenes, and chromosomal aberrations. However, it has become apparent that cancer is also driven by epigenetic alterations. Epigenetic alterations refer to functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence. Examples of such modifications are changes in DNA methylation (hypermethylation and hypomethylation) and histone modification, changes in chromosomal architecture (caused by inappropriate expression of proteins such as HMGA2 or HMGA1) and changes caused by microRNAs. Each of these epigenetic alterations serves to regulate gene expression without altering the underlying DNA sequence. These changes usually remain through cell divisions, last for multiple cell generations, and can be considered to be epimutations (equivalent to mutations). While large numbers of epigenetic alterations are found in cancers, the epigenetic alterations in DNA repair genes, causing reduced expression of DNA repair proteins, appear to be particularly important. Such alterations are thought to occur early in progression to cancer and to be a likely cause of the genetic instability characteristic of cancers. Reduced expression of DNA repair genes causes deficient DNA repair. When DNA repair is deficient DNA damages remain in cells at a higher than usual level and these excess damages cause increased frequencies of mutation or epimutation. Mutation rates increase substantially in cells defective in DNA mismatch repair  or in homologous recombinational repair (HRR). Chromosomal rearrangements and aneuploidy also increase in HRR defective cells. Higher levels of DNA damage not only cause increased mutation, but also cause increased epimutation. During repair of DNA double strand breaks, or repair of other DNA damages, incompletely cleared sites of repair can cause epigenetic gene silencing. At least 169 enzymes are either directly employed in DNA repair or influence DNA repair processes. Of these, 83 are directly employed in the 5 types of DNA repair processes illustrated
Lymphoid cells were thought to be uniquely susceptible to excess 2'-deoxyadenosine (dAdo), when exposed to inhibitors of adenosine deaminase (ADA). However, we now find that human monocytes are as sensitive as lymphocytes to dAdo or to the ADA-resistant congener 2-chloro-2'-deoxyadenosine (CldAdo). Monocytes exposed in vitro to CldAdo, or to dAdo plus deoxycoformycin rapidly developed DNA strand breaks. Both the DNA
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The MGMT mean promoter (see Additional file 2) methylation was significantly higher (0.337% ± 0.468, P = 0.023) after the intervention (Figure 4 and Table 3). The percentages of methylated cytosine were generally low (≤ 5%). No correlation of CpG methylation with the MLH1 expression could be found. DNA methylation dif- ferences between baseline and T1 were not significantly different in the intervention groups. Patient number 76 (IFG) showed a baseline methylation level of 11% and 2% after intervention at CpG number 8, leading to a dis- torted statistic at this position. Excluding patient 76 from pooled statistics leads to + 0.733% overall methylation (SE = 0.146, P < 0.001). A European Molecular Biology Open Software Suite (EMBOSS) transcription factor pre- diction within shared motifs (> 5 bp; 50-AGCCCG-30 and 50-GGACAGC-30) with MLH1 region 1 was negative.
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The ability to maintain genomic integrity is probably most important for postmitotic cells that are long lived such as neurons. Unable to divide, and for the most part irreplaceable, the majority of neurons have to rely on their genetic material for the lifetime of an organism. Furthermore, neurons have a highly active metabolism and produce large quantities of free radicals, which can cause oxidative DNA damage . Notably, increased levels of DNA damage in aging brains are associated with the reduced expression of essential genes, including genes involved in neuronal plasticity . Neuronal DNA damage is further exacerbated in many neuro- degenerative disorders [6, 42, 65, 92], which may contri- bute to the extensive changes in gene expression and neuronal loss found in these conditions.
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Previous work on echinoid embryos revealed significant effects of UVR on cell division, development, morphology and DNA damage (Rustad, 1960; Eima et al., 1984; Akimoto and Shiroya, 1987). All of these studies, however, utilized artificial light sources containing germicidal UV-C (254·nm) radiation that is not ecologically relevant, as UVR below 290·nm does not reach sea level, but would result in significant damage to DNA, as indicated by the biological weighting function for DNA damage (Setlow, 1974). An action spectrum for cleavage delay after exposure to UVR showed a significant effect of wavelengths below 310·nm (Giese, 1939), again consistent with the biological weighting function for DNA damage (Setlow, 1974). More recent studies (Adams and Shick, 1996, 2001) have shown similar effects on urchin embryos after exposure to environmentally relevant wavelengths of UV-A and UV-B. Additionally, Lesser and Barry (2003) reported that environmentally relevant UVR had significant effects on echinoid survivorship, development, morphology and DNA damage. What has been generally lacking is an understanding of the underlying mechanism(s) causing cleavage delay, developmental abnormalities and cellular death after exposure to UVR.
ABSTRACT: The objective of the present study was to i) Extract ursolic acid , a pentacyclic triterpenoid from Ocimum sanctum; ii) develop a HPTLC based method for its quantification; and iii) investigate ursolic acid dependent adaptive response to cytotoxic effect of hydrogen peroxide. To elucidate the possible underlying mechanism(s), lymphocytes were co-incubated with hydrogen peroxide (0, 10, 25 and 100 M) and ursolic acid (20 and 40 g/ml incubation medium). After 2 h of incubation we investigated the profile of DNA strand breaks, intracellular reactive oxygen species (ROS), apurinic/apyrimidinic sites (AP sites) and 8-hydroxy-2'- deoxyguanosine (8-OH-dG) residues in DNA. Results showed that ursolic acid inhibited hydrogen peroxide induced DNA strand breaks. Ursolic also showed significant free radical scavenging as indicated by reduced ROS and decreased AP sites. The formation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in lymphocyte DNA was also inhibited. These findings clearly showed mechanistic basis for protective effect of ursolic acid against intracellular ROS and consequent generation of AP sites in genomic DNA. In conclusion, our studies clearly suggest that ursolic acid might be a possible chemo-preventive phytochemical against oxidative stress.
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