DNA Damage by Ionizing Radiation: Tandem Double Lesions by Charged Particles

Science.gov (United States)

Huo, Winifred M.; Chaban, Galina M.; Wang, Dunyou; Dateo, Christopher E.

2005-01-01

Oxidative damages by ionizing radiation are the source of radiation-induced carcinogenesis, damage to the central nervous system, lowering of the immune response, as well as other radiation-induced damages to human health. Monte Carlo track simulations and kinetic modeling of radiation damages to the DNA employ available molecular and cellular data to simulate the biological effect of high and low LET radiation io the DNA. While the simulations predict single and double strand breaks and base damages, so far all complex lesions are the result of stochastic coincidence from independent processes. Tandem double lesions have not yet been taken into account. Unlike the standard double lesions that are produced by two separate attacks by charged particles or radicals, tandem double lesions are produced by one single attack. The standard double lesions dominate at the high dosage regime. On the other hand, tandem double lesions do not depend on stochastic coincidences and become important at the low dosage regime of particular interest to NASA. Tandem double lesions by hydroxyl radical attack of guanine in isolated DNA have been reported at a dosage of radiation as low as 10 Gy. The formation of two tandem base lesions was found to be linear with the applied doses, a characteristic of tandem lesions. However, tandem double lesions from attack by a charged particle have not been reported.

DNA lesions induced by replication stress trigger mitotic aberration and tetraploidy development.

Directory of Open Access Journals (Sweden)

Yosuke Ichijima

Full Text Available During tumorigenesis, cells acquire immortality in association with the development of genomic instability. However, it is still elusive how genomic instability spontaneously generates during the process of tumorigenesis. Here, we show that precancerous DNA lesions induced by oncogene acceleration, which induce situations identical to the initial stages of cancer development, trigger tetraploidy/aneuploidy generation in association with mitotic aberration. Although oncogene acceleration primarily induces DNA replication stress and the resulting lesions in the S phase, these lesions are carried over into the M phase and cause cytokinesis failure and genomic instability. Unlike directly induced DNA double-strand breaks, DNA replication stress-associated lesions are cryptogenic and pass through cell-cycle checkpoints due to limited and ineffective activation of checkpoint factors. Furthermore, since damaged M-phase cells still progress in mitotic steps, these cells result in chromosomal mis-segregation, cytokinesis failure and the resulting tetraploidy generation. Thus, our results reveal a process of genomic instability generation triggered by precancerous DNA replication stress.

Traveling Rocky Roads: The Consequences of Transcription-Blocking DNA Lesions on RNA Polymerase II.

Science.gov (United States)

Steurer, Barbara; Marteijn, Jurgen A

2017-10-27

The faithful transcription of eukaryotic genes by RNA polymerase II (RNAP2) is crucial for proper cell function and tissue homeostasis. However, transcription-blocking DNA lesions of both endogenous and environmental origin continuously challenge the progression of elongating RNAP2. The stalling of RNAP2 on a transcription-blocking lesion triggers a series of highly regulated events, including RNAP2 processing to make the lesion accessible for DNA repair, R-loop-mediated DNA damage signaling, and the initiation of transcription-coupled DNA repair. The correct execution and coordination of these processes is vital for resuming transcription following the successful repair of transcription-blocking lesions. Here, we outline recent insights into the molecular consequences of RNAP2 stalling on transcription-blocking DNA lesions and how these lesions are resolved to restore mRNA synthesis. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Modified jailed balloon technique for bifurcation lesions.

Science.gov (United States)

Saito, Shigeru; Shishido, Koki; Moriyama, Noriaki; Ochiai, Tomoki; Mizuno, Shingo; Yamanaka, Futoshi; Sugitatsu, Kazuya; Tobita, Kazuki; Matsumi, Junya; Tanaka, Yutaka; Murakami, Masato

2017-12-04

We propose a new systematic approach in bifurcation lesions, modified jailed balloon technique (M-JBT), and report the first clinical experience. Side branch occlusion brings with a serious complication and occurs in more than 7.0% of cases during bifurcation stenting. A jailed balloon (JB) is introduced into the side branch (SB), while a stent is placed in the main branch (MB) as crossing SB. The size of the JB is half of the MB stent size. While the proximal end of JB attaching to MB stent, both stent and JB are simultaneously inflated with same pressure. JB is removed and then guidewires are recrossed. Kissing balloon dilatation (KBD) and/or T and protrusion (TAP) stenting are applied as needed. Between February 2015 and February 2016, 233 patients (254 bifurcation lesions including 54 left main trunk disease) underwent percutaneous coronary intervention (PCI) using this technique. Procedure success was achieved in all cases. KBD was performed for 183 lesions and TAP stenting was employed for 31 lesions. Occlusion of SV was not observed in any of the patients. Bench test confirmed less deformity of MB stent in M-JBT compared with conventional-JBT. This is the first report for clinical experiences by using modified jailed balloon technique. This novel M-JBT is safe and effective in the preservation of SB patency during bifurcation stenting. © 2017 Wiley Periodicals, Inc.

Automated DNA extraction from genetically modified maize using aminosilane-modified bacterial magnetic particles.

Science.gov (United States)

Ota, Hiroyuki; Lim, Tae-Kyu; Tanaka, Tsuyoshi; Yoshino, Tomoko; Harada, Manabu; Matsunaga, Tadashi

2006-09-18

A novel, automated system, PNE-1080, equipped with eight automated pestle units and a spectrophotometer was developed for genomic DNA extraction from maize using aminosilane-modified bacterial magnetic particles (BMPs). The use of aminosilane-modified BMPs allowed highly accurate DNA recovery. The (A(260)-A(320)):(A(280)-A(320)) ratio of the extracted DNA was 1.9+/-0.1. The DNA quality was sufficiently pure for PCR analysis. The PNE-1080 offered rapid assay completion (30 min) with high accuracy. Furthermore, the results of real-time PCR confirmed that our proposed method permitted the accurate determination of genetically modified DNA composition and correlated well with results obtained by conventional cetyltrimethylammonium bromide (CTAB)-based methods.

Lesion Orientation of O4-Alkylthymidine Influences Replication by Human DNA Polymerase η

OpenAIRE

O’Flaherty, D. K.; Patra, A.; Su, Y.; Guengerich, F. P.; Egli, M.; Wilds, C. J.

2016-01-01

DNA lesions that elude repair may undergo translesion synthesis catalyzed by Y-family DNA polymerases. O4-Alkylthymidines, persistent adducts that can result from carcinogenic agents, may be encountered by DNA polymerases. The influence of lesion orientation around the C4-O4 bond on processing by human DNA polymerase η (hPol η) was studied for oligonucleotides containing O4-methylthymidine, O4-ethylthymidine, and analogs restricting the O4-methylene group in an anti-orientation. Primer extens...

DNA oxidation and DNA repair in gills of zebra mussels exposed to cadmium and benzo(a)pyrene.

Science.gov (United States)

Michel, Cécile; Vincent-Hubert, Françoise

2015-11-01

Freshwater bivalve molluscs are considered as effective indicators of environmental pollution. The comet assay allows the detection of DNA damage such as DNA strand breaks and alkali-labile sites. The main oxidative lesion, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), which is a pre-mutagenic lesion, can be detected by the comet assay coupled with the hOGG1 DNA repair enzyme. With this modified assay we recently observed that BaP induced 8-oxodG lesions and with the modified comet-Fpg assay we observed that Cd induced oxidative DNA damage. The aim of this study was to determine the stability of DNA lesions in Cd and BaP exposed zebra mussels using the comet-hOGG1 assay. Mussels were exposed for 24 h to these two chemicals and then placed in clean water for 6 days. We observed that BaP (7, 12 and 18 µg/L) induced an increase of DNA strand break levels as soon as 6 h of exposure and that the two highest concentrations of BaP induced a low level of hOGG1-sensitive sites. After 2 days of depuration, BaP induced DNA lesions returned to the basal level, indicating an effective DNA repair. Cd (3, 32 and 81 µg/L) induced an increase of the DNA strand break levels and a low level of hOGG1-sensitive sites. This study revealed that BaP-induced DNA lesions are repaired more efficiently than Cd-induced DNA lesions. As the level of hOGG1 sensitive sites was increased in Cd and BaP exposed mussels, it seems that these chemicals induce 8-oxo-dG.

Repair of model compounds of photoinduced lesions in DNA. Electrochemical approaches

International Nuclear Information System (INIS)

Boussicault, F.

2006-09-01

The goal of this work is to better understand the repair mechanism of photoinduced lesions in DNA (cyclobutane dimers and pyrimidine (6-4) pyrimidone adducts) by photolyase redox enzymes, using tools and concepts of molecular electrochemistry. Thanks to the study of model compounds of cyclobutane lesions by cyclic voltametry, we have been able to mimic the key step of the enzymatic repair (dissociative electron transfer) and to monitor the repair of model compounds by Escherichia coli DNA photolyase. From these results, we have discussed the repair mechanism, especially the stepwise or concerted character of the process. Repair mechanism of (6-4) adducts is not known now, but a possible pathway implies an electron transfer coupled to the cleavage of two bonds in the closed form of the lesions (oxetanes). Voltammetric study of reduction and oxidation of model oxetanes and their repair by E. coli DNA photolyase gave some experimental evidence confirming the proposed mechanism and allowing a better understanding of it. (author)

Clustered DNA lesions containing 5-formyluracil and AP site: repair via the BER system.

Directory of Open Access Journals (Sweden)

Ekaterina A Belousova

Full Text Available Lesions in the DNA arise under ionizing irradiation conditions or various chemical oxidants as a single damage or as part of a multiply damaged site within 1-2 helical turns (clustered lesion. Here, we explored the repair opportunity of the apurinic/apyrimidinic site (AP site composed of the clustered lesion with 5-formyluracil (5-foU by the base excision repair (BER proteins. We found, that if the AP site is shifted relative to the 5-foU of the opposite strand, it could be repaired primarily via the short-patch BER pathway. In this case, the cleavage efficiency of the AP site-containing DNA strand catalyzed by human apurinic/apyrimidinic endonuclease 1 (hAPE1 decreased under AP site excursion to the 3'-side relative to the lesion in the other DNA strand. DNA synthesis catalyzed by DNA polymerase lambda was more accurate in comparison to the one catalyzed by DNA polymerase beta. If the AP site was located exactly opposite 5-foU it was expected to switch the repair to the long-patch BER pathway. In this situation, human processivity factor hPCNA stimulates the process.

Detection and repair of a UV-induced photosensitive lesion in the DNA of human cells

International Nuclear Information System (INIS)

Francis, A.A.; Regan, J.D.

1986-01-01

Irradiation with UV light results in damage to the DNA of human cells. The most numerous lesions are pyrimidine dimers; however, other lesions are known to occur and may contribute to the overall deleterious effect of UV irradiation. The authors have observed evidence of a UV-induced lesion other than pyrimidine dimers in the DNA of human cells by measuring DNA strand breaks induced by irradiating with 313-nm light following UV (254-nm) irradiation. The data suggest that, in normal cells, the lesion responsible for this effect is rapidly repaired or altered; whereas, in xeroderma pigmentosum variant cells it seems to remain unchanged. Some change apparently occurs in the DNA of xeroderma pigmentosum group A cells which results in an increase in photolability. These data indicate a deficiency in DNA repair of xeroderma pigmentosum variant cells as well as in xeroderma pigmentosum group A cells. (Auth.)

Carboxymethyl chitin-glucan (CM-CG) protects human HepG2 and HeLa cells against oxidative DNA lesions and stimulates DNA repair of lesions induced by alkylating agents.

Science.gov (United States)

Slamenová, Darina; Kováciková, Ines; Horváthová, Eva; Wsólová, Ladislava; Navarová, Jana

2010-10-01

A large number of functional foods, including those that contain β-d-glucans, have been shown to prevent human DNA against genotoxic effects and associated development of cancer and other chronic diseases. In this paper, carboxymethyl chitin-glucan (CM-CG) isolated from Aspergillus niger was investigated from two standpoints: (1) DNA-protective effects against oxidative DNA damage induced by H(2)O(2) and alkylating DNA damage induced by MMS and MNNG, and (2) a potential effect on rejoining of MMS- and MNNG-induced single strand DNA breaks. The results obtained by the comet assay in human cells cultured in vitro showed that CM-CG reduced significantly the level of oxidative DNA lesions induced by H(2)O(2) but did not change the level of alkylating DNA lesions induced by MMS or MNNG. On the other side, the efficiency of DNA-rejoining of single strand DNA breaks induced by MMS and MNNG was significantly higher in HepG2 cells pre-treated with CM-CG. The antioxidative activity of carboxymethyl chitin-glucan was confirmed by the DPPH assay. Copyright © 2010 Elsevier Ltd. All rights reserved.

Recombinational DNA repair is regulated by compartmentalization of DNA lesions at the nuclear pore complex

DEFF Research Database (Denmark)

Géli, Vincent; Lisby, Michael

2015-01-01

and colleagues shows that also physiological threats to genome integrity such as DNA secondary structure-forming triplet repeat sequences relocalize to the NPC during DNA replication. Mutants that fail to reposition the triplet repeat locus to the NPC cause repeat instability. Here, we review the types of DNA...... lesions that relocalize to the NPC, the putative mechanisms of relocalization, and the types of recombinational repair that are stimulated by the NPC, and present a model for NPC-facilitated repair....

Trans-Lesion DNA Polymerases May Be Involved in Yeast Meiosis

Science.gov (United States)

Arbel-Eden, Ayelet; Joseph-Strauss, Daphna; Masika, Hagit; Printzental, Oxana; Rachi, Eléanor; Simchen, Giora

2013-01-01

Trans-lesion DNA polymerases (TLSPs) enable bypass of DNA lesions during replication and are also induced under stress conditions. Being only weakly dependent on their template during replication, TLSPs introduce mutations into DNA. The low processivity of these enzymes ensures that they fall off their template after a few bases are synthesized and are then replaced by the more accurate replicative polymerase. We find that the three TLSPs of budding yeast Saccharomyces cerevisiae Rev1, PolZeta (Rev3 and Rev7), and Rad30 are induced during meiosis at a time when DNA double-strand breaks (DSBs) are formed and homologous chromosomes recombine. Strains deleted for one or any combination of the three TLSPs undergo normal meiosis. However, in the triple-deletion mutant, there is a reduction in both allelic and ectopic recombination. We suggest that trans-lesion polymerases are involved in the processing of meiotic double-strand breaks that lead to mutations. In support of this notion, we report significant yeast two-hybrid (Y2H) associations in meiosis-arrested cells between the TLSPs and DSB proteins Rev1-Spo11, Rev1-Mei4, and Rev7-Rec114, as well as between Rev1 and Rad30. We suggest that the involvement of TLSPs in processing of meiotic DSBs could be responsible for the considerably higher frequency of mutations reported during meiosis compared with that found in mitotically dividing cells, and therefore may contribute to faster evolutionary divergence than previously assumed. PMID:23550131

Spontaneous mutation by mutagenic repair of spontaneous lesions in DNA

International Nuclear Information System (INIS)

Hastings, P.J.; Quah, S.-K.; Borstel, R.C. von

1976-01-01

It is stated that strains of yeast carrying mutations in many of the steps in pathways repairing radiation-induced damage to DNA have enhanced spontaneous mutation rates. Most strains isolated because they have enhanced spontaneous mutation carry mutations in DNA repair systems. This suggests that much spontaneous mutation arises by mutagenic repair of spontaneous lesions. (author)

Kinetic Analysis of the Bypass of a Bulky DNA Lesion Catalyzed by Human Y-family DNA Polymerases

Science.gov (United States)

Sherrer, Shanen M.; Sanman, Laura E.; Xia, Cynthia X.; Bolin, Eric R.; Malik, Chanchal K.; Efthimiopoulos, Georgia; Basu, Ashis K.; Suo, Zucai

2012-01-01

1-Nitropyrene (1-NP), a mutagen and potential carcinogen, is the most abundant nitro polyaromatic hydrocarbon in diesel exhaust, which reacts with DNA to form predominantly N-(deoxyguanosin-8-yl)-1-aminopyrene (dGAP). If not repaired, this DNA lesion is presumably bypassed in vivo by any of human Y-family DNA polymerases kappa (hPolκ), iota (hPolτ), eta (hPolη), and Rev1 (hRev1). Our running start assays demonstrated that each of these enzymes was indeed capable of traversing a site-specifically placed dGAP on a synthetic DNA template but hRev1 was stopped after lesion bypass. The time required to bypass 50% of the dGAP sites (t50bypass ) encountered by hPolη, hPolκ and hPolτ was determined to be 2.5 s, 4.1 s, and 106.5 s, respectively. The efficiency order of catalyzing translesion synthesis of dGAP (hPolη > hPolκ > hPolτ >> hRev1) is the same as the order for these human Y-family enzymes to elongate undamaged DNA. Although hPolη bypassed dGAP efficiently, replication by both hPolκ and hPolτ was strongly stalled at the lesion site and at a site immediately downstream from dGAP. By employing pre-steady state kinetic methods, a kinetic basis was established for polymerase pausing at these DNA template sites. Besides efficiency of bypass, the fidelity of those low-fidelity polymerases at these pause sites was also significantly decreased. Thus, if the translesion DNA synthesis of dGAP in vivo is catalyzed by a human Y-family DNA polymerase, e.g. hPolη, the process is certainly mutagenic. PMID:22324639

DNA immobilization on polymer-modified Si surface by controlling pH

International Nuclear Information System (INIS)

Demirel, Goekcen Birlik; Caykara, Tuncer

2009-01-01

A novel approach based on polymer-modified Si surface as DNA sensor platforms is presented. The polymer-modified Si surface was prepared by using 3-(methacryloxypropyl)trimethoxysilane [γ-MPS] and poly(acrylamide) [PAAm]. Firstly, a layer of γ-MPS was formed on the hydroxylated silicon surface as a monolayer and then modified with different molecular weight of PAAm to form polymer-modified surface. The polymer-modified Si surface was used for dsDNA immobilization. All steps about formation of layer structure were characterized by ellipsometry, atomic force microscopy (AFM), attenuated total reflectance Fourier transformed infrared (ATR-FTIR), and contact angle (CA) measurements. We found that in this case the amount of dsDNA immobilized onto the surface was dictated by the electrostatic interaction between the substrate surface and the DNA. Our results thus demonstrated that DNA molecules could be immobilized differently onto the polymer-modified support surface via electrostatic interactions.

Folic Acid Supplementation Delays Atherosclerotic Lesion Development by Modulating MCP1 and VEGF DNA Methylation Levels In Vivo and In Vitro

Science.gov (United States)

Cui, Shanshan; Li, Wen; Lv, Xin; Wang, Pengyan; Gao, Yuxia; Huang, Guowei

2017-01-01

The pathogenesis of atherosclerosis has been partly acknowledged to result from aberrant epigenetic mechanisms. Accordingly, low folate levels are considered to be a contributing factor to promoting vascular disease because of deregulation of DNA methylation. We hypothesized that increasing the levels of folic acid may act via an epigenetic gene silencing mechanism to ameliorate atherosclerosis. Here, we investigated the atheroprotective effects of folic acid and the resultant methylation status in high-fat diet-fed ApoE knockout mice and in oxidized low-density lipoprotein-treated human umbilical vein endothelial cells. We analyzed atherosclerotic lesion histology, folate concentration, homocysteine concentration, S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), and DNA methyltransferase activity, as well as monocyte chemotactic protein-1 (MCP1) and vascular endothelial growth factor (VEGF) expression and promoter methylation. Folic acid reduced atherosclerotic lesion size in ApoE knockout mice. The underlying folic acid protective mechanism appears to operate through regulating the normal homocysteine state, upregulating the SAM: SAH ratio, elevating DNA methyltransferase activity and expression, altering MCP1 and VEGF promoter methylation, and inhibiting MCP1 and VEGF expression. We conclude that folic acid supplementation effectively prevented atherosclerosis by modifying DNA methylation through the methionine cycle, improving DNA methyltransferase activity and expression, and thus changing the expression of atherosclerosis-related genes. PMID:28475147

Liquid biopsy in the diagnosis of HPV DNA in breast lesions.

Science.gov (United States)

Carolis, Sabrina De; Pellegrini, Alice; Santini, Donatella; Ceccarelli, Claudio; De Leo, Antonio; Alessandrini, Federica; Arienti, Chiara; Pignatta, Sara; Tesei, Anna; Mantovani, Vilma; Zamagni, Claudio; Taffurelli, Mario; Sansone, Pasquale; Bonafé, Massimiliano; Cricca, Monica

2018-02-01

HPV DNA has never been investigated in nipple discharges (ND) and serum-derived extracellular vesicles, although its presence has been reported in ductal lavage fluids and blood specimens. We analyzed 50 ND, 22 serum-derived extracellular vesicles as well as 51 pathologic breast tissues for the presence of 16 HPV DNA types. We show that the presence of HPV DNA in the ND is predictive of HPV DNA-positive breast lesions and that HPV DNA is more represented in intraductal papillomas. We also show the presence of HPV DNA in the serum-derived extracellular vesicles. Our data supports the use of liquid biopsy to detect HPV DNA in breast pathology.

Theoretical approach of complex DNA lesions: from formation to repair

International Nuclear Information System (INIS)

Bignon, Emmanuelle

2017-01-01

This thesis work is focused on the theoretical modelling of DNA damages, from formation to repair. Several projects have been led in this framework, which can be sorted into three different parts. One on hand, we studied complex DNA reactivity. It included a study about 8-oxo-7,8-dihydro-guanine (8oxoG) mechanisms of formation, a project concerning the UV-induced pyrimidine 6-4 pyrimidone (6-4PP) endogenous photo-sensitizer features, and another one about DNA photo-sensitization by nonsteroidal anti-inflammatory drugs (i.e. ketoprofen and ibuprofen). On the other hand, we investigated mechanical properties of damaged DNA. The structural signature of a DNA lesion is of major importance for their repair, unfortunately only few NMR and X-ray structures of such systems are available. In order to gain insights into their dynamical structure, we investigated a series of complex damages: clustered abasic sites, interstrand cross-links, and the 6-4PP photo-lesion. Likewise, we studied the interaction modes DNA with several polyamines, which are well known to interact with the double helix, but also with the perspective to model DNA-protein cross-linking. The third part concerned the study of DNA interactions with repair enzymes. In line with the structural study about clustered abasic sites, we investigated the dynamics of the same system, but this time interacting with the APE1 endonuclease. We also studied interactions between the Fpg glycosylase with an oligonucleotides containing tandem 8-oxoG on one hand and 8-oxoG - abasic site as multiply damaged sites. Thus, we shed new lights on damaged DNA reactivity, structure and repair, which provides perspectives for biomedicine and life's mechanisms understanding as we begin to describe nucleosomal DNA. (author)

[Endonuclease modified comet assay for oxidative DNA damage induced by detection of genetic toxicants].

Science.gov (United States)

Zhao, Jian; Li, Hongli; Zhai, Qingfeng; Qiu, Yugang; Niu, Yong; Dai, Yufei; Zheng, Yuxin; Duan, Huawei

2014-03-01

The aim of this study was to investigate the use of the lesion-specific endonucleases-modified comet assay for analysis of DNA oxidation in cell lines. DNA breaks and oxidative damage were evaluated by normal alkaline and formamidopyrimidine-DNA-glycosylase (FPG) modified comet assays. Cytotoxicity were assessed by MTT method. The human bronchial epithelial cell (16HBE) were treated with benzo (a) pyrene (B(a)P), methyl methanesulfonate (MMS), colchicine (COL) and vincristine (VCR) respectively, and the dose is 20 µmol/L, 25 mg/ml, 5 mg/L and 0.5 mg/L for 24 h, respectively. Oxidative damage was also detected by levels of reactive oxygen species in treated cells. Four genotoxicants give higher cytotoxicity and no significant changes on parameters of comet assay treated by enzyme buffer. Cell survival rate were (59.69 ± 2.60) %, (54.33 ± 2.81) %, (53.11 ± 4.00) %, (51.43 ± 3.92) % in four groups, respectively. There was the direct DNA damage induced by test genotoxicants presented by tail length, Olive tail moment (TM) and tail DNA (%) in the comet assay. The presence of FPG in the assays increased DNA migration in treated groups when compared to those without it, and the difference was statistically significant which indicated that the clastogen and aneugen could induce oxidative damage in DNA strand. In the three parameters, the Olive TM was changed most obviously after genotoxicants treatment. In the contrast group, the Olive TM of B(a) P,MMS, COL,VCR in the contrast groups were 22.99 ± 17.33, 31.65 ± 18.86, 19.86 ± 9.56 and 17.02 ± 9.39, respectively, after dealing with the FPG, the Olive TM were 34.50 ± 17.29, 43.80 ± 10.06, 33.10 ± 12.38, 28.60 ± 10.53, increased by 58.94%, 38.48%, 66.86% and 68.21%, respectively (t value was 3.91, 3.89, 6.66 and 3.87, respectively, and all P comet assay appears more specific for detecting oxidative DNA damage induced by genotoxicants exposure, and the application of comet assay will be expanded. The endonuclease

Poly(ADP-ribose) polymerase 1 escorts XPC to UV-induced DNA lesions during nucleotide excision repair.

Science.gov (United States)

Robu, Mihaela; Shah, Rashmi G; Purohit, Nupur K; Zhou, Pengbo; Naegeli, Hanspeter; Shah, Girish M

2017-08-15

Xeroderma pigmentosum C (XPC) protein initiates the global genomic subpathway of nucleotide excision repair (GG-NER) for removal of UV-induced direct photolesions from genomic DNA. The XPC has an inherent capacity to identify and stabilize at the DNA lesion sites, and this function is facilitated in the genomic context by UV-damaged DNA-binding protein 2 (DDB2), which is part of a multiprotein UV-DDB ubiquitin ligase complex. The nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) has been shown to facilitate the lesion recognition step of GG-NER via its interaction with DDB2 at the lesion site. Here, we show that PARP1 plays an additional DDB2-independent direct role in recruitment and stabilization of XPC at the UV-induced DNA lesions to promote GG-NER. It forms a stable complex with XPC in the nucleoplasm under steady-state conditions before irradiation and rapidly escorts it to the damaged DNA after UV irradiation in a DDB2-independent manner. The catalytic activity of PARP1 is not required for the initial complex formation with XPC in the nucleoplasm but it enhances the recruitment of XPC to the DNA lesion site after irradiation. Using purified proteins, we also show that the PARP1-XPC complex facilitates the handover of XPC to the UV-lesion site in the presence of the UV-DDB ligase complex. Thus, the lesion search function of XPC in the genomic context is controlled by XPC itself, DDB2, and PARP1. Our results reveal a paradigm that the known interaction of many proteins with PARP1 under steady-state conditions could have functional significance for these proteins.

On the biophysical interpretation of lethal DNA lesions induced by ionising radiation

Czech Academy of Sciences Publication Activity Database

Kundrát, Pavel; Stewart, R.D.

2006-01-01

Roč. 122, 1-4 (2006), s. 169-172 ISSN 0144-8420 R&D Projects: GA ČR GA202/05/2728 Institutional research plan: CEZ:AV0Z10100502 Keywords : clustered DNA lesions * V79 cells * proton tracks * DNA damage * DNA repair * radiobiological modelling Subject RIV: BF - Elementary Particles and High Energy Physics Impact factor: 0.446, year: 2006

Lesion Orientation of O4-Alkylthymidine Influences Replication by Human DNA Polymerase η.

Science.gov (United States)

O'Flaherty, D K; Patra, A; Su, Y; Guengerich, F P; Egli, M; Wilds, C J

2016-08-01

DNA lesions that elude repair may undergo translesion synthesis catalyzed by Y-family DNA polymerases. O 4 -Alkylthymidines, persistent adducts that can result from carcinogenic agents, may be encountered by DNA polymerases. The influence of lesion orientation around the C4- O 4 bond on processing by human DNA polymerase η (hPol η ) was studied for oligonucleotides containing O 4 -methylthymidine, O 4 -ethylthymidine, and analogs restricting the O 4 -methylene group in an anti -orientation. Primer extension assays revealed that the O 4 -alkyl orientation influences hPol η bypass. Crystal structures of hPol η •DNA•dNTP ternary complexes with O 4 -methyl- or O 4 -ethylthymidine in the template strand showed the nucleobase of the former lodged near the ceiling of the active site, with the syn - O 4 -methyl group engaged in extensive hydrophobic interactions. This unique arrangement for O 4 -methylthymidine with hPol η , inaccessible for the other analogs due to steric/conformational restriction, is consistent with differences observed for nucleotide incorporation and supports the concept that lesion conformation influences extension across DNA damage. Together, these results provide mechanistic insights on the mutagenicity of O 4 MedT and O 4 EtdT when acted upon by hPol η .

Modified DNA extraction for rapid PCR detection of methicillin-resistant staphylococci

International Nuclear Information System (INIS)

Japoni, A.; Alborzi, A.; Rasouli, M.; Pourabbas, B.

2004-01-01

Nosocomial infection caused by methicillin-resistant staphylococci poses a serious problem in many countries. The aim of this study was to rapidly and reliably detect methicillin-resistant-staphylococci in order to suggest appropriate therapy. The presence or absence of the methicillin-resistance gene in 115 clinical isolates of staphylococcus aureus and 50 isolates of coagulase negative staphylococci was examined by normal PCR. DNA extraction for PCR performance was then modified by omission of achromopeptadiase and proteinase K digestion, phenol/chloroform extraction and ethanol precipitation. All isolates with Mic>8 μ g/ml showed positive PCR. No differences in PCR detection have been observed when normal and modified DNA extractions have been performed. Our modified DNA extraction can quickly detect methicillin-resistant staphylococci by PCR. The advantage of rapid DNA extraction extends to both reduction of time and cost of PCR performance. This modified DNA extraction is suitable for different PCR detection, when staphylococci are the subject of DNA analysis

DNA-Mediated Electrochemistry

Science.gov (United States)

Gorodetsky, Alon A.; Buzzeo, Marisa C.

2009-01-01

The base pair stack of DNA has been demonstrated as a medium for long range charge transport chemistry both in solution and at DNA-modified surfaces. This chemistry is exquisitely sensitive to structural perturbations in the base pair stack as occur with lesions, single base mismatches, and protein binding. We have exploited this sensitivity for the development of reliable electrochemical assays based on DNA charge transport at self-assembled DNA monolayers. Here we discuss the characteristic features, applications, and advantages of DNA-mediated electrochemistry. PMID:18980370

Association of thymine glycol lesioned DNA with repair enzyme endonuclease III-molecular dynamics study

International Nuclear Information System (INIS)

Pinak, Miroslav

2001-07-01

The 2 nanoseconds molecular dynamics (MD) simulation has been performed for the system consisting of repair enzyme and DNA 30-mer with native thymine at position 16 replaced by thymine glycol (TG) solvated in water environment. After 950 picoseconds of MD the enzyme and DNA associated together to form complex that lasted stable up to 2 ns when simulation was terminated. At the contact area of enzyme and DNA there is glutamic acid located as close as 1.6 A to the C3' atom of phosphodiester bond of TG. Initial B-DNA molecule was bent and kinked at the TG during MD. This distortion caused that phosphodiester bond was easier accessible by amino acids of enzyme. The negative value of electrostatic energy (-26 kcal/mol) discriminates TG from nearly neutral native thymine and contributes to the specific recognition of this lesion. Higher number of close water molecules at TG site before formation of complex (compared with other nucleotides) indicates that glycosyl bond of the lesion is easily approached by repair enzyme during scanning of DNA surface and suggests the importance of specific hydration at the lesion during recognition process. (author)

Association of thymine glycol lesioned DNA with repair enzyme endonuclease III-molecular dynamics study

Energy Technology Data Exchange (ETDEWEB)

Pinak, Miroslav [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

2001-07-01

The 2 nanoseconds molecular dynamics (MD) simulation has been performed for the system consisting of repair enzyme and DNA 30-mer with native thymine at position 16 replaced by thymine glycol (TG) solvated in water environment. After 950 picoseconds of MD the enzyme and DNA associated together to form complex that lasted stable up to 2 ns when simulation was terminated. At the contact area of enzyme and DNA there is glutamic acid located as close as 1.6 A to the C3' atom of phosphodiester bond of TG. Initial B-DNA molecule was bent and kinked at the TG during MD. This distortion caused that phosphodiester bond was easier accessible by amino acids of enzyme. The negative value of electrostatic energy (-26 kcal/mol) discriminates TG from nearly neutral native thymine and contributes to the specific recognition of this lesion. Higher number of close water molecules at TG site before formation of complex (compared with other nucleotides) indicates that glycosyl bond of the lesion is easily approached by repair enzyme during scanning of DNA surface and suggests the importance of specific hydration at the lesion during recognition process. (author)

Inducible DNA-repair systems in yeast: competition for lesions.

Science.gov (United States)

Mitchel, R E; Morrison, D P

1987-03-01

DNA lesions may be recognized and repaired by more than one DNA-repair process. If two repair systems with different error frequencies have overlapping lesion specificity and one or both is inducible, the resulting variable competition for the lesions can change the biological consequences of these lesions. This concept was demonstrated by observing mutation in yeast cells (Saccharomyces cerevisiae) exposed to combinations of mutagens under conditions which influenced the induction of error-free recombinational repair or error-prone repair. Total mutation frequency was reduced in a manner proportional to the dose of 60Co-gamma- or 254 nm UV radiation delivered prior to or subsequent to an MNNG exposure. Suppression was greater per unit radiation dose in cells gamma-irradiated in O2 as compared to N2. A rad3 (excision-repair) mutant gave results similar to wild-type but mutation in a rad52 (rec-) mutant exposed to MNNG was not suppressed by radiation. Protein-synthesis inhibition with heat shock or cycloheximide indicated that it was the mutation due to MNNG and not that due to radiation which had changed. These results indicate that MNNG lesions are recognized by both the recombinational repair system and the inducible error-prone system, but that gamma-radiation induction of error-free recombinational repair resulted in increased competition for the lesions, thereby reducing mutation. Similarly, gamma-radiation exposure resulted in a radiation dose-dependent reduction in mutation due to MNU, EMS, ENU and 8-MOP + UVA, but no reduction in mutation due to MMS. These results suggest that the number of mutational MMS lesions recognizable by the recombinational repair system must be very small relative to those produced by the other agents. MNNG induction of the inducible error-prone systems however, did not alter mutation frequencies due to ENU or MMS exposure but, in contrast to radiation, increased the mutagenic effectiveness of EMS. These experiments demonstrate

Selective detection and quantification of modified DNA with solid-state nanopores.

Science.gov (United States)

Carlsen, Autumn T; Zahid, Osama K; Ruzicka, Jan A; Taylor, Ethan W; Hall, Adam R

2014-10-08

We demonstrate a solid-state nanopore assay for the unambiguous discrimination and quantification of modified DNA. Individual streptavidin proteins are employed as high-affinity tags for DNA containing a single biotin moiety. We establish that the rate of translocation events corresponds directly to relative concentration of protein-DNA complexes and use the selectivity of our approach to quantify modified oligonucleotides from among a background of unmodified DNA in solution.

Are lesions induced by ionizing radiation direct blocks to DNA chain elongation

International Nuclear Information System (INIS)

Painter, R.B.

1983-01-01

Ionizing radiation blocks DNA chain elongation in normal diploid fibroblasts but not in fibroblasts from patients with ataxia-telangiectasia, even though there are no differences in the damage induced between the two cell types. This difference suggests that radiation-induced lesions in DNA are not themselves blocks to chain elongation in ataxia cells and raises the possibility that in normal cells a mediator exists between DNA damage and chain termination

Identification of potentially cytotoxic lesions induced by UVA photoactivation of DNA 4-thiothymidine in human cells

Science.gov (United States)

Reelfs, Olivier; Macpherson, Peter; Ren, Xiaolin; Xu, Yao-Zhong; Karran, Peter; Young, Antony R.

2011-01-01

Photochemotherapy—in which a photosensitizing drug is combined with ultraviolet or visible radiation—has proven therapeutic effectiveness. Existing approaches have drawbacks, however, and there is a clinical need to develop alternatives offering improved target cell selectivity. DNA substitution by 4-thiothymidine (S4TdR) sensitizes cells to killing by ultraviolet A (UVA) radiation. Here, we demonstrate that UVA photoactivation of DNA S4TdR does not generate reactive oxygen or cause direct DNA breakage and is only minimally mutagenic. In an organotypic human skin model, UVA penetration is sufficiently robust to kill S4TdR-photosensitized epidermal cells. We have investigated the DNA lesions responsible for toxicity. Although thymidine is the predominant UVA photoproduct of S4TdR in dilute solution, more complex lesions are formed when S4TdR-containing oligonucleotides are irradiated. One of these, a thietane/S5-(6-4)T:T, is structurally related to the (6-4) pyrimidine:pyrimidone [(6-4) Py:Py] photoproducts induced by UVB/C radiation. These lesions are detectable in DNA from S4TdR/UVA-treated cells and are excised from DNA more efficiently by keratinocytes than by leukaemia cells. UVA irradiation also induces DNA interstrand crosslinking of S4TdR-containing duplex oligonucleotides. Cells defective in repairing (6-4) Py:Py DNA adducts or processing DNA crosslinks are extremely sensitive to S4TdR/UVA indicating that these lesions contribute significantly to S4TdR/UVA cytotoxicity. PMID:21890905

Analysis of damaged DNA / proteins interactions: Methodological optimizations and applications to DNA lesions induced by platinum anticancer drugs

International Nuclear Information System (INIS)

Bounaix Morand du Puch, Ch

2010-10-01

DNA lesions contribute to the alteration of DNA structure, thereby inhibiting essential cellular processes. Such alterations may be beneficial for chemotherapies, for example in the case of platinum anticancer agents. They generate bulky adducts that, if not repaired, ultimately cause apoptosis. A better understanding of the biological response to such molecules can be obtained through the study of proteins that directly interact with the damages. These proteins constitute the DNA lesions interactome. This thesis presents the development of tools aiming at increasing the list of platinum adduct-associated proteins. Firstly, we designed a ligand fishing system made of damaged plasmids immobilized onto magnetic beads. Three platinum drugs were selected for our study: cisplatin, oxali-platin and satra-platin. Following exposure of the trap to nuclear extracts from HeLa cancer cells and identification of retained proteins by proteomics, we obtained already known candidates (HMGB1, hUBF, FACT complex) but also 29 new members of the platinated-DNA interactome. Among them, we noted the presence of PNUTS, TOX4 and WDR82, which associate to form the recently-discovered PTW/PP complex. Their capture was then confirmed with a second model, namely breast cancer cell line MDA MB 231, and the biological consequences of such an interaction now need to be elucidated. Secondly, we adapted a SPRi bio-chip to the study of platinum-damaged DNA/proteins interactions. Affinity of HMGB1 and newly characterized TOX4 for adducts generated by our three platinum drugs could be validated thanks to the bio-chip. Finally, we used our tools, as well as analytical chemistry and biochemistry methods, to evaluate the role of DDB2 (a factor involved in the recognition of UV-induced lesions) in the repair of cisplatin adducts. Our experiments using MDA MB 231 cells differentially expressing DDB2 showed that this protein is not responsible for the repair of platinum damages. Instead, it appears to act

Synthesis and structural characterization of piperazino-modified DNA that favours hybridization towards DNA over RNA

DEFF Research Database (Denmark)

Skov, Joan; Bryld, Torsten; Lindegaard, Dorthe

2011-01-01

We report the synthesis of two C4'-modified DNA analogues and characterize their structural impact on dsDNA duplexes. The 4'-C-piperazinomethyl modification stabilizes dsDNA by up to 5°C per incorporation. Extension of the modification with a butanoyl-linked pyrene increases the dsDNA stabilizati...

Role of DNA lesions and repair in the transformation of human cells

International Nuclear Information System (INIS)

Maher, V.M.; McCormick, J.J.

1987-01-01

Results of studies on the transformation of diploid human fibroblasts in culture into tumor-forming cells by exposure to chemical carcinogens or radiation indicate that such transformation is multi-stepped process that at least one step, acquisition of anchorage independence, occurs as a mutagenic event. Studies comparing normal-repairing human cells with DNA repair-deficient cells, such as those derived from cancer-prone xeroderma pigmentosum patients, indicate that excision repair in human fibroblasts is essentially an error-free process that the ability to excise potentially cytotoxic, mutagenic, or transforming lesions induced DNA by carcinogens determines their ultimate biological consequences. Cells deficient in excision repair are abnormally sensitive to these agents. Studies with cells treated at various times in the cell cycle show that there is a certain limited amount of time available for DNA repair between the initial exposure and the onset of the cellular event responsible for mutation induction and transformation to anchorage independence. The data suggest that DNA replication on a template containing unexcised lesions (photoproducts, adducts) is the critical event

Nucleotide Excision Repair Lesion-Recognition Protein Rad4 Captures a Pre-Flipped Partner Base in a Benzo[a]pyrene-Derived DNA Lesion: How Structure Impacts the Binding Pathway.

Science.gov (United States)

Mu, Hong; Geacintov, Nicholas E; Min, Jung-Hyun; Zhang, Yingkai; Broyde, Suse

2017-06-19

The xeroderma pigmentosum C protein complex (XPC) recognizes a variety of environmentally induced DNA lesions and is the key in initiating their repair by the nucleotide excision repair (NER) pathway. When bound to a lesion, XPC flips two nucleotide pairs that include the lesion out of the DNA duplex, yielding a productively bound complex that can lead to successful lesion excision. Interestingly, the efficiencies of NER vary greatly among different lesions, influencing their toxicity and mutagenicity in cells. Though differences in XPC binding may influence NER efficiency, it is not understood whether XPC utilizes different mechanisms to achieve productive binding with different lesions. Here, we investigated the well-repaired 10R-(+)-cis-anti-benzo[a]pyrene-N 2 -dG (cis-B[a]P-dG) DNA adduct in a duplex containing normal partner C opposite the lesion. This adduct is derived from the environmental pro-carcinogen benzo[a]pyrene and is likely to be encountered by NER in the cell. We have extensively investigated its binding to the yeast XPC orthologue, Rad4, using umbrella sampling with restrained molecular dynamics simulations and free energy calculations. The NMR solution structure of this lesion in duplex DNA has shown that the dC complementary to the adducted dG is flipped out of the DNA duplex in the absence of XPC. However, it is not known whether the "pre-flipped" base would play a role in its recognition by XPC. Our results show that Rad4 first captures the displaced dC, which is followed by a tightly coupled lesion-extruding pathway for productive binding. This binding path differs significantly from the one deduced for the small cis-syn cyclobutane pyrimidine dimer lesion opposite mismatched thymines [ Mu , H. , ( 2015 ) Biochemistry , 54 ( 34 ), 5263 - 7 ]. The possibility of multiple paths that lead to productive binding to XPC is consistent with the versatile lesion recognition by XPC that is required for successful NER.

Distribution of ultraviolet-induced lesions in Simian Virus 40 DNA

International Nuclear Information System (INIS)

Bourre, F.; Renault, G.; Sarasin, A.; Seawell, P.C.

1985-01-01

In order to analyze the molecular mechanisms of mutagenesis in mammalian cells, we devised an analytical assay using Simian Virus 40 as biological probe. To study the possible correlations between the distribution of the lesions on the treated DNA and the distribution of mutations, we have located and quantified the lesions induced by ultraviolet light (254 nm) on a SV40 DNA fragment. At a fluence of 2,000J/m 2 , our results show that the formation frequency of thymine-thymine dimers (TT) is three to four times higher than the formation frequency of the other types of dimers (TC, CT, CC). On the other hand, the formation frequency of a dimer is influenced by the adjacent sequence. In particular, a pyrimidine in the 5' position of a thymine-thymine dimer enhances its formation frequency. At the dose used the formation frequency of the pyrimidine (6-4) pyrimidone photoproducts is twenty times less than the formation frequency of pyrimidine dimers. This paper shows the distribution of the major lesions induced by UV-light on a defined fragment of SV40 genome after UV irradiation. This work is necessary to get an insight in the molecular mechanisms of UV-mutagenesis

Influence of the complexity of radiation-induced DNA damage on enzyme recognition

International Nuclear Information System (INIS)

Palmer, Philip

2002-01-01

Ionising radiation is unique in inducing DNA clustered damage together with the simple isolated lesions. Understanding how these complex lesions are recognised and repaired by the cell is key to understanding the health risks associated with radiation exposure. This study focuses on whether ionising radiation-induced complex single-strand breaks (SSB) are recognised by DNA-PK and PARP, and whether the complexity of DSB influence their ligation by either DNA ligase lV/XRCC4 (LX) complex or T4 DNA ligase. Plasmid DNA, irradiated in aqueous solution using sparsely ionising γ-rays and densely ionising α-particles produce different yields of complex DNA damages, used as substrates for in vitro DNA-PK and PARP activity assays. The activity of DNA-PK to phosphorylate a peptide was determined using HF19 cell nuclear extracts as a source of DNA-PK. PARP ADP-ribosylation activity was determined using purified PARP enzyme. The activation of DNA-PK and PARP by irradiated DNA is due to SSB and not the low yield of DSB (linear plasmid DNA <10%). A ∼2 fold increase in DNA-PK activation and a ∼3-fold reduction in PARP activity seen on increasing the ionising density of the radiation (proportion of complex damage) are proposed to reflect changes in the complexity of SSB and may relate to damage signalling. Complex DSB synthesised as double-stranded oligonucleotides, with a 2 bp 5'-overhang, and containing modified lesions, 8-oxoguanine and abasic sites, at known positions relative to the termini were used as substrates for in vitro ligation by DNA ligase IV/XRCC4 or T4 ligase. The presence of a modified lesion 2 or 3 bp but not 4 bp from the 3'-termini and 2 or 6 bp from the 5'-termini caused a drastic reduction in the extent of ligation. Therefore, the presence of modified lesions near to the termini of a DSB may compromise their rejoining by non-homologous end-joining (NHEJ) involving the LX complex. (author)

Repair of model compounds of photoinduced lesions in DNA. Electrochemical approaches; Reparation de modeles de lesions photoinduites de l'ADN. Approches electrochimiques

Energy Technology Data Exchange (ETDEWEB)

Boussicault, F

2006-09-15

The goal of this work is to better understand the repair mechanism of photoinduced lesions in DNA (cyclobutane dimers and pyrimidine (6-4) pyrimidone adducts) by photolyase redox enzymes, using tools and concepts of molecular electrochemistry. Thanks to the study of model compounds of cyclobutane lesions by cyclic voltametry, we have been able to mimic the key step of the enzymatic repair (dissociative electron transfer) and to monitor the repair of model compounds by Escherichia coli DNA photolyase. From these results, we have discussed the repair mechanism, especially the stepwise or concerted character of the process. Repair mechanism of (6-4) adducts is not known now, but a possible pathway implies an electron transfer coupled to the cleavage of two bonds in the closed form of the lesions (oxetanes). Voltammetric study of reduction and oxidation of model oxetanes and their repair by E. coli DNA photolyase gave some experimental evidence confirming the proposed mechanism and allowing a better understanding of it. (author)

DNA-modified electrodes (Ⅶ)——Preparation and characterization of DNA-bonded and DNA-adsorbed SAM/Au electrodes

Institute of Scientific and Technical Information of China (English)

陆琪; 庞代文; 胡深; 程介克; 蔡雄伟; 施财辉; 毛秉伟; 戴鸿平

1999-01-01

Two kinds of DNA-modified electrodes were prepared by covalent and adsorptive immobilization of DNA onto self-assembled monolayers of 2, 2’-dithiodiethanol on gold electrodes and characterized by cyclic voltammetry, Xray photoelectron spectroscopy and scanning tunneling microscopy. The results suggest that the methods are satisfactory for the immobilization of DNA on electrodes.

Characterization of non-dimer DNA lesions and cellular damages caused by ultraviolet light

International Nuclear Information System (INIS)

Nakao, Kumi

1989-01-01

To understand the mechanisms of carcinogenicity and cytotoxicity induced by ultraviolet (UV) light, non-dimer DNA damages produced by near UV light (wave-length: 290∼320 nm) were examined by alkaline elution using Chinese hamster V-79 cells. UV exposure produced a dose-dependent induction of DNA single strand breaks and DNA-protein crosslinks. However, neither of these DNA lesions were repaired within a 24 hr incubation of the cells following UV exposure. Rather the number of these lesions increased. Also, UV exposure inhibited DNA and RNA synthesis. In addition, UV induced both cytotoxicity and chromosomal aberration. Electron spin resornance (ESR) studies showed that the exposure of cells to UV light resulted in the appearance of an ESR signal at -120degC. The roles of glutathione, vitamin E and vitamin B 2 , which were celluar antioxidant, on the induction of cytotoxicity by UV exposure were also examined. Pretreatment with vitamin E reduced the cytotoxicty caused by UV, whereas neither preteatment with vitamin B 2 nor the alteration of cellular gluthaione content affected the cytotoxicity. These results suggest that non-dimer DNA damages, such as DNA single strand breaks and DNA-protein crosslinks play an important role in inducing UV-carcinogenicity and UV-cytotoxicity, and that the mechanisms of these damages may be associated with the generation of free radicals. (author)

Does trans-lesion synthesis explain the UV-radiation resistance of DNA synthesis in C.elegans embryos?

International Nuclear Information System (INIS)

Hartman, Phil; Reddy, Jennifer; Svendsen, Betty-Ann

1991-01-01

Over 10-fold larger fluences were required to inhibit both DNA synthesis and cell division in wild-type C.elegans embryos as compared with other model systems or C.elegans rad mutants. In addition, unlike in other organisms, the molecular weight of daughter DNA strands was reduced only after large, superlethal fluences. The molecular weight of nascent DNA fragments exceeded the interdimer distance by up to 19-fold, indicating that C.elegans embryos can replicate through non-instructional lesions. This putative trans-lesion synthetic capability may explain the refractory nature of UV-radiation on embryonic DNA synthesis and nuclear division in C.elegans. (author). 42 refs.; 7 figs

Does trans-lesion synthesis explain the UV-radiation resistance of DNA synthesis in C. elegans embryos

Energy Technology Data Exchange (ETDEWEB)

Hartman, Phil; Reddy, Jennifer; Svendsen, Betty-Ann [Texas Christian Univ., Fort Worth, TX (United States). Dept. of Biology

1991-09-01

Over 10-fold larger fluences were required to inhibit both DNA synthesis and cell division in wild-type C.elegans embryos as compared with other model systems or C.elegans rad mutants. In addition, unlike in other organisms, the molecular weight of daughter DNA strands was reduced only after large, superlethal fluences. The molecular weight of nascent DNA fragments exceeded the interdimer distance by up to 19-fold, indicating that C.elegans embryos can replicate through non-instructional lesions. This putative trans-lesion synthetic capability may explain the refractory nature of UV-radiation on embryonic DNA synthesis and nuclear division in C.elegans. (author). 42 refs.; 7 figs.

The (6-4) Dimeric Lesion as a DNA Photosensitizer.

Science.gov (United States)

Vendrell-Criado, Victoria; Rodríguez-Muñiz, Gemma M; Lhiaubet-Vallet, Virginie; Cuquerella, M Consuelo; Miranda, Miguel A

2016-07-04

Based on our previous investigations into the photophysical properties of the 5-methyl-2-pyrimidone (Pyo) chromophore, we now extend our studies to the photobehavior of the dimeric (6-4) thymine photoproducts (6-4 PP) to evaluate their capability to act as instrinsic DNA photosensitizers. The lesion presents significant absorption in the UVB/UVA region, weak fluorescence emission, a singlet-excited-state energy of approximately 351 kJ mol(-1) , and a triplet-excited-state energy of 297 kJ mol(-1) . Its triplet transient absorption has a maximum at 420-440 nm, a lifetime of around 7 μs, and a high formation quantum yield, ΦISC =0.86. This species is efficiently quenched by thymidine. Its DNA photosensitizing properties are demonstrated by a series of experiments run on a pBR322 plasmid. The lesion photoinduces both single-strand breaks and the formation of cyclobutane thymine dimers. Altogether, these results show that, the substitution of the pyrimidone ring at C4 by a 5-hydroxy-5,6-dihydrothymine does not cancel out the photosensitization properties of the chromophore. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Monoclonal antibodies to DNA modified with cis- or trans-diamminedichloroplatinum(II)

International Nuclear Information System (INIS)

Sundquist, W.I.; Lippard, S.J.; Stollar, B.D.

1987-01-01

Murine monoclonal antibodies that bind selectively to adducts formed on DNA by the antitumor drug cis-diamminedichloroplatinum(II), cis-DDP, or to the chemothrapeutically inactive trans isomer trans-DDP were elicited by immunization with calf thymus DNA modified with either cis- or trans-DDP at ratios of bound platinum per nucleotide, (D/N)/sub b/, of 0.06-0.08. The binding of two monoclonal antibodies to cis-DDP-modified DNA was competitively inhibited in an enzyme-linked immunosorbent assay (ELISA) by 4-6 nM concentrations of cis-DDP bound to DNA. Adducts formed by cis-DDP on other synthetic DNA polymers did not inhibit antibody binding to cis-DDP-DNA. The biologically active compounds [Pt(en)Cl 2 ], [Pt(dach)Cl 2 ], and [Pt(NH 3 ) 2 (cbdca)] (carboplatin) all formed antibody-detectable adducts on DNA, whereas the inactive platinum complexes trans-DDP and [Pt(dien)Cl]Cl (dien, diethylenetriamine) did not. The monoclonal antibodies therefore recognize a bifunctional Pt-DNA adduct with cis stereochemistry in which platinum is coordinated by two adjacent guanines or, to a lesser degree, by adjacent adenine and guanine. A monoclonal antibody raised against trans-DDP-DNA was competitively inhibited in an ELISA by 40 nM trans-DDP bound to DNA. This antibody crossreacted with unmodified, denatured DNA. The recognition of cis- or trans-DDP-modified DNAs by monoclonal antibodies thus parallels the known modes of DNA binding of these compounds and may correlate with their biological activities

Role of DNA lesions and DNA repair in mutagenesis by carcinogens in diploid human fibroblasts

International Nuclear Information System (INIS)

Maher, V.M.; McCormick, J.J.

1986-01-01

The authors investigated the cytotoxicity, mutagenicity, and transforming activity of carcinogens and radiation in diploid human fibroblasts, using cells which differ in their DNA repair capacity. The results indicate that cell killing and induction of mutations are correlated with the number of specific lesions remaining unrepaired in the cells at a particular time posttreatment. DNA excision repair acts to eliminate potentially cytotoxic and mutagenic (and transforming) damage from DNA before these can be converted into permanent cellular effects. Normal human fibroblasts were derived from skin biopsies or circumcision material. Skin fibroblasts from xeroderma pigmentosum (XP) patients provided cells deficient in nucleotide excision repair of pyrimidine dimers or DNA adducts formed by bulky ring structures. Cytotoxicity was determined from loss of ability to form a colony. The genetic marker used was resistance to 6-thioguanine (TG). Transformation was measured by determining the frequency of anchorage-independent cells

Modes of DNA repair and replication

International Nuclear Information System (INIS)

Hanawalt, P.; Kondo, S.

1979-01-01

Modes of DNA repair and replication require close coordination as well as some overlap of enzyme functions. Some classes of recovery deficient mutants may have defects in replication rather than repair modes. Lesions such as the pyrimidine dimers produced by ultraviolet light irradiation are the blocks to normal DNA replication in vivo and in vitro. The DNA synthesis by the DNA polymerase 1 of E. coli is blocked at one nucleotide away from the dimerized pyrimidines in template strands. Thus, some DNA polymerases seem to be unable to incorporate nucleotides opposite to the non-pairing lesions in template DNA strands. The lesions in template DNA strands may block the sequential addition of nucleotides in the synthesis of daughter strands. Normal replication utilizes a constitutive ''error-free'' mode that copies DNA templates with high fidelity, but which may be totally blocked at a lesion that obscures the appropriate base pairing specificity. It might be expected that modified replication system exhibits generally high error frequency. The error rate of DNA polymerases may be controlled by the degree of phosphorylation of the enzyme. Inducible SOS system is controlled by recA genes that also control the pathways for recombination. It is possible that SOS system involves some process other than the modification of a blocked replication apparatus to permit error-prone transdimer synthesis. (Yamashita, S.)

Fundamental study of the radiation monitoring system based on evaluation of DNA lesions

International Nuclear Information System (INIS)

Shimizu, K.; Matuo, Y.; Izumi, Y.; Ikeda, T.

2011-01-01

The biological dosemeter that measures biological responses to ionising radiation is useful for radiation protection. This paper presents the development and characterisation of a gamma ray irradiation dosimetry system based on real-time PCR (polymerase chain reaction) methodology. Real-time PCR is used to amplify and simultaneously quantify a targeted DNA molecule. If there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment. The essential point of this assay is that DNA lesions caused by ionising radiation block DNA synthesis by DNA polymerase, resulting in a decrease in the amplification of a damaged DNA template compared with that of non-damaged DNA templates. (authors)

Modifying and adapting a plant-based DNA extraction protocol for ...

African Journals Online (AJOL)

... a 100 apparently healthy individuals residing in Calabar. The modified DNA procedure yielded good quality genomic DNA which was used in carrying out allele specific polymerase chain reaction which also yielded good quality amplicons. This method is simple and suitable for the extraction of DNA from human red cell.

Immunological probes for lesions and repoair patches in DNA

International Nuclear Information System (INIS)

Leadon, S.A.

1988-01-01

This paper describes two immunological approaches for the detection of DNA damage and its repair. The first uses a monoclonal antibody to directly measure the production and removal of one type of oxidized base, thymine glycol; the second uses an antibody to detect the repair synthesis event itself and, when combined with the use of molecular biological techniques, can be used to monitor the production and removal of lesions in specific sequences within the genome

Mitochondrial DNA mutations in preneoplastic lesions of the gastrointestinal tract: A biomarker for the early detection of cancer

Directory of Open Access Journals (Sweden)

Montgomery Elizabeth A

2006-12-01

Full Text Available Abstract Background Somatic mutations of mitochondrial DNA (mtDNA are common in many human cancers. We have described an oligonucleotide microarray ("MitoChip" for rapid sequencing of the entire mitochondrial genome (Zhou et al, J Mol Diagn 2006, facilitating the analysis of mtDNA mutations in preneoplastic lesions. We examined 14 precancerous lesions, including seven Barrett esophagus biopsies, with or without associated dysplasia; four colorectal adenomas; and three inflammatory colitis-associated dysplasia specimens. In all cases, matched normal tissues from the corresponding site were obtained as germline control. MitoChip analysis was performed on DNA obtained from cryostat-embedded specimens. Results A total of 513,639 bases of mtDNA were sequenced in the 14 samples, with 490,224 bases (95.4% bases assigned by the automated genotyping software. All preneoplastic lesions examined demonstrated at least one somatic mtDNA sequence alteration. Of the 100 somatic mtDNA alterations observed in the 14 cases, 27 were non-synonymous coding region mutations (i.e., resulting in an amino acid change, 36 were synonymous, and 37 involved non-coding mtDNA. Overall, somatic alterations most commonly involved the COI, ND4 and ND5 genes. Notably, somatic mtDNA alterations were observed in preneoplastic lesions of the gastrointestinal tract even in the absence of histopathologic evidence of dysplasia, suggesting that the mitochondrial genome is susceptible at the earliest stages of multistep cancer progression. Conclusion Our findings further substantiate the rationale for exploring the mitochondrial genome as a biomarker for the early diagnosis of cancer, and confirm the utility of a high-throughput array-based platform for this purpose from a clinical applicability standpoint.

DNA-modified electrodes fabricated using copper-free click chemistry for enhanced protein detection.

Science.gov (United States)

Furst, Ariel L; Hill, Michael G; Barton, Jacqueline K

2013-12-31

A method of DNA monolayer formation has been developed using copper-free click chemistry that yields enhanced surface homogeneity and enables variation in the amount of DNA assembled; extremely low-density DNA monolayers, with as little as 5% of the monolayer being DNA, have been formed. These DNA-modified electrodes (DMEs) were characterized visually, with AFM, and electrochemically, and were found to facilitate DNA-mediated reduction of a distally bound redox probe. These low-density monolayers were found to be more homogeneous than traditional thiol-modified DNA monolayers, with greater helix accessibility through an increased surface area-to-volume ratio. Protein binding efficiency of the transcriptional activator TATA-binding protein (TBP) was also investigated on these surfaces and compared to that on DNA monolayers formed with standard thiol-modified DNA. Our low-density monolayers were found to be extremely sensitive to TBP binding, with a signal decrease in excess of 75% for 150 nM protein. This protein was detectable at 4 nM, on the order of its dissociation constant, with our low-density monolayers. The improved DNA helix accessibility and sensitivity of our low-density DNA monolayers to TBP binding reflects the general utility of this method of DNA monolayer formation for DNA-based electrochemical sensor development.

Interaction of DNA-lesions induced by sodium fluoride and radiation and its influence in apoptotic induction in cancer cell lines

Directory of Open Access Journals (Sweden)

Santosh Podder

2015-01-01

Full Text Available Fluoride is an essential trace element but also an environmental contaminant with major sources of exposure being drinking water, food and pesticides. Previous studies showed that sodium fluoride (NaF at 5 mM or more is required to induce apoptosis and chromosome aberrations and proposed that DNA damage and apoptosis play an important role in toxicity of excessive fluoride. The aim of this study is directed to understand the nature of DNA-lesions induced by NaF by allowing its interaction with radiation induced DNA-lesions. NaF 5 mM was used after observing inability to induce DNA damages and apoptosis by single exposure with 50 μM or 1 mM NaF. Co-exposure to NaF and radiation significantly increased the frequency of aberrant metaphases and exchange aberrations in human lymphocytes and arrested the cells in G1 stage instead of apoptotic death. Flow cytometric analysis, DNA fragmentation and PARP-cleavage analysis clearly indicated that 5 mM NaF together with radiation (1 Gy induced apoptosis in both U87 and K562 cells due to down regulation of expression of anti-apoptotic proteins, like Bcl2 in U87 and inhibitors of apoptotic proteins like survivin and cIAP in K562 cells. This study herein suggested that single exposure with extremely low concentration of NaF unable to induce DNA lesions whereas higher concentration induced DNA lesions interact with the radiation-induced DNA lesions. Both are probably repaired rapidly thus showed increased interactive effect. Coexposure to NaF and radiation induces more apoptosis in cancer cell lines which could be due to increased exchange aberrations through lesions interaction and downregulating anti-apoptotic genes.

Genotoxic effect and antigen binding characteristics of SLE auto-antibodies to peroxynitrite-modified human DNA.

Science.gov (United States)

Khan, Md Asad; Alam, Khursheed; Mehdi, Syed Hassan; Rizvi, M Moshahid A

2017-12-01

Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease characterized by auto-antibodies against native deoxyribonucleic acid after modification and is one of the reasons for the development of SLE. Here, we have evaluated the structural perturbations in human placental DNA by peroxynitrite using spectroscopy, thermal denaturation and high-performance liquid chromatography (HPLC). Peroxynitrite is a powerful potent bi-functional oxidative/nitrative agent that is produced both endogenously and exogenously. In experimental animals, the peroxynitrite-modified DNA was found to be highly immunogenic. The induced antibodies showed cross-reactions with different types of DNA and nitrogen bases that were modified with peroxynitrite by inhibition ELISA. The antibody activity was inhibited by approximately 89% with its immunogen as the inhibitor. The antigen-antibodies interaction between induced antibodies with peroxynitrite-modified DNA showed retarded mobility as compared to the native form. Furthermore, significantly increased binding was also observed in SLE autoantibodies with peroxynitrite-modified DNA than native form. Moreover, DNA isolated from lymphocyte of SLE patients revealed significant recognition of anti-peroxynitrite-modified DNA immunoglobulin G (IgG). Our data indicates that DNA modified with peroxynitrite presents unique antigenic determinants that may induce autoantibody response in SLE. Copyright © 2017 Elsevier Inc. All rights reserved.

SUMO-2 Orchestrates Chromatin Modifiers in Response to DNA Damage

DEFF Research Database (Denmark)

Hendriks, Ivo A; Treffers, Louise W; Verlaan-de Vries, Matty

2015-01-01

dynamically SUMOylated interaction networks of chromatin modifiers, transcription factors, DNA repair factors, and nuclear body components. SUMOylated chromatin modifiers include JARID1B/KDM5B, JARID1C/KDM5C, p300, CBP, PARP1, SetDB1, and MBD1. Whereas SUMOylated JARID1B was ubiquitylated by the SUMO......-targeted ubiquitin ligase RNF4 and degraded by the proteasome in response to DNA damage, JARID1C was SUMOylated and recruited to the chromatin to demethylate histone H3K4....

Effects of radiation quality and oxygen on clustered DNA lesions and cell death.

Science.gov (United States)

Stewart, Robert D; Yu, Victor K; Georgakilas, Alexandros G; Koumenis, Constantinos; Park, Joo Han; Carlson, David J

2011-11-01

Radiation quality and cellular oxygen concentration have a substantial impact on DNA damage, reproductive cell death and, ultimately, the potential efficacy of radiation therapy for the treatment of cancer. To better understand and quantify the effects of radiation quality and oxygen on the induction of clustered DNA lesions, we have now extended the Monte Carlo Damage Simulation (MCDS) to account for reductions in the initial lesion yield arising from enhanced chemical repair of DNA radicals under hypoxic conditions. The kinetic energy range and types of particles considered in the MCDS have also been expanded to include charged particles up to and including (56)Fe ions. The induction of individual and clustered DNA lesions for arbitrary mixtures of different types of radiation can now be directly simulated. For low-linear energy transfer (LET) radiations, cells irradiated under normoxic conditions sustain about 2.9 times as many double-strand breaks (DSBs) as cells irradiated under anoxic conditions. New experiments performed by us demonstrate similar trends in the yields of non-DSB (Fpg and Endo III) clusters in HeLa cells irradiated by γ rays under aerobic and hypoxic conditions. The good agreement among measured and predicted DSBs, Fpg and Endo III cluster yields suggests that, for the first time, it may be possible to determine nucleotide-level maps of the multitude of different types of clustered DNA lesions formed in cells under reduced oxygen conditions. As particle LET increases, the MCDS predicts that the ratio of DSBs formed under normoxic to hypoxic conditions by the same type of radiation decreases monotonically toward unity. However, the relative biological effectiveness (RBE) of higher-LET radiations compared to (60)Co γ rays (0.24 keV/μm) tends to increase with decreasing oxygen concentration. The predicted RBE of a 1 MeV proton (26.9 keV/μm) relative to (60)Co γ rays for DSB induction increases from 1.9 to 2.3 as oxygen concentration

Detection and analysis of human papillomavirus 16 and 18 homologous DNA sequences in oral lesions.

Science.gov (United States)

Wen, S; Tsuji, T; Li, X; Mizugaki, Y; Hayatsu, Y; Shinozaki, F

1997-01-01

The prevalence of human papillomavirus (HPV) 16 and 18 was investigated in oral lesions of the population of northeast China including squamous cell carcinomas (SCCs), candida leukoplakias, lichen planuses and papillomas, by southern blot hybridization with polymerase chain reaction (PCR). Amplified HPV16 and 18 E6 DNA was analyzed by cycle sequence. HPV DNA was detected in 14 of 45 SCCs (31.1%). HPV18 E6 DNA and HPV16 E6. DNA were detected in 24.4% and 20.0% of SCCs. respectively. Dual infection of both HPV 16 and HPV 18 was detected in 6 of 45 SCCs (13.3%), but not in other oral lesions. HPV 18 E6 DNA was also detected in 2 of 3 oral candida leukoplakias, but in none of the 5 papillomas. Our study indicated that HPV 18 infection might be more frequent than HPV 16 infection in oral SCCs in northeast Chinese, dual infection of high risk HPV types was restricted in oral SCCs, and that HPV infection might be involved in the pathogenesis of oral candida leukoplakia.

[Application of DNA extraction kit, 'GM quicker' for detection of genetically modified soybeans].

Science.gov (United States)

Sato, Noriko; Sugiura, Yoshitsugu; Tanaka, Toshitsugu

2012-01-01

Several DNA extraction methods have been officially introduced to detect genetically modified soybeans, but the choice of DNA extraction kits depend on the nature of the samples, such as grains or processed foods. To overcome this disadvantage, we examined whether the GM quicker kit is available for both grains and processed foods. We compared GM quicker with four approved DNA extraction kits in respect of DNA purity, copy numbers of lectin gene, and working time. We found that the DNA quality of GM quicker was superior to that of the other kits for grains, and the procedure was faster. However, in the case of processed foods, GM quicker was not superior to the other kits. We therefore investigated an unapproved GM quicker 3 kit, which is available for DNA extraction from processed foods, such as tofu and boiled soybeans. The GM quicker 3 kit provided good DNA quality from both grains and processed foods, so we made a minor modification of the GM quicker-based protocol that was suitable for processed foods, using GM quicker and its reagents. The modified method enhanced the performance of GM quicker with processed foods. We believe that GM quicker with the modified protocol is an excellent tool to obtain high-quality DNA from grains and processed foods for detection of genetically modified soybeans.

Detection of DNA of genetically modified maize by a silicon nanowire field-effect transistor

International Nuclear Information System (INIS)

Pham, Van Binh; Tung Pham, Xuan Thanh; Duong Dang, Ngoc Thuy; Tuyen Le, Thi Thanh; Tran, Phu Duy; Nguyen, Thanh Chien; Nguyen, Van Quoc; Dang, Mau Chien; Tong, Duy Hien; Van Rijn, Cees J M

2011-01-01

A silicon nanowire field-effect transistor based sensor (SiNW-FET) has been proved to be the most sensitive and powerful device for bio-detection applications. In this paper, SiNWs were first fabricated by using our recently developed deposition and etching under angle technique (DEA), then used to build up the complete SiNW device based biosensor. The fabricated SiNW biosensor was used to detect DNA of genetically modified maize. As the DNA of the genetically modified maize has particular DNA sequences of 35S promoter, we therefore designed 21 mer DNA oligonucleotides, which are used as a receptor to capture the transferred DNA of maize. In our work, the SiNW biosensor could detect DNA of genetically modified maize with concentrations down to about 200 pM

The (6-4) Dimeric Lesion as a DNA Photosensitizer

OpenAIRE

Vendrell Criado, Victoria; Rodríguez Muñiz, Gemma María; Lhiaubet ., Virginie Lyria; Cuquerella Alabort, Maria Consuelo; Miranda Alonso, Miguel Ángel

2016-01-01

[EN] Based on our previous investigations into the photophysical properties of the 5-methyl-2-pyrimidone (Pyo) chromophore, we now extend our studies to the photobehavior of the dimeric (6-4) thymine photoproducts (6-4 PP) to evaluate their capability to act as instrinsic DNA photosensitizers. The lesion presents significant absorption in the UVB/UVA region, weak fluorescence emission, a singlet-excited-state energy of approximately 351 kJ mol(-1), and a triplet-excited-state energy of 297 kJ...

Molecular dynamics of formation of TD lesioned DNA complexed with repair enzyme - onset of the enzymatic repair process

Energy Technology Data Exchange (ETDEWEB)

Pinak, Miroslav [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

1999-12-01

To describe the first step of the enzymatic repair process (formation of complex enzyme-DNA), in which the thymine dimer (TD) part is removed from DNA, the 500 picosecond (ps) molecular dynamics (MD) simulation of TD lesioned DNA and part of repair enzyme cell (inclusive of catalytic center - Arg-22, Glu-23, Arg-26 and Thr-2) was performed. TD is UV originated lesion in DNA and T4 Endonuclease V is TD specific repair enzyme. Both molecules were located in the same simulation cell and their relative movement was examined. During the simulation the research was focused on the role of electrostatic energy in formation of complex enzyme-DNA. It is found, that during the first 100 ps of MD, the part of enzyme approaches the DNA surface at the TD lesion, interacts extensively by electrostatic and van der Walls interactions with TD part of DNA and forms complex that lasts stabile for 500 ps of MD. In the beginning of MD, the positive electrostatic interaction energy between part of enzyme and TD ({approx} +10 kcal/mol) drives enzyme towards the DNA molecule. Water-mediated hydrogen bonds between enzyme and DNA help to keep complex stabile. As a reference, the MD simulation of the identical system with native DNA molecule (two native thymines (TT) instead of TD) was performed. In this system the negative electrostatic interaction energy between part of enzyme and TT ({approx} -11 kcal/mol), in contrary to the positive one in the system with TD, doesn't drive enzyme towards DNA and complex is not formed. (author)

Molecular dynamics of formation of TD lesioned DNA complexed with repair enzyme - onset of the enzymatic repair process

International Nuclear Information System (INIS)

Pinak, Miroslav

1999-12-01

To describe the first step of the enzymatic repair process (formation of complex enzyme-DNA), in which the thymine dimer (TD) part is removed from DNA, the 500 picosecond (ps) molecular dynamics (MD) simulation of TD lesioned DNA and part of repair enzyme cell (inclusive of catalytic center - Arg-22, Glu-23, Arg-26 and Thr-2) was performed. TD is UV originated lesion in DNA and T4 Endonuclease V is TD specific repair enzyme. Both molecules were located in the same simulation cell and their relative movement was examined. During the simulation the research was focused on the role of electrostatic energy in formation of complex enzyme-DNA. It is found, that during the first 100 ps of MD, the part of enzyme approaches the DNA surface at the TD lesion, interacts extensively by electrostatic and van der Walls interactions with TD part of DNA and forms complex that lasts stabile for 500 ps of MD. In the beginning of MD, the positive electrostatic interaction energy between part of enzyme and TD (∼ +10 kcal/mol) drives enzyme towards the DNA molecule. Water-mediated hydrogen bonds between enzyme and DNA help to keep complex stabile. As a reference, the MD simulation of the identical system with native DNA molecule (two native thymines (TT) instead of TD) was performed. In this system the negative electrostatic interaction energy between part of enzyme and TT (∼ -11 kcal/mol), in contrary to the positive one in the system with TD, doesn't drive enzyme towards DNA and complex is not formed. (author)

From DNA lesions to tissue malfunction

International Nuclear Information System (INIS)

Denekamp, J.

1989-01-01

After large doses of radiation, tissues fail to function when the proliferating cells lose their clonogenic ability. This results from unrepaired or misrepaired double strand breaks in the DNA. The lesions are inflicted immediately but there is a variable latent period before tissue damage is expressed. This ranges from a few days in intestine, to weeks in skin, and to months or years in deep visceral tissues, e.g. heart, lung, kidney, spinal cord. The latency relates to the proliferation kinetics of each tissue component. Doses of 10-30 Gy do not cause serious functional defects in differentiated cells, but they prevent successful mitosis in proliferating cells. Thus each tissue continues to function until its differentiated cells are lost by normal wear and tear processes. After a time which relates to the natural lifespan of the differentiated cells, failure to provide replacement cells from the proliferating compartment becomes important and the tissue shows atrophy and eventually a functional deficit. If the radiation exposure is divided into a series of smaller exposures or is given at a low dose-rate, the biochemical repair of DNA is more effective and less damage is observed. After high LET ionizing radiation, e.g. neutrons or α particles, the response is almost linear and is not affected by doserate or fractionation. (author)

5-year clinical performance of resin composite versus resin modified glass ionomer restorative system in non-carious cervical lesions

DEFF Research Database (Denmark)

Franco, Eduardo Batista; Benetti, Ana Raquel; Ishikiriama, Sérgio Kiyoshi

2006-01-01

To comparatively assess the 5-year clinical performance of a 1-bottle adhesive and resin composite system with a resin-modified glass ionomer restorative in non-carious cervical lesions.......To comparatively assess the 5-year clinical performance of a 1-bottle adhesive and resin composite system with a resin-modified glass ionomer restorative in non-carious cervical lesions....

Molecular dynamics simulation studies of radiation damaged DNA. Molecules and repair enzymes

International Nuclear Information System (INIS)

Pinak, Miroslav

2004-12-01

Molecular dynamics (MD) studies on several radiation damages to DNA and their recognition by repair enzymes are introduced in order to describe the stepwise description of molecular process observed at radiation lesion sites. MD studies were performed on pyrimidine (thymine dimer, thymine glycol) and purine (8-oxoguanine) lesions using an MD simulation code AMBER 5.0. The force field was modified for each lesion. In all cases the significant structural changes in the DNA double helical structure were observed; a) the breaking of hydrogen bond network between complementary bases and resulting opening of the double helix (8-oxoguanine); b) the sharp bending of the DNA helix centered at the lesion site (thymine dimer, thymine glycol); and c) the flipping-out base on the strand complementary to the lesion (8-oxoguanine). These changes were related to the overall collapsing double helical structure around the lesion and might facilitate the docking of the repair enzyme into the DNA and formation of DNA-enzyme complex. In addition to the structural changes, at lesion sites there were found electrostatic interaction energy values different from those at native sites (thymine dimer -10 kcal/mol, thymine glycol -26 kcal/mol, 8-oxoguanine -48 kcal/mol). These values of electrostatic energy may discriminate lesion from values at native sites (thymine 0 kcal/mol, guanine -37 kcal/mol) and enable a repair enzyme to recognize a lesion during scanning DNA surface. The observed specific structural conformation and energetic properties at the lesions sites are factors that guide a repair enzyme to discriminate lesions from non-damaged native DNA segments. (author)

In vitro enzymatic studies on the nature and repair of x-ray induced lesions in DNA

International Nuclear Information System (INIS)

Wallace, S.S.

1979-01-01

Areas studied include: purification and properties of enzyme probes for x-ray induced DNA lesions using E. Coli x-ray endonuclease and S. cerevisiae endonuclease E; use of enzymes probes; and use of physical, chemical and enzymatic probes to quantify x-ray-induced lesions in viruses and cells

Visualization of complex DNA damage along accelerated ions tracks

Science.gov (United States)

Kulikova, Elena; Boreyko, Alla; Bulanova, Tatiana; Ježková, Lucie; Zadneprianetc, Mariia; Smirnova, Elena

2018-04-01

The most deleterious DNA lesions induced by ionizing radiation are clustered DNA double-strand breaks (DSB). Clustered or complex DNA damage is a combination of a few simple lesions (single-strand breaks, base damage etc.) within one or two DNA helix turns. It is known that yield of complex DNA lesions increases with increasing linear energy transfer (LET) of radiation. For investigation of the induction and repair of complex DNA lesions, human fibroblasts were irradiated with high-LET 15N ions (LET = 183.3 keV/μm, E = 13MeV/n) and low-LET 60Co γ-rays (LET ≈ 0.3 keV/μm) radiation. DNA DSBs (γH2AX and 53BP1) and base damage (OGG1) markers were visualized by immunofluorecence staining and high-resolution microscopy. The obtained results showed slower repair kinetics of induced DSBs in cells irradiated with accelerated ions compared to 60Co γ-rays, indicating induction of more complex DNA damage. Confirming previous assumptions, detailed 3D analysis of γH2AX/53BP1 foci in 15N ions tracks revealed more complicated structure of the foci in contrast to γ-rays. It was shown that proteins 53BP1 and OGG1 involved in repair of DNA DSBs and modified bases, respectively, were colocalized in tracks of 15N ions and thus represented clustered DNA DSBs.

A high-throughput and quantitative method to assess the mutagenic potential of translesion DNA synthesis

Science.gov (United States)

Taggart, David J.; Camerlengo, Terry L.; Harrison, Jason K.; Sherrer, Shanen M.; Kshetry, Ajay K.; Taylor, John-Stephen; Huang, Kun; Suo, Zucai

2013-01-01

Cellular genomes are constantly damaged by endogenous and exogenous agents that covalently and structurally modify DNA to produce DNA lesions. Although most lesions are mended by various DNA repair pathways in vivo, a significant number of damage sites persist during genomic replication. Our understanding of the mutagenic outcomes derived from these unrepaired DNA lesions has been hindered by the low throughput of existing sequencing methods. Therefore, we have developed a cost-effective high-throughput short oligonucleotide sequencing assay that uses next-generation DNA sequencing technology for the assessment of the mutagenic profiles of translesion DNA synthesis catalyzed by any error-prone DNA polymerase. The vast amount of sequencing data produced were aligned and quantified by using our novel software. As an example, the high-throughput short oligonucleotide sequencing assay was used to analyze the types and frequencies of mutations upstream, downstream and at a site-specifically placed cis–syn thymidine–thymidine dimer generated individually by three lesion-bypass human Y-family DNA polymerases. PMID:23470999

Base-pairing preferences, physicochemical properties and mutational behaviour of the DNA lesion 8-nitroguanine.

Science.gov (United States)

Bhamra, Inder; Compagnone-Post, Patricia; O'Neil, Ian A; Iwanejko, Lesley A; Bates, Andrew D; Cosstick, Richard

2012-11-01

8-Nitro-2'-deoxyguanosine (8-nitrodG) is a relatively unstable, mutagenic lesion of DNA that is increasingly believed to be associated with tissue inflammation. Due to the lability of the glycosidic bond, 8-nitrodG cannot be incorporated into oligodeoxynucleotides (ODNs) by chemical DNA synthesis and thus very little is known about its physicochemical properties and base-pairing preferences. Here we describe the synthesis of 8-nitro-2'-O-methylguanosine, a ribonucleoside analogue of this lesion, which is sufficiently stable to be incorporated into ODNs. Physicochemical studies demonstrated that 8-nitro-2'-O-methylguanosine adopts a syn conformation about the glycosidic bond; thermal melting studies and molecular modelling suggest a relatively stable syn-8-nitroG·anti-G base pair. Interestingly, when this lesion analogue was placed in a primer-template system, extension of the primer by either avian myeloblastosis virus reverse transcriptase (AMV-RT) or human DNA polymerase β (pol β), was significantly impaired, but where incorporation opposite 8-nitroguanine did occur, pol β showed a 2:1 preference to insert dA over dC, while AMV-RT incorporated predominantly dC. The fact that no 8-nitroG·G base pairing is seen in the primer extension products suggests that the polymerases may discriminate against this pairing system on the basis of its poor geometric match to a Watson-Crick pair.

Base-pairing preferences, physicochemical properties and mutational behaviour of the DNA lesion 8-nitroguanine†

Science.gov (United States)

Bhamra, Inder; Compagnone-Post, Patricia; O’Neil, Ian A.; Iwanejko, Lesley A.; Bates, Andrew D.; Cosstick, Richard

2012-01-01

8-Nitro-2′-deoxyguanosine (8-nitrodG) is a relatively unstable, mutagenic lesion of DNA that is increasingly believed to be associated with tissue inflammation. Due to the lability of the glycosidic bond, 8-nitrodG cannot be incorporated into oligodeoxynucleotides (ODNs) by chemical DNA synthesis and thus very little is known about its physicochemical properties and base-pairing preferences. Here we describe the synthesis of 8-nitro-2′-O-methylguanosine, a ribonucleoside analogue of this lesion, which is sufficiently stable to be incorporated into ODNs. Physicochemical studies demonstrated that 8-nitro-2′-O-methylguanosine adopts a syn conformation about the glycosidic bond; thermal melting studies and molecular modelling suggest a relatively stable syn-8-nitroG·anti-G base pair. Interestingly, when this lesion analogue was placed in a primer-template system, extension of the primer by either avian myeloblastosis virus reverse transcriptase (AMV-RT) or human DNA polymerase β (pol β), was significantly impaired, but where incorporation opposite 8-nitroguanine did occur, pol β showed a 2:1 preference to insert dA over dC, while AMV-RT incorporated predominantly dC. The fact that no 8-nitroG·G base pairing is seen in the primer extension products suggests that the polymerases may discriminate against this pairing system on the basis of its poor geometric match to a Watson–Crick pair. PMID:22965127

Accumulation of premutagenic DNA lesions in mice defective in removal of oxidative base damage

Science.gov (United States)

Klungland, Arne; Rosewell, Ian; Hollenbach, Stephan; Larsen, Elisabeth; Daly, Graham; Epe, Bernd; Seeberg, Erling; Lindahl, Tomas; Barnes, Deborah E.

1999-01-01

DNA damage generated by oxidant byproducts of cellular metabolism has been proposed as a key factor in cancer and aging. Oxygen free radicals cause predominantly base damage in DNA, and the most frequent mutagenic base lesion is 7,8-dihydro-8-oxoguanine (8-oxoG). This altered base can pair with A as well as C residues, leading to a greatly increased frequency of spontaneous G·C→T·A transversion mutations in repair-deficient bacterial and yeast cells. Eukaryotic cells use a specific DNA glycosylase, the product of the OGG1 gene, to excise 8-oxoG from DNA. To assess the role of the mammalian enzyme in repair of DNA damage and prevention of carcinogenesis, we have generated homozygous ogg1−/− null mice. These animals are viable but accumulate abnormal levels of 8-oxoG in their genomes. Despite this increase in potentially miscoding DNA lesions, OGG1-deficient mice exhibit only a moderately, but significantly, elevated spontaneous mutation rate in nonproliferative tissues, do not develop malignancies, and show no marked pathological changes. Extracts of ogg1 null mouse tissues cannot excise the damaged base, but there is significant slow removal in vivo from proliferating cells. These findings suggest that in the absence of the DNA glycosylase, and in apparent contrast to bacterial and yeast cells, an alternative repair pathway functions to minimize the effects of an increased load of 8-oxoG in the genome and maintain a low endogenous mutation frequency. PMID:10557315

[Mechanistic modelling allows to assess pathways of DNA lesion interactions underlying chromosome aberration formation].

Science.gov (United States)

Eĭdel'man, Iu A; Slanina, S V; Sal'nikov, I V; Andreev, S G

2012-12-01

The knowledge of radiation-induced chromosomal aberration (CA) mechanisms is required in many fields of radiation genetics, radiation biology, biodosimetry, etc. However, these mechanisms are yet to be quantitatively characterised. One of the reasons is that the relationships between primary lesions of DNA/chromatin/chromosomes and dose-response curves for CA are unknown because the pathways of lesion interactions in an interphase nucleus are currently inaccessible for direct experimental observation. This article aims for the comparative analysis of two principally different scenarios of formation of simple and complex interchromosomal exchange aberrations: by lesion interactions at chromosome territories' surface vs. in the whole space of the nucleus. The analysis was based on quantitative mechanistic modelling of different levels of structures and processes involved in CA formation: chromosome structure in an interphase nucleus, induction, repair and interactions of DNA lesions. It was shown that the restricted diffusion of chromosomal loci, predicted by computational modelling of chromosome organization, results in lesion interactions in the whole space of the nucleus being impossible. At the same time, predicted features of subchromosomal dynamics agrees well with in vivo observations and does not contradict the mechanism of CA formation at the surface of chromosome territories. On the other hand, the "surface mechanism" of CA formation, despite having certain qualities, proved to be insufficient to explain high frequency of complex exchange aberrations observed by mFISH technique. The alternative mechanism, CA formation on nuclear centres is expected to be sufficient to explain frequent complex exchanges.

Hydrophobically modified chitosan/gold nanoparticles for DNA delivery

International Nuclear Information System (INIS)

Bhattarai, Shanta Raj; Remant Bahadur, K.C.; Aryal, Santosh; Bhattarai, Narayan; Kim, Sun Young; Yi, Ho Keun; Hwang, Pyoung Han; Kim, Hak Yong

2008-01-01

Present study dealt an application of modified chitosan gold nanoparticles (Nac-6-Au) for the immobilization of necked plasmid DNA. Gold nanoparticles stabilized with N-acylated chitosan were prepared by graft-onto approach. The stabilized gold nanoparticles were characterized by different physico-chemical techniques such as UV-vis, TEM, ELS and DLS. MTT assay was used for in vitro cytotoxicity of the nanoparticles into three different cell lines (NIH 3T3, CT-26 and MCF-7). The formulation of plasmid DNA with the nanoparticles corresponds to the complex forming capacity and in-vitro/in-vivo transfection efficiency was studied via gel electrophoresis and transfection methods, respectively. Results showed the modified chitosan gold nanoparticles were well-dispersed and spherical in shape with average size around 10∼12 nm in triple distilled water at pH 7.4, and showed relatively no cytotoxicity at low concentration. Addition of plasmid DNA on the aqueous solution of the nanoparticles markedly reduced surface potential (50.0∼66.6%) as well as resulted in a 13.33% increase in hydrodynamic diameters of the formulated nanoparticles. Transfection efficiency of Nac-6-Au/DNA was dependent on cell type, and higher β-galactosidase activity was observed on MCF-7 breast cancer cell. Typically, this activity was 5 times higher in 4.5 mg/ml nanoparticles concentration than that achieved by the nanoparticles of other concentrations (and/or control). However, this activity was lower in in-vitro and dramatically higher in in-vivo than that of commercially available transfection kit (Lipofectin (registered) ) and DNA. From these results, it can be expected to develop alternative new vectors for gene delivery

Redox-sensitive structural change in the A-domain of HMGB1 and its implication for the binding to cisplatin modified DNA

International Nuclear Information System (INIS)

Wang, Jing; Tochio, Naoya; Takeuchi, Aya; Uewaki, Jun-ichi; Kobayashi, Naohiro; Tate, Shin-ichi

2013-01-01

Highlights: •The structure of the oxidized A-domain of human HMGB1 was solved. •Phe38 ring was flipped in the oxidized structure from that in the reduced form. •The flipped ring disables the intercalation into the cisplatinated lesions. •The functionally relevant redox-dependent structural change was described. -- Abstract: HMGB1 (high-mobility group B1) is a ubiquitously expressed bifunctional protein that acts as a nuclear protein in cells and also as an inflammatory mediator in the extracellular space. HMGB1 changes its functions according to the redox states in both intra- and extra-cellular environments. Two cysteines, Cys23 and Cys45, in the A-domain of HMGB1 form a disulfide bond under oxidative conditions. The A-domain with the disulfide bond shows reduced affinity to cisplatin modified DNA. We have solved the oxidized A-domain structure by NMR. In the structure, Phe38 has a flipped ring orientation from that found in the reduced form; the phenyl ring in the reduced form intercalates into the platinated lesion in DNA. The phenyl ring orientation in the oxidized form is stabilized through intramolecular hydrophobic contacts. The reorientation of the Phe38 ring by the disulfide bond in the A-domain may explain the reduced HMGB1 binding affinity towards cisplatinated DNA

Thickness, morphology, and optoelectronic characteristics of pristine and surfactant-modified DNA thin films

International Nuclear Information System (INIS)

Arasu, Velu; Reddy Dugasani, Sreekantha; Son, Junyoung; Gnapareddy, Bramaramba; Ha Park, Sung; Jeon, Sohee; Jeong, Jun-Ho

2017-01-01

Although the preparation of DNA thin films with well-defined thicknesses controlled by simple physical parameters is crucial for constructing efficient, stable, and reliable DNA-based optoelectronic devices and sensors, it has not been comprehensively studied yet. Here, we construct DNA and surfactant-modified DNA thin films by drop-casting and spin-coating techniques. The DNA thin films formed with different control parameters, such as drop-volume and spin-speed at given DNA concentrations, exhibit characteristic thickness, surface roughness, surface potential, and absorbance, which are measured by a field emission scanning electron microscope, a surface profilometer, an ellipsometer, an atomic force microscope, a Kelvin probe force microscope, and an UV–visible spectroscope. From the observations, we realized that thickness significantly affects the physical properties of DNA thin films. This comprehensive study of thickness-dependent characteristics of DNA and surfactant-modified DNA thin films provides insight into the choice of fabrication techniques in order for the DNA thin films to have desired physical characteristics in further applications, such as optoelectronic devices and sensors. (paper)

Lesion-induced DNA weak structural changes detected by pulsed EPR spectroscopy combined with site-directed spin labelling.

Science.gov (United States)

Sicoli, Giuseppe; Mathis, Gérald; Aci-Sèche, Samia; Saint-Pierre, Christine; Boulard, Yves; Gasparutto, Didier; Gambarelli, Serge

2009-06-01

Double electron-electron resonance (DEER) was applied to determine nanometre spin-spin distances on DNA duplexes that contain selected structural alterations. The present approach to evaluate the structural features of DNA damages is thus related to the interspin distance changes, as well as to the flexibility of the overall structure deduced from the distance distribution. A set of site-directed nitroxide-labelled double-stranded DNA fragments containing defined lesions, namely an 8-oxoguanine, an abasic site or abasic site analogues, a nick, a gap and a bulge structure were prepared and then analysed by the DEER spectroscopic technique. New insights into the application of 4-pulse DEER sequence are also provided, in particular with respect to the spin probes' positions and the rigidity of selected systems. The lesion-induced conformational changes observed, which were supported by molecular dynamics studies, confirm the results obtained by other, more conventional, spectroscopic techniques. Thus, the experimental approaches described herein provide an efficient method for probing lesion-induced structural changes of nucleic acids.

XRCC1 and PCNA are loading platforms with distinct kinetic properties and different capacities to respond to multiple DNA lesions

Directory of Open Access Journals (Sweden)

Leonhardt Heinrich

2007-09-01

Full Text Available Abstract Background Genome integrity is constantly challenged and requires the coordinated recruitment of multiple enzyme activities to ensure efficient repair of DNA lesions. We investigated the dynamics of XRCC1 and PCNA that act as molecular loading platforms and play a central role in this coordination. Results Local DNA damage was introduced by laser microirradation and the recruitment of fluorescent XRCC1 and PCNA fusion proteins was monitored by live cell microscopy. We found an immediate and fast recruitment of XRCC1 preceding the slow and continuous recruitment of PCNA. Fluorescence bleaching experiments (FRAP and FLIP revealed a stable association of PCNA with DNA repair sites, contrasting the high turnover of XRCC1. When cells were repeatedly challenged with multiple DNA lesions we observed a gradual depletion of the nuclear pool of PCNA, while XRCC1 dynamically redistributed even to lesions inflicted last. Conclusion These results show that PCNA and XRCC1 have distinct kinetic properties with functional consequences for their capacity to respond to successive DNA damage events.

XRCC1 and PCNA are loading platforms with distinct kinetic properties and different capacities to respond to multiple DNA lesions

Science.gov (United States)

Mortusewicz, Oliver; Leonhardt, Heinrich

2007-01-01

Background Genome integrity is constantly challenged and requires the coordinated recruitment of multiple enzyme activities to ensure efficient repair of DNA lesions. We investigated the dynamics of XRCC1 and PCNA that act as molecular loading platforms and play a central role in this coordination. Results Local DNA damage was introduced by laser microirradation and the recruitment of fluorescent XRCC1 and PCNA fusion proteins was monitored by live cell microscopy. We found an immediate and fast recruitment of XRCC1 preceding the slow and continuous recruitment of PCNA. Fluorescence bleaching experiments (FRAP and FLIP) revealed a stable association of PCNA with DNA repair sites, contrasting the high turnover of XRCC1. When cells were repeatedly challenged with multiple DNA lesions we observed a gradual depletion of the nuclear pool of PCNA, while XRCC1 dynamically redistributed even to lesions inflicted last. Conclusion These results show that PCNA and XRCC1 have distinct kinetic properties with functional consequences for their capacity to respond to successive DNA damage events. PMID:17880707

Prevalence of human papillomavirus DNA in female cervical lesions from Rio de Janeiro, Brazil

Directory of Open Access Journals (Sweden)

S. M. B. Cavalcanti

1994-12-01

Full Text Available A hundred-sixty paraffin-embedded specimens from female cervical lesions were examined for human papillomavirus (HPV types 6, 11, 16 and 18 infections by non-isotopic in situ hybridization. The data were compared with histologic diagnosis. Eighty-eight (55 biopsies contained HPV DNA sequences. In low grade cervical intraepithelial neoplasias (CIN I, HPV infection was detected in 78.7 of the cases, the benign HPV 6 was the most prevalent type. HPV DNA was detected in 58 of CIN II and CIN III cases and in 41.8 of squamous cell carcinomas (SCC. Histologically normal women presented 20 of HPV infection. Oncogenic HPV was found in 10 of these cases, what may indicate a higher risk of developing CINs and cancer. Twenty-five percent of the infected tissues contained mixed infections. HPV 16 was the most common type infecting the cervix and its prevalence raised significantly with the severity of the lesions, pointing its role in cancer pathogenesis. White women presented twice the cervical lesions of mulatto and African origin women, although HPV infection rates were nearly the same for the three groups (approximately 50. Our results showed that HPV typing by in situ hybridization is a useful tool for distinguishing between low and high risk cervical lesions. Further studies are required to elucidate risk factors associated with HPV infection and progression to malignancy in Brazilian population.

Preferential recognition of auto-antibodies against 4-hydroxynonenal modified DNA in the cancer patients.

Science.gov (United States)

Faisal, Mohammad; Shahab, Uzma; Alatar, Abdulrahman A; Ahmad, Saheem

2017-11-01

The structural perturbations in DNA molecule may be caused by a break in a strand, a missing base from the backbone, or a chemically changed base. These alterations in DNA that occurs naturally can result from metabolic or hydrolytic processes. DNA damage plays a major role in the mutagenesis, carcinogenesis, aging and various other patho-physiological conditions. DNA damage can be induced through hydrolysis, exposure to reactive oxygen species (ROS) and other reactive carbonyl metabolites including 4-hydroxynonenal (HNE). 4-HNE is an important lipid peroxidation product which has been implicated in the mutagenesis and carcinogenesis processes. The present study examines to probe the presence of auto-antibodies against 4-hydroxynonenal damaged DNA (HNE-DNA) in various cancer subjects. In this study, the purified calf thymus DNA was damaged by the action of 4-HNE. The DNA was incubated with 4-HNE for 24 h at 37°C temperature. The binding characteristics of cancer auto-antibodies were assessed by direct binding and competitive inhibition ELISA. DNA modifications produced hyperchromicity in UV spectrum and decreased fluorescence intensity. Cancer sera exhibited enhanced binding with the 4-HNE modified calf thymus DNA as compared to its native conformer. The 4-HNE modified DNA presents unique epitopes which may be one of the factors for the auto-antibody induction in cancer patients. The HNE modified DNA presents unique epitopes which may be one of the factors for the autoantibody induction in cancer patients. © 2017 Wiley Periodicals, Inc.

Sex determination based on amelogenin DNA by modified electrode with gold nanoparticle.

Science.gov (United States)

Mazloum-Ardakani, Mohammad; Rajabzadeh, Nooshin; Benvidi, Ali; Heidari, Mohammad Mehdi

2013-12-15

We have developed a simple and renewable electrochemical biosensor based on carbon paste electrode (CPE) for the detection of DNA synthesis and hybridization. CPE was modified with gold nanoparticles (AuNPs), which are helpful for immobilization of thiolated bioreceptors. AuNPs were characterized by scanning electron microscopy (SEM). Self-assembled monolayers (SAMs) of thiolated single-stranded DNA (SH-ssDNA) of the amelogenin gene was formed on CPE. The immobilization of the probe and its hybridization with the target DNA was optimized using different experimental conditions. The modified electrode was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The electrochemical response of ssDNA hybridization and DNA synthesis was measured using differential pulse voltammetry (DPV) with methylene blue (MB) as an electroactive indicator. The new biosensor can distinguish between complementary and non-complementary strands of amelogenin ssDNA. Genomic DNA was extracted from blood and was detected based on changes in the MB reduction signal. These results demonstrated that the new biosensor could be used for sex determination. The proposed biosensor in this study could be used for detection and discrimination of polymerase chain reaction (PCR) products of amelogenin DNA. Copyright © 2013 Elsevier Inc. All rights reserved.

Repair of UVC induced DNA lesions in erythrocytes from Carassius auratus gibelio

International Nuclear Information System (INIS)

Bagdonas, E.; Zukas, K.

2004-01-01

The kinetics of UVC (254 nm) irradiation induced DNA single-strand breaks generated during the excision repair of UV induced DNA damage in erythrocytes from Carassius auratus gibelio were studied using alkaline comet assay. Nucleotide excision repair recognised DNA lesions such as UVC induced cyclobutane pyrimidine dimers and 6-4 pyrimidine-pyrimidone photoproducts and produced DNA single-stranded breaks that were easily detected by comet assay. After irradiation of erythrocytes with 58 j/m 2 UVC dose, there was an increase in comet tail moment (CTM) at 2 hours post-radiation, whereas at 4 hours post-radiation CTM decreased and did not differ significantly from the control level (P=0,127). When erythrocytes were exposed to 173 J/m 2 UVC dose, the excision repair delayed in the beginning (0 hours), reached maximum level at 2 hours post-radiation (CTM-54,8) and showed slightly decreased level at 4 hours post-radiation (CTM=18,5). (author)

Chemistry of the 8-Nitroguanine DNA Lesion: Reactivity, Labelling and Repair.

Science.gov (United States)

Alexander, Katie J; McConville, Matthew; Williams, Kathryn R; Luzyanin, Konstantin V; O'Neil, Ian A; Cosstick, Richard

2018-02-26

The 8-nitroguanine lesion in DNA is increasingly associated with inflammation-related carcinogenesis, whereas the same modification on guanosine 3',5'-cyclic monophosphate generates a second messenger in NO-mediated signal transduction. Very little is known about the chemistry of 8-nitroguanine nucleotides, despite the fact that their biological effects are closely linked to their chemical properties. To this end, a selection of chemical reactions have been performed on 8-nitroguanine nucleosides and oligodeoxynucleotides. Reactions with alkylating reagents reveal how the 8-nitro substituent affects the reactivity of the purine ring, by significantly decreasing the reactivity of the N2 position, whilst the relative reactivity at N1 appears to be enhanced. Interestingly, the displacement of the nitro group with thiols results in an efficient and specific method of labelling this lesion and is demonstrated in oligodeoxynucleotides. Additionally, the repair of this lesion is also shown to be a chemically feasible reaction through a reductive denitration with a hydride source. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bioorthogonal Metabolic DNA Labelling using Vinyl Thioether-Modified Thymidine and o-Quinolinone Quinone Methide.

Science.gov (United States)

Gubu, Amu; Li, Long; Ning, Yan; Zhang, Xiaoyun; Lee, Seonghyun; Feng, Mengke; Li, Qiang; Lei, Xiaoguang; Jo, Kyubong; Tang, Xinjing

2018-04-17

Bioorthogonal metabolic DNA labeling with fluorochromes is a powerful strategy to visualize DNA molecules and their functions. Here, we report the development of a new DNA metabolic labeling strategy enabled by the catalyst-free bioorthogonal ligation using vinyl thioether modified thymidine and o-quinolinone quinone methide. With the newly designed vinyl thioether-modified thymidine (VTdT), we added labeling tags on cellular DNA, which could further be linked to fluorochromes in cells. Therefore, we successfully visualized the DNA localization within cells as well as single DNA molecules without other staining reagents. In addition, we further characterized this bioorthogonal DNA metabolic labeling using DNase I digestion, MS characterization of VTdT as well as VTdT-oQQF conjugate in cell nuclei or mitochondria. This technique provides a powerful strategy to study DNA in cells, which paves the way to achieve future spatiotemporal deciphering of DNA synthesis and functions. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Traveling Rocky Roads: The Consequences of Transcription-Blocking DNA Lesions on RNA Polymerase II

NARCIS (Netherlands)

B. Steurer (Barbara); J.A. Marteijn (Jurgen)

2016-01-01

textabstractThe faithful transcription of eukaryotic genes by RNA polymerase II (RNAP2) is crucial for proper cell function and tissue homeostasis. However, transcription-blocking DNA lesions of both endogenous and environmental origin continuously challenge the progression of elongating RNAP2. The

Modified surface of titanium dioxide nanoparticles-based biosensor for DNA detection

Science.gov (United States)

Nadzirah, Sh.; Hashim, U.; Rusop, M.

2018-05-01

A new technique was used to develop a simple and selective picoammeter DNA biosensor for identification of E. coli O157:H7. This biosensor was fabricated from titanium dioxide nanoparticles that was synthesized by sol-gel method and spin-coated on silicon dioxide substrate via spinner. 3-Aminopropyl triethoxy silane (APTES) was used to modify the surface of TiO2. Simple surface modification approach has been applied; which is single dropping of APTES onto the TiO2 nanoparticles surface. Carboxyl modified probe DNA has been bind onto the surface of APTES/TiO2 without any amplifier element. Electrical signal has been used as the indicator to differentiate each step (surface modification of TiO2 and probe DNA immobilization). The I-V measurements indicate extremely low current (pico-ampere) flow through the device which is 2.8138E-10 A for pure TiO2 nanoparticles, 2.8124E-10 A after APTES modification and 3.5949E-10 A after probe DNA immobilization.

Expression of DNA repair proteins MSH2, MLH1 and MGMT in human benign and malignant thyroid lesions: An immunohistochemical study

Science.gov (United States)

Giaginis, Constantinos; Michailidi, Christina; Stolakis, Vasileios; Alexandrou, Paraskevi; Tsourouflis, Gerasimos; Klijanienko, Jerzy; Delladetsima, Ioanna; Theocharis, Stamatios

2011-01-01

Summary Background DNA repair is a major defense mechanism, which contributes to the maintenance of genetic sequence, and minimizes cell death, mutation rates, replication errors, DNA damage persistence and genomic instability. Alterations in the expression levels of proteins participating in DNA repair mechanisms have been associated with several aspects of cancer biology. The present study aimed to evaluate the clinical significance of DNA repair proteins MSH2, MLH1 and MGMT in benign and malignant thyroid lesions. Material/Methods MSH2, MLH1 and MGMT protein expression was assessed immunohistochemically on paraffin-embedded thyroid tissues from 90 patients with benign and malignant lesions. Results The expression levels of MLH1 was significantly upregulated in cases with malignant compared to those with benign thyroid lesions (p=0.038). The expression levels of MGMT was significantly downregulated in malignant compared to benign thyroid lesions (p=0.001). Similar associations for both MLH1 and MGMT between cases with papillary carcinoma and hyperplastic nodules were also noted (p=0.014 and p=0.026, respectively). In the subgroup of malignant thyroid lesions, MSH2 downregulation was significantly associated with larger tumor size (p=0.031), while MLH1 upregulation was significantly associated with the presence of lymphatic and vascular invasion (p=0.006 and p=0.002, respectively). Conclusions Alterations in the mismatch repair proteins MSH2 and MLH1 and the direct repair protein MGMT may result from tumor development and/or progression. Further studies are recommended to draw definite conclusions on the clinical significance of DNA repair proteins in thyroid neoplasia. PMID:21358597

Relationship of DNA lesions and their repair to chromosomal aberration production

International Nuclear Information System (INIS)

Bender, M.A.

1979-01-01

Recent work on the roles of specific kinds of DNA lesions and their enzymatic repair systems in the production of chromosomal aberrations seems consistent with a simple molecular model of chromosomal aberrations formation. Evidence from experiments with the human repair-deficient genetic diseases xeroderma pigmentosom, ataxia telangiectasia, and Fanconi's anemia is reviewed in the light of the contributions to aberration production of single and double polynucleotide strand breaks, base damage, polynucleotide strand crosslinks, and pyrimidine cyclobutane dimers

Relationship of DNA lesions and their repair to chromosomal aberration production

Energy Technology Data Exchange (ETDEWEB)

Bender, M.A.

1979-01-01

Recent work on the roles of specific kinds of DNA lesions and their enzymatic repair systems in the production of chromosomal aberrations seems consistent with a simple molecular model of chromosomal aberrations formation. Evidence from experiments with the human repair-deficient genetic diseases xeroderma pigmentosom, ataxia telangiectasia, and Fanconi's anemia is reviewed in the light of the contributions to aberration production of single and double polynucleotide strand breaks, base damage, polynucleotide strand crosslinks, and pyrimidine cyclobutane dimers.

Excision repair of gamma-ray-induced alkali-stable DNA lesions with the help of γ-endonuclease from Micrococcus luteus

International Nuclear Information System (INIS)

Tomilin, N.V.; Barenfeld, L.S.

1979-01-01

γ-endonuclease Y, an enzyme that hydrolyses phosphodiester bonds at alkali-stable lesions in γ-irradiated (N 2 , tris buffer) DNA, has been partially purified from Micrococcus luteus. The enzyme has a molecular weight of about 19 000, induces single-strand breaks with 3'OH-5'PO 4 termini and contains endonuclease activity towards DNA treated with 7-bromomethylbenz(a)anthracene. γ-endonuclease Y induces breaks in OsO 4 -treated poly(dA-dT) and apparently is specific towards γ-ray-induced base lesions of the t' type. The complete excision repair of γ-endonuclease Y substrate sites has been performed in vitro by γ-endonuclease Y, DNA polymerase and ligase. (author)

Excision repair of gamma-ray-induced alkali-stable DNA lesions with the help of. gamma. -endonuclease from Micrococcus luteus

Energy Technology Data Exchange (ETDEWEB)

Tomilin, N V; Barenfeld, L S [AN SSSR, Leningrad. Inst. Tsitologii

1979-03-01

..gamma..-endonuclease Y, an enzyme that hydrolyses phosphodiester bonds at alkali-stable lesions in ..gamma..-irradiated (N/sub 2/, tris buffer) DNA, has been partially purified from Micrococcus luteus. The enzyme has a molecular weight of about 19 000, induces single-strand breaks with 3'OH-5'PO/sub 4/ termini and contains endonuclease activity towards DNA treated with 7-bromomethylbenz(a)anthracene. ..gamma..-endonuclease Y induces breaks in OsO/sub 4/-treated poly(dA-dT) and apparently is specific towards ..gamma..-ray-induced base lesions of the t' type. The complete excision repair of ..gamma..-endonuclease Y substrate sites has been performed in vitro by ..gamma..-endonuclease Y, DNA polymerase and ligase.

An immunochemical approach to the study of DNA damage and repair

International Nuclear Information System (INIS)

Wallace, S.S.; Erlanger, B.F.

1992-05-01

The overall objective of this project has been to develop immunochemical methods to quantitate unique DNA base damages in order to facilitate studies on radiation-induced damage production and repair. Specifically, we have been using antibodies raised to damaged bases to quantitate unique lesions in model systems in order to evaluate their potential biological consequences. Our approach has been to synthesize modified nucleotides or nucleosides, conjugate them to protein carriers, and use the conjugates as immunogens in rabbits or to prepare monoclonal antibodies. We have been studying damages that are stable radiolysis products found in X-irradiated DNA and thus of potential biological consequence. Our aim is to build an in vitro and in vivo data base on the interactions between model DNA lesions and such cellular enzymes as DNA polymerases and repair endonucleases. Initial studies have focused on pyrimidine ring saturation products (thymine glycol.and dihydrothymine), products resulting from ring fragmentation or base loss (urea, Β-ureidoisobutyric acid, abasic sites), 7-hydro-8-oxopurines, and more recently, cytosine radiolysis products. These modified bases serve as useful models for examining the potential lethal and/or mutagenic (carcinogenic) effects of the products of DNA radiolysis

Genomic and functional integrity of the hematopoietic system requires tolerance of oxidative DNA lesions

DEFF Research Database (Denmark)

Martín-Pardillos, Ana; Tsaalbi-Shtylik, Anastasia; Chen, Si

2017-01-01

-distorting nucleotide lesions, resulted in the perinatal loss of hematopoietic stem cells, progressive loss of bone marrow, and fatal aplastic anemia between 3 and 4 months of age. This was associated with replication stress, genomic breaks, DNA damage signaling, senescence, and apoptosis in bone marrow. Surprisingly...

An immunochemical approach to the study of DNA damage and repair

International Nuclear Information System (INIS)

Wallace, S.S.

1991-08-01

The overall objective of this project is to produce antibodies to unique modified DNA bases and develop immunochemical assays to quantitate these lesions in damaged DNA. During this past year we have characterized antibodies to 8-oxopurines, produced novel antibodies to 5-hydroxyuracil and developed new methodologies to increase our level of sensitivity of detection. 7 refs., 5 figs

53BP1 nuclear bodies form around DNA lesions generated by mitotic transmission of chromosomes under replication stress

DEFF Research Database (Denmark)

Lukas, Claudia; Savic, Velibor; Bekker-Jensen, Simon

2011-01-01

stress increases the frequency of chromosomal lesions that are transmitted to daughter cells. Throughout G1, these lesions are sequestered in nuclear compartments marked by p53-binding protein 1 (53BP1) and other chromatin-associated genome caretakers. We show that the number of such 53BP1 nuclear bodies...... increases after genetic ablation of BLM, a DNA helicase associated with dissolution of entangled DNA. Conversely, 53BP1 nuclear bodies are partially suppressed by knocking down SMC2, a condensin subunit required for mechanical stability of mitotic chromosomes. Finally, we provide evidence that 53BP1 nuclear...... bodies shield chromosomal fragile sites sequestered in these compartments against erosion. Together, these data indicate that restoration of DNA or chromatin integrity at loci prone to replication problems requires mitotic transmission to the next cell generations....

DNA Methylation of MMP9 Is Associated with High Levels of MMP-9 Messenger RNA in Periapical Inflammatory Lesions.

Science.gov (United States)

Campos, Kelma; Gomes, Carolina Cavalieri; Farias, Lucyana Conceição; Silva, Renato Menezes; Letra, Ariadne; Gomez, Ricardo Santiago

2016-01-01

Matrix metalloproteinases (MMPs) are the major class of enzymes responsible for degradation of extracellular matrix components and participate in the pathogenesis of periapical inflammatory lesions. MMP expression may be regulated by DNA methylation. The purpose of the present investigation was to analyze the expression of MMP2 and MMP9 in periapical granulomas and radicular cysts and to test the hypothesis that, in these lesions, their transcription may be modulated by DNA methylation. Methylation-specific polymerase chain reaction was used to evaluate the DNA methylation pattern of the MMP2 gene in 13 fresh periapical granuloma samples and 10 fresh radicular cyst samples. Restriction enzyme digestion was used to assess methylation of the MMP9 gene in 12 fresh periapical granuloma samples and 10 fresh radicular cyst samples. MMP2 and MMP9 messenger RNA transcript levels were measured by quantitative real-time polymerase chain reaction. All periapical lesions and healthy mucosa samples showed partial methylation of the MMP2 gene; however, periapical granulomas showed higher MMP2 mRNA expression levels than healthy mucosa (P = .014). A higher unmethylated profile of the MMP9 gene was found in periapical granulomas and radicular cysts compared with healthy mucosa. In addition, higher MMP9 mRNA expression was observed in the periapical lesions compared with healthy tissues. The present study suggests that the unmethylated status of the MMP9 gene in periapical lesions may explain the observed up-regulation of messenger RNA transcription in these lesions. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Formamidopyrimidines in DNA: mechanisms of formation, repair, and biological effects.

Science.gov (United States)

Dizdaroglu, Miral; Kirkali, Güldal; Jaruga, Pawel

2008-12-15

Oxidatively induced damage to DNA results in a plethora of lesions comprising modified bases and sugars, DNA-protein cross-links, tandem lesions, strand breaks, and clustered lesions. Formamidopyrimidines, 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), are among the major lesions generated in DNA by hydroxyl radical attack, UV radiation, or photosensitization under numerous in vitro and in vivo conditions. They are formed by one-electron reduction of C8-OH-adduct radicals of purines and thus have a common precursor with 8-hydroxypurines generated upon one-electron oxidation. Methodologies using mass spectrometry exist to accurately measure FapyAde and FapyGua in vitro and in vivo. Formamidopyrimidines are repaired by base excision repair. Numerous prokaryotic and eukaryotic DNA glycosylases are highly specific for removal of these lesions from DNA in the first step of this repair pathway, indicating their biological importance. FapyAde and FapyGua are bypassed by DNA polymerases with the insertion of the wrong intact base opposite them, leading to mutagenesis. In mammalian cells, the mutagenicity of FapyGua exceeds that of 8-hydroxyguanine, which is thought to be the most mutagenic of the oxidatively induced lesions in DNA. The background and formation levels of the former in vitro and in vivo equal or exceed those of the latter under various conditions. FapyAde and FapyGua exist in living cells at significant background levels and are abundantly generated upon exposure to oxidative stress. Mice lacking the genes that encode specific DNA glycosylases accumulate these lesions in different organs and, in some cases, exhibit a series of pathological conditions including metabolic syndrome and cancer. Animals exposed to environmental toxins accumulate formamidopyrimidines in their organs. Here, we extensively review the mechanisms of formation, measurement, repair, and biological effects of formamidopyrimidines

Base-pairing preferences, physicochemical properties and mutational behaviour of the DNA lesion 8-nitroguanine †

OpenAIRE

Bhamra, Inder; Compagnone-Post, Patricia; O’Neil, Ian A.; Iwanejko, Lesley A.; Bates, Andrew D.; Cosstick, Richard

2012-01-01

8-Nitro-2′-deoxyguanosine (8-nitrodG) is a relatively unstable, mutagenic lesion of DNA that is increasingly believed to be associated with tissue inflammation. Due to the lability of the glycosidic bond, 8-nitrodG cannot be incorporated into oligodeoxynucleotides (ODNs) by chemical DNA synthesis and thus very little is known about its physicochemical properties and base-pairing preferences. Here we describe the synthesis of 8-nitro-2′-O-methylguanosine, a ribonucleoside analogue of this lesi...

RECENT ADVANCES IN STRATEGIES FOR IMMUNOTHERAPY OF HUMAN PAPILLOMAVIRUS-INDUCED LESIONS

Science.gov (United States)

Kanodia, Shreya; Da Silva, Diane M.; Kast, W. Martin

2016-01-01

Human papillomavirus (HPV)-induced lesions are distinct in that they have targetable foreign antigens, the expression of which is necessary to maintain the cancerous phenotype. Hence, they pose as a very attractive target for “proof of concept” studies in the development of therapeutic vaccines. This review will focus on the most recent clinical trials for the immunotherapy of mucosal and cutaneous HPV-induced lesions as well as emerging therapeutic strategies that have been tested in pre-clinical models for HPV-induced lesions. Progress in peptide-based vaccines, DNA-based vaccines, viral/bacterial vector-based vaccines, immune response modifiers, photodynamic therapy and T cell receptor based therapy for HPV will be discussed. PMID:17973257

The impact of arginine-modified chitosan-DNA nanoparticles on the function of macrophages

Energy Technology Data Exchange (ETDEWEB)

Liu Lanxia; Bai Yuanyuan; Song Chunni; Zhu Dunwan; Song Liping; Zhang Hailing; Dong Xia; Leng Xigang, E-mail: lengxg@bme.org.c [Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Laboratory of Bioengineering (China)

2010-06-15

It has been demonstrated that incorporation of arginine moieties into chitosan significantly elevates the transgenic efficacy of the chitosan. However, little is known about the impact of arginine-modified chitosan on the function of macrophages, which play a vitally important role in the inflammatory response of the body to foreign substances, especially particulate substances. This study was designed to investigate the impact of arginine-modified chitosan/DNA nanoparticles on the function of the murine macrophage through observation of phagocytic activity and production of pro-inflammatory cytokines (IL-1{beta}, IL-6, IL-10, IL-12, and TNF-{alpha}). Results showed that both chitosan/DNA nanoparticles and arginine-modified chitosan/DNA nanoparticles, containing 20 {mu}g/mL DNA, were internalized by almost all the macrophages in contact. This led to no significant changes, compared to the non-exposure group, in production of cytokines and phagocytic activity of the macrophages 24 h post co-incubation, whereas exposure to LPS induced obviously elevated cytokine production and phagocytic activity, suggesting that incorporation of arginine moieties into chitosan does not have a negative impact on the function of the macrophages.

Ex vivo assessment of protective effects of carvacrol against DNA lesions induced in primary rat cells by visible light excited methylene blue (VL+MB).

Science.gov (United States)

Slamenova, D; Horvathova, E; Chalupa, I; Wsolova, L; Navarova, J

2011-01-01

Carvacrol belongs to frequently occurring phenolic components of essential oils (EOs) and it is present in many kinds of plants. Biological effect of this phenol derivative on human beings is however not sufficiently known. The present study was undertaken to evaluate the level of VL+MB-induced oxidative DNA lesions in hepatocytes and testicular cells (freshly isolated from control or carvacrol-watered rats) by the modified single cell gel electrophoresis (SCGE). The results showed that carvacrol significantly reduced the level of VL+MB-induced oxidized bases (EndoIII- and Fpg-sensitive sites) only in hepatocytes but not in testicular cells. Chromosomal aberration assay of primary hepatocytes, isolated from control or carvacrol-watered rats did not testify any genotoxic activity of carvacrol. We suggest that in vivo applied synthetic carvacrol, whose antioxidative activity was confirmed by DPPH assay, exhibits primarily a strong hepatoprotective activity against oxidative damage to DNA.

Synthesis of base-modified 2'-deoxyribonucleoside triphosphates and their use in enzymatic synthesis of modified DNA for applications in bioanalysis and chemical biology.

Science.gov (United States)

Hocek, Michal

2014-11-07

The synthesis of 2'-deoxyribonucleoside triphosphates (dNTPs) either by classical triphosphorylation of nucleosides or by aqueous cross-coupling reactions of halogenated dNTPs is discussed. Different enzymatic methods for synthesis of modified oligonucleotides and DNA by polymerase incorporation of modified nucleotides are summarized, and the applications in redox or fluorescent labeling, as well as in bioconjugations and modulation of interactions of DNA with proteins, are outlined.

Pyrimidine dimers are not the principal pre-mutagenic lesions induced in lambda phage DNA by ultraviolet light

International Nuclear Information System (INIS)

Wood, R.D.

1985-01-01

Experiments were performed to examine the role of cyclobutyl pyrimidine dimers in the process of mutagenesis by ultraviolet light. Lambda phage DNA was irradiated with u.v. and then incubated with an Escherichia coli photoreactivating enzyme, which monomerizes cyclobutyl pyrimidine dimers upon exposure to visible light. The photoreactivated DNA was packaged into lambda phage particles, which were used to infect E. coli uvr - host cells that had been induced for SOS functions by ultraviolet irradiation. Photoreactivation removed most toxic lesions from irradiated phage, but did not change the frequency of induction of mutations to the clear-plaque phenotype. This implies that cyclobutyl pyrimidine dimers can be lethal, but usually do not serve as sites of mutations in the phage. The DNA sequences of mutants, derived from photoreactivated DNA showed that almost two-thirds (16/28) were transitions, the same fraction found for u.v. mutagenesis without photoreactivation. Thus the lesion inducing transitions is not the cyclobutyl pyrimidine dimer. Photoreactivation of SOS-induced host cells before infection with u.v.-irradiated phage reduced mutagenesis substantially. In this case, photoreversal of cyclobutyl dimers serves to reduce expression of the SOS functions that are required in targeted u.v. mutagenesis. (author)

One simple DNA extraction device and its combination with modified visual loop-mediated isothermal amplification for rapid on-field detection of genetically modified organisms.

Science.gov (United States)

Zhang, Miao; Liu, Yinan; Chen, Lili; Quan, Sheng; Jiang, Shimeng; Zhang, Dabing; Yang, Litao

2013-01-02

Quickness, simplicity, and effectiveness are the three major criteria for establishing a good molecular diagnosis method in many fields. Herein we report a novel detection system for genetically modified organisms (GMOs), which can be utilized to perform both on-field quick screening and routine laboratory diagnosis. In this system, a newly designed inexpensive DNA extraction device was used in combination with a modified visual loop-mediated isothermal amplification (vLAMP) assay. The main parts of the DNA extraction device included a silica gel membrane filtration column and a modified syringe. The DNA extraction device could be easily operated without using other laboratory instruments, making it applicable to an on-field GMO test. High-quality genomic DNA (gDNA) suitable for polymerase chain reaction (PCR) and isothermal amplification could be quickly isolated from plant tissues using this device within 15 min. In the modified vLAMP assay, a microcrystalline wax encapsulated detection bead containing SYBR green fluorescent dye was introduced to avoid dye inhibition and cross-contaminations from post-LAMP operation. The system was successfully applied and validated in screening and identification of GM rice, soybean, and maize samples collected from both field testing and the Grain Inspection, Packers, and Stockyards Administration (GIPSA) proficiency test program, which demonstrated that it was well-adapted to both on-field testing and/or routine laboratory analysis of GMOs.

Radiation chemical and photochemical study of Z-DNA modified by 2-aminopurine and 8- bromodeoxyguanosine

International Nuclear Information System (INIS)

Kimura, T.; Kawai, K.; Majima, T.

2003-01-01

DNA is able to take a number of local conformations. (CG) n repeats have the highest potential to Z-DNA which has a left-handed zig-zag backbone and unusual syn-conformation purine base. Because of the polymorphic nature of dinucleotide repeats, it seems possible that Z-DNA forming sequences may provide a source of genetic variation if they occur in regions that are important for the regulation of gene activity. Here, we investigated structural properties of Z-DNA compared with those of B-DNA with respects to one-electron attachment reaction of 8-bromodeoxyguanosine (dBrG) and fluorescence properties of 2-aminopurine (Ap). To investigate one-electron attachment reaction of Z-DNA, we synthesized oligodeoxynucleotides modified by dBrG in which syn-conformation deoxyguanosine was stabilized by steric repulsion between 8-bromo group of dBrG and sugar moiety in Z-DNA. Debromination from the dBrG modified oligodeoxynucleotides occurred from the one-electron attachment during the gamma-ray irradiation. The structural dependence of B- and Z-DNA was observed in the one-electron attachment reaction. Interestingly, the higher conversion of dBrG were observed in the Z-DNA than in the B-DNA. Since the solvent accessibility to purine base in Z-DNA increases compared with that in B-DNA, it is suggested that the electron attachment is enhanced in Z-DNA than in B-DNA. Next we studied the fluorescence properties of Ap in left-handed Z-DNA and compared with those in B-DNA. Since photoexcited adenine analogue Ap can serve as a sensitive probe of DNA structural dynamics, we synthesized Ap- and dBrG-modified oligodeoxynucleotides. Higher intensity was observed in the steady-state fluorescence of Ap in Z-DNA than in B-DNA. A new peak at 275 nm was observed in the excitation spectrum measured at the Ap emission wavelength 370 nm in Z-DNA. This has been explained by the energy transfer from the excited nucleobases to Ap. It is found that Ap is a useful fluorescence probe of Z-DNA

Atherosclerotic lesions and mitochondria DNA deletions in brain microvessels: implication in the pathogenesis of Alzheimer's disease.

Science.gov (United States)

Aliev, Gjumrakch; Gasimov, Eldar; Obrenovich, Mark E; Fischbach, Kathryn; Shenk, Justin C; Smith, Mark A; Perry, George

2008-01-01

The pathogenesis that is primarily responsible for Alzheimer's disease (AD) and cerebrovascular accidents (CVA) appears to involve chronic hypoperfusion. We studied the ultrastructural features of vascular lesions and mitochondria in brain vascular wall cells from human AD biopsy samples and two transgenic mouse models of AD, yeast artificial chromosome (YAC) and C57B6/SJL Tg (+), which overexpress human amyloid beta precursor protein (AbetaPP). In situ hybridization using probes for normal and 5 kb deleted human and mouse mitochondrial DNA (mtDNA) was performed along with immunocytochemistry using antibodies against the Abeta peptide processed from AbetaPP, 8-hydroxy-2'-guanosine (8OHG), and cytochrome c oxidase (COX). More amyloid deposition, oxidative stress markers as well as mitochondrial DNA deletions and structural abnormalities were present in the vascular walls of the human AD samples and the AbetaPP-YAC and C57B6/SJL Tg (+) transgenic mice compared to age-matched controls. Ultrastructural damage in perivascular cells highly correlated with endothelial lesions in all samples. Therefore, pharmacological interventions, directed at correcting the chronic hypoperfusion state, may change the natural course of the development of dementing neurodegeneration.

DNA Polymerases λ and β: The Double-Edged Swords of DNA Repair

Directory of Open Access Journals (Sweden)

Elisa Mentegari

2016-08-01

Full Text Available DNA is constantly exposed to both endogenous and exogenous damages. More than 10,000 DNA modifications are induced every day in each cell’s genome. Maintenance of the integrity of the genome is accomplished by several DNA repair systems. The core enzymes for these pathways are the DNA polymerases. Out of 17 DNA polymerases present in a mammalian cell, at least 13 are specifically devoted to DNA repair and are often acting in different pathways. DNA polymerases β and λ are involved in base excision repair of modified DNA bases and translesion synthesis past DNA lesions. Polymerase λ also participates in non-homologous end joining of DNA double-strand breaks. However, recent data have revealed that, depending on their relative levels, the cell cycle phase, the ratio between deoxy- and ribo-nucleotide pools and the interaction with particular auxiliary proteins, the repair reactions carried out by these enzymes can be an important source of genetic instability, owing to repair mistakes. This review summarizes the most recent results on the ambivalent properties of these enzymes in limiting or promoting genetic instability in mammalian cells, as well as their potential use as targets for anticancer chemotherapy.

DNA Polymerases λ and β: The Double-Edged Swords of DNA Repair.

Science.gov (United States)

Mentegari, Elisa; Kissova, Miroslava; Bavagnoli, Laura; Maga, Giovanni; Crespan, Emmanuele

2016-08-31

DNA is constantly exposed to both endogenous and exogenous damages. More than 10,000 DNA modifications are induced every day in each cell's genome. Maintenance of the integrity of the genome is accomplished by several DNA repair systems. The core enzymes for these pathways are the DNA polymerases. Out of 17 DNA polymerases present in a mammalian cell, at least 13 are specifically devoted to DNA repair and are often acting in different pathways. DNA polymerases β and λ are involved in base excision repair of modified DNA bases and translesion synthesis past DNA lesions. Polymerase λ also participates in non-homologous end joining of DNA double-strand breaks. However, recent data have revealed that, depending on their relative levels, the cell cycle phase, the ratio between deoxy- and ribo-nucleotide pools and the interaction with particular auxiliary proteins, the repair reactions carried out by these enzymes can be an important source of genetic instability, owing to repair mistakes. This review summarizes the most recent results on the ambivalent properties of these enzymes in limiting or promoting genetic instability in mammalian cells, as well as their potential use as targets for anticancer chemotherapy.

Damage of DNA and plasma membranes in murine lymphoma cells irradiated under aerobic or hypoxic conditions

International Nuclear Information System (INIS)

Wlodek, D.

1983-01-01

A review of the knowledge of radiation effects on cell membranes and DNA and of repair mechanisms of radiation lesions is given. Investigations of properties of plasma membranes in L5178Y-S and L5178Y-R cells (surface charge, fluidity, transport of amino acids) indicate that there is no direct connection between membrane lesions and reproductive death. It was also found that in irradiated cells of both L5178Y-strains the rate of DNA chain elongation is the same, similarly as the amount of the initial DNA lesions and the rate of repair processes. Difference in the level of DNA synthesis inhibition is not proportional to the lethal effect. The results are also reported point to the difference between L5178Y-S and L5178Y-R cells in susceptibility of post-irradiation DNA synthesis to factors modifying chromatin conformation, such as inhibitors of (ADP-ribose) n polymerase. 221 refs. (author)

Electrochemical impedance-based DNA sensor using a modified single walled carbon nanotube electrode

Energy Technology Data Exchange (ETDEWEB)

Weber, Jessica E. [Department of Mechanical Engineering, University of South Florida, Tampa, FL (United States); Nanomaterials and Nanomanufacturing Research Center, University of South Florida, Tampa, FL (United States); Pillai, Shreekumar [Center for NanoBiotechnology Research, Alabama State University, Montgomery, AL (United States); Ram, Manoj Kumar, E-mail: mkram@usf.edu [Department of Mechanical Engineering, University of South Florida, Tampa, FL (United States); Nanomaterials and Nanomanufacturing Research Center, University of South Florida, Tampa, FL (United States); Kumar, Ashok [Department of Mechanical Engineering, University of South Florida, Tampa, FL (United States); Nanomaterials and Nanomanufacturing Research Center, University of South Florida, Tampa, FL (United States); Singh, Shree R. [Center for NanoBiotechnology Research, Alabama State University, Montgomery, AL (United States)

2011-07-20

Carbon nanotubes have become promising functional materials for the development of advanced electrochemical biosensors with novel features which could promote electron-transfer with various redox active biomolecules. This paper presents the detection of Salmonella enterica serovar Typhimurium using chemically modified single walled carbon nanotubes (SWNTs) with single stranded DNA (ssDNA) on a polished glassy carbon electrode. Hybridization with the corresponding complementary ssDNA has shown a shift in the impedance studies due to a higher charge transfer in ssDNA. The developed biosensor has revealed an excellent specificity for the appropriate targeted DNA strand. The methodologies to prepare and functionalize the electrode could be adopted in the development of DNA hybridization biosensor.

On-bead fluorescent DNA nanoprobes to analyze base excision repair activities

International Nuclear Information System (INIS)

Gines, Guillaume; Saint-Pierre, Christine; Gasparutto, Didier

2014-01-01

Graphical abstract: -- Highlights: •On magnetic beads fluorescent enzymatic assays. •Simple, easy, non-radioactive and electrophoresis-free functional assay. •Lesion-containing hairpin DNA probes are selective for repair enzymes. •The biosensing platform allows the measurement of DNA repair activities from purified enzymes or within cell free extracts. -- Abstract: DNA integrity is constantly threatened by endogenous and exogenous agents that can modify its physical and chemical structure. Changes in DNA sequence can cause mutations sparked by some genetic diseases or cancers. Organisms have developed efficient defense mechanisms able to specifically repair each kind of lesion (alkylation, oxidation, single or double strand break, mismatch, etc). Here we report the adjustment of an original assay to detect enzymes’ activity of base excision repair (BER), that supports a set of lesions including abasic sites, alkylation, oxidation or deamination products of bases. The biosensor is characterized by a set of fluorescent hairpin-shaped nucleic acid probes supported on magnetic beads, each containing a selective lesion targeting a specific BER enzyme. We have studied the DNA glycosylase alkyl-adenine glycosylase (AAG) and the human AP-endonuclease (APE1) by incorporating within the DNA probe a hypoxanthine lesion or an abasic site analog (tetrahydrofuran), respectively. Enzymatic repair activity induces the formation of a nick in the damaged strand, leading to probe's break, that is detected in the supernatant by fluorescence. The functional assay allows the measurement of DNA repair activities from purified enzymes or in cell-free extracts in a fast, specific, quantitative and sensitive way, using only 1 pmol of probe for a test. We recorded a detection limit of 1 μg mL −1 and 50 μg mL −1 of HeLa nuclear extracts for APE1 and AAG enzymes, respectively. Finally, the on-bead assay should be useful to screen inhibitors of DNA repair activities

Application of DNA Hybridization Biosensor as a Screening Method for the Detection of Genetically Modified Food Components

Directory of Open Access Journals (Sweden)

Marian Filipiak

2008-03-01

Full Text Available An electrochemical biosensor for the detection of genetically modified food components is presented. The biosensor was based on 21-mer single-stranded oligonucleotide (ssDNA probe specific to either 35S promoter or nos terminator, which are frequently present in transgenic DNA cassettes. ssDNA probe was covalently attached by 5’-phosphate end to amino group of cysteamine self-assembled monolayer (SAM on gold electrode surface with the use of activating reagents – water soluble 1-ethyl-3(3’- dimethylaminopropyl-carbodiimide (EDC and N-hydroxy-sulfosuccinimide (NHS. The hybridization reaction on the electrode surface was detected via methylene blue (MB presenting higher affinity to ssDNA probe than to DNA duplex. The electrode modification procedure was optimized using 19-mer oligoG and oligoC nucleotides. The biosensor enabled distinction between DNA samples isolated from soybean RoundupReady® (RR soybean and non-genetically modified soybean. The frequent introduction of investigated DNA sequences in other genetically modified organisms (GMOs give a broad perspectives for analytical application of the biosensor.

Use of avidin-biotin-peroxidase complex for measurement of UV lesions in human DNA by microELISA

Energy Technology Data Exchange (ETDEWEB)

Leipold, B [Technischen Universitaet Muenchen (Germany, F.R.). Dermatologische Klinik; Remy, W [Max-Planck-Institut fuer Biochemie, Muenchen (Germany, F.R.)

1984-02-10

The avidin/biotin system was introduced into the standard enzyme-linked immunosorbent assay (ELISA) to increase its sensitivity for detecting UV lesions in human DNA. Goat anti-rabbit IgG-peroxidase used in the standard ELISA as second antibody was replaced by biotinylated goat anti-rabbit IgG plus the avidin-biotin-peroxidase complex (ABC) reagent. Sensitivity of detection of plate-fixed UV-DNA-antibody complexes was increased about 8-fold and photolesions in human DNA samples irradiated with as low a dose as 1 J/m/sup 2/ UVC or a suberythermal dose of UVB light could be detected.

Processing of UV-induced DNA damage in diverse biological systems

International Nuclear Information System (INIS)

Galloway, A.M.

1992-01-01

A novel protocol has been developed allowing direct evaluation and accurate quantitation of UV lesions contained with both genomic DNA and the small oligonucleotides excised by a living cell during nucleotide excision repair. Using this methodology, the repair capacity of normal and UV-sensitive cells of human, Chinese hamster, and Escherichia coli origin, has been assessed. Several conclusions have been reached: (1) severage of the interpyrimidine phosphodiester linkage of cyclobutane dimers appears to be an evolutionarily conserved phenomenon; (2) the kinetics of cyclobutane dimer repair differ markedly from both (6-4) photoproduct and TA* lesion removal; (3) the ability to excise cyclobutane dimers is independent of (6-4) photoproduct repair capacity, suggesting that the lesions are not repaired/recognized by identical mechanisms; (4) fibroblast strains representing the eight xeroderma pigmentosum complementation groups each show a unique proficiency/deficiency to repair the different photolesions under study, implicating that a defect in a different locus underlies each genetic form of this disease; (5) the repair deficiency in UV-sensitive strains of trichothiodystrophy appears to be completely unrelated to that of non-complementing XP-D cells. To allow direct assessment of an IDP-altered photoproduct, substrates have been constructed which contain, at a defined dithymidine site, no lesion, a conventional cyclobutane dimer, or a cyclobutane dimer modified by severage of the intradimer phosphodiester bond. Bacteriophage T4 UV endonuclease has no activity towards a modified lesion, questioning the interpretation of experiments which utilize the strand-incising activity of this enzyme to monitor repair. Furthermore, although this altered lesion acts as a block to E. coli DNA polymerase I, it allows SP6 RNA polymerase to bypass the otherwise RNA polymerase-blocking lesion

Anchoring of self-assembled plasmid DNA/ anti-DNA antibody/cationic lipid micelles on bisphosphonate-modified stent for cardiovascular gene delivery

Directory of Open Access Journals (Sweden)

Ma G

2013-03-01

Full Text Available Guilei Ma,1,# Yong Wang,1,# Ilia Fishbein,2 Mei Yu,1 Linhua Zhang,1 Ivan S Alferiev,2 Jing Yang,1 Cunxian Song,1 Robert J Levy2 1Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China; 2Children's Hospital of Philadelphia, Abramson Research Building, Philadelphia, PA, USA #These authors contributed equally to this work Purpose: To investigate the anchoring of plasmid DNA/anti-DNA antibody/cationic lipid tri-complex (DAC micelles onto bisphosphonate-modified 316 L coronary stents for cardiovascular site-specific gene delivery. Methods: Stents were first modified with polyallylamine bisphosphonate (PAA-BP, thereby enabling the retention of a PAA-BP molecular monolayer that permits the anchoring (via vector-binding molecules of DAC micelles. DAC micelles were then chemically linked onto the PAA-BP-modified stents by using N-succinimidyl-3-(2-pyridyldithiol-propionate (SPDP as a crosslinker. Rhodamine-labeled DNA was used to assess the anchoring of DAC micelles, and radioactive-labeled antibody was used to evaluate binding capacity and stability. DAC micelles (encoding green fluorescent protein were tethered onto the PAA-BP-modified stents, which were assessed in cell culture. The presence of a PAA-BP molecular monolayer on the steel surface was confirmed by X-ray photoelectron spectroscopy and atomic force microscope analysis. Results: The anchoring of DAC micelles was generally uniform and devoid of large-scale patches of defects. Isotopic quantification confirmed that the amount of antibody chemically linked on the stents was 17-fold higher than that of the physical adsorbed control stents and its retention time was also significantly longer. In cell culture, numerous green fluorescent protein-positive cells were found on the PAA-BP modified stents, which demonstrated high localization and efficiency of gene delivery. Conclusion: The DAC micelle

Viewing Human DNA Polymerase β Faithfully and Unfaithfully Bypass an Oxidative Lesion by Time-Dependent Crystallography

Science.gov (United States)

Vyas, Rajan; Reed, Andrew J.; Tokarsky, E. John; Suo, Zucai

2015-01-01

One common oxidative DNA lesion, 8-oxo-7,8-dihydro-2′-deoxyguanine (8-oxoG), is highly mutagenic in vivo due to its anti-conformation forming a Watson–Crick base pair with correct deoxycytidine 5′-triphosphate (dCTP) and its syn-conformation forming a Hoogsteen base pair with incorrect deoxyadenosine 5′-triphosphate (dATP). Here, we utilized time-resolved X-ray crystallography to follow 8-oxoG bypass by human DNA polymerase β (hPolβ). In the 12 solved structures, both Watson–Crick (anti-8-oxoG:anti-dCTP) and Hoogsteen (syn-8-oxoG:anti-dATP) base pairing were clearly visible and were maintained throughout the chemical reaction. Additionally, a third Mg2+ appeared during the process of phosphodiester bond formation and was located between the reacting α- and β-phosphates of the dNTP, suggesting its role in stabilizing reaction intermediates. After phosphodiester bond formation, hPolβ reopened its conformation, pyrophosphate was released, and the newly incorporated primer 3′-terminal nucleotide stacked, rather than base paired, with 8-oxoG. These structures provide the first real-time pictures, to our knowledge, of how a polymerase correctly and incorrectly bypasses a DNA lesion. PMID:25825995

The beta subunit modulates bypass and termination at UV lesions during in vitro replication with DNA polymerase III holoenzyme of Escherichia coli

International Nuclear Information System (INIS)

Shavitt, O.; Livneh, Z.

1989-01-01

The cycling time of DNA polymerase III holoenzyme during replication of UV-irradiated single-stranded (ss) DNA was longer than with unirradiated DNA (8 versus 3 min, respectively), most likely due to slow dissociation from lesion-terminated nascent DNA strands. Initiation of elongation on primed ssDNA was not significantly inhibited by the presence of UV lesions as indicated by the identical distribution of replication products synthesized at early and late reaction times and by the identical duration of the initial synthesis bursts on both unirradiated and UV-irradiated DNA templates. When replication was performed with DNA polymerase III* supplemented with increasing quantities of purified beta 2 subunit, the cycling time on UV-irradiated DNA decreased from 14.8 min at 1.7 nM beta 2 down to 6 min at 170 nM beta 2, a concentration in which beta 2 was in large excess over the polymerase. In parallel to the reduction in cycling time, also the bypass frequency of cyclobutane-photodimers decreased with increasing beta 2 concentration, and at 170 nM beta 2, bypass of photodimers was essentially eliminated. It has been shown that polymerase complexes with more than one beta 2 per polymerase molecule were formed at high beta 2 concentrations. It is plausible that polymerase complexes obtained under high beta 2 concentration dissociate from lesion-terminated primers faster than polymerase complexes formed at a low beta 2 concentration. This is expected to favor termination over bypass at pyrimidine photodimers and thus decrease their bypass frequency. These results suggest that the beta 2 subunit might act as a sensor for obstacles to replication caused by DNA damage, and that it terminates elongation at these sites by promoting dissociation. The intracellular concentration of beta 2 was estimated to be 250 nM

Human papillomavirus in oral lesions Virus papiloma humano en lesiones orales

OpenAIRE

Joaquín V. Gónzalez; Rafael A. Gutiérrez; Alicia Keszler; Maria Del Carmen Colacino; Lidia V. Alonio; Angélica R. Teyssie; Maria Alejandra Picconi

2007-01-01

Growing evidence suggests a role for human papillomavirus (HPV) in oral cancer; however its involvement is still controversial. This study evaluates the frequency of HPV DNA in a variety of oral lesions in patients from Argentina. A total of 77 oral tissue samples from 66 patients were selected (cases); the clinical-histopathological diagnoses corresponded to: 11 HPV- associated benign lesions, 8 non-HPV associated benign lesions, 33 premalignant lesions and 25 cancers. Sixty exfoliated cell ...

The role of the HCR system in the repair of lethal lesions of Bacillus subtilis phages and their transfecting DNA damaged by radiation and alkylating agents

International Nuclear Information System (INIS)

Vizdalova, M.; Janovska, E.; Zhestyanikov, V.D.

1980-01-01

The role of the HCR system in the repair of prelethal lesions induced by UV light, γ radiation and alkylating agents was studied in the Bacillus subtilis SPP1 phage, its heat sensitive mutants (N3, N73 nad ts 1 ) and corresponding infectious DNA. The survival of phages and their transfecting DNA after treatment with UV light is substantially higher in hcr + cells than in hcr cells, the differences being more striking in intact phages than in their transfecting DNA's. Repair inhibitors reduce survival in hcr + cells: caffeine lowers the survival of UV-irradiated phage SPP1 in exponentially growing hcr + cells but has no effect on its survival in competent hcr + cells; acriflavin and ethidium bromide decrease the survival of the UV-irradiated SPP1 phage in both exponentially growing and competent hcr + cells to the level of survival observed in hcr cells; moreover, ethidium bromide lowers the number of infective centres in hcr + cells of the UV-irradiated DNA of the SPP1 phage. Repair inhibitors do not lower the survival of the UV-irradiated phages or their DNA in hcr cells. The repair mechanism under study also effectively repairs lesions induced by polyfunctional alkylating agents in the transfecting DNA's of B. subtilis phages but is not functional with lesions induced by these agents in free phages and lesions caused in the phages and their DNA by ethyl methanesulphonate or γ radiation. (author)

The effect of temperature and wavelength on production and photolysis of a UV-induced photosensitive DNA lesion which is not repaired in xeroderma pigmentosum variant cells

International Nuclear Information System (INIS)

Francis, A.A.; Carrier, W.L.; Regan, J.D.

1988-01-01

Ultraviolet light causes a type of damage to the DNA of human cells that results in a DNA strand break upon subsequent irradiation with wavelengths around 300 nm. This DNA damage disappears from normal human fibroblasts within 5 h, but not from pyrimidine dimer excision repair deficient xeroderma pigmentosum group A cells or from excision proficient xeroderma pigmentosum variant cells. The apparent lack of repair of the ultraviolet light DNA damage described here may contribute to the cancer prone nature of xeroderma pigmentosum variant individuals. These experiments show that the same amount of damage was produced at 0 0 C and 37 0 C indicating a photodynamic effect and not an enzymatic reaction. The disappearance of the photosensitive lesions from the DNA is probably enzymatic since none of the damage was removed at 0 0 C. Both the formation of the lesion and its photolysis by near ultraviolet light were wavelength dependent. An action spectrum for the formation of photosensitive lesions was similar to that for the formation of pyrimidine dimers and (6-4) photoproducts and included wavelengths found in sunlight. The DNA containing the lesions was sensitive to wavelengths from 304 to 340 nm with a maximum at 313 to 317 nm. This wavelength dependence of photolysis is similar to the absorption and photolysis spectra of the pyrimidine (6-4) photoproducts. (author)

Mechanisms of Insertion of dCTP and dTTP Opposite the DNA Lesion O6-Methyl-2′-deoxyguanosine by Human DNA Polymerase η*

Science.gov (United States)

Patra, Amitraj; Zhang, Qianqian; Guengerich, F. Peter; Egli, Martin

2016-01-01

O6-Methyl-2′-deoxyguanosine (O6-MeG) is a ubiquitous DNA lesion, formed not only by xenobiotic carcinogens but also by the endogenous methylating agent S-adenosylmethionine. It can introduce mutations during DNA replication, with different DNA polymerases displaying different ratios of correct or incorrect incorporation opposite this nucleoside. Of the “translesion” Y-family human DNA polymerases (hpols), hpol η is most efficient in incorporating equal numbers of correct and incorrect C and T bases. However, the mechanistic basis for this specific yet indiscriminate activity is not known. To explore this question, we report biochemical and structural analysis of the catalytic core of hpol η. Activity assays showed the truncated form displayed similar misincorporation properties as the full-length enzyme, incorporating C and T equally and extending from both. X-ray crystal structures of both dC and dT paired with O6-MeG were solved in both insertion and extension modes. The structures revealed a Watson-Crick-like pairing between O6-MeG and 2"-deoxythymidine-5"-[(α, β)-imido]triphosphate (approximating dT) at both the insertion and extension stages with formation of two H-bonds. Conversely, both the structures with O6- MeG opposite dCTP and dC display sheared configuration of base pairs but to different degrees, with formation of two bifurcated H-bonds and two single H-bonds in the structures trapped in the insertion and extension states, respectively. The structural data are consistent with the observed tendency of hpol η to insert both dC and dT opposite the O6-MeG lesion with similar efficiencies. Comparison of the hpol η active site configurations with either O6-MeG:dC or O6-MeG:dT bound compared with the corresponding situations in structures of complexes of Sulfolobus solfataricus Dpo4, a bypass pol that favors C relative to T by a factor of ∼4, helps rationalize the more error-prone synthesis opposite the lesion by hpol η. PMID:27694439

Base sequence effects on DNA replication influenced by bulky adducts. Final report, March 1, 1995--February 28, 1997

Energy Technology Data Exchange (ETDEWEB)

Geacintov, N.E.

1997-05-31

Polycyclic aromatic hydrocarbons (PAH) are environmental pollutants that are present in air, food, and water. While PAH compounds are chemically inert and are sparingly soluble in aqueous solutions, in living cells they are metabolized to a variety of oxygenated derivatives, including the high mutagenic and tumorigenic diol epoxide derivatives. The diol epoxides of the sterically hindered fjord region compound benzo[c]phenanthrene (B[c]PhDE) are among the most powerful tumorigenic compounds in animal model test systems. In this project, site-specifically modified oligonucleotides containing single B[c]PhDE-N{sup 6}-dA lesions derived from the reactions of the 1S,2R,3R,4S and 1R,2S,3S,4R diol epoxides of B[c]PhDE with dA residues were synthesized. The replication of DNA catalyzed by a prokaryotic DNA polymerase (the exonuclease-free Klenow fragment E. Coli Po1 I) in the vicinity of the lesion at base-specific sites on B[c]PhDE-modified template strands was investigated in detail. The Michaelis-Menten parameters for the insertion of single deoxynucleotide triphosphates into growing DNA (primer) strands using the modified dA* and the bases just before and after the dA* residue as templates, depend markedly on the stereochemistry of the B[c]PhDE-modified dA residues. These observations provide novel insights into the mechanisms by which bulky PAH-DNA adducts affect normal DNA replication.

Role of exonucleolytic processing and polymerase-DNA association in bypass of lesions during replication in vitro. Significance for SOS-targeted mutagenesis

International Nuclear Information System (INIS)

Shwartz, H.; Shavitt, O.; Livneh, Z.

1988-01-01

The role of exonuclease activity in trans-lesion DNA replication with Escherichia coli DNA polymerase III holoenzyme was investigated. RecA protein inhibited the 3'----5' exonuclease activity of the polymerase 2-fold when assayed in the absence of replication and had no effect on turnover of dNTPs into dNMPs. In contrast, single-stranded DNA-binding protein, which had no effect on the exonuclease activity in the absence of replication, showed a pronounced 7-fold suppression of the 3'----5' exonuclease activity during replication. The excision of incorporated dNMP alpha S residues from DNA by the 3'----5' exonuclease activity of DNA polymerase III holoenzyme was inhibited 10-20-fold; still no increase in bypass of pyrimidine photodimers was observed. Thus, in agreement with our previous results in which the exonuclease activity was inhibited at the protein level, inhibition at the DNA level also did not increase bypass of photodimers. Fractionation of the replication mixture after termination of DNA synthesis on a Bio-Gel A-5m column under conditions which favor polymerase-DNA binding yielded a termination complex which could perform turnover of dNTPs into dNMPs. Adding challenge-primed single-stranded DNA to the complex yielded a burst of DNA synthesis which was promoted most likely by DNA polymerase III holoenzyme molecules transferred from the termination complex to the challenge DNA thus demonstrating the instability of the polymerase-DNA association. Addition of a fresh sample of DNA polymerase III holoenzyme to purified termination products, which consist primarily of partially replicated molecules with nascent chains terminated at UV lesions, did not result in any net DNA synthesis as expected

The proofreading 3'→5' exonuclease activity of DNA polymerases: a kinetic barrier to translesion DNA synthesis

International Nuclear Information System (INIS)

Khare, Vineeta; Eckert, Kristin A.

2002-01-01

The 3'→5' exonuclease activity intrinsic to several DNA polymerases plays a primary role in genetic stability; it acts as a first line of defense in correcting DNA polymerase errors. A mismatched basepair at the primer terminus is the preferred substrate for the exonuclease activity over a correct basepair. The efficiency of the exonuclease as a proofreading activity for mispairs containing a DNA lesion varies, however, being dependent upon both the DNA polymerase/exonuclease and the type of DNA lesion. The exonuclease activities intrinsic to the T4 polymerase (family B) and DNA polymerase γ (family A) proofread DNA mispairs opposite endogenous DNA lesions, including alkylation, oxidation, and abasic adducts. However, the exonuclease of the Klenow polymerase cannot discriminate between correct and incorrect bases opposite alkylation and oxidative lesions. DNA damage alters the dynamics of the intramolecular partitioning of DNA substrates between the 3'→5' exonuclease and polymerase activities. Enzymatic idling at lesions occurs when an exonuclease activity efficiently removes the same base that is preferentially incorporated by the DNA polymerase activity. Thus, the exonuclease activity can also act as a kinetic barrier to translesion synthesis (TLS) by preventing the stable incorporation of bases opposite DNA lesions. Understanding the downstream consequences of exonuclease activity at DNA lesions is necessary for elucidating the mechanisms of translesion synthesis and damage-induced cytotoxicity

High-affinity triplex targeting of double stranded DNA using chemically modified peptide nucleic acid oligomers

DEFF Research Database (Denmark)

Hansen, Mads E; Bentin, Thomas; Nielsen, Peter E

2009-01-01

While sequence-selective dsDNA targeting by triplex forming oligonucleotides has been studied extensively, only very little is known about the properties of PNA-dsDNA triplexes-mainly due to the competing invasion process. Here we show that when appropriately modified using pseudoisocytosine subs...

On-bead fluorescent DNA nanoprobes to analyze base excision repair activities

Energy Technology Data Exchange (ETDEWEB)

Gines, Guillaume; Saint-Pierre, Christine; Gasparutto, Didier, E-mail: didier.gasparutto@cea.fr

2014-02-17

Graphical abstract: -- Highlights: •On magnetic beads fluorescent enzymatic assays. •Simple, easy, non-radioactive and electrophoresis-free functional assay. •Lesion-containing hairpin DNA probes are selective for repair enzymes. •The biosensing platform allows the measurement of DNA repair activities from purified enzymes or within cell free extracts. -- Abstract: DNA integrity is constantly threatened by endogenous and exogenous agents that can modify its physical and chemical structure. Changes in DNA sequence can cause mutations sparked by some genetic diseases or cancers. Organisms have developed efficient defense mechanisms able to specifically repair each kind of lesion (alkylation, oxidation, single or double strand break, mismatch, etc). Here we report the adjustment of an original assay to detect enzymes’ activity of base excision repair (BER), that supports a set of lesions including abasic sites, alkylation, oxidation or deamination products of bases. The biosensor is characterized by a set of fluorescent hairpin-shaped nucleic acid probes supported on magnetic beads, each containing a selective lesion targeting a specific BER enzyme. We have studied the DNA glycosylase alkyl-adenine glycosylase (AAG) and the human AP-endonuclease (APE1) by incorporating within the DNA probe a hypoxanthine lesion or an abasic site analog (tetrahydrofuran), respectively. Enzymatic repair activity induces the formation of a nick in the damaged strand, leading to probe's break, that is detected in the supernatant by fluorescence. The functional assay allows the measurement of DNA repair activities from purified enzymes or in cell-free extracts in a fast, specific, quantitative and sensitive way, using only 1 pmol of probe for a test. We recorded a detection limit of 1 μg mL{sup −1} and 50 μg mL{sup −1} of HeLa nuclear extracts for APE1 and AAG enzymes, respectively. Finally, the on-bead assay should be useful to screen inhibitors of DNA repair

Repriming by PrimPol is critical for DNA replication restart downstream of lesions and chain-terminating nucleosides.

Science.gov (United States)

Kobayashi, Kaori; Guilliam, Thomas A; Tsuda, Masataka; Yamamoto, Junpei; Bailey, Laura J; Iwai, Shigenori; Takeda, Shunichi; Doherty, Aidan J; Hirota, Kouji

2016-08-02

PrimPol is a DNA damage tolerance enzyme possessing both translesion synthesis (TLS) and primase activities. To uncover its potential role in TLS-mediated IgVλ hypermutation and define its interplay with other TLS polymerases, PrimPol(-/-) and PrimPol(-/-)/Polη(-/-)/Polζ (-/-) gene knockouts were generated in avian cells. Loss of PrimPol had no significant impact on the rate of hypermutation or the mutation spectrum of IgVλ. However, PrimPol(-/-) cells were sensitive to methylmethane sulfonate, suggesting that it may bypass abasic sites at the IgVλ segment by repriming DNA synthesis downstream of these sites. PrimPol(-/-) cells were also sensitive to cisplatin and hydroxyurea, indicating that it assists in maintaining / restarting replication at a variety of lesions. To accurately measure the relative contribution of the TLS and primase activities, we examined DNA damage sensitivity in PrimPol(-/-) cells complemented with polymerase or primase-deficient PrimPol. Polymerase-defective, but not primase-deficient, PrimPol suppresses the hypersensitivity of PrimPol(-/-) cells. This indicates that its primase, rather than TLS activity, is pivotal for DNA damage tolerance. Loss of TLS polymerases, Polη and Polζ has an additive effect on the sensitivity of PrimPol(-/-) cells. Moreover, we found that PrimPol and Polη-Polζ redundantly prevented cell death and facilitated unperturbed cell cycle progression. PrimPol(-/-) cells also exhibited increased sensitivity to a wide variety of chain-terminating nucleoside analogs (CTNAs). PrimPol could perform close-coupled repriming downstream of CTNAs and oxidative damage in vitro. Together, these results indicate that PrimPol's repriming activity plays a central role in reinitiating replication downstream from CTNAs and other specific DNA lesions.

Unusual hydrogen bonding patterns in AF [aminofluorene] and AAF [acetylaminofluorene] modified DNA

International Nuclear Information System (INIS)

Broyde, S.; Hingerty, B.E.; Shapiro, R.; Norman, D.; Oak Ridge National Lab., TN; New York Univ., NY; Columbia Univ., New York, NY

1989-01-01

New structures are presented for AF and AAF modified DNAs that place the carcinogen in the minor groove of a B-DNA helix. These structures employ non-Watson-Crick base pairing schemes with syn guanine at the modification site. 32 refs., 9 figs

DNA Repair Mechanisms and the Bypass of DNA Damage in Saccharomyces cerevisiae

Science.gov (United States)

Boiteux, Serge; Jinks-Robertson, Sue

2013-01-01

DNA repair mechanisms are critical for maintaining the integrity of genomic DNA, and their loss is associated with cancer predisposition syndromes. Studies in Saccharomyces cerevisiae have played a central role in elucidating the highly conserved mechanisms that promote eukaryotic genome stability. This review will focus on repair mechanisms that involve excision of a single strand from duplex DNA with the intact, complementary strand serving as a template to fill the resulting gap. These mechanisms are of two general types: those that remove damage from DNA and those that repair errors made during DNA synthesis. The major DNA-damage repair pathways are base excision repair and nucleotide excision repair, which, in the most simple terms, are distinguished by the extent of single-strand DNA removed together with the lesion. Mistakes made by DNA polymerases are corrected by the mismatch repair pathway, which also corrects mismatches generated when single strands of non-identical duplexes are exchanged during homologous recombination. In addition to the true repair pathways, the postreplication repair pathway allows lesions or structural aberrations that block replicative DNA polymerases to be tolerated. There are two bypass mechanisms: an error-free mechanism that involves a switch to an undamaged template for synthesis past the lesion and an error-prone mechanism that utilizes specialized translesion synthesis DNA polymerases to directly synthesize DNA across the lesion. A high level of functional redundancy exists among the pathways that deal with lesions, which minimizes the detrimental effects of endogenous and exogenous DNA damage. PMID:23547164

Radiation and non-radiation damage to DNA. Onset of molecular instability and carcinogenesis. Theoretical explorations on DNA damage and repair

International Nuclear Information System (INIS)

Pinak, Miroslay; Bunta, J.K.

2006-01-01

The current work is focused on results of molecular dynamics simulations performed on two DNA damages: 8-oxoguanine as the most significant oxidative damage leading to transversion mutation cytosine-guanine→adenine-thymine', which is common mutation found in human cancer cells; and on the DNA strand break, the type of damage that is considered to be one of the most significant damage leading to genetic instability that may result in enhanced cell proliferation or carcinogenesis. Except the structural changes induced by these two lesions the role and importance of electrostatic energy in recognition process in which a respective repair enzyme recognizes damaged DNA site is also described. Among the significant results can be included the fact, that most of the damages on DNA alternate locally electronic state by modifying chemical and electron orbital configuration. This modified configuration may be represented outside DNA molecule as an enhanced electrostatic interaction with surrounding environment, that may signal the presence of the damaged site toward the repair enzyme. Work on the DNA strand break shows that open valences at broken strand ends are quickly filled by the electrons generated during radiolysis. Results of simulation indicate a local instability of hydrogen bonds between complementary bases. (author)

Label-free, electrochemical detection of methicillin-resistant staphylococcus aureus DNA with reduced graphene oxide-modified electrodes

KAUST Repository

Wang, Zhijuan

2011-05-01

Reduced graphene oxide (rGO)-modified glassy carbon electrode is used to detect the methicillin-resistant Staphylococcus aureus (MRSA) DNA by using electrochemical impedance spectroscopy. Our experiments confirm that ssDNA, before and after hybridization with target DNA, are successfully anchored on the rGO surface. After the probe DNA, pre-adsorbed on rGO electrode, hybridizes with target DNA, the measured impedance increases dramatically. It provides a new method to detect DNA with high sensitivity (10-13M, i.e., 100 fM) and selectivity. © 2011 Elsevier B.V.

Are dinucleoside monophosphates relevant models for the study of DNA intrastrand cross-link lesions? The example of g[8-5m]T.

Science.gov (United States)

Garrec, Julian; Dumont, Elise

2014-07-21

Oxidatively generated tandem lesions such as G[8-5m]T pose a potent threat to genome integrity. Direct experimental studies of the kinetics and thermodynamics of a specific lesion within DNA are very challenging, mostly due to the variety of products that can be formed in oxidative conditions. Dinucleoside monophosphates (DM) involving only the reactive nucleobases in water represent appealing alternative models on which most physical chemistry and structural techniques can be applied. However, it is not yet clear how relevant these models are. Here, we present QM/MM MD simulations of the cyclization step involved in the formation of G[8-5m]T from the guanine-thymine (GpT) DM in water, with the aim of comparing our results to our previous investigation of the same reaction in DNA ( Garrec , J. , Patel , C. , Rothlisberger , U. , and Dumont , E. ( 2012 ) J. Am. Chem. Soc. 134 , 2111 - 2119 ). We show that, despite the different levels of preorganization of the two systems, the corresponding reactions share many energetic and structural characteristics. The main difference lies in the angle between the G and T bases, which is slightly higher in the transition state (TS) and product of the reaction in water than in the reaction in DNA. This effect is due to the Watson-Crick H-bonds, which are absent in the {GpT+water} system and restrain the relative positioning of the reactive nucleobases in DNA. However, since the lesion is accommodated easily in the DNA macromolecule, the induced energetic penalty is relatively small. The high similarity between the two reactions strongly supports the use of GpT in water as a model of the corresponding reaction in DNA.

Fish DNA-modified clays: Towards highly flame retardant polymer nanocomposite with improved interfacial and mechanical performance

Science.gov (United States)

Zabihi, Omid; Ahmadi, Mojtaba; Khayyam, Hamid; Naebe, Minoo

2016-12-01

Deoxyribonucleic Acid (DNA) has been recently found to be an efficient renewable and environmentally-friendly flame retardant. In this work, for the first time, we have used waste DNA from fishing industry to modify clay structure in order to increase the clay interactions with epoxy resin and take benefit of its additional thermal property effect on thermo-physical properties of epoxy-clay nanocomposites. Intercalation of DNA within the clay layers was accomplished in a one-step approach confirmed by FT-IR, XPS, TGA, and XRD analyses, indicating that d-space of clay layers was expanded from ~1.2 nm for pristine clay to ~1.9 nm for clay modified with DNA (d-clay). Compared to epoxy nanocomposite containing 2.5%wt of Nanomer I.28E organoclay (m-clay), it was found that at 2.5%wt d-clay loading, significant enhancements of ~14%, ~6% and ~26% in tensile strength, tensile modulus, and fracture toughness of epoxy nanocomposite can be achieved, respectively. Effect of DNA as clay modifier on thermal performance of epoxy nanocomposite containing 2.5%wt d-clay was evaluated using TGA and cone calorimetry analysis, revealing significant decreases of ~4000 kJ/m2 and ~78 kW/m2 in total heat release and peak of heat release rate, respectively, in comparison to that containing 2.5%wt of m-clay.

Inhibiting DNA Polymerases as a Therapeutic Intervention against Cancer

Directory of Open Access Journals (Sweden)

Anthony J. Berdis

2017-11-01

Full Text Available Inhibiting DNA synthesis is an important therapeutic strategy that is widely used to treat a number of hyperproliferative diseases including viral infections, autoimmune disorders, and cancer. This chapter describes two major categories of therapeutic agents used to inhibit DNA synthesis. The first category includes purine and pyrmidine nucleoside analogs that directly inhibit DNA polymerase activity. The second category includes DNA damaging agents including cisplatin and chlorambucil that modify the composition and structure of the nucleic acid substrate to indirectly inhibit DNA synthesis. Special emphasis is placed on describing the molecular mechanisms of these inhibitory effects against chromosomal and mitochondrial DNA polymerases. Discussions are also provided on the mechanisms associated with resistance to these therapeutic agents. A primary focus is toward understanding the roles of specialized DNA polymerases that by-pass DNA lesions produced by DNA damaging agents. Finally, a section is provided that describes emerging areas in developing new therapeutic strategies targeting specialized DNA polymerases.

Comparison of QuadrapolarTM radiofrequency lesions produced by standard versus modified technique: an experimental model

Directory of Open Access Journals (Sweden)

Safakish R

2017-06-01

Full Text Available Ramin Safakish Allevio Pain Management Clinic, Toronto, ON, Canada Abstract: Lower back pain (LBP is a global public health issue and is associated with substantial financial costs and loss of quality of life. Over the years, different literature has provided different statistics regarding the causes of the back pain. The following statistic is the closest estimation regarding our patient population. The sacroiliac (SI joint pain is responsible for LBP in 18%–30% of individuals with LBP. Quadrapolar™ radiofrequency ablation, which involves ablation of the nerves of the SI joint using heat, is a commonly used treatment for SI joint pain. However, the standard Quadrapolar radiofrequency procedure is not always effective at ablating all the sensory nerves that cause the pain in the SI joint. One of the major limitations of the standard Quadrapolar radiofrequency procedure is that it produces small lesions of ~4 mm in diameter. Smaller lesions increase the likelihood of failure to ablate all nociceptive input. In this study, we compare the standard Quadrapolar radiofrequency ablation technique to a modified Quadrapolar ablation technique that has produced improved patient outcomes in our clinic. The methodology of the two techniques are compared. In addition, we compare results from an experimental model comparing the lesion sizes produced by the two techniques. Taken together, the findings from this study suggest that the modified Quadrapolar technique provides longer lasting relief for the back pain that is caused by SI joint dysfunction. A randomized controlled clinical trial is the next step required to quantify the difference in symptom relief and quality of life produced by the two techniques. Keywords: lower back pain, radiofrequency ablation, sacroiliac joint, Quadrapolar radiofrequency ablation

Direct voltammetric analysis of DNA modified with enzymatically incorporated 7-deazapurines

Czech Academy of Sciences Publication Activity Database

Pivoňková, Hana; Horáková Brázdilová, Petra; Fojtová, Miloslava; Fojta, Miroslav

2010-01-01

Roč. 82, č. 16 (2010), s. 6807-6813 ISSN 0003-2700 R&D Projects: GA AV ČR(CZ) IAA400040901; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : modified DNA * 7-deazapurines * voltammetric analysis Subject RIV: BO - Biophysics Impact factor: 5.874, year: 2010

Structural basis for the inhibition of human alkyladenine DNA glycosylase (AAG) by 3,N4-ethenocytosine-containing DNA.

Science.gov (United States)

Lingaraju, Gondichatnahalli M; Davis, C Ainsley; Setser, Jeremy W; Samson, Leona D; Drennan, Catherine L

2011-04-15

Reactive oxygen and nitrogen species, generated by neutrophils and macrophages in chronically inflamed tissues, readily damage DNA, producing a variety of potentially genotoxic etheno base lesions; such inflammation-related DNA damage is now known to contribute to carcinogenesis. Although the human alkyladenine DNA glycosylase (AAG) can specifically bind DNA containing either 1,N(6)-ethenoadenine (εA) lesions or 3,N(4)-ethenocytosine (εC) lesions, it can only excise εA lesions. AAG binds very tightly to DNA containing εC lesions, forming an abortive protein-DNA complex; such binding not only shields εC from repair by other enzymes but also inhibits AAG from acting on other DNA lesions. To understand the structural basis for inhibition, we have characterized the binding of AAG to DNA containing εC lesions and have solved a crystal structure of AAG bound to a DNA duplex containing the εC lesion. This study provides the first structure of a DNA glycosylase in complex with an inhibitory base lesion that is induced endogenously and that is also induced upon exposure to environmental agents such as vinyl chloride. We identify the primary cause of inhibition as a failure to activate the nucleotide base as an efficient leaving group and demonstrate that the higher binding affinity of AAG for εC versus εA is achieved through formation of an additional hydrogen bond between Asn-169 in the active site pocket and the O(2) of εC. This structure provides the basis for the design of AAG inhibitors currently being sought as an adjuvant for cancer chemotherapy.

The AID-induced DNA damage response in chromatin

DEFF Research Database (Denmark)

Daniel, Jeremy A; Nussenzweig, André

2013-01-01

Chemical modifications to the DNA and histone protein components of chromatin can modulate gene expression and genome stability. Understanding the physiological impact of changes in chromatin structure remains an important question in biology. As one example, in order to generate antibody diversity...... with somatic hypermutation and class switch recombination, chromatin must be made accessible for activation-induced cytidine deaminase (AID)-mediated deamination of cytosines in DNA. These lesions are recognized and removed by various DNA repair pathways but, if not handled properly, can lead to formation...... of oncogenic chromosomal translocations. In this review, we focus the discussion on how chromatin-modifying activities and -binding proteins contribute to the native chromatin environment in which AID-induced DNA damage is targeted and repaired. Outstanding questions remain regarding the direct roles...

Isolation/separation of plasmid DNA using hemoglobin modified magnetic nanocomposites as solid-phase adsorbent.

Science.gov (United States)

Chen, Xu-Wei; Mao, Quan-Xing; Liu, Jia-Wei; Wang, Jian-Hua

2012-10-15

Hemoglobin (Hb) modified magnetic nanocomposites are prepared by immobilization of Hb onto the surface of amino-functionalized Fe(3)O(4)@SiO(2) magnetic nanoparticles via covalent bonding with glutaraldehyde as cross-linker. The obtained nanocomposites are characterized with FT-IR, SEM, XRD and surface charge analysis. A direct solid-phase extraction procedure for the isolation/separation of plasmid DNA using this nanocomposite as a novel adsorbent is thus developed. Some important experimental parameters governing the sorption efficiency, i.e., the pH of sample solution and the ionic strength, are investigated. The Hb modified magnetic nanocomposites provide a sorption capacity of 27.86 mg g(-1) for DNA. By using 2.0mg of the nanocomposites as sorption medium and a suitable acidity of pH 6.1, a sorption efficiency of 93% is achieved for 25 μg mL(-1) of DNA in 1.0 mL of sample solution. Afterwards, the absorbed DNA could be readily recovered by using 1.0 mL of Tris-HCl buffer (pH 8.9, 0.01 mol L(-1)), giving rise to a recovery of ca. 68.3%. The present solid-phased extraction protocol is applied for the isolation of plasmid DNA from Escherichia coli culture, resulting in comparable yield and purity of plasmid DNA with respect to those obtained by using commercial kits. Copyright © 2012 Elsevier B.V. All rights reserved.

Cleavage and protection of locked nucleic acid-modified DNA by restriction endonucleases

DEFF Research Database (Denmark)

Crouzier, Lucile; Dubois, Camille; Wengel, Jesper

2012-01-01

Locked nucleic acid (LNA) is one of the most prominent nucleic acid analogues reported so far. We herein for the first time report cleavage by restriction endonuclease of LNA-modified DNA oligonucleotides. The experiments revealed that RsaI is an efficient enzyme capable of recognizing and cleaving...

Correspondence between radioactive and functional methods in the quality control of DNA restriction and modifying enzymes

DEFF Research Database (Denmark)

Trujillo, L E; Pupo, E; Miranda, F

1996-01-01

We evaluated the use of two radiolabeled lambda DNA/Hpa II substrates to detect 5'-->3', 3'-->5' single and double stranded DNA dependent exonuclease and phosphatase activities found as contaminants in restriction and modifying enzyme preparations. Looking for the meaning of the radioactive assay...

Cryopreservation of human blood for alkaline and Fpg-modified comet assay.

Science.gov (United States)

Pu, Xinzhu; Wang, Zemin; Klaunig, James E

2016-01-01

The Comet assay is a reproducible and sensitive assay for the detection of DNA damage in eukaryotic cells and tissues. Incorporation of lesion specific, oxidative DNA damage repair enzymes (for example, Fpg, OGG1 and EndoIII) in the standard alkaline Comet assay procedure allows for the detection and measurement of oxidative DNA damage. The Comet assay using white blood cells (WBC) has proven useful in monitoring DNA damage from environmental agents in humans. However, it is often impractical to performance Comet assay immediately after blood sampling. Thus, storage of blood sample is required. In this study, we developed and tested a simple storage method for very small amount of whole blood for standard and Fpg-modified modified Comet assay. Whole blood was stored in RPMI 1640 media containing 10% FBS, 10% DMSO and 1 mM deferoxamine at a sample to media ratio of 1:50. Samples were stored at -20 °C and -80 °C for 1, 7, 14 and 28 days. Isolated lymphocytes from the same subjects were also stored under the same conditions for comparison. Direct DNA strand breakage and oxidative DNA damage in WBC and lymphocytes were analyzed using standard and Fpg-modified alkaline Comet assay and compared with freshly analyzed samples. No significant changes in either direct DNA strand breakage or oxidative DNA damage was seen in WBC and lymphocytes stored at -20 °C for 1 and 7 days compared to fresh samples. However, significant increases in both direct and oxidative DNA damage were seen in samples stored at -20 °C for 14 and 28 days. No changes in direct and oxidative DNA damage were observed in WBC and lymphocytes stored at -80 °C for up to 28 days. These results identified the proper storage conditions for storing whole blood or isolated lymphocytes to evaluate direct and oxidative DNA damage using standard and Fpg-modified alkaline Comet assay.

Modified dorsal root entry zone lesioning for intractable pain relief in patients with root avulsion injury.

Science.gov (United States)

Takai, Keisuke; Taniguchi, Makoto

2017-08-01

OBJECTIVE Dorsal root entry zone (DREZ) lesioning has been the most effective surgical treatment for the relief of intractable pain due to root avulsion injury, but residual pain and a decrease in pain relief in the follow-up period have been reported in 23%-70% of patients. Based on pain topography in the most recent studies on neuropathic pain, the authors modified the conventional DREZ lesioning procedure to improve clinical outcomes. The presumed rationale for this procedure is to eliminate the spontaneous discharges of neurons in the superficial spinal dorsal horn as well as wide dynamic range neurons in the deep spinal dorsal horn. METHODS Ten patients with avulsion-related pain underwent surgery between 2011 and 2015. The surgical procedure was described and postoperative pain relief was assessed as follows: excellent (residual pain never exceeded 3 on the visual analog scale [VAS] without medication), good (residual pain never exceeded 5 on the VAS with medication), and poor (residual pain was greater than 5 with medication). Specific perioperative complications were assessed. RESULTS The aim of this surgical procedure was to destroy the deeper layers of the posterior horn of spinal gray matter, which was in contrast to the procedures of Nashold and Sindou, which were to destroy the superficial layers. All patients achieved excellent (n = 7, pain relief without medication) or good (n = 3, pain relief with medication) pain relief postoperatively, and the recurrence of pain was not reported in any patients (median 29 months after surgery, range 12-64 months). Nine patients (90%) achieved complete pain relief (a score of 0 or 1 on the VAS) with or without medication. No surgical site complications such as infection or CSF leakage were noted. No motor deficit was observed in any patient. A sensory deficit was observed in 2 patients and disappeared within 1 month in 1 patient. New pain at the adjacent level of DREZ lesioning was observed in 3 patients and

A component of DNA double-strand break repair is dependent on the spatial orientation of the lesions within the higher-order structures of chromatin

Energy Technology Data Exchange (ETDEWEB)

Johnston, P.J.; Bryant, P.E. (Saint Andrews Univ. (United Kingdom))

1994-11-01

By the use of a modified neutral filter elution procedure variations in the repair of DNA dsb have been observed between the ionising radiation sensitive mutant xrs-5 and the parent cell line CHO-K1. Conventional neutral filter elution requires harsh lysis conditions to remove higher-order chromatin structures which interfere with elution of DNA containing dsb. By lysing cells with non-ionic detergent in the presence of 2 mol dm[sup -3] salt, histone-depleted structures that retain the higher-order nuclear matrix organization, including chromatin loops, can be produced. Elution from these structures will only occur if two or more dsb lie within a single-looped domain delineated by points of attachment to the nuclear matrix. Repair experiments indicate that in CHO cells repair of dsb in loops containing multiple dsb are repaired with slow kinetics whilst dsb occurring in loops containing single dsb are repaired with fast kinetics. Xrs-5 cells are defective in the repair of multiply damaged loops. This work indicates that the spatial orientation of dsb in the higher-order structures of chromatin are a possible factor in the repair of these lesions. (Author).

A component of DNA double-strand break repair is dependent on the spatial orientation of the lesions within the higher-order structures of chromatin

International Nuclear Information System (INIS)

Johnston, P.J.; Bryant, P.E.

1994-01-01

By the use of a modified neutral filter elution procedure variations in the repair of DNA dsb have been observed between the ionising radiation sensitive mutant xrs-5 and the parent cell line CHO-K1. Conventional neutral filter elution requires harsh lysis conditions to remove higher-order chromatin structures which interfere with elution of DNA containing dsb. By lysing cells with non-ionic detergent in the presence of 2 mol dm -3 salt, histone-depleted structures that retain the higher-order nuclear matrix organization, including chromatin loops, can be produced. Elution from these structures will only occur if two or more dsb lie within a single-looped domain delineated by points of attachment to the nuclear matrix. Repair experiments indicate that in CHO cells repair of dsb in loops containing multiple dsb are repaired with slow kinetics whilst dsb occurring in loops containing single dsb are repaired with fast kinetics. Xrs-5 cells are defective in the repair of multiply damaged loops. This work indicates that the spatial orientation of dsb in the higher-order structures of chromatin are a possible factor in the repair of these lesions. (Author)

Hydrogen peroxide biosensor based on DNA-Hb modified gold electrode

International Nuclear Information System (INIS)

Kafi, A.K.M.; Fan Yin; Shin, Hoon-Kyu; Kwon, Young-Soo

2006-01-01

A hydrogen peroxide (H 2 O 2 ) biosensor based on DNA-hemoglobin (Hb) modified electrode is described in this paper. The sensor was designed by DNA and hemoglobin dropletting onto gold electrode surface layer by layer. The sensor based on the direct electron transfer of iron of hemoglobin showed a well electrocatalytic response to the reduction of the H 2 O 2 . This sensor offered an excellent electrochemical response for H 2 O 2 concentration below micromole level with high sensitivity and selectivity and short response time. Experimental conditions influencing the biosensor performance such as, pH, potential were optimized and assessed. The levels of the RSD's ( 2 O 2 was observed from 10 to 120 μM with the detection limit of 0.4 μM (based on the S/N = 3)

DNA extraction methods for detecting genetically modified foods: A comparative study.

Science.gov (United States)

Elsanhoty, Rafaat M; Ramadan, Mohamed Fawzy; Jany, Klaus Dieter

2011-06-15

The work presented in this manuscript was achieved to compare six different methods for extracting DNA from raw maize and its derived products. The methods that gave higher yield and quality of DNA were chosen to detect the genetic modification in the samples collected from the Egyptian market. The different methods used were evaluated for extracting DNA from maize kernels (without treatment), maize flour (mechanical treatment), canned maize (sweet corn), frozen maize (sweet corn), maize starch, extruded maize, popcorn, corn flacks, maize snacks, and bread made from corn flour (mechanical and thermal treatments). The quality and quantity of the DNA extracted from the standards, containing known percentages of GMO material and from the different food products were evaluated. For qualitative detection of the GMO varieties in foods, the GMOScreen 35S/NOS test kit was used, to screen the genetic modification in the samples. The positive samples for the 35S promoter and/or the NOS terminator were identified by the standard methods adopted by EU. All of the used methods extracted yielded good DNA quality. However, we noted that the purest DNA extract were obtained using the DNA extraction kit (Roche) and this generally was the best method for extracting DNA from most of the maize-derived foods. We have noted that the yield of DNA extracted from maize-derived foods was generally lower in the processed products. The results indicated that 17 samples were positive for the presence of 35S promoter, while 34% from the samples were positive for the genetically modified maize line Bt-176. Copyright © 2010 Elsevier Ltd. All rights reserved.

Photodynamic DNA damage induced by phycocyanin and its repair in Saccharomyces cerevisiae

Directory of Open Access Journals (Sweden)

M. Pádula

1999-09-01

Full Text Available In the present study, we analyzed DNA damage induced by phycocyanin (PHY in the presence of visible light (VL using a set of repair endonucleases purified from Escherichia coli. We demonstrated that the profile of DNA damage induced by PHY is clearly different from that induced by molecules that exert deleterious effects on DNA involving solely singlet oxygen as reactive species. Most of PHY-induced lesions are single strand breaks and, to a lesser extent, base oxidized sites, which are recognized by Nth, Nfo and Fpg enzymes. High pressure liquid chromatography coupled to electrochemical detection revealed that PHY photosensitization did not induce 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo at detectable levels. DNA repair after PHY photosensitization was also investigated. Plasmid DNA damaged by PHY photosensitization was used to transform a series of Saccharomyces cerevisiae DNA repair mutants. The results revealed that plasmid survival was greatly reduced in rad14 mutants, while the ogg1 mutation did not modify the plasmid survival when compared to that in the wild type. Furthermore, plasmid survival in the ogg1 rad14 double mutant was not different from that in the rad14 single mutant. The results reported here indicate that lethal lesions induced by PHY plus VL are repaired differently by prokaryotic and eukaryotic cells. Morever, nucleotide excision repair seems to play a major role in the recognition and repair of these lesions in Saccharomyces cerevisiae.

Direct on-chip DNA synthesis using electrochemically modified gold electrodes as solid support

Science.gov (United States)

Levrie, Karen; Jans, Karolien; Schepers, Guy; Vos, Rita; Van Dorpe, Pol; Lagae, Liesbet; Van Hoof, Chris; Van Aerschot, Arthur; Stakenborg, Tim

2018-04-01

DNA microarrays have propelled important advancements in the field of genomic research by enabling the monitoring of thousands of genes in parallel. The throughput can be increased even further by scaling down the microarray feature size. In this respect, microelectronics-based DNA arrays are promising as they can leverage semiconductor processing techniques with lithographic resolutions. We propose a method that enables the use of metal electrodes for de novo DNA synthesis without the need for an insulating support. By electrochemically functionalizing gold electrodes, these electrodes can act as solid support for phosphoramidite-based synthesis. The proposed method relies on the electrochemical reduction of diazonium salts, enabling site-specific incorporation of hydroxyl groups onto the metal electrodes. An automated DNA synthesizer was used to couple phosphoramidite moieties directly onto the OH-modified electrodes to obtain the desired oligonucleotide sequence. Characterization was done via cyclic voltammetry and fluorescence microscopy. Our results present a valuable proof-of-concept for the integration of solid-phase DNA synthesis with microelectronics.

NAA-modified DNA oligonucleotides with zwitterionic backbones: stereoselective synthesis of A-T phosphoramidite building blocks.

Science.gov (United States)

Schmidtgall, Boris; Höbartner, Claudia; Ducho, Christian

2015-01-01

Modifications of the nucleic acid backbone are essential for the development of oligonucleotide-derived bioactive agents. The NAA-modification represents a novel artificial internucleotide linkage which enables the site-specific introduction of positive charges into the otherwise polyanionic backbone of DNA oligonucleotides. Following initial studies with the introduction of the NAA-linkage at T-T sites, it is now envisioned to prepare NAA-modified oligonucleotides bearing the modification at X-T motifs (X = A, C, G). We have therefore developed the efficient and stereoselective synthesis of NAA-linked 'dimeric' A-T phosphoramidite building blocks for automated DNA synthesis. Both the (S)- and the (R)-configured NAA-motifs were constructed with high diastereoselectivities to furnish two different phosphoramidite reagents, which were employed for the solid phase-supported automated synthesis of two NAA-modified DNA oligonucleotides. This represents a significant step to further establish the NAA-linkage as a useful addition to the existing 'toolbox' of backbone modifications for the design of bioactive oligonucleotide analogues.

The SRS2 suppressor of rad6 mutations of Saccharomyces cerevisiae acts by channeling DNA lesions into the RAD52 DNA repair pathway

International Nuclear Information System (INIS)

Schiestl, R.H.; Prakash, S.; Prakash, L.

1990-01-01

rad6 mutants of Saccharomyces cerevisiae are defective in the repair of damaged DNA, DNA damage induced mutagenesis, and sporulation. In order to identify genes that can substitute for RAD6 function, the authors have isolated genomic suppressors of the UV sensitivity of rad6 deletion (rad6Δ) mutations and show that they also suppress the γ-ray sensitivity but not the UV mutagenesis or sporulation defects of rad6. The suppressors show semidominance for suppression of UV sensitivity and dominance for suppression of γ-ray sensitivity. The six suppressor mutations they isolated are all alleles of the same locus and are also allelic to a previously described suppressor of the rad6-1 nonsense mutation, SRS2. They show that suppression of rad6Δ is dependent on the RAD52 recombinational repair pathway since suppression is not observed in the rad6Δ SRS2 strain containing an additional mutation in either the RAD51, RAD52, RAD54, RAD55 or RAD57 genes. Possible mechanisms by which SRS2 may channel unrepaired DNA lesions into the RAD52 DNA repair pathway are discussed

Aneuploidy in benign tumors and nonneoplastic lesions of musculoskeletal tissues.

Science.gov (United States)

Alho, A; Skjeldal, S; Pettersen, E O; Melvik, J E; Larsen, T E

1994-02-15

Aneuploidy in DNA flow cytometry (FCM) of musculoskeletal tumors is generally considered to be a sign of malignancy. Previously, giant cell tumor of the bone has been reported to contain aneuploid (near-diploid) DNA stemlines. Otherwise, only spordic cases have been reported. The authors wanted to study the relationships among DNA FCM, histology, and clinical course of nonmalignant musculoskeletal lesions. Twenty-eight histologically benign tumors and seven nonneoplastic lesions were subjected to DNA FCM: After tissue preparation mechanically and with ribonuclease and trypsin, the isolated nuclei were stained with propidium iodine using chicken and rainbow trout erythrocytes as controls. In the DNA FCM histograms, ploidy and cell cycle fractions were determined using a computerized mathematical model. The histologic diagnoses were made without knowledge of the DNA FCM results. Aneuploidy was found in eight lesions. A shoulder in the diploid peak, suggesting a diploid and a near-diploid population, was found in DNA histograms of a condensing osteitis of the clavicle (a benign inflammatory process) and of a giant cell tumor of bone. The latter lesion also had a tetraploid population. Six benign tumors--two enchondromas, one osteochondroma, one subcutaneous and one intramuscular lipoma, and a calcifying aponeurotic fibroma--showed clear aneuploidy with separate peaks. The S-phase fraction was less than 10% in all cases. The highest aneuploid population, DNA index = 1.70, in a subcutaneous lipoma, was small, with an undetectable S phase. Despite nonradical operations in seven lesions, no recurrences were observed during a median follow-up of 49 months (range, 28-73 months). Small aneuploid populations with low DNA synthetic activity may be compatible with a benign histologic picture and uneventful clinical course of the musculoskeletal lesion.

UVA-induced DNA double-strand breaks result from the repair of clustered oxidative DNA damages

Science.gov (United States)

Greinert, R.; Volkmer, B.; Henning, S.; Breitbart, E. W.; Greulich, K. O.; Cardoso, M. C.; Rapp, Alexander

2012-01-01

UVA (320–400 nm) represents the main spectral component of solar UV radiation, induces pre-mutagenic DNA lesions and is classified as Class I carcinogen. Recently, discussion arose whether UVA induces DNA double-strand breaks (dsbs). Only few reports link the induction of dsbs to UVA exposure and the underlying mechanisms are poorly understood. Using the Comet-assay and γH2AX as markers for dsb formation, we demonstrate the dose-dependent dsb induction by UVA in G1-synchronized human keratinocytes (HaCaT) and primary human skin fibroblasts. The number of γH2AX foci increases when a UVA dose is applied in fractions (split dose), with a 2-h recovery period between fractions. The presence of the anti-oxidant Naringin reduces dsb formation significantly. Using an FPG-modified Comet-assay as well as warm and cold repair incubation, we show that dsbs arise partially during repair of bi-stranded, oxidative, clustered DNA lesions. We also demonstrate that on stretched chromatin fibres, 8-oxo-G and abasic sites occur in clusters. This suggests a replication-independent formation of UVA-induced dsbs through clustered single-strand breaks via locally generated reactive oxygen species. Since UVA is the main component of solar UV exposure and is used for artificial UV exposure, our results shine new light on the aetiology of skin cancer. PMID:22941639

A simple approach for producing highly efficient DNA carriers with reduced toxicity based on modified polyallylamine

Energy Technology Data Exchange (ETDEWEB)

Oskuee, Reza Kazemi [Neurogenic Inflammation Research Center, Mashhad University of Medical Sciences, Mashhad (Iran, Islamic Republic of); Department of Medical Biotechnology, School of Medicine, Mashhad University of Medical Sciences, Mashhad (Iran, Islamic Republic of); Dosti, Fatemeh [School of Pharmacy, Mashhad University of Medical Sciences, Mashhad (Iran, Islamic Republic of); Gholami, Leila [Targeted Drug Delivery Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad (Iran, Islamic Republic of); Malaekeh-Nikouei, Bizhan, E-mail: malaekehb@mums.ac.ir [Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad (Iran, Islamic Republic of)

2015-04-01

Nowadays gene delivery is a topic in many research studies. Non-viral vectors have many advantages over viral vectors in terms of safety, immunogenicity and gene carrying capacity but they suffer from low transfection efficiency and high toxicity. In this study, polyallylamine (PAA), the cationic polymer, has been modified with hydrophobic branches to increase the transfection efficiency of the polymer. Polyallylamine with molecular weights of 15 and 65 kDa was selected and grafted with butyl, hexyl and decyl acrylate at percentages of 10, 30 and 50. The ability of the modified polymer to condense DNA was examined by ethidium bromide test. The complex of modified polymer and DNA (polyplex) was characterized for size, zeta potential, transfection efficiency and cytotoxicity in Neuro2A cell lines. The results of ethidium bromide test showed that grafting of PAA decreased its ability for DNA condensation but vectors could still condense DNA at moderate and high carrier to DNA ratios. Most of polyplexes had particle size between 150 and 250 nm. The prepared vectors mainly showed positive zeta potential but carriers composed of PAA with high percentage of grafting had negative zeta potential. The best transfection activity was observed in vectors with hexyl acrylate chain. Grafting of polymer reduced its cytotoxicity especially at percentages of 30 and 50. The vectors based of PAA 15 kDa had better transfection efficiency than the vectors made of PAA 65 kDa. In conclusion, results of the present study indicated that grafting PAA 15 kDa with high percentages of hexyl acrylate can help to prepare vectors with better transfection efficiency and less cytotoxicity. - Highlights: • The modified polyallylamine was synthesized as a gene carrier. • Modification of polyallylamine (15 kDa) with high percentages of hexyl acrylate improved transfection activity remarkably. • Grafting of polymer with acrylate derivatives reduced polymer cytotoxicity especially at percentages of

Direct assay of radiation-induced DNA base lesions in mammalian cells

International Nuclear Information System (INIS)

1992-01-01

Adenine (Ade), 2'-deoxyadenosine (dAdo), 5'-deoxyadenosine monophosphate (dAUT), single stranded poly adenylic acid [poly (dA)], double stranded deoxyadenylic-thymidylic acid [ds poly (dA-T)] and salmon testis DNA were irradiated with 500 Gy under oxic and anoxic conditions. The major damage products were analyzed by BPLC with optical detection and quantitated in terms of the percentage of the adenosine in each model compound found as a specific damage product. Outside of the Ade free base, 8-OH-dAdo was the major oxic damage product from each model compound. The type and quantity of the major damage products depended on the sequence and conformation of the model compounds under anoxic conditions. When dAdo and dAMP were irradiated under anoxic conditions, the major damage product was either the R or S isomer of 8,5'cdAdo and little Ade or α-dAdo was observed. However, when poly(dA), poly(dA-dT), and salmon testis DNA were γ-irradiated under nitrogen, the major deoxyadenosine damage product was identified as the α-anomer of deoxyadenosine. No α-deoxyadenosine was detected after irradiation under oxic conditions. The presence of nucleotides with the α-configuration at the anomeric carbon atom in the DNA chain may have a significant effect on its tertiary structure and possibly modify its biological activity

Carborane-linked 2'-deoxyuridine 5'-O-triphosphate as building block for polymerase synthesis of carborane-modified DNA.

Science.gov (United States)

Balintová, Jana; Simonova, Anna; Białek-Pietras, Magdalena; Olejniczak, Agnieszka; Lesnikowski, Zbigniew J; Hocek, Michal

2017-11-01

5-[(p-Carborane-2-yl)ethynyl]-2'-deoxyuridine 5'-O-triphosphate was synthesized and used as a good substrate in enzymatic construction of carborane-modified DNA or oligonucleotides containing up to 21 carborane moieties in primer extension reactions by DNA polymerases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mechanism of Error-Free DNA Replication Past Lucidin-Derived DNA Damage by Human DNA Polymerase κ.

Science.gov (United States)

Yockey, Oliver P; Jha, Vikash; Ghodke, Pratibha P; Xu, Tianzuo; Xu, Wenyan; Ling, Hong; Pradeepkumar, P I; Zhao, Linlin

2017-11-20

DNA damage impinges on genetic information flow and has significant implications in human disease and aging. Lucidin-3-O-primeveroside (LuP) is an anthraquinone derivative present in madder root, which has been used as a coloring agent and food additive. LuP can be metabolically converted to genotoxic compound lucidin, which subsequently forms lucidin-specific N 2 -2'-deoxyguanosine (N 2 -dG) and N 6 -2'-deoxyadenosine (N 6 -dA) DNA adducts. Lucidin is mutagenic and carcinogenic in rodents but has low carcinogenic risks in humans. To understand the molecular mechanism of low carcinogenicity of lucidin in humans, we performed DNA replication assays using site-specifically modified oligodeoxynucleotides containing a structural analogue (LdG) of lucidin-N 2 -dG DNA adduct and determined the crystal structures of DNA polymerase (pol) κ in complex with LdG-bearing DNA and an incoming nucleotide. We examined four human pols (pol η, pol ι, pol κ, and Rev1) in their efficiency and accuracy during DNA replication with LdG; these pols are key players in translesion DNA synthesis. Our results demonstrate that pol κ efficiently and accurately replicates past the LdG adduct, whereas DNA replication by pol η, pol ι is compromised to different extents. Rev1 retains its ability to incorporate dCTP opposite the lesion albeit with decreased efficiency. Two ternary crystal structures of pol κ illustrate that the LdG adduct is accommodated by pol κ at the enzyme active site during insertion and postlesion-extension steps. The unique open active site of pol κ allows the adducted DNA to adopt a standard B-form for accurate DNA replication. Collectively, these biochemical and structural data provide mechanistic insights into the low carcinogenic risk of lucidin in humans.

Degradation of transgene DNA in genetically modified herbicide-tolerant rice during food processing.

Science.gov (United States)

Song, Shangxin; Zhou, Guanghong; Gao, Feng; Zhang, Wei; Qiu, Liangyan; Dai, Sifa; Xu, Xinglian; Xiao, Hongmei

2011-12-01

In order to assess the effect of food processing on the degradation of exogenous DNA components in sweet rice wine and rice crackers made from genetically modified (GM) rice (Oryza sativa L.), we developed genomic DNA extraction methods and compared the effect of different food processing procedures on DNA degradation. It was found that the purity, quantity and quality of DNA by alkaline lysis method were higher than by CTAB (cetyltrimethylammonium bromide) method. For sweet rice wine, CAMV35S (cauliflower mosaic virus 35S) promoter and NOS (nopaline synthase) terminator were degraded by the third day, whereas the exogenous gene Bar (bialaphos resistance) remained unaffected. For rice crackers, boiling, drying and microwaving contributed to the initial degradations of DNA. Baking resulted in further degradations, and frying led to the most severe changes. These results indicated that the stability of DNA in GM rice was different under different processing conditions. For sweet rice wine, Bar was most stable, followed by NOS, CAMV35S, and SPS. For rice crackers, CAMV35S was most stable, followed by SPS, NOS, and Bar. Copyright © 2011 Elsevier Ltd. All rights reserved.

Evaluation of a Microculture Method for Isolation of Leishmania Parasites from Cutaneous Lesions of Patients in Peru▿

Science.gov (United States)

Boggild, Andrea K.; Miranda-Verastegui, Cesar; Espinosa, Diego; Arevalo, Jorge; Adaui, Vanessa; Tulliano, Gianfranco; Llanos-Cuentas, Alejandro; Low, Donald E.

2007-01-01

Traditional culture of Leishmania spp. is labor intensive and has poor sensitivity. We evaluated a microculture method for the diagnosis of cutaneous leishmaniasis in consecutive patients presenting to the Leishmaniasis Clinic at the Instituto de Medicina Tropical Alexander von Humboldt, Peru, for evaluation of skin lesions. Lesion aspirates were cultured in duplicate and parallel in traditional culture tubes containing modified Novy-MacNeal-Nicolle (NNN) medium or Roswell Park Memorial Institute medium 1640 with 10% fetal bovine serum (10% RPMI) and in 70-μl capillary tubes containing a mixture of lesion aspirate and 10% RPMI. For sensitivity analysis, the consensus standard was considered to be a positive result in any two of the following four tests: Giemsa-stained lesion smear, culture, kinetoplast DNA PCR, or leishmanin skin test. The outcome measures were sensitivity and time to culture positivity. Forty-five patients with 62 skin lesions were enrolled in the study, of which 53 lesions fulfilled the consensus criteria for a final diagnosis of cutaneous leishmaniasis. Of these 53 lesions, 39 were culture positive: 38 in capillary tubes, 29 in traditional culture tubes with modified NNN medium, and 19 in traditional culture tubes with 10% RPMI medium. The sensitivity of microculture was 71.7%, versus 54.7% for traditional culture with NNN (P, 0.038) and 35.8% with 10% RPMI (P, microculture, 5.2 days in NNN, and 6 days in 10% RPMI (P, 0.009). We have demonstrated that microculture is a more sensitive and time-efficient means of isolating Leishmania parasites from cutaneous lesions than traditional culture. PMID:17881557

Evaluation of a microculture method for isolation of Leishmania parasites from cutaneous lesions of patients in Peru.

Science.gov (United States)

Boggild, Andrea K; Miranda-Verastegui, Cesar; Espinosa, Diego; Arevalo, Jorge; Adaui, Vanessa; Tulliano, Gianfranco; Llanos-Cuentas, Alejandro; Low, Donald E

2007-11-01

Traditional culture of Leishmania spp. is labor intensive and has poor sensitivity. We evaluated a microculture method for the diagnosis of cutaneous leishmaniasis in consecutive patients presenting to the Leishmaniasis Clinic at the Instituto de Medicina Tropical Alexander von Humboldt, Peru, for evaluation of skin lesions. Lesion aspirates were cultured in duplicate and parallel in traditional culture tubes containing modified Novy-MacNeal-Nicolle (NNN) medium or Roswell Park Memorial Institute medium 1640 with 10% fetal bovine serum (10% RPMI) and in 70-microl capillary tubes containing a mixture of lesion aspirate and 10% RPMI. For sensitivity analysis, the consensus standard was considered to be a positive result in any two of the following four tests: Giemsa-stained lesion smear, culture, kinetoplast DNA PCR, or leishmanin skin test. The outcome measures were sensitivity and time to culture positivity. Forty-five patients with 62 skin lesions were enrolled in the study, of which 53 lesions fulfilled the consensus criteria for a final diagnosis of cutaneous leishmaniasis. Of these 53 lesions, 39 were culture positive: 38 in capillary tubes, 29 in traditional culture tubes with modified NNN medium, and 19 in traditional culture tubes with 10% RPMI medium. The sensitivity of microculture was 71.7%, versus 54.7% for traditional culture with NNN (P, 0.038) and 35.8% with 10% RPMI (P, microculture, 5.2 days in NNN, and 6 days in 10% RPMI (P, 0.009). We have demonstrated that microculture is a more sensitive and time-efficient means of isolating Leishmania parasites from cutaneous lesions than traditional culture.

Heavy ion-induced lesions in DNA: A theoretical model for the initial induction of DNA strand breaks and chromatin breaks

International Nuclear Information System (INIS)

Schmidt, J.B.

1993-01-01

A theoretical model has been developed and used to calculate yields and spatial distributions of DNA strand breaks resulting from the interactions of heavy ions with chromatin in aqueous systems. The three dimensional spatial distribution of ionizing events has been modeled for charged particles as a function of charge and velocity. Chromatin has been modeled as a 30 nm diameter solenoid of nucleosomal DNA. The Monte Carlo methods used by Chatterjee et al. have been applied to DNA in a chromatin conformation. Refinements to their methods include: a combined treatment of primary and low energy (<2 keV) secondary electron interactions, an improved low energy delta ray model, and the combined simulation of direct energy deposition on the DNA and attack by diffusing hydroxyl radicals. Individual particle tracks are treated independently, which is assumed to be applicable to low fluence irradiations in which multiple particle effects are negligible. Single strand break cross section open-quotes hooksclose quotes seen in experiments at very high LET appear to be due to the collapsing radial extent of the track, as predicted in the open-quotes deep sieveclose quotes hypothesis proposed by Tobias et al. Spatial distributions of lesions produced by particles have been found to depend on chromatin structure. In the future, heavy ions may be used as a tool to probe the organization of DNA in chromatin. A Neyman A-binomial variation of the open-quotes cluster modelclose quotes for the distribution of chromatin breaks per irradiated cell has been theoretically tested. The model includes a treatment of the chromatin fragment detection technique's resolution, which places a limitation on the minimum size of fragments which can be detected. The model appears to fit some of the experimental data reasonably well. However, further experimental and theoretical refinements are desirable

Influence of DNA Lesions on Polymerase-Mediated DNA Replication at Single-Molecule Resolution.

Science.gov (United States)

Gahlon, Hailey L; Romano, Louis J; Rueda, David

2017-11-20

Faithful replication of DNA is a critical aspect in maintaining genome integrity. DNA polymerases are responsible for replicating DNA, and high-fidelity polymerases do this rapidly and at low error rates. Upon exposure to exogenous or endogenous substances, DNA can become damaged and this can alter the speed and fidelity of a DNA polymerase. In this instance, DNA polymerases are confronted with an obstacle that can result in genomic instability during replication, for example, by nucleotide misinsertion or replication fork collapse. It is important to know how DNA polymerases respond to damaged DNA substrates to understand the mechanism of mutagenesis and chemical carcinogenesis. Single-molecule techniques have helped to improve our current understanding of DNA polymerase-mediated DNA replication, as they enable the dissection of mechanistic details that can otherwise be lost in ensemble-averaged experiments. These techniques have also been used to gain a deeper understanding of how single DNA polymerases behave at the site of the damage in a DNA substrate. In this review, we evaluate single-molecule studies that have examined the interaction between DNA polymerases and damaged sites on a DNA template.

AFFINITY BIOSENSOR BASED ON SCREEN-PRINTED ELECTRODE MODIFIED WITH DNA FOR GENOTOXIC COMPOUNDS DETECTION

Directory of Open Access Journals (Sweden)

Bambang Kuswandi

2010-06-01

Full Text Available An electrochemical method for the detection of the genotoxic compounds using a DNA-modified electrode was developed. This electrode was successfully used for the electrochemical detection of genotoxic compounds in water samples. The electrochemical results clearly demonstrated that, the development is related to the molecular interaction between the surface-linked DNA obtained from calf thymus and the target compounds, such as pollutants, in order to develop a simple device for rapid screening of genotoxic compounds in environmental samples. The detection of such compounds was measured by their effect on the oxidation signal of the guanine peak of the DNA immobilised on the surface of carbon based Screen-Printed Electrode (SPE in disposable mode, and monitored by square-wave voltametric analysis. The DNA biosensor is able to detect known intercalating and groove-binding genotoxic compounds such as Dioxin, Bisphenol A, PCBs, and Phtalates. Application to real water samples is discussed and reported.   Keywords: electrochemical, screen-printed electrode, DNA biosensor, genotoxic compounds

Towards observing the encounter of the T7 DNA replication fork with a lesion site at the Single molecule level

KAUST Repository

Shirbini, Afnan

2017-05-01

Single-molecule DNA flow-stretching assays have been a powerful approach to study various aspects on the mechanism of DNA replication for more than a decade. This technique depends on flow-induced force on a bead attached to a surface-tethered DNA. The difference in the elastic property between double-strand DNA (long) and single-strand DNA (short) at low regime force allows the observation of the beads motion when the dsDNA is converted to ssDNA by the replisome machinery during DNA replication. Here, I aim to develop an assay to track in real-time the encounter of the bacteriophage T7 replisome with abasic lesion site inserted on the leading strand template. I optimized methods to construct the DNA substrate that contains the abasic site and established the T7 leading strand synthesis at the single molecule level. I also optimized various control experiments to remove any interference from the nonspecific interactions of the DNA with the surface. My work established the foundation to image the encounter of the T7 replisome with abasic site and to characterize how the interactions between the helicase and the polymerase could influence the polymerase proofreading ability and its direct bypass of this highly common DNA damage type.

Detection, characterization and measure of a new radiation-induced damage in isolated and cellular DNA

International Nuclear Information System (INIS)

Regulus, P.

2006-10-01

Deoxyribonucleic acid (DNA) contains the genetic information and chemical injury to this macromolecule may have severe biological consequences. We report here the detection of 4 new radiation-induced DNA lesions by using a high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) approach. For that purpose, the characteristic fragmentation of most 2'-deoxy-ribo nucleosides, the loss of 116 Da corresponding to the loss of the 2-deoxyribose moiety, was used in the so-called neutral loss mode of the HPLC-MS/MS. One of the newly detected lesions, named dCyd341 because it is a 2'-deoxycytidine modification exhibiting a molecular weight of 341 Da, was also detected in cellular DNA. Characterization of this modified nucleoside was performed using NMR and exact mass determination of the product obtained by chemical synthesis. A mechanism of formation was then proposed, in which the first event is the H-abstraction at the C4 position of a 2-deoxyribose moiety. Then, the sugar modification produced exhibits a reactive aldehyde that, through reaction with a vicinal cytosine base, gives rise to dCyd341. dCyd341 could be considered as a complex damage since its formation involves a DNA strand break and a cross-link between a damaged sugar residue and a vicinal cytosine base located most probably on the complementary DNA strand. In addition to its characterization, preliminary biological studies revealed that cells are able to remove the lesion from DNA. Repair studies have revealed the ability of cells to excise the lesion. Identification of the repair systems involved could represent an interesting challenge. (author)

Chemically modified DNA : methods and applications

NARCIS (Netherlands)

Gjonaj, Lorina

2013-01-01

Over het toepassen van DNA in de chemie DNA is een fascinerend biopolymeer dat gemakkelijk gemanipuleerd kan worden. Doel van het onderzoek van Lorina Gjonaj was het ontwikkelen van nieuwe methodologie om DNA chemisch te modificeren. Verschillende functionaliteiten zijn aan DNA gekoppeld. Deze

Cytogenetic status and oxidative DNA-damage induced by atorvastatin in human peripheral blood lymphocytes: Standard and Fpg-modified comet assay

International Nuclear Information System (INIS)

Gajski, Goran; Garaj-Vrhovac, Vera; Orescanin, Visnja

2008-01-01

To investigate the genotoxic potential of atorvastatin on human lymphocytes in vitro standard comet assay was used in the evaluation of basal DNA damage and to investigate possible oxidative DNA damage produced by reactive oxygen species (ROS) Fpg-modified version of comet assay was also conducted. In addition to these techniques the new criteria for scoring micronucleus test were applied for more complete detection of baseline damage in binuclear lymphocytes exposed to atorvastatin 80 mg/day in different time periods by virtue of measuring the frequency of micronuclei, nucleoplasmic bridges and nuclear buds. All parameters obtained with the standard comet assay and Fpg-modified comet assay were significantly higher in the treated than in control lymphocytes. The Fpg-modified comet assay showed a significantly greater tail length, tail intensity, and tail moment in all treated lymphocytes than did the standard comet assay, which suggests that oxidative stress is likely to be responsible for DNA damage. DNA damage detected by the standard comet assay indicates that some other mechanism is also involved. In addition to the comet assay, a total number of micronuclei, nucleoplasmic bridges and nuclear buds were significantly higher in the exposed than in controlled lymphocytes. Regression analyses showed a positive correlation between the results obtained by the comet (Fpg-modified and standard) and micronucleus assay. Overall, the study demonstrated that atorvastatin in its highest dose is capable of producing damage on the level of DNA molecule and cell

Cytogenetic status and oxidative DNA-damage induced by atorvastatin in human peripheral blood lymphocytes: Standard and Fpg-modified comet assay

Energy Technology Data Exchange (ETDEWEB)

Gajski, Goran [Institute for Medical Research and Occupational Health, Mutagenesis Unit, 10000 Zagreb (Croatia); Garaj-Vrhovac, Vera [Institute for Medical Research and Occupational Health, Mutagenesis Unit, 10000 Zagreb (Croatia); Orescanin, Visnja [Ruder Boskovic Institute, 10000 Zagreb (Croatia)

2008-08-15

To investigate the genotoxic potential of atorvastatin on human lymphocytes in vitro standard comet assay was used in the evaluation of basal DNA damage and to investigate possible oxidative DNA damage produced by reactive oxygen species (ROS) Fpg-modified version of comet assay was also conducted. In addition to these techniques the new criteria for scoring micronucleus test were applied for more complete detection of baseline damage in binuclear lymphocytes exposed to atorvastatin 80 mg/day in different time periods by virtue of measuring the frequency of micronuclei, nucleoplasmic bridges and nuclear buds. All parameters obtained with the standard comet assay and Fpg-modified comet assay were significantly higher in the treated than in control lymphocytes. The Fpg-modified comet assay showed a significantly greater tail length, tail intensity, and tail moment in all treated lymphocytes than did the standard comet assay, which suggests that oxidative stress is likely to be responsible for DNA damage. DNA damage detected by the standard comet assay indicates that some other mechanism is also involved. In addition to the comet assay, a total number of micronuclei, nucleoplasmic bridges and nuclear buds were significantly higher in the exposed than in controlled lymphocytes. Regression analyses showed a positive correlation between the results obtained by the comet (Fpg-modified and standard) and micronucleus assay. Overall, the study demonstrated that atorvastatin in its highest dose is capable of producing damage on the level of DNA molecule and cell.

Repair of Clustered Damage and DNA Polymerase Iota.

Science.gov (United States)

Belousova, E A; Lavrik, O I

2015-08-01

Multiple DNA lesions occurring within one or two turns of the DNA helix known as clustered damage are a source of double-stranded DNA breaks, which represent a serious threat to the cells. Repair of clustered lesions is accomplished in several steps. If a clustered lesion contains oxidized bases, an individual DNA lesion is repaired by the base excision repair (BER) mechanism involving a specialized DNA polymerase after excising DNA damage. Here, we investigated DNA synthesis catalyzed by DNA polymerase iota using damaged DNA templates. Two types of DNA substrates were used as model DNAs: partial DNA duplexes containing breaks of different length, and DNA duplexes containing 5-formyluracil (5-foU) and uracil as a precursor of apurinic/apyrimidinic sites (AP) in opposite DNA strands. For the first time, we showed that DNA polymerase iota is able to catalyze DNA synthesis using partial DNA duplexes having breaks of different length as substrates. In addition, we found that DNA polymerase iota could catalyze DNA synthesis during repair of clustered damage via the BER system by using both undamaged and 5-foU-containing templates. We found that hPCNA (human proliferating cell nuclear antigen) increased efficacy of DNA synthesis catalyzed by DNA polymerase iota.

Cancerous hyper-mutagenesis in p53 genes is possibly associated with transcriptional bypass of DNA lesions

International Nuclear Information System (INIS)

Rodin, S.N.; Rodin, A.S.; Juhasz, A.; Holmquist, G.P.

2002-01-01

The database of tumor-associated p53 base substitutions includes about 5% of tumors with two or more base substitutions. These multiplet base substitutions in one tumor are evidence for hyper-mutagenesis. Our retrospective analysis of this database indicates that most multiplets arise from a single transient hyper-mutagenic event in one cell that subsequently proliferated into a clonal tumor. The hyper-mutagenesis, 1.8x10 -4 substitutions per base pair, is detected as multiple mutations in p53 genes of tumors. It requires one strongly tumorigenic p53 substitution, usually missense, called the driver mutation. The occurrence frequencies of ancillary base substitutions, those that hitch-hike along with the driver mutation, are independent of their amino acid coding properties. In this respect, they act like neutral mutations. In support of this neutrality, we find that the frequency distribution of hitch-hiking CpG transitions along the p53 exons, their mutational spectrum, approximates the spontaneous pre-selection mutational spectrum of most human tissues and is correlated with the mutational spectrum of p53 pseudogenes in mammalian germ cells. The driver substitutions of multiplets predominantly originate along the transcribed strand while the ancillary substitutions tend to originate along the non-transcribed strand. This data is consistent with a model of time-dependent mutagenesis in non-dividing stem cells for generating multiple strand-asymmetric p53 mutations in tumors. By transcriptional bypass of DNA lesions with concomitant misincorporation, transcriptional mutagenesis generates a transient mutant p53 mRNA. The associated mutant p53 protein could allow the host cell a growth advantage, release from G 1 -arrest. Then, during subsequent DNA replication and misreading of the same lesion, the damaged base along the transcribed DNA strand would serve as the origin of the p53 base substitution that drives the hyper-mutagenic event leading to tumors with

A plasma modified cellulose-chitosan porous membrane allows efficient DNA binding and provides antibacterial properties: A step towards developing a new DNA collecting card.

Science.gov (United States)

Chumwangwapee, Sasiwimon; Chingsungnoen, Artit; Siri, Sineenat

2016-11-01

In forensic DNA analyses, biological specimens are collected and stored for subsequent recovery and analysis of DNA. A cost-effective and efficient DNA recovery approach is therefore a need. This study aims to produce a plasma modified cellulose-chitosan membrane (pCE-CS) that efficiently binds and retains DNA as a potential DNA collecting card. The pCE-CS membrane was produced by a phase separation of ionic liquid dissolving CE and CS in water with subsequent surface-modification by a two-step exposure of argon plasma and nitrogen gas. Through plasma modification, the pCE-CS membrane demonstrated better DNA retention after a washing process and higher rate of DNA recovery as compared with the original CE-CS membrane and the commercial FTA card. In addition, the pCE-CS membrane exhibited anti-bacterial properties against both Escherichia coli and Staphylococcus aureus. The results of this work suggest a potential function of the pCE-CS membrane as a DNA collecting card with a high recovery rate of captured DNA. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Persistent and heritable structural damage induced in heterochromatic DNA from rat liver by N-nitrosodimethylamine

International Nuclear Information System (INIS)

Ward, E.J.; Stewart, B.W.

1987-01-01

Analysis, by benzoylated DEAE-cellulose chromatography, has been made of structural change in eu- and heterochromatic DNA from rat liver following administration of the carcinogen N-nitrosodimethylamine. Either hepatic DNA was prelabeled with [ 3 H]thymidine administered 2-3 weeks before injection of the carcinogen or the labeled precursor was given during regenerative hyperplasia in rats treated earlier with N-nitrosodimethylamine. Following phenol extraction of either whole liver homogenate or nuclease-fractionated eu- and heterochromatin, carcinogen-modified DNA was examined by stepwise or caffeine gradient elution from benzoylated DEAE-cellulose. In whole DNA, nitrosamine-induced single-stranded character was maximal 4-24 h after treatment, declining rapidly thereafter; gradient elution of these DNA preparations also provided short-term evidence of structural change. Caffeine gradient chromatography suggested short-term nitrosamine-induced structural change in euchromatic DNA, while increased binding of heterochromatic DNA was evident for up to 3 months after carcinogen treatment. Preparations of newly synthesized heterochromatic DNA from animals subjected to hepatectomy up to 2 months after carcinogen treatment provided evidence of heritable structural damage. Carcinogen-induced binding of heterochromatic DNA to benzoylated DEAE-cellulose was indicative of specific structural lesions whose affinity equalled that of single-stranded DNA up to 1.0 kilobase in length. The data suggest that structural lesions in heterochromatin, which may be a consequence of incomplete repair, are preferentially degraded by endogenous nuclease(s)

Bypass of a 5',8-cyclopurine-2'-deoxynucleoside by DNA polymerase β during DNA replication and base excision repair leads to nucleotide misinsertions and DNA strand breaks.

Science.gov (United States)

Jiang, Zhongliang; Xu, Meng; Lai, Yanhao; Laverde, Eduardo E; Terzidis, Michael A; Masi, Annalisa; Chatgilialoglu, Chryssostomos; Liu, Yuan

2015-09-01

5',8-Cyclopurine-2'-deoxynucleosides including 5',8-cyclo-dA (cdA) and 5',8-cyclo-dG (cdG) are induced by hydroxyl radicals resulting from oxidative stress such as ionizing radiation. 5',8-cyclopurine-2'-deoxynucleoside lesions are repaired by nucleotide excision repair with low efficiency, thereby leading to their accumulation in the human genome and lesion bypass by DNA polymerases during DNA replication and base excision repair (BER). In this study, for the first time, we discovered that DNA polymerase β (pol β) efficiently bypassed a 5'R-cdA, but inefficiently bypassed a 5'S-cdA during DNA replication and BER. We found that cell extracts from pol β wild-type mouse embryonic fibroblasts exhibited significant DNA synthesis activity in bypassing a cdA lesion located in replication and BER intermediates. However, pol β knock-out cell extracts exhibited little DNA synthesis to bypass the lesion. This indicates that pol β plays an important role in bypassing a cdA lesion during DNA replication and BER. Furthermore, we demonstrated that pol β inserted both a correct and incorrect nucleotide to bypass a cdA at a low concentration. Nucleotide misinsertion was significantly stimulated by a high concentration of pol β, indicating a mutagenic effect induced by pol β lesion bypass synthesis of a 5',8-cyclopurine-2'-deoxynucleoside. Moreover, we found that bypass of a 5'S-cdA by pol β generated an intermediate that failed to be extended by pol β, resulting in accumulation of single-strand DNA breaks. Our study provides the first evidence that pol β plays an important role in bypassing a 5',8-cyclo-dA during DNA replication and repair, as well as new insight into mutagenic effects and genome instability resulting from pol β bypassing of a cdA lesion. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparison of impedimetric detection of DNA hybridization on the various biosensors based on modified glassy carbon electrodes with PANHS and nanomaterials of RGO and MWCNTs.

Science.gov (United States)

Benvidi, Ali; Tezerjani, Marzieh Dehghan; Jahanbani, Shahriar; Mazloum Ardakani, Mohammad; Moshtaghioun, Seyed Mohammad

2016-01-15

In this research, we have developed lable free DNA biosensors based on modified glassy carbon electrodes (GCE) with reduced graphene oxide (RGO) and carbon nanotubes (MWCNTs) for detection of DNA sequences. This paper compares the detection of BRCA1 5382insC mutation using independent glassy carbon electrodes (GCE) modified with RGO and MWCNTs. A probe (BRCA1 5382insC mutation detection (ssDNA)) was then immobilized on the modified electrodes for a specific time. The immobilization of the probe and its hybridization with the target DNA (Complementary DNA) were performed under optimum conditions using different electrochemical techniques such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The proposed biosensors were used for determination of complementary DNA sequences. The non-modified DNA biosensor (1-pyrenebutyric acid-N- hydroxysuccinimide ester (PANHS)/GCE), revealed a linear relationship between ∆Rct and logarithm of the complementary target DNA concentration ranging from 1.0×10(-16)molL(-1) to 1.0×10(-10)mol L(-1) with a correlation coefficient of 0.992, for DNA biosensors modified with multi-wall carbon nanotubes (MWCNTs) and reduced graphene oxide (RGO) wider linear range and lower detection limit were obtained. For ssDNA/PANHS/MWCNTs/GCE a linear range 1.0×10(-17)mol L(-1)-1.0×10(-10)mol L(-1) with a correlation coefficient of 0.993 and for ssDNA/PANHS/RGO/GCE a linear range from 1.0×10(-18)mol L(-1) to 1.0×10(-10)mol L(-1) with a correlation coefficient of 0.985 were obtained. In addition, the mentioned biosensors were satisfactorily applied for discriminating of complementary sequences from noncomplementary sequences, so the mentioned biosensors can be used for the detection of BRCA1-associated breast cancer. Copyright © 2015. Published by Elsevier B.V.

Induction of DNA damage in γ-irradiated nuclei stripped of nuclear protein classes: differential modulation of double-strand break and DNA-protein crosslink formation

International Nuclear Information System (INIS)

Xue, L.-Y.; Friedman, L.R.; Oleinick, N.L.; Chiu, S.-M.

1994-01-01

The influence of chromatin proteins on the induction of DNA double-strand breaks (dsb) and DNA-protein crosslinks (dpc) by γ-radiation was investigated. Low molecular weight non-histone proteins and classes of histones were extracted with increasing concentrations of NaC1, whereas nuclear matrix proteins were not extractable even by 2.0 M NACl. The yield of dsb increased with progressive removal of proteins from chromatin. The data support our previous conclusion that nuclear matrix protein rather than the majority of the histones are the predominant substrates for dpc production, although the involvement of a subset of tightly bound histones (H3 and H4) has not been excluded. This finding demonstrates that chromatin proteins can differentially modify the yield of two types of radiation-induced DNA lesions. (author)

The utilization of SiNWs/AuNPs-modified indium tin oxide (ITO) in fabrication of electrochemical DNA sensor.

Science.gov (United States)

Rashid, Jahwarhar Izuan Abdul; Yusof, Nor Azah; Abdullah, Jaafar; Hashim, Uda; Hajian, Reza

2014-12-01

This work describes the incorporation of SiNWs/AuNPs composite as a sensing material for DNA detection on indium tin-oxide (ITO) coated glass slide. The morphology of SiNWs/AuNPs composite as the modifier layer on ITO was studied by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX). The morphological studies clearly showed that SiNWs were successfully decorated with 20 nm-AuNPs using self-assembly monolayer (SAM) technique. The effective surface area for SiNWs/AuNPs-modified ITO enhanced about 10 times compared with bare ITO electrode. SiNWs/AuNPs nanocomposite was further explored as a matrix for DNA probe immobilization in detection of dengue virus as a bio-sensing model to evaluate its performance in electrochemical sensors. The hybridization of complementary DNA was monitored by differential pulse voltammetry (DPV) using methylene blue (MB) as the redox indicator. The fabricated biosensor was able to discriminate significantly complementary, non-complementary and single-base mismatch oligonucleotides. The electrochemical biosensor was sensitive to target DNA related to dengue virus in the range of 9.0-178.0 ng/ml with detection limit of 3.5 ng/ml. In addition, SiNWs/AuNPs-modified ITO, regenerated up to 8 times and its stability was up to 10 weeks at 4°C in silica gel. Copyright © 2014 Elsevier B.V. All rights reserved.

SUMO E3 ligase Mms21 prevents spontaneous DNA damage induced genome rearrangements.

Directory of Open Access Journals (Sweden)

Jason Liang

2018-03-01

Full Text Available Mms21, a subunit of the Smc5/6 complex, possesses an E3 ligase activity for the Small Ubiquitin-like MOdifier (SUMO. Here we show that the mms21-CH mutation, which inactivates Mms21 ligase activity, causes increased accumulation of gross chromosomal rearrangements (GCRs selected in the dGCR assay. These dGCRs are formed by non-allelic homologous recombination between divergent DNA sequences mediated by Rad52-, Rrm3- and Pol32-dependent break-induced replication. Combining mms21-CH with sgs1Δ caused a synergistic increase in GCRs rates, indicating the distinct roles of Mms21 and Sgs1 in suppressing GCRs. The mms21-CH mutation also caused increased rates of accumulating uGCRs mediated by breakpoints in unique sequences as revealed by whole genome sequencing. Consistent with the accumulation of endogenous DNA lesions, mms21-CH mutants accumulate increased levels of spontaneous Rad52 and Ddc2 foci and had a hyper-activated DNA damage checkpoint. Together, these findings support that Mms21 prevents the accumulation of spontaneous DNA lesions that cause diverse GCRs.

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

DEFF Research Database (Denmark)

Skarpengland, Tonje; B. Dahl, Tuva; Skjelland, Mona

2016-01-01

Lesional and systemic oxidative stress has been implicated in the pathogenesis of atherosclerosis, potentially leading to accumulation of DNA base lesions within atherosclerotic plaques. Although base excision repair (BER) is a major pathway counteracting oxidative DNA damage, our knowledge on BER...

Functions of Ubiquitin and SUMO in DNA Replication and Replication Stress

Science.gov (United States)

García-Rodríguez, Néstor; Wong, Ronald P.; Ulrich, Helle D.

2016-01-01

Complete and faithful duplication of its entire genetic material is one of the essential prerequisites for a proliferating cell to maintain genome stability. Yet, during replication DNA is particularly vulnerable to insults. On the one hand, lesions in replicating DNA frequently cause a stalling of the replication machinery, as most DNA polymerases cannot cope with defective templates. This situation is aggravated by the fact that strand separation in preparation for DNA synthesis prevents common repair mechanisms relying on strand complementarity, such as base and nucleotide excision repair, from working properly. On the other hand, the replication process itself subjects the DNA to a series of hazardous transformations, ranging from the exposure of single-stranded DNA to topological contortions and the generation of nicks and fragments, which all bear the risk of inducing genomic instability. Dealing with these problems requires rapid and flexible responses, for which posttranslational protein modifications that act independently of protein synthesis are particularly well suited. Hence, it is not surprising that members of the ubiquitin family, particularly ubiquitin itself and SUMO, feature prominently in controlling many of the defensive and restorative measures involved in the protection of DNA during replication. In this review we will discuss the contributions of ubiquitin and SUMO to genome maintenance specifically as they relate to DNA replication. We will consider cases where the modifiers act during regular, i.e., unperturbed stages of replication, such as initiation, fork progression, and termination, but also give an account of their functions in dealing with lesions, replication stalling and fork collapse. PMID:27242895

The utilization of SiNWs/AuNPs-modified indium tin oxide (ITO) in fabrication of electrochemical DNA sensor

Energy Technology Data Exchange (ETDEWEB)

Rashid, Jahwarhar Izuan Abdul [Institute of Advanced Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Department of Chemistry and Biology, Centre for Defense Foundation Studies, National Defense University of Malaysia, Sungai Besi Camp, 57000 Kuala Lumpur (Malaysia); Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Yusof, Nor Azah, E-mail: azahy@upm.edu.my [Institute of Advanced Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Abdullah, Jaafar [Institute of Advanced Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Hashim, Uda [Institute of Nanoelectronic Engineering, Universiti Malaysia Perlis, 01000 Kangar, Perlis (Malaysia); Hajian, Reza, E-mail: rezahajian@upm.edu.my [Institute of Advanced Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor (Malaysia)

2014-12-01

This work describes the incorporation of SiNWs/AuNPs composite as a sensing material for DNA detection on indium tin-oxide (ITO) coated glass slide. The morphology of SiNWs/AuNPs composite as the modifier layer on ITO was studied by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX). The morphological studies clearly showed that SiNWs were successfully decorated with 20 nm-AuNPs using self-assembly monolayer (SAM) technique. The effective surface area for SiNWs/AuNPs-modified ITO enhanced about 10 times compared with bare ITO electrode. SiNWs/AuNPs nanocomposite was further explored as a matrix for DNA probe immobilization in detection of dengue virus as a bio-sensing model to evaluate its performance in electrochemical sensors. The hybridization of complementary DNA was monitored by differential pulse voltammetry (DPV) using methylene blue (MB) as the redox indicator. The fabricated biosensor was able to discriminate significantly complementary, non-complementary and single-base mismatch oligonucleotides. The electrochemical biosensor was sensitive to target DNA related to dengue virus in the range of 9.0–178.0 ng/ml with detection limit of 3.5 ng/ml. In addition, SiNWs/AuNPs-modified ITO, regenerated up to 8 times and its stability was up to 10 weeks at 4 °C in silica gel. - Highlights: • A sensitive biosensor is presented for detection of dengue virus. • SiNWs and AuNPs used as nanocomposite layers on ITO for construction of biosensor • The detection mechanism is based on the interaction of MB with DNA bonded on AuNPs. • The reduction signal of MB decreases upon complementary hybridization.

The utilization of SiNWs/AuNPs-modified indium tin oxide (ITO) in fabrication of electrochemical DNA sensor

International Nuclear Information System (INIS)

Rashid, Jahwarhar Izuan Abdul; Yusof, Nor Azah; Abdullah, Jaafar; Hashim, Uda; Hajian, Reza

2014-01-01

This work describes the incorporation of SiNWs/AuNPs composite as a sensing material for DNA detection on indium tin-oxide (ITO) coated glass slide. The morphology of SiNWs/AuNPs composite as the modifier layer on ITO was studied by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX). The morphological studies clearly showed that SiNWs were successfully decorated with 20 nm-AuNPs using self-assembly monolayer (SAM) technique. The effective surface area for SiNWs/AuNPs-modified ITO enhanced about 10 times compared with bare ITO electrode. SiNWs/AuNPs nanocomposite was further explored as a matrix for DNA probe immobilization in detection of dengue virus as a bio-sensing model to evaluate its performance in electrochemical sensors. The hybridization of complementary DNA was monitored by differential pulse voltammetry (DPV) using methylene blue (MB) as the redox indicator. The fabricated biosensor was able to discriminate significantly complementary, non-complementary and single-base mismatch oligonucleotides. The electrochemical biosensor was sensitive to target DNA related to dengue virus in the range of 9.0–178.0 ng/ml with detection limit of 3.5 ng/ml. In addition, SiNWs/AuNPs-modified ITO, regenerated up to 8 times and its stability was up to 10 weeks at 4 °C in silica gel. - Highlights: • A sensitive biosensor is presented for detection of dengue virus. • SiNWs and AuNPs used as nanocomposite layers on ITO for construction of biosensor • The detection mechanism is based on the interaction of MB with DNA bonded on AuNPs. • The reduction signal of MB decreases upon complementary hybridization

Modified method for combined DNA and RNA isolation from peanut and other oil seeds.

Science.gov (United States)

Dang, Phat M; Chen, Charles Y

2013-02-01

Isolation of good quality RNA and DNA from seeds is difficult due to high levels of polysaccharides, polyphenols, and lipids that can degrade or co-precipitate with nucleic acids. Standard RNA extraction methods utilizing guanidinium-phenol-chloroform extraction has not shown to be successful. RNA isolation from plant seeds is a prerequisite for many seed specific gene expression studies and DNA is necessary in marker-assisted selection and other genetic studies. We describe a modified method to isolate both RNA and DNA from the same seed tissue and have been successful with several oil seeds including peanut, soybean, sunflower, canola, and oil radish. An additional LiCl precipitation step was added to isolate both RNA and DNA from the same seed tissues. High quality nucleic acids were observed based on A(260)/A(280) and A(260)/A(230) ratios above 2.0 and distinct bands on gel-electrophoresis. RNA was shown to be suitable for reverse transcriptase polymerase chain reaction based on actin or 60S ribosomal primer amplification and DNA was shown to have a single band on gel-electrophoresis analysis. This result shows that RNA and DNA isolated using this method can be appropriate for molecular studies in peanut and other oil containing seeds.

Conductivity of Pedot-Pss with Gold and Silver Nanocomposites Modified Gold Electrodes for Ganoderma Boninense DNA Detection

Directory of Open Access Journals (Sweden)

Sabo Wada Dutse

2015-08-01

Full Text Available The conductivity of a designed electrochemical DNA biosensor was improved using gold and or silver nanoparticles. A gold electrode modified with a conductive nanocomposite of poly(3,4-ethylene dioxythiophen–poly (styrenesulfonate (Pedot-Pss and gold or silver nano particles enhanced the conductivity of the electrode surface area. Bare and modified gold electrode surfaces were characterized using cyclic voltammetry (CV technique in ethylenediaminetetraacetic acid (TE supporting electrolyte. Immobilization of a 20-mer DNA probe was achieved by covalent attachment of the amine group of the capture probe to a carboxylic group of an activated 3,3’-dithiodipropionic acid layer using EDC/NHSS for Hybridization. The effect of hybridization temperature and time was optimized and the sensor demonstrated specific detection for the target concentration ranged between 1.0´10-15 M to 1.0´10-9 M with a detection limit of 9.70´10-19 M. Control experiments verified the specificity of the biosensor in the presence of mismatched DNA sequence. The DNA hybridization was monitored using a new ruthenium complex [Ru(dppz2(qtpyCl2; dppz = dipyrido [3,2–a:2’,3’-c] phenazine; qtpy=2,2’,-4,4”.4’4”’-quarterpyridyl redox indicator.

The caretakers of the genome. Repair of DNA lesions induced by ultraviolet-light and ionizing radiation

International Nuclear Information System (INIS)

Boiteux, S.; Radicella, J.P.

2000-01-01

The DNA contained in the nucleus of each of our cells daily suffers of thousand damages caused by solar ultraviolet radiations or ionizing radiations, with a natural or not origin, agents able to modify the genetic information. This information stays stable. True caretakers of the genome repair the DNA, provided that the cell is not over-taken by the level of the attack. Alterations of the repair mechanism are at the origin of extremely severe syndromes. The failure of one of these caretakers of the genome, the O.G.G.1 gene, seems implicated in the cancer development. It can be a lead to discover a predisposition to radioinduced or caused by other toxic agents cancers. (N.C.)

The contribution of thermally labile sugar lesions to DNA double-strand break formation in cells grown in the presence of BrdU.

Science.gov (United States)

Li, Fanghua; Cheng, Yanlei; Iliakis, George

2015-04-01

Radiosensitization by bromodeoxyuridine (BrdU) is commonly attributed to an increase in the yield of double-strand breaks (DSB) in the DNA and an associated decrease in the reparability of these lesions. Radiation chemistry provides a mechanism for the increased yield of DSB through the generation, after bromine loss, of a highly reactive uracilyl radical that attacks the sugar moiety of the nucleotide to produce a single-strand break (SSB). The effects underpinning DSB repair inhibition remain, in contrast, incompletely characterized. A possible source of reduced reparability is a change in the nature or complexity of the DSB in BrdU-substituted DNA. Recent studies show that DSB-complexity or DSB-nature may also be affected by the presence within the cluster of thermally labile sugar lesions (TLSL) that break the DNA backbone only if they chemically evolve to SSB, a process thought to occur within the first hour post-irradiation. Since BrdU radiosensitization might be associated with increased yields and reduced reparability of DSB, we investigated whether BrdU underpins these effects by shifting the balance in the generation of TLSL. We employed asymmetric-field-inversion gel electrophoresis (AFIGE), a pulsed-field gel electrophoresis (PFGE) method to quantitate DSB in a battery of five cells lines grown in the presence of different concentrations of BrdU. We measured specifically the yields of promptly forming DSB (prDSB) using low temperature lysis protocols, and the yields of total DSB (tDSB = prDSB + tlDSB; tlDSB form after evolution to SSB of TLSL) using high temperature lysis protocols. We report that incorporation of BrdU generates similar increases in the formation of tlDSB and prDSB, but variations are noted among the different cell lines tested. The similar increase in the yields of tlDSB and prDSB in BrdU substituted DNA showed that shifts in the yields of these forms of lesions could not be invoked to explain BrdU radiosensitization.

Graphene electrode modified with electrochemically reduced graphene oxide for label-free DNA detection.

Science.gov (United States)

Li, Bing; Pan, Genhua; Avent, Neil D; Lowry, Roy B; Madgett, Tracey E; Waines, Paul L

2015-10-15

A novel printed graphene electrode modified with electrochemically reduced graphene oxide was developed for the detection of a specific oligonucleotide sequence. The graphene oxide was immobilized onto the surface of a graphene electrode via π-π bonds and electrochemical reduction of graphene oxide was achieved by cyclic voltammetry. A much higher redox current was observed from the reduced graphene oxide-graphene double-layer electrode, a 42% and 36.7% increase, respectively, in comparison with that of a bare printed graphene or reduced graphene oxide electrode. The good electron transfer activity is attributed to a combination of the large number of electroactive sites in reduced graphene oxide and the high conductivity nature of graphene. The probe ssDNA was further immobilized onto the surface of the reduced graphene oxide-graphene double-layer electrode via π-π bonds and then hybridized with its target cDNA. The change of peak current due to the hybridized dsDNA could be used for quantitative sensing of DNA concentration. It has been demonstrated that a linear range from 10(-7)M to 10(-12)M is achievable for the detection of human immunodeficiency virus 1 gene with a detection limit of 1.58 × 10(-13)M as determined by three times standard deviation of zero DNA concentration. Copyright © 2015 Elsevier B.V. All rights reserved.

Molecular characterization of poxviruses associated with tattoo skin lesions in UK cetaceans.

Directory of Open Access Journals (Sweden)

Barbara A Blacklaws

Full Text Available There is increasing concern for the well-being of cetacean populations around the UK. Tattoo skin disease (characterised by irregular, grey, black or yellowish, stippled cutaneous lesions caused by poxvirus infection is a potential health indicatora potential health indicator for cetaceans. Limited sequence data indicates that cetacean poxviruses (CPVs belong to an unassigned genus of the Chordopoxvirinae. To obtain further insight into the phylogenetic relationships between CPV and other Chordopoxvirinae members we partially characterized viral DNA originating from tattoo lesions collected in Delphinidae and Phocoenidae stranded along the UK coastline in 1998-2008. We also evaluated the presence of CPV in skin lesions other than tattoos to examine specificity and sensitivity of visual diagnosis. After DNA extraction, regions of the DNA polymerase and DNA topoisomerase I genes were amplified by PCR, sequenced and compared with other isolates. The presence of CPV DNA was demonstrated in tattoos from one striped dolphin (Stenella coeruleoalba, eight harbour porpoises (Phocoena phocoena and one short-beaked common dolphin (Delphinus delphis and in one 'dubious tattoo' lesion detected in one other porpoise. Seventeen of the 18 PCR positive skin lesions had been visually identified as tattoos and one as a dubious tattoo. None of the other skin lesions were PCR positive. Thus, visual identification had a 94.4% sensitivity and 100% specificity. The DNA polymerase PCR was most effective in detecting CPV DNA. Limited sequence phylogeny grouped the UK samples within the odontocete poxviruses (CPV group 1 and indicated that two different poxvirus lineages infect the Phocoenidae and the Delphinidae. The phylogenetic tree had three major branches: one with the UK Phocoenidae viruses, one with the Delphinidae isolates and one for the mysticete poxvirus (CPV group 2. This implies a radiation of poxviruses according to the host suborder and the families within

Degradation and half-life of DNA present in biomass from a genetically-modified organism during land application.

Science.gov (United States)

Halter, Mathew C; Zahn, James A

2017-02-01

White biotechnology has made a positive impact on the chemical industry by providing safer, more efficient chemical manufacturing processes that have reduced the use of toxic chemicals, harsh reaction conditions, and expensive metal catalysts, which has improved alignment with the principles of Green Chemistry. The genetically-modified (GM) biocatalysts that are utilized in these processes are typically separated from high-value products and then recycled, or eliminated. Elimination routes include disposal in sanitary landfills, incineration, use as a fuel, animal feed, or reuse as an agricultural soil amendment or other value-added products. Elimination routes that have the potential to impact the food chain or environment have been more heavily scrutinized for the fate and persistence of biological products. In this study, we developed and optimized a method for monitoring the degradation of strain-specific DNA markers from a genetically-modified organism (GMO) used for the commercial production of 1,3-propanediol. Laboratory and field tests showed that a marker for heterologous DNA in the GM organism was no longer detectable by end-point polymerase chain reaction (PCR) after 14 days. The half-life of heterologous DNA was increased by 17% (from 42.4 to 49.7 h) after sterilization of the soil from a field plot, which indicated that abiotic factors were important in degradation of DNA under field conditions. There was no evidence for horizontal transfer of DNA target sequences from the GMO to viable organisms present in the soil.

Analysis of damaged DNA / proteins interactions: Methodological optimizations and applications to DNA lesions induced by platinum anticancer drugs; Analyse des interactions ADN lese / proteines: Optimisations methodologiques et applications aux dommages de l'ADN engendres par les derives du platine

Energy Technology Data Exchange (ETDEWEB)

Bounaix Morand du Puch, Ch

2010-10-15

DNA lesions contribute to the alteration of DNA structure, thereby inhibiting essential cellular processes. Such alterations may be beneficial for chemotherapies, for example in the case of platinum anticancer agents. They generate bulky adducts that, if not repaired, ultimately cause apoptosis. A better understanding of the biological response to such molecules can be obtained through the study of proteins that directly interact with the damages. These proteins constitute the DNA lesions interactome. This thesis presents the development of tools aiming at increasing the list of platinum adduct-associated proteins. Firstly, we designed a ligand fishing system made of damaged plasmids immobilized onto magnetic beads. Three platinum drugs were selected for our study: cisplatin, oxali-platin and satra-platin. Following exposure of the trap to nuclear extracts from HeLa cancer cells and identification of retained proteins by proteomics, we obtained already known candidates (HMGB1, hUBF, FACT complex) but also 29 new members of the platinated-DNA interactome. Among them, we noted the presence of PNUTS, TOX4 and WDR82, which associate to form the recently-discovered PTW/PP complex. Their capture was then confirmed with a second model, namely breast cancer cell line MDA MB 231, and the biological consequences of such an interaction now need to be elucidated. Secondly, we adapted a SPRi bio-chip to the study of platinum-damaged DNA/proteins interactions. Affinity of HMGB1 and newly characterized TOX4 for adducts generated by our three platinum drugs could be validated thanks to the bio-chip. Finally, we used our tools, as well as analytical chemistry and biochemistry methods, to evaluate the role of DDB2 (a factor involved in the recognition of UV-induced lesions) in the repair of cisplatin adducts. Our experiments using MDA MB 231 cells differentially expressing DDB2 showed that this protein is not responsible for the repair of platinum damages. Instead, it appears to act

DNA damage and autophagy

International Nuclear Information System (INIS)

Rodriguez-Rocha, Humberto; Garcia-Garcia, Aracely; Panayiotidis, Mihalis I.; Franco, Rodrigo

2011-01-01

Both exogenous and endogenous agents are a threat to DNA integrity. Exogenous environmental agents such as ultraviolet (UV) and ionizing radiation, genotoxic chemicals and endogenous byproducts of metabolism including reactive oxygen species can cause alterations in DNA structure (DNA damage). Unrepaired DNA damage has been linked to a variety of human disorders including cancer and neurodegenerative disease. Thus, efficient mechanisms to detect DNA lesions, signal their presence and promote their repair have been evolved in cells. If DNA is effectively repaired, DNA damage response is inactivated and normal cell functioning resumes. In contrast, when DNA lesions cannot be removed, chronic DNA damage triggers specific cell responses such as cell death and senescence. Recently, DNA damage has been shown to induce autophagy, a cellular catabolic process that maintains a balance between synthesis, degradation, and recycling of cellular components. But the exact mechanisms by which DNA damage triggers autophagy are unclear. More importantly, the role of autophagy in the DNA damage response and cellular fate is unknown. In this review we analyze evidence that supports a role for autophagy as an integral part of the DNA damage response.

Evaluation of plasmid and genomic DNA calibrants used for the quantification of genetically modified organisms.

Science.gov (United States)

Caprioara-Buda, M; Meyer, W; Jeynov, B; Corbisier, P; Trapmann, S; Emons, H

2012-07-01

The reliable quantification of genetically modified organisms (GMOs) by real-time PCR requires, besides thoroughly validated quantitative detection methods, sustainable calibration systems. The latter establishes the anchor points for the measured value and the measurement unit, respectively. In this paper, the suitability of two types of DNA calibrants, i.e. plasmid DNA and genomic DNA extracted from plant leaves, for the certification of the GMO content in reference materials as copy number ratio between two targeted DNA sequences was investigated. The PCR efficiencies and coefficients of determination of the calibration curves as well as the measured copy number ratios for three powder certified reference materials (CRMs), namely ERM-BF415e (NK603 maize), ERM-BF425c (356043 soya), and ERM-BF427c (98140 maize), originally certified for their mass fraction of GMO, were compared for both types of calibrants. In all three systems investigated, the PCR efficiencies of plasmid DNA were slightly closer to the PCR efficiencies observed for the genomic DNA extracted from seed powders rather than those of the genomic DNA extracted from leaves. Although the mean DNA copy number ratios for each CRM overlapped within their uncertainties, the DNA copy number ratios were significantly different using the two types of calibrants. Based on these observations, both plasmid and leaf genomic DNA calibrants would be technically suitable as anchor points for the calibration of the real-time PCR methods applied in this study. However, the most suitable approach to establish a sustainable traceability chain is to fix a reference system based on plasmid DNA.

Ionization versus indirect effects of ionizing radiation on cellular DNA

International Nuclear Information System (INIS)

Cadet, Jean; Ravanat, Jean-Luc; Douki, Thierry

2012-01-01

Emphasis has been placed in the last decade on the elucidation of the main degradation pathways of isolated DNA mediated by hydroxyl radical (OH) and one-electron oxidation reactions as the result of indirect and direct effects of ionizing radiation respectively. This has led to the isolation and characterization of about 100 oxidized purine and pyrimidine nucleosides if hydroperoxide precursors and diastereomers are included. However, far less information is available on the mechanisms of radiation-induced degradation of bases in cellular DNA mostly due partly to analytical difficulties. It may be reminded that the measurement of oxidized nucleosides and bases in nuclear DNA is still a challenging issue which until recently has been hampered by the use of inappropriate methods such as the GC-MS that have led to overestimated values of the lesions by factors varying between two and three orders of magnitude. At the present, using the accurate and sensitive HPLC/MS/MS assay, 11 single modified nucleosides and bases were found to be generated in cellular DNA upon exposure to gamma rays and heavy ions. This validates several of the OH-mediated oxidation pathways of thymine, guanine and adenine that were previously inferred from model studies. The concomitant decrease in the yields of oxidized bases with the increase in the LET of heavy ions is accounted for by the preponderance of indirect effects in the damaging action of ionizing radiation on DNA. Further evidence for the major role played by .OH was provided by the results of exposure of cells to high intensity 266 nm laser pulses. Under these conditions 8-oxo-7,8-dihydroguanine is mostly produced by biphotonic ionization of DNA nucleobases and subsequent hole migration to guanine bases. It is likely that some of the oxidized bases that have been isolated as single lesions are in fact involved in clustered damage. Interestingly it was recently shown that a single oxidation hit is capable of generating complex

DNA damage signaling and apoptosis in preinvasive tubal lesions of ovarian carcinoma.

Science.gov (United States)

Chene, Gautier; Ouellet, Veronique; Rahimi, Kurosh; Barres, Veronique; Caceres, Katia; Meunier, Liliane; Cyr, Louis; De Ladurantaye, Manon; Provencher, Diane; Mes Masson, Anne Marie

2015-06-01

High-grade serous ovarian cancer (HGSC) is the most life-threatening gynecological malignancy despite surgery and chemotherapy. A better understanding of the molecular basis of the preinvasive stages might be helpful in early detection and diagnosis. Genetic instability is 1 of the characteristics shared by most human cancers, and its level is variable through precancerous lesions to advanced cancer. Because DNA damage response (DDR) has been described as 1 of the first phases in genomic instability, we investigated the level of DDR activation and the apoptosis pathway in serous tubal intraepithelial carcinoma (STIC), the potential precursor of HGSC. A tissue microarray including 21 benign fallopian tubes, 21 STICs, 17 HGSCs from patients with STICs (associated ovarian cancer [AOC]) from the same individuals, and 30 HGSCs without STICs (non-AOC) was used in this study.Immunohistochemistry was performed to evaluate the level of DDR proteins (pATM, pChk2, γH2AX, 53BP1, and TRF2), apoptosis proteins (Bcl2, BAX, and BIM), and cyclin E. The expression of all DDR proteins increased from benign fallopian tubes to STICs. The level of expression of pATM, pChk2, γH2AX, and TRF2 was also increased in STICs in comparison with AOC. BAX, BIM, and cyclin E expressions were high in STICs, whereas Bcl2 expression was low. Immunohistochemical profiles of AOC and non-AOC were also different. These results suggest an activation of the DDR and apoptosis pathways in STICs, indicating that genomic instability may occur early in the precancerous lesions of HGSC.

Chemical repair activity of free radical scavenger edaravone. Reduction reactions with dGMP hydroxyl radical adducts and suppression of base lesions and AP sites on irradiated plasmid DNA

International Nuclear Information System (INIS)

Hata, Kuniki; Katsumura, Yosuke; Urushibara, Ayumi; Yamashita, Shinichi; Lin Mingzhang; Muroya, Yusa; Shikazono, Naoya; Yokoya, Akinari; Fu Haiying

2015-01-01

Reactions of edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) with deoxyguanosine monophosphate (dGMP) hydroxyl radical adducts were investigated by pulse radiolysis technique. Edaravone was found to reduce the dGMP hydroxyl radical adducts through electron transfer reactions. The rate constants of the reactions were greater than 4 × 10 8 dm 3 mol -1 s -1 and similar to those of the reactions of ascorbic acid, which is a representative antioxidant. Yields of single-strand breaks, base lesions, and abasic sites produced in pUC18 plasmid DNA by gamma ray irradiation in the presence of low concentrations (10–1000 μmol dm -3 ) of edaravone were also quantified, and the chemical repair activity of edaravone was estimated by a method recently developed by the authors. By comparing suppression efficiencies to the induction of each DNA lesion, it was found that base lesions and abasic sites were suppressed by the chemical repair activity of edaravone, although the suppression of single-strand breaks was not very effective. This phenomenon was attributed to the chemical repair activity of edaravone toward base lesions and abasic sites. However, the chemical repair activity of edaravone for base lesions was lower than that of ascorbic acid. (author)

Nuclear DNA-Content in Mesenchymal Lesions in Dogs: Its Value as Marker of Malignancy and Extent of Genomic Instability

International Nuclear Information System (INIS)

Boerkamp, Kim M.; Rutteman, Gerard R.; Kik, Marja J. L.; Kirpensteijn, Jolle; Schulze, Christoph; Grinwis, Guy C. M.

2012-01-01

DNA-aneuploidy may reflect the malignant nature of mesenchymal proliferations and herald gross genomic instability as a mechanistic factor in tumor genesis. DNA-ploidy and -index were determined by flow cytometry in canine inflammatory or neoplastic mesenchymal tissues and related to clinico-pathological features, biological behavior and p53 gene mutational status. Half of all sarcomas were aneuploid. Benign mesenchymal neoplasms were rarely aneuploid and inflammatory lesions not at all. The aneuploidy rate was comparable to that reported for human sarcomas with significant variation amongst subtypes. DNA-ploidy status in canines lacked a relation with histological grade of malignancy, in contrast to human sarcomas. While aneuploidy was related to the development of metastases in soft tissue sarcomas it was not in osteosarcomas. No relation amongst sarcomas was found between ploidy status and presence of P53 gene mutations. Heterogeneity of the DNA index between primary and metastatic sarcoma sites was present in half of the cases examined. Hypoploidy is more common in canine sarcomas and hyperploid cases have less deviation of the DNA index than human sarcomas. The variation in the presence and extent of aneuploidy amongst sarcoma subtypes indicates variation in genomic instability. This study strengthens the concept of interspecies variation in the evolution of gross chromosomal aberrations during cancer development

Nuclear DNA-Content in Mesenchymal Lesions in Dogs: Its Value as Marker of Malignancy and Extent of Genomic Instability

Science.gov (United States)

Boerkamp, Kim M.; Rutteman, Gerard R.; Kik, Marja J. L.; Kirpensteijn, Jolle; Schulze, Christoph; Grinwis, Guy C. M.

2012-01-01

DNA-aneuploidy may reflect the malignant nature of mesenchymal proliferations and herald gross genomic instability as a mechanistic factor in tumor genesis. DNA-ploidy and -index were determined by flow cytometry in canine inflammatory or neoplastic mesenchymal tissues and related to clinico-pathological features, biological behavior and p53 gene mutational status. Half of all sarcomas were aneuploid. Benign mesenchymal neoplasms were rarely aneuploid and inflammatory lesions not at all. The aneuploidy rate was comparable to that reported for human sarcomas with significant variation amongst subtypes. DNA-ploidy status in canines lacked a relation with histological grade of malignancy, in contrast to human sarcomas. While aneuploidy was related to the development of metastases in soft tissue sarcomas it was not in osteosarcomas. No relation amongst sarcomas was found between ploidy status and presence of P53 gene mutations. Heterogeneity of the DNA index between primary and metastatic sarcoma sites was present in half of the cases examined. Hypoploidy is more common in canine sarcomas and hyperploid cases have less deviation of the DNA index than human sarcomas. The variation in the presence and extent of aneuploidy amongst sarcoma subtypes indicates variation in genomic instability. This study strengthens the concept of interspecies variation in the evolution of gross chromosomal aberrations during cancer development. PMID:24213507

Nuclear DNA-Content in Mesenchymal Lesions in Dogs: Its Value as Marker of Malignancy and Extent of Genomic Instability

Energy Technology Data Exchange (ETDEWEB)

Boerkamp, Kim M., E-mail: K.M.Boerkamp@uu.nl; Rutteman, Gerard R. [Department of Clinical Science of Companion Animals, Faculty of Veterinary Medicine, UU, Yalelaan 104, 3584 CM, Utrecht (Netherlands); Kik, Marja J. L. [Department of Pathobiology, Faculty of Veterinary Medicine, UU, Yalelaan 1, 3508 TD, Utrecht (Netherlands); Kirpensteijn, Jolle [Department of Clinical Science of Companion Animals, Faculty of Veterinary Medicine, UU, Yalelaan 104, 3584 CM, Utrecht (Netherlands); Schulze, Christoph; Grinwis, Guy C. M. [Department of Pathobiology, Faculty of Veterinary Medicine, UU, Yalelaan 1, 3508 TD, Utrecht (Netherlands)

2012-12-03

DNA-aneuploidy may reflect the malignant nature of mesenchymal proliferations and herald gross genomic instability as a mechanistic factor in tumor genesis. DNA-ploidy and -index were determined by flow cytometry in canine inflammatory or neoplastic mesenchymal tissues and related to clinico-pathological features, biological behavior and p53 gene mutational status. Half of all sarcomas were aneuploid. Benign mesenchymal neoplasms were rarely aneuploid and inflammatory lesions not at all. The aneuploidy rate was comparable to that reported for human sarcomas with significant variation amongst subtypes. DNA-ploidy status in canines lacked a relation with histological grade of malignancy, in contrast to human sarcomas. While aneuploidy was related to the development of metastases in soft tissue sarcomas it was not in osteosarcomas. No relation amongst sarcomas was found between ploidy status and presence of P53 gene mutations. Heterogeneity of the DNA index between primary and metastatic sarcoma sites was present in half of the cases examined. Hypoploidy is more common in canine sarcomas and hyperploid cases have less deviation of the DNA index than human sarcomas. The variation in the presence and extent of aneuploidy amongst sarcoma subtypes indicates variation in genomic instability. This study strengthens the concept of interspecies variation in the evolution of gross chromosomal aberrations during cancer development.

Nuclear DNA-Content in Mesenchymal Lesions in Dogs: Its Value as Marker of Malignancy and Extent of Genomic Instability

Directory of Open Access Journals (Sweden)

Christoph Schulze

2012-12-01

Full Text Available DNA-aneuploidy may reflect the malignant nature of mesenchymal proliferations and herald gross genomic instability as a mechanistic factor in tumor genesis. DNA-ploidy and -index were determined by flow cytometry in canine inflammatory or neoplastic mesenchymal tissues and related to clinico-pathological features, biological behavior and p53 gene mutational status. Half of all sarcomas were aneuploid. Benign mesenchymal neoplasms were rarely aneuploid and inflammatory lesions not at all. The aneuploidy rate was comparable to that reported for human sarcomas with significant variation amongst subtypes. DNA-ploidy status in canines lacked a relation with histological grade of malignancy, in contrast to human sarcomas. While aneuploidy was related to the development of metastases in soft tissue sarcomas it was not in osteosarcomas. No relation amongst sarcomas was found between ploidy status and presence of P53 gene mutations. Heterogeneity of the DNA index between primary and metastatic sarcoma sites was present in half of the cases examined. Hypoploidy is more common in canine sarcomas and hyperploid cases have less deviation of the DNA index than human sarcomas. The variation in the presence and extent of aneuploidy amongst sarcoma subtypes indicates variation in genomic instability. This study strengthens the concept of interspecies variation in the evolution of gross chromosomal aberrations during cancer development.

An active site aromatic triad in Escherichia coli DNA Pol IV coordinates cell survival and mutagenesis in different DNA damaging agents.

Directory of Open Access Journals (Sweden)

Ryan W Benson

Full Text Available DinB (DNA Pol IV is a translesion (TLS DNA polymerase, which inserts a nucleotide opposite an otherwise replication-stalling N(2-dG lesion in vitro, and confers resistance to nitrofurazone (NFZ, a compound that forms these lesions in vivo. DinB is also known to be part of the cellular response to alkylation DNA damage. Yet it is not known if DinB active site residues, in addition to aminoacids involved in DNA synthesis, are critical in alkylation lesion bypass. It is also unclear which active site aminoacids, if any, might modulate DinB's bypass fidelity of distinct lesions. Here we report that along with the classical catalytic residues, an active site "aromatic triad", namely residues F12, F13, and Y79, is critical for cell survival in the presence of the alkylating agent methyl methanesulfonate (MMS. Strains expressing dinB alleles with single point mutations in the aromatic triad survive poorly in MMS. Remarkably, these strains show fewer MMS- than NFZ-induced mutants, suggesting that the aromatic triad, in addition to its role in TLS, modulates DinB's accuracy in bypassing distinct lesions. The high bypass fidelity of prevalent alkylation lesions is evident even when the DinB active site performs error-prone NFZ-induced lesion bypass. The analyses carried out with the active site aromatic triad suggest that the DinB active site residues are poised to proficiently bypass distinctive DNA lesions, yet they are also malleable so that the accuracy of the bypass is lesion-dependent.

(UVB)-induced DNA damage

African Journals Online (AJOL)

Jane

2011-08-17

dependent cytogenetic lesions were assessed by the micronucleus test (MNT). It was found that POE effectively reduced the extent of DNA breakages and cytogenetic lesions upon exposure to UVB (erythemal ultraviolet (EUV);.

DNA damage and repair in plants

International Nuclear Information System (INIS)

Britt, A.B.

1996-01-01

The biological impact of any DNA damaging agent is a combined function of the chemical nature of the induced lesions and the efficiency and accuracy of their repair. Although much has been learned frommicrobes and mammals about both the repair of DNA damage and the biological effects of the persistence of these lesions, much remains to be learned about the mechanism and tissue-specificity of repair in plants. This review focuses on recent work on the induction and repair of DNA damage in higher plants, with special emphasis on UV-induced DNA damage products. (author)

Identification of genetically modified DNA found in Roundup Ready soybean using gold nanoparticles

International Nuclear Information System (INIS)

Jang, Huisoo; Kwak, Cheol Hwan; Kim, Gibum; Kim, Sun Min; Huh, Yun Suk; Jeon, Tae-Joon

2016-01-01

The authors describe an SPR sensor chip coated with gold nanoparticles (AuNPs) that enables highly sensitive determination of genetically modified (GM) crops. Detection is based on localized surface plasmon resonance (LSPR) with its known sensitivity to even minute changes in refractive index. The device consists of a halogen light source, a light detector, and a cuvette cell that contains a sensor chip coated with AuNPs. It is operated in the transmission mode of the optical path to enhance the plasmonic signal. The sample solution containing target DNA (e.g. from the GM crop) is introduced into the cuvette with the sensor chip whose surface was functionalized with a capture DNA. Following a 30-min hybridization, the changes of the signal are recorded at 540 nm. The chip responds to target DNA in the 1 to 100 nM concentration range and has a 1 nM detection limit. Features of this sensor chip include a short reaction time, ease of handling, and portability, and this enables on-site detection and in-situ testing. (author)

The essential DNA polymerases δ and ε are involved in repair of UV-damaged DNA in the yeast Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Halas, A.; Policinska, Z.; Baranowska, H.; Jachymczyk, W.J.

1999-01-01

We have studied the ability of yeast DNA polymerases to carry out repair of lesions caused by UV irradiation in Saccharomyces cerevisiae. By the analysis of postirradiation relative molecular mass changes in cellular DNA of different DNA polymerases mutant strains, it was established that mutations in DNA polymerases δ and ε showed accumulation of single-strand breaks indicating defective repair. Mutations in other DNA polymerase genes exhibited no defects in DNA repair. Thus, the data obtained suggest that DNA polymerases δ and ε are both necessary for DNA replication and for repair of lesions caused by UV irradiation. The results are discussed in the light of current concepts concerning the specificity of DNA polymerases in DNA repair. (author)

Glycosylase-mediated repair of radiation-induced DNA bases: substrate specificities and mechanisms

International Nuclear Information System (INIS)

D'ham, Cedric

1998-01-01

Cellular DNA is subject to permanent damage and repair processes. One way to restore the integrity of DNA involves the base excision repair pathway. Glycosylases are the key-enzymes of this process. The present work deals with the determination of the substrate specificity and the mechanism of action of three glycosylases: endonuclease III and Fpg of Escherichia coli and Ogg1 of Saccharomyces cerevisiae. The present manuscript is divided into four parts: Endonuclease III-mediated excision of 5,6-dihydro-thymine and 5-hydroxy-5,6-dihydro-thymine from γ-irradiated DNA was analyzed by a gas chromatography-mass spectrometry assay, including a liquid chromatography pre-purification step. This was found to be necessary in order to separate the cis and trans isomers of 6-hydroxy-5,6-dihydro-thymine from the 5-hydroxy-5,6-dihydro-thymine. Modified oligonucleotides that contained a unique lesion, including thymine glycol, 5,6-dihydro-thymine and 5-hydroxy-cytosine were synthesized to assess the substrate specificity of endonuclease III and Fpg. The order of preference of the enzymes for the substrates was determined by the measurement of the Michaelis constants of the kinetics. Furthermore, the mechanism of action of endonuclease III has been reconsidered, after analysis using the MALDI mass spectrometry technique. These studies reveal that hydrolysis is the main pathway by which endonuclease III cleaves the DNA backbone. Using a modified oligonucleotide, 8-oxo-7,8-dihydro-adenine was shown to be a product of excision of the Ogg1 enzyme. The role of the complementary base towards the lesion was found to be preponderant in the damage excision. A last chapter concerns the synthesis and the characterization of the four isomers of 5(6)-hydroxy-6(5)-hydroperoxides of thymine. These products may be substrates for endonuclease III or Fpg. (author) [fr

DNA repair and cancer

International Nuclear Information System (INIS)

Rathore, Shakuntla; Joshi, Pankaj Kumar; Gaur, Sudha

2012-01-01

DNA repair refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecule that encode it's genome. In human cells, both normal metabolic activities and environmental factors such as UV light and radiation can cause DNA damage, resulting in as many one million individual molecular lesions per day. Many of these lesions cause structural damage to the DNA molecule and can alter or eliminate the cell's ability to transcribe the gene that the affected DNA encodes. Other lesions include potentially harmful mutation in cell's genome which affect the survival of it's daughter cells after it undergoes mitosis. As a consequence, the DNA repair process is constantly active as it responds to damage in the DNA structure. Inherited mutation that affect DNA repair genes are strongly associated with high cancer risks in humans. Hereditary non polyposis colorectal cancer (HNPCC) is strongly associated with specific mutation in the DNA mismatch repair pathway. BRCA1, BRCA2 two famous mutation conferring a hugely increased risk of breast cancer on carrier, are both associated with a large number of DNA repair pathway, especially NHEJ and homologous recombination. Cancer therapy procedures such as chemotherapy and radiotherapy work by overwhelming the capacity of the cell to repair DNA damage, resulting in cell death. Cells that are most rapidly dividing most typically cancer cells are preferentially affected. The side effect is that other non-cancerous but rapidly dividing cells such as stem cells in the bone marrow are also affected. Modern cancer treatment attempt to localize the DNA damage to cells and tissue only associated with cancer, either by physical means (concentrating the therapeutic agent in the region of the tumor) or by biochemical means (exploiting a feature unique to cancer cells in the body). (author)

Development of Ionic Liquid Modified Disposable Graphite Electrodes for Label-Free Electrochemical Detection of DNA Hybridization Related to Microcystis spp.

Directory of Open Access Journals (Sweden)

Ceren Sengiz

2015-09-01

Full Text Available In this present study, ionic liquid (1-butyl-3-methylimidazolium hexafluorophosphate (IL modified pencil graphite electrode (IL-PGEs was developed for electrochemical monitoring of DNA hybridization related to Microcystis spp. (MYC. The characterization of IL-PGEs was performed using microscopic and electrochemical techniques. DNA hybridization related to MYC was then explored at the surface of IL-PGEs using differential pulse voltammetry (DPV technique. After the experimental parameters were optimized, the sequence-selective DNA hybridization related to MYC was performed in the case of hybridization between MYC probe and its complementary DNA target, noncomplementary (NC or mismatched DNA sequence (MM, or and in the presence of mixture of DNA target: NC (1:1 and DNA target: MM (1:1.

Electrochemical label-free and sensitive nanobiosensing of DNA hybridization by graphene oxide modified pencil graphite electrode.

Science.gov (United States)

Ahour, F; Shamsi, A

2017-09-01

Based on the strong interaction between single-stranded DNA (ss-DNA) and graphene material, we have constructed a novel label-free electrochemical biosensor for rapid and facile detection of short sequences ss-DNA molecules related to hepatitis C virus 1a using graphene oxide modified pencil graphite electrode. The sensing mechanism is based on the superior adsorption of single-stranded DNA to GO over double stranded DNA (ds-DNA). The intrinsic guanine oxidation signal measured by differential pulse voltammetry (DPV) has been used for duplex DNA formation detection. The probe ss-DNA adsorbs onto the surface of GO via the π- π* stacking interactions leading to a strong background guanine oxidation signal. In the presence of complementary target, formation of helix which has weak binding ability to GO induced ds-DNA to release from the electrode surface and significant variation in differential pulse voltammetric response of guanine bases. The results indicated that the oxidation peak current was proportional to the concentration of complementary strand in the range of 0.1 nM-0.5 μM with a detection limit of 4.3 × 10 -11  M. The simple fabricated electrochemical biosensor has high sensitivity, good selectivity, and could be applied as a new platform for a range of target molecules in future. Copyright © 2017 Elsevier Inc. All rights reserved.

DNA repair in bacterial cultures and plasmid DNA exposed to infrared laser for treatment of pain

International Nuclear Information System (INIS)

Canuto, K S; Sergio, L P S; Marciano, R S; Guimarães, O R; Polignano, G A C; Geller, M; Fonseca, A S; Paoli, F

2013-01-01

Biostimulation of tissues by low intensity lasers has been described on a photobiological basis and clinical protocols are recommended for treatment of various diseases, but their effects on DNA are controversial. The objective of this work was to evaluate effects of low intensity infrared laser exposure on survival and bacterial filamentation in Escherichia coli cultures, and induction of DNA lesions in bacterial plasmids. In E. coli cultures and plasmids exposed to an infrared laser at fluences used to treat pain, bacterial survival and filamentation and DNA lesions in plasmids were evaluated by electrophoretic profile. Data indicate that the infrared laser (i) increases survival of E. coli wild type in 24 h of stationary growth phase, (ii) induces bacterial filamentation, (iii) does not alter topological forms of plasmids and (iv) does not alter the electrophoretic profile of plasmids incubated with exonuclease III or formamidopyrimidine DNA glycosylase. A low intensity infrared laser at the therapeutic fluences used to treat pain can alter survival of E. coli wild type, induce filamentation in bacterial cells, depending on physiologic conditions and DNA repair, and induce DNA lesions other than single or double DNA strand breaks or alkali-labile sites, which are not targeted by exonuclease III or formamidopyrimidine DNA glycosylase. (letter)

DNA repair is indispensable for survival after acute inflammation

Science.gov (United States)

Calvo, Jennifer A.; Meira, Lisiane B.; Lee, Chun-Yue I.; Moroski-Erkul, Catherine A.; Abolhassani, Nona; Taghizadeh, Koli; Eichinger, Lindsey W.; Muthupalani, Sureshkumar; Nordstrand, Line M.; Klungland, Arne; Samson, Leona D.

2012-01-01

More than 15% of cancer deaths worldwide are associated with underlying infections or inflammatory conditions, therefore understanding how inflammation contributes to cancer etiology is important for both cancer prevention and treatment. Inflamed tissues are known to harbor elevated etheno-base (ε-base) DNA lesions induced by the lipid peroxidation that is stimulated by reactive oxygen and nitrogen species (RONS) released from activated neutrophils and macrophages. Inflammation contributes to carcinogenesis in part via RONS-induced cytotoxic and mutagenic DNA lesions, including ε-base lesions. The mouse alkyl adenine DNA glycosylase (AAG, also known as MPG) recognizes such base lesions, thus protecting against inflammation-associated colon cancer. Two other DNA repair enzymes are known to repair ε-base lesions, namely ALKBH2 and ALKBH3; thus, we sought to determine whether these DNA dioxygenase enzymes could protect against chronic inflammation-mediated colon carcinogenesis. Using established chemically induced colitis and colon cancer models in mice, we show here that ALKBH2 and ALKBH3 provide cancer protection similar to that of the DNA glycosylase AAG. Moreover, Alkbh2 and Alkbh3 each display apparent epistasis with Aag. Surprisingly, deficiency in all 3 DNA repair enzymes confers a massively synergistic phenotype, such that animals lacking all 3 DNA repair enzymes cannot survive even a single bout of chemically induced colitis. PMID:22684101

Immunological detection of O6-methylguanine in alkylated DNA

International Nuclear Information System (INIS)

Briscoe, W.T.; Spizizen, J.; Tan, E.M.

1978-01-01

Antibodies to O 6 -methyldeoxyguanosine were produced in rabbits and utilized in a radioimmunoassay to detect this nucleoside at picomole levels. The specificity of the antibodies was demonstrated by the use of nucleoside analogues as inhibitors in the radioimmunoassay. The antibodies cross-reacted with O 6 -methylguanosine, O 6 -methylguanine, and O 6 -ethylguanosine. There was 10 4 to 10 6 times less sensitivity to inhibition by deoxyadenosine, deoxyguanosine, and guanosine than by O 6 -methyldeoxyguanosine. The radioimmunoassay also detected O 6 -methylguanine in DNA alkylated by agents known to produce O 6 -methylguanine, such as N'-methyl-N-nitrosourea. DNA alkylated with dimethyl sulfate, which does not produce O 6 -methylguanine in DNA, cross-reacted with the antibodies to a very limited extent. Such an assay system for modified nucleic acid components would be very useful in following the production, persistence, and repair of these lesions in a variety of cells and tissues treated with a broad spectrum of carcinogens and suspected carcinogens

Electrochemical sensor for multiplex screening of genetically modified DNA: identification of biotech crops by logic-based biomolecular analysis.

Science.gov (United States)

Liao, Wei-Ching; Chuang, Min-Chieh; Ho, Ja-An Annie

2013-12-15

Genetically modified (GM) technique, one of the modern biomolecular engineering technologies, has been deemed as profitable strategy to fight against global starvation. Yet rapid and reliable analytical method is deficient to evaluate the quality and potential risk of such resulting GM products. We herein present a biomolecular analytical system constructed with distinct biochemical activities to expedite the computational detection of genetically modified organisms (GMOs). The computational mechanism provides an alternative to the complex procedures commonly involved in the screening of GMOs. Given that the bioanalytical system is capable of processing promoter, coding and species genes, affirmative interpretations succeed to identify specified GM event in terms of both electrochemical and optical fashions. The biomolecular computational assay exhibits detection capability of genetically modified DNA below sub-nanomolar level and is found interference-free by abundant coexistence of non-GM DNA. This bioanalytical system, furthermore, sophisticates in array fashion operating multiplex screening against variable GM events. Such a biomolecular computational assay and biosensor holds great promise for rapid, cost-effective, and high-fidelity screening of GMO. Copyright © 2013 Elsevier B.V. All rights reserved.

Detection of the Epstein-Barr Virus and DNA-Topoisomerase II-α in Recurrent and Nonrecurrent Giant Cell Lesion of the Jawbones

Directory of Open Access Journals (Sweden)

Manal M. Zyada

2013-01-01

Full Text Available The aims of this study were to determine whether the expression of Topo II- correlates with presence of EBV in giant cell lesion of the jawbones and whether it is predictive of clinical biologic behavior of these lesions. Paraffin-embedded tissues from 8 recurrent and 7 nonrecurrent cases of bony GCLs and 9 peripheral giant cell lesions (PGCLs as a control group were assessed for the expression of EBV and Topo II- using immunohistochemistry. The results showed positive staining for Topo II- in mononuclear stromal cells (MSCs and multinucleated giant cells (MGCs. Student t-test showed that mean Topo II- labelling index (LI in recurrent cases was significantly higher than that in non-recurrent cases (. Moreover, Spearman's correlation coefficients method showed a significant correlation between DNA Topo II- LI and both of gender and site in these lesions. Moderate EBV expression in relation to the highest Topo II- LI was observed in two cases of GCT. It was concluded that high Topo II- LIs could be identified as reliable predicators for the clinical behavior of GCLs. Moreover, EBV has no etiological role in the benign CGCLs in contrast to its role in the pathogenesis of GCTs.

Oxidative Glial Cell Damage Associated with White Matter Lesions in the Aging Human Brain.

Science.gov (United States)

Al-Mashhadi, Sufana; Simpson, Julie E; Heath, Paul R; Dickman, Mark; Forster, Gillian; Matthews, Fiona E; Brayne, Carol; Ince, Paul G; Wharton, Stephen B

2015-09-01

White matter lesions (WML) are common in brain aging and are associated with dementia. We aimed to investigate whether oxidative DNA damage and occur in WML and in apparently normal white matter in cases with lesions. Tissue from WML and control white matter from brains with lesions (controls lesional) and without lesions (controls non-lesional) were obtained, using post-mortem magnetic resonance imaging-guided sampling, from the Medical Research Council Cognitive Function and Ageing Study. Oxidative damage was assessed by immunohistochemistry to 8-hydroxy-2'-deoxoguanosine (8-OHdG) and Western blotting for malondialdehyde. DNA response was assessed by phosphorylated histone H2AX (γH2AX), p53, senescence markers and by quantitative Reverse transcription polymerase chain reaction (RT-PCR) panel for candidate DNA damage-associated genes. 8-OHdG was expressed in glia and endothelium, with increased expression in both WML and controls lesional compared with controls non-lesional (P glial dysfunction. Their expression in apparently normal white matter in cases with WML suggests that white matter dysfunction is not restricted to lesions. The role of this field-effect lesion pathogenesis and cognitive impairment are areas to be defined. © 2014 The Authors. Brain Pathology published by John Wiley & Sons Ltd on behalf of International Society of Neuropathology.

Chemical repair activity of free radical scavenger edaravone: reduction reactions with dGMP hydroxyl radical adducts and suppression of base lesions and AP sites on irradiated plasmid DNA.

Science.gov (United States)

Hata, Kuniki; Urushibara, Ayumi; Yamashita, Shinichi; Lin, Mingzhang; Muroya, Yusa; Shikazono, Naoya; Yokoya, Akinari; Fu, Haiying; Katsumura, Yosuke

2015-01-01

Reactions of edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) with deoxyguanosine monophosphate (dGMP) hydroxyl radical adducts were investigated by pulse radiolysis technique. Edaravone was found to reduce the dGMP hydroxyl radical adducts through electron transfer reactions. The rate constants of the reactions were greater than 4 × 10(8) dm(3) mol(-1) s(-1) and similar to those of the reactions of ascorbic acid, which is a representative antioxidant. Yields of single-strand breaks, base lesions, and abasic sites produced in pUC18 plasmid DNA by gamma ray irradiation in the presence of low concentrations (10-1000 μmol dm(-3)) of edaravone were also quantified, and the chemical repair activity of edaravone was estimated by a method recently developed by the authors. By comparing suppression efficiencies to the induction of each DNA lesion, it was found that base lesions and abasic sites were suppressed by the chemical repair activity of edaravone, although the suppression of single-strand breaks was not very effective. This phenomenon was attributed to the chemical repair activity of edaravone toward base lesions and abasic sites. However, the chemical repair activity of edaravone for base lesions was lower than that of ascorbic acid. © The Author 2014. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

DNA-Conjugated Organic Chromophores in DNA Stacking Interactions

DEFF Research Database (Denmark)

Filichev, Vyacheslav V.; Pedersen, Erik Bjerregaard

2009-01-01

Since the discovery of the intercalation of acridine derivatives into DNA (1961), chemists have synthesized many intercalators tethered to DNA. Advances in the chemical synthesis of modified nucleosides along with progress in oligonucleotide synthesis have made it possible to introduce organic ch...... review presents those efforts in the design of intercalators/organic chromophores as oligonucleotide conjugates that form a foundation for the generation of novel nucleic acid architectures......Since the discovery of the intercalation of acridine derivatives into DNA (1961), chemists have synthesized many intercalators tethered to DNA. Advances in the chemical synthesis of modified nucleosides along with progress in oligonucleotide synthesis have made it possible to introduce organic...

Detection of DNA damage in cells exposed to ionizing radiation by use of antisingle-stranded-DNA monoclonal antibody

International Nuclear Information System (INIS)

Schans, G.P. van der; Loon, A.A.W.M. van; Groenendijk, R.H.; Baan, R.A.

1989-03-01

An immunochemical method has been developed for quantitative detection of DNA damage in mammalian cells. The method is based on the binding of a monoclonal antibody to single-stranded DNA. The clone producing this antibody, D1B, was obtained as a by-product from fusion of mouse myeloma cells with spleen cells isolated from a mouse immunized with chemically modified DNA. The technique is based upon the determination of the percentage single-strandedness resulting from the partial umwinding of cellular DNA under alkaline conditions, a time-dependent process. Single-strand and double-strand DNA breaks, or lesions converted into such breaks in alkaline medium, form initiation points for the unwinding. The extent of unwinding under controlled conditions is a measure, therefore, of the amount of such sites. The method is rapid, does not require radioactive labelling of DNA or physical separation of single- from double-stranded molecules, is sufficiently sensitive to detect damage induced by 1 Gu of ionizing radiation and needs only small amounts of cells. The usefulness of the technique was demonstrated in a study on the induction of damage and its repair in unlabelled cultured Chinese hamster cells and in DNA-containing cells of human blood, both after exposure to 60 Co-γ-rays, and in white blood cells and bone marrow cells of X-irradiated mice. A dose-related degree of unwinding was observed and repair could be observed up to 60 min after irradiation. (author). 19 refs.; 3 figs.; 1 tab

Mechanistic Investigation of the Bypass of a Bulky Aromatic DNA Adduct Catalyzed by a Y-family DNA Polymerase

Science.gov (United States)

Gadkari, Varun V.; Tokarsky, E. John; Malik, Chanchal K.; Basu, Ashis K.; Suo, Zucai

2014-01-01

3-Nitrobenzanthrone (3-NBA), a nitropolyaromatic hydrocarbon (NitroPAH) pollutant in diesel exhaust, is a potent mutagen and carcinogen. After metabolic activation, the primary metabolites of 3-NBA react with DNA to form dG and dA adducts. One of the three major adducts identified is N-(2’-deoxyguanosin-8-yl)-3-aminobenzanthrone (dGC8-N-ABA). This bulky adduct likely stalls replicative DNA polymerases but can be traversed by lesion bypass polymerases in vivo. Here, we employed running start assays to show that a site-specifically placed dGC8-N-ABA is bypassed in vitro by Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase. However, the nucleotide incorporation rate of Dpo4 was significantly reduced opposite both the lesion and the template position immediately downstream from the lesion site, leading to two strong pause sites. To investigate the kinetic effect of dGC8-N-ABA on polymerization, we utilized pre-steady-state kinetic methods to determine the kinetic parameters for individual nucleotide incorporations upstream, opposite, and downstream from the dGC8-N-ABA lesion. Relative to the replication of the corresponding undamaged DNA template, both nucleotide incorporation efficiency and fidelity of Dpo4 were considerably decreased during dGC8-N-ABA lesion bypass and the subsequent extension step. The lower nucleotide incorporation efficiency caused by the lesion is a result of a significantly reduced dNTP incorporation rate constant and modestly weaker dNTP binding affinity. At both pause sites, nucleotide incorporation followed biphasic kinetics with a fast and a slow phase and their rates varied with nucleotide concentration. In contrast, only the fast phase was observed with undamaged DNA. A kinetic mechanism was proposed for the bypass of dGC8-N-ABA bypass catalyzed by Dpo4. PMID:25048879

Fabrication of endothelial progenitor cell capture surface via DNA aptamer modifying dopamine/polyethyleneimine copolymer film

Energy Technology Data Exchange (ETDEWEB)

Li, Xin; Deng, Jinchuan; Yuan, Shuheng; Wang, Juan; Luo, Rifang; Chen, Si [Key Lab. of Advanced Technology for Materials of Education Ministry, Southwest Jiaotong University, Chengdu 610031 (China); School of Material Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Wang, Jin, E-mail: jinxxwang@263.net [Key Lab. of Advanced Technology for Materials of Education Ministry, Southwest Jiaotong University, Chengdu 610031 (China); Huang, Nan [Key Lab. of Advanced Technology for Materials of Education Ministry, Southwest Jiaotong University, Chengdu 610031 (China); School of Material Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China)

2016-11-15

Highlights: • The dopamine/PEI film with controlled amine density was successfully prepared. • The DNA aptamer was assembled onto the film via electrostatic incorporation. • The A@DPfilmscanspecificallyandeffectivelycaptureEPCs. • The A@DP film can support the survival of ECs, control the hyperplasia of SMCs. • The dynamic/co-culture models are useful for studying cells competitive adhesion. - Abstract: Endothelial progenitor cells (EPCs) are mainly located in bone marrow and circulate, and play a crucial role in repairmen of injury endothelium. One of the most promising strategies of stents designs were considered to make in-situ endothelialization in vivo via EPC-capture biomolecules on a vascular graft to capture EPCs directly from circulatory blood. In this work, an EPC specific aptamer with a 34 bases single strand DNA sequence was conjugated onto the stent surface via dopamine/polyethyleneimine copolymer film as a platform and linker. The assembled density of DNA aptamer could be regulated by controlling dopamine percentage in this copolymer film. X-ray photoelectron spectroscopy (XPS), water contact angle (WCA) and fluorescence test confirmed the successful immobilization of DNA aptamer. To confirm its biofunctionality and cytocompatibility, the capturing cells ability of the aptamer modified surface and the effects on the growth behavior of human umbilical vein endothelial cells (HUVECs), smooth muscle cells (SMCs) were investigated. The aptamer functionalized sample revealed a good EPC-capture ability, and had a cellular friendly feature for both EPC and EC growth, while not stimulated the hyperplasia of SMCs. And, the co-culture experiment of three types of cells confirmed the specificity capturing of EPCs to aptamer modified surface, rather than ECs and SMCs. These data suggested that this aptamer functionalized surface may have a large potentiality for the application of vascular grafts with targeted endothelialization.

Fabrication of endothelial progenitor cell capture surface via DNA aptamer modifying dopamine/polyethyleneimine copolymer film

International Nuclear Information System (INIS)

Li, Xin; Deng, Jinchuan; Yuan, Shuheng; Wang, Juan; Luo, Rifang; Chen, Si; Wang, Jin; Huang, Nan

2016-01-01

Highlights: • The dopamine/PEI film with controlled amine density was successfully prepared. • The DNA aptamer was assembled onto the film via electrostatic incorporation. • The A@DPfilmscanspecificallyandeffectivelycaptureEPCs. • The A@DP film can support the survival of ECs, control the hyperplasia of SMCs. • The dynamic/co-culture models are useful for studying cells competitive adhesion. - Abstract: Endothelial progenitor cells (EPCs) are mainly located in bone marrow and circulate, and play a crucial role in repairmen of injury endothelium. One of the most promising strategies of stents designs were considered to make in-situ endothelialization in vivo via EPC-capture biomolecules on a vascular graft to capture EPCs directly from circulatory blood. In this work, an EPC specific aptamer with a 34 bases single strand DNA sequence was conjugated onto the stent surface via dopamine/polyethyleneimine copolymer film as a platform and linker. The assembled density of DNA aptamer could be regulated by controlling dopamine percentage in this copolymer film. X-ray photoelectron spectroscopy (XPS), water contact angle (WCA) and fluorescence test confirmed the successful immobilization of DNA aptamer. To confirm its biofunctionality and cytocompatibility, the capturing cells ability of the aptamer modified surface and the effects on the growth behavior of human umbilical vein endothelial cells (HUVECs), smooth muscle cells (SMCs) were investigated. The aptamer functionalized sample revealed a good EPC-capture ability, and had a cellular friendly feature for both EPC and EC growth, while not stimulated the hyperplasia of SMCs. And, the co-culture experiment of three types of cells confirmed the specificity capturing of EPCs to aptamer modified surface, rather than ECs and SMCs. These data suggested that this aptamer functionalized surface may have a large potentiality for the application of vascular grafts with targeted endothelialization.

Prevalent Bacterial Species and Novel Phylotypes in Advanced Noma Lesions

OpenAIRE

Paster, B. J.; Falkler, Jr., W. A.; Enwonwu, C. O.; Idigbe, E. O.; Savage, K. O.; Levanos, V. A.; Tamer, M. A.; Ericson, R. L.; Lau, C. N.; Dewhirst, F. E.

2002-01-01

The purpose of this study was to determine the bacterial diversity in advanced noma lesions using culture-independent molecular methods. 16S ribosomal DNA bacterial genes from DNA isolated from advanced noma lesions of four Nigerian children were PCR amplified with universally conserved primers and spirochetal selective primers and cloned into Escherichia coli. Partial 16S rRNA sequences of approximately 500 bases from 212 cloned inserts were used initially to determine species identity or cl...

DNA Adducts aand Human Atherosclerotis Lesions

Czech Academy of Sciences Publication Activity Database

Strejc, Přemysl; Boubelík, O.; Stávková, Zdena; Chvátalová, Irena; Šrám, Radim

2001-01-01

Roč. 42, - (2001), s. 662 ISSN 0008-5472. [Annual Meeting of Proceedings /92./. 24.03.2001-28.03.2001, New Orleans] R&D Projects: GA MZd NM10 Keywords : DNA adducts * LDL cholesterol Subject RIV: DN - Health Impact of the Environment Quality

Comparative study of DNA encapsulation into PLGA microparticles using modified double emulsion methods and spray drying techniques.

Science.gov (United States)

Oster, C G; Kissel, T

2005-05-01

Recently, several research groups have shown the potential of microencapsulated DNA as adjuvant for DNA immunization and in tissue engineering approaches. Among techniques generally used for microencapsulation of hydrophilic drug substances into hydrophobic polymers, modified WOW double emulsion method and spray drying of water-in-oil dispersions take a prominent position. The key parameters for optimized microspheres are particle size, encapsulation efficiency, continuous DNA release and stabilization of DNA against enzymatic and mechanical degradation. This study investigates the possibility to encapsulate DNA avoiding shear forces which readily degrade DNA during this microencapsulation. DNA microparticles were prepared with polyethylenimine (PEI) as a complexation agent for DNA. Polycations are capable of stabilizing DNA against enzymatic, as well as mechanical degradation. Further, complexation was hypothesized to facilitate the encapsulation by reducing the size of the macromolecule. This study additionally evaluated the possibility of encapsulating lyophilized DNA and lyophilized DNA/PEI complexes. For this purpose, the spray drying and double emulsion techniques were compared. The size of the microparticles was characterized by laser diffractometry and the particles were visualized by scanning electron microscopy (SEM). DNA encapsulation efficiencies were investigated photometrically after complete hydrolysis of the particles. Finally, the DNA release characteristics from the particles were studied. Particles with a size of <10 microm which represent the threshold for phagocytic uptake could be prepared with these techniques. The encapsulation efficiency ranged from 100-35% for low theoretical DNA loadings. DNA complexation with PEI 25?kDa prior to the encapsulation process reduced the initial burst release of DNA for all techniques used. Spray-dried particles without PEI exhibited high burst releases, whereas double emulsion techniques showed continuous

Detection of Genetically Modified Food: Has Your Food Been Genetically Modified?

Science.gov (United States)

Brandner, Diana L.

2002-01-01

Explains the benefits and risks of genetically-modified foods and describes methods for genetically modifying food. Presents a laboratory experiment using a polymerase chain reaction (PCR) test to detect foreign DNA in genetically-modified food. (Contains 18 references.) (YDS)

Aging and DNA repair capability. [Review

Energy Technology Data Exchange (ETDEWEB)

Tice, R R

1977-01-01

A review of the literature on DNA repair processes in relation to aging is presented under the following headings: DNA repair processes; age-related occurrence of unrepaired DNA lesions; DNA repair capability as a function of age; tissue-specific DNA repair capability; acceleration of the aging process by exposure to DNA damaging agents; human genetic syndromes; and longevity and DNA repair processes. (HLW)

DNA repair deficiency in neurodegeneration

DEFF Research Database (Denmark)

Jeppesen, Dennis Kjølhede; Bohr, Vilhelm A; Stevnsner, Tinna V.

2011-01-01

Deficiency in repair of nuclear and mitochondrial DNA damage has been linked to several neurodegenerative disorders. Many recent experimental results indicate that the post-mitotic neurons are particularly prone to accumulation of unrepaired DNA lesions potentially leading to progressive...... neurodegeneration. Nucleotide excision repair is the cellular pathway responsible for removing helix-distorting DNA damage and deficiency in such repair is found in a number of diseases with neurodegenerative phenotypes, including Xeroderma Pigmentosum and Cockayne syndrome. The main pathway for repairing oxidative...... base lesions is base excision repair, and such repair is crucial for neurons given their high rates of oxygen metabolism. Mismatch repair corrects base mispairs generated during replication and evidence indicates that oxidative DNA damage can cause this pathway to expand trinucleotide repeats, thereby...

Formation of monofunctional cisplatin-DNA adducts in carbonate buffer.

Science.gov (United States)

Binter, Alexandra; Goodisman, Jerry; Dabrowiak, James C

2006-07-01

Carbonate in its various forms is an important component in blood and the cytosol. Since, under conditions that simulate therapy, carbonate reacts with cisplatin to form carbonato complexes, one of which is taken up and/or modified by the cell [C.R. Centerwall, J. Goodisman, D.J. Kerwood, J. Am. Chem. Soc., 127 (2005) 12768-12769], cisplatin-carbonato complexes may be important in the mechanism of action of cisplatin. In this report we study the binding of cisplatin to pBR322 DNA in two different buffers, using gel electrophoresis. In 23.8mM HEPES, N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid, 5mM NaCl, pH 7.4 buffer, cisplatin produces aquated species, which react with DNA to unwind supercoiled Form I DNA, increasing its mobility, and reducing the binding of ethidium to DNA. This behavior is consistent with the formation of the well-known intrastrand crosslink on DNA. In 23.8mM carbonate buffer, 5mM NaCl, pH 7.4, cisplatin forms carbonato species that produce DNA-adducts which do not significantly change supercoiling but enhance binding of ethidium to DNA. This behavior is consistent with the formation of a monofunctional cisplatin adduct on DNA. These results show that aquated cisplatin and carbonato complexes of cisplatin produce different types of lesions on DNA and they underscore the importance of carrying out binding studies with cisplatin and DNA using conditions that approximate those found in the cell.

Selective recognition of DNA from olive leaves and olive oil by PNA and modified-PNA microarrays

Science.gov (United States)

Rossi, Stefano; Calabretta, Alessandro; Tedeschi, Tullia; Sforza, Stefano; Arcioni, Sergio; Baldoni, Luciana; Corradini, Roberto; Marchelli, Rosangela

2012-01-01

PNA probes for the specific detection of DNA from olive oil samples by microarray technology were developed. The presence of as low as 5% refined hazelnut (Corylus avellana) oil in extra-virgin olive oil (Olea europaea L.) could be detected by using a PNA microarray. A set of two single nucleotide polymorphisms (SNPs) from the Actin gene of Olive was chosen as a model for evaluating the ability of PNA probes for discriminating olive cultivars. Both unmodified and C2-modified PNAs bearing an arginine side-chain were used, the latter showing higher sequence specificity. DNA extracted from leaves of three different cultivars (Ogliarola leccese, Canino and Frantoio) could be easily discriminated using a microarray with unmodified PNA probes, whereas discrimination of DNA from oil samples was more challenging, and could be obtained only by using chiral PNA probes. PMID:22772038

Characterization of DNA repair phenotypes of Xeroderma pigmentosum cell lines by a paralleled in vitro test

International Nuclear Information System (INIS)

Raffin, A.L.

2009-06-01

DNA is constantly damaged modifying the genetic information for which it encodes. Several cellular mechanisms as the Base Excision Repair (BER) and the Nucleotide Excision Repair (NER) allow recovering the right DNA sequence. The Xeroderma pigmentosum is a disease characterised by a deficiency in the NER pathway. The aim of this study was to propose an efficient and fast test for the diagnosis of this disease as an alternative to the currently available UDS test. DNA repair activities of XP cell lines were quantified using in vitro miniaturized and paralleled tests in order to establish DNA repair phenotypes of XPA and XPC deficient cells. The main advantage of the tests used in this study is the simultaneous measurement of excision or excision synthesis (ES) of several lesions by only one cellular extract. We showed on one hand that the relative ES of the different lesions depend strongly on the protein concentration of the nuclear extract tested. Working at high protein concentration allowed discriminating the XP phenotype versus the control one, whereas it was impossible under a certain concentration's threshold. On the other hand, while the UVB irradiation of control cells stimulated their repair activities, this effect was not observed in XP cells. This study brings new information on the XPA and XPC protein roles during BER and NER and underlines the complexity of the regulations of DNA repair processes. (author)

Design of a DNA chip for detection of unknown genetically modified organisms (GMOs).

Science.gov (United States)

Nesvold, Håvard; Kristoffersen, Anja Bråthen; Holst-Jensen, Arne; Berdal, Knut G

2005-05-01

Unknown genetically modified organisms (GMOs) have not undergone a risk evaluation, and hence might pose a danger to health and environment. There are, today, no methods for detecting unknown GMOs. In this paper we propose a novel method intended as a first step in an approach for detecting unknown genetically modified (GM) material in a single plant. A model is designed where biological and combinatorial reduction rules are applied to a set of DNA chip probes containing all possible sequences of uniform length n, creating probes capable of detecting unknown GMOs. The model is theoretically tested for Arabidopsis thaliana Columbia, and the probabilities for detecting inserts and receiving false positives are assessed for various parameters for this organism. From a theoretical standpoint, the model looks very promising but should be tested further in the laboratory. The model and algorithms will be available upon request to the corresponding author.

Tumor‐associated DNA mutation detection in individuals undergoing colonoscopy

OpenAIRE

Fleshner, Phillip; Braunstein, Glenn D.; Ovsepyan, Gayane; Tonozzi, Theresa R.; Kammesheidt, Anja

2017-01-01

Abstract The majority of colorectal cancers (CRC) harbor somatic mutations and epigenetic modifications in the tumor tissue, and some of these mutations can be detected in plasma as circulating tumor DNA (ctDNA). Precancerous colorectal lesions also contain many of these same mutations. This study examined plasma for ctDNA from patients undergoing a screening or diagnostic colonoscopy to determine the sensitivity and specificity of the ctDNA panel for detecting CRC and precancerous lesions. T...

Correction of the lack of commutability between plasmid DNA and genomic DNA for quantification of genetically modified organisms using pBSTopas as a model.

Science.gov (United States)

Zhang, Li; Wu, Yuhua; Wu, Gang; Cao, Yinglong; Lu, Changming

2014-10-01

Plasmid calibrators are increasingly applied for polymerase chain reaction (PCR) analysis of genetically modified organisms (GMOs). To evaluate the commutability between plasmid DNA (pDNA) and genomic DNA (gDNA) as calibrators, a plasmid molecule, pBSTopas, was constructed, harboring a Topas 19/2 event-specific sequence and a partial sequence of the rapeseed reference gene CruA. Assays of the pDNA showed similar limits of detection (five copies for Topas 19/2 and CruA) and quantification (40 copies for Topas 19/2 and 20 for CruA) as those for the gDNA. Comparisons of plasmid and genomic standard curves indicated that the slopes, intercepts, and PCR efficiency for pBSTopas were significantly different from CRM Topas 19/2 gDNA for quantitative analysis of GMOs. Three correction methods were used to calibrate the quantitative analysis of control samples using pDNA as calibrators: model a, or coefficient value a (Cva); model b, or coefficient value b (Cvb); and the novel model c or coefficient formula (Cf). Cva and Cvb gave similar estimated values for the control samples, and the quantitative bias of the low concentration sample exceeded the acceptable range within ±25% in two of the four repeats. Using Cfs to normalize the Ct values of test samples, the estimated values were very close to the reference values (bias -13.27 to 13.05%). In the validation of control samples, model c was more appropriate than Cva or Cvb. The application of Cf allowed pBSTopas to substitute for Topas 19/2 gDNA as a calibrator to accurately quantify the GMO.

High-performance liquid chromatography/electrospray mass spectrometry for the analysis of modified bases in DNA: 7-(2-hydroxyethyl)guanine, the major ethylene oxide-DNA adduct.

Science.gov (United States)

Leclercq, L; Laurent, C; De Pauw, E

1997-05-15

A method was developed for the analysis of 7-(2-hydroxyethyl)guanine (7HEG), the major DNA adduct formed after exposure to ethylene oxide (EO). The method is based on DNA neutral thermal hydrolysis, adduct micro-concentration, and final characterization and quantification by HPLC coupled to single-ion monitoring electrospray mass spectrometry (HPLC/SIR-ESMS). The method was found to be selective, sensitive, and easy to handle with no need for enzymatic digestion or previous sample derivatization. Detection limit was found to be close to 1 fmol of adduct injected (10(-10) M), thus allowing the detection of approximately three modified bases on 10(8) intact nucleotides in blood sample analysis. Quantification results are shown for 7HEG after calf thymus DNA and blood exposure to various doses of EO, in both cases obtaining clear dose-response relationships.

Immunohistochemical study of DNA topoisomerase I, DNA topoisomerase II alpha, p53, and Ki-67 in oral preneoplastic lesions and oral squamous cell carcinomas.

Science.gov (United States)

Hafian, Hilal; Venteo, Lydie; Sukhanova, Alyona; Nabiev, Igor; Lefevre, Benoît; Pluot, Michel

2004-06-01

Human DNA topoisomerase I (topo I) is the molecular target of the camptothecin group of anticancer drugs. Laboratory studies have shown that the cellular response to topo I-targeted drugs depends on the topo I expression and DNA replication rate and the apoptotic pathway activity. In this study, we tested potential indicators of the sensitivity of topo I-targeted drugs in 36 cases of oral squamous cell carcinoma (OSCC). Formalin-fixed, paraffin-embedded tissue sections were immunostained with monoclonal antibodies against Ki-67, p53, and topo I, and with polyclonal antibodies against DNA topoisomerase II-alpha (topo II-alpha). These markers were also tested in 18 epithelial hyperplastic lesions and 18 mild dysplasias. Immunostaining was quantified by the percentage of stained nuclei in each sample (the labeling index); 200 immunoreactive epithelial nuclei were counted per case for each antibody. The results support the possibility of using topo II-alpha staining for assessing the proliferative activity. High expression of topo II-alpha and topo I in OSCCs suggests that they may serve as potential indicators of sensitivity to topo I inhibitors. However, the apoptotic pathway assessed by p53 immunostaining was found to be uninformative. Analysis of the relationship between immunohistochemical results and clinical and pathologic parameters (the T and N stages and differentiation) showed that only the differentiation parameter correlated with the topo I expression rate. Thus, significant increase in the topo I expression in the poorly differentiated OSCCs suggests their higher sensitivity to drug treatment.

Lethal and mutagenic properties of MMS-generated DNA lesions in Escherichia coli cells deficient in BER and AlkB-directed DNA repair.

Science.gov (United States)

Sikora, Anna; Mielecki, Damian; Chojnacka, Aleksandra; Nieminuszczy, Jadwiga; Wrzesinski, Michal; Grzesiuk, Elzbieta

2010-03-01

Methylmethane sulphonate (MMS), an S(N)2-type alkylating agent, generates DNA methylated bases exhibiting cytotoxic and mutagenic properties. Such damaged bases can be removed by a system of base excision repair (BER) and by oxidative DNA demethylation catalysed by AlkB protein. Here, we have shown that the lack of the BER system and functional AlkB dioxygenase results in (i) increased sensitivity to MMS, (ii) elevated level of spontaneous and MMS-induced mutations (measured by argE3 --> Arg(+) reversion) and (iii) induction of the SOS response shown by visualization of filamentous growth of bacteria. In the xth nth nfo strain additionally mutated in alkB gene, all these effects were extreme and led to 'error catastrophe', resulting from the presence of unrepaired apurinic/apyrimidinic (AP) sites and 1-methyladenine (1meA)/3-methylcytosine (3meC) lesions caused by deficiency in, respectively, BER and AlkB dioxygenase. The decreased level of MMS-induced Arg(+) revertants in the strains deficient in polymerase V (PolV) (bearing the deletion of the umuDC operon), and the increased frequency of these revertants in bacteria overproducing PolV (harbouring the pRW134 plasmid) indicate the involvement of PolV in the error-prone repair of 1meA/3meC and AP sites. Comparison of the sensitivity to MMS and the induction of Arg(+) revertants in the double nfo alkB and xth alkB, and the quadruple xth nth nfo alkB mutants showed that the more AP sites there are in DNA, the stronger the effect of the lack of AlkB protein. Since the sum of MMS-induced Arg(+) revertants in xth, nfo and nth xth nfo and alkB mutants is smaller than the frequency of these revertants in the BER(-) alkB(-) strain, we consider two possibilities: (i) the presence of AP sites in DNA results in relaxation of its structure that facilitates methylation and (ii) additional AP sites are formed in the BER(-) alkB(-) mutants.

Close encounters for the first time: Helicase interactions with DNA damage.

Science.gov (United States)

Khan, Irfan; Sommers, Joshua A; Brosh, Robert M

2015-09-01

DNA helicases are molecular motors that harness the energy of nucleoside triphosphate hydrolysis to unwinding structured DNA molecules that must be resolved during cellular replication, DNA repair, recombination, and transcription. In vivo, DNA helicases are expected to encounter a wide spectrum of covalent DNA modifications to the sugar phosphate backbone or the nitrogenous bases; these modifications can be induced by endogenous biochemical processes or exposure to environmental agents. The frequency of lesion abundance can vary depending on the lesion type. Certain adducts such as oxidative base modifications can be quite numerous, and their effects can be helix-distorting or subtle perturbations to DNA structure. Helicase encounters with specific DNA lesions and more novel forms of DNA damage will be discussed. We will also review the battery of assays that have been used to characterize helicase-catalyzed unwinding of damaged DNA substrates. Characterization of the effects of specific DNA adducts on unwinding by various DNA repair and replication helicases has proven to be insightful for understanding mechanistic and biological aspects of helicase function in cellular DNA metabolism. Published by Elsevier B.V.

Replisome-mediated Translesion Synthesis and Leading Strand Template Lesion Skipping Are Competing Bypass Mechanisms*

Science.gov (United States)

Gabbai, Carolina B.; Yeeles, Joseph T. P.; Marians, Kenneth J.

2014-01-01

A number of different enzymatic pathways have evolved to ensure that DNA replication can proceed past template base damage. These pathways include lesion skipping by the replisome, replication fork regression followed by either correction of the damage and origin-independent replication restart or homologous recombination-mediated restart of replication downstream of the lesion, and bypass of the damage by a translesion synthesis DNA polymerase. We report here that of two translesion synthesis polymerases tested, only DNA polymerase IV, not DNA polymerase II, could engage productively with the Escherichia coli replisome to bypass leading strand template damage, despite the fact that both enzymes are shown to be interacting with the replicase. Inactivation of the 3′ → 5′ proofreading exonuclease of DNA polymerase II did not enable bypass. Bypass by DNA polymerase IV required its ability to interact with the β clamp and act as a translesion polymerase but did not require its “little finger” domain, a secondary region of interaction with the β clamp. Bypass by DNA polymerase IV came at the expense of the inherent leading strand lesion skipping activity of the replisome, indicating that they are competing reactions. PMID:25301949

Enhancement of fluorescence quenching and exciplex formation in DNA major groove by double incorporation of modified fluorescent deoxyuridines.

Science.gov (United States)

Tanaka, Makiko; Oguma, Kazuhiro; Saito, Yoshio; Saito, Isao

2012-06-15

5-(1-Naphthalenylethynyl)-2'-deoxyuridine ((N)U) and 5-[(4-cyano-1-naphthalenyl)ethynyl]-2'-deoxyuridine ((CN)U) were synthesized and incorporated into oligodeoxynucleotides. Fluorescence emissions of modified duplexes containing double (N)U were efficiently quenched depending upon the sequence pattern of the naphthalenes in DNA major groove, as compared to the duplex possessing single (N)U. When one of the naphthalene moieties has a cyano substituent, the exciplex emission from the chromophores in DNA major groove was observed at longer wavelength. Copyright © 2012 Elsevier Ltd. All rights reserved.

NMR solution structure of an N2-guanine DNA adduct derived from the potent tumorigen dibenzo[a,l]pyrene: Intercalation from the minor groove with ruptured Watson-Crick base pairing

Science.gov (United States)

Tang, Yijin; Liu, Zhi; Ding, Shuang; Lin, Chin H.; Cai, Yuqin; Rodriguez, Fabian A.; Sayer, Jane M.; Jerina, Donald M.; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E.

2012-01-01

The most potent tumorigen identified among the polycyclic aromatic hydrocarbons (PAH) is the non-planar fjord region dibenzo[a,l]pyrene (DB[a,l]P). It is metabolically activated in vivo through the widely-studied diol epoxide (DE) pathway to form covalent adducts with DNA bases, predominantly guanine and adenine. The (+)-11S,12R,13R,14S DE enantiomer forms adducts via its C14-position with the exocyclic amino group of guanine. Here, we present the first NMR solution structure of a DB[a,l]P-derived adduct, the 14R (+)-trans-anti-DB[a,l]P–N2-dG (DB[a,l]P-dG) lesion in double-stranded DNA. In contrast to the stereochemically identical benzo[a]pyrene-derived N2-dG adduct (B[a]P-dG) in which the B[a]P rings reside in the B-DNA minor groove on the 3’-side of the modifed deoxyguanosine, in the DB[a,l]P-derived adduct the DB[a,l]P rings intercalate into the duplex on the 3’-side of the modified base from the sterically crowded minor groove. Watson-Crick base pairing of the modified guanine with the partner cytosine is broken, but these bases retain some stacking with the bulky DB[a,l]P ring system. This new theme in PAH DE - DNA adduct conformation differs from: (1) the classical intercalation motif where Watson-Crick base-pairing is intact at the lesion site, and (2) the base-displaced intercalation motif in which the damaged base and its partner are extruded from the helix . The structural considerations that lead to the intercalated conformation of the DB[a,l]P-dG lesion in contrast to the minor groove alignment of the B[a]P-dG adduct, and the implications of the DB[a,l]P-dG conformational motif for the recognition of such DNA lesions by the human nucleotide excision repair apparatus, are discussed. PMID:23121427

DNA polymerase η modulates replication fork progression and DNA damage responses in platinum-treated human cells

Science.gov (United States)

Sokol, Anna M.; Cruet-Hennequart, Séverine; Pasero, Philippe; Carty, Michael P.

2013-11-01

Human cells lacking DNA polymerase η (polη) are sensitive to platinum-based cancer chemotherapeutic agents. Using DNA combing to directly investigate the role of polη in bypass of platinum-induced DNA lesions in vivo, we demonstrate that nascent DNA strands are up to 39% shorter in human cells lacking polη than in cells expressing polη. This provides the first direct evidence that polη modulates replication fork progression in vivo following cisplatin and carboplatin treatment. Severe replication inhibition in individual platinum-treated polη-deficient cells correlates with enhanced phosphorylation of the RPA2 subunit of replication protein A on serines 4 and 8, as determined using EdU labelling and immunofluorescence, consistent with formation of DNA strand breaks at arrested forks in the absence of polη. Polη-mediated bypass of platinum-induced DNA lesions may therefore represent one mechanism by which cancer cells can tolerate platinum-based chemotherapy.

The DNA damage response during mitosis

NARCIS (Netherlands)

Heijink, Anne Margriet; Krajewska, Malgorzata; van Vugt, Marcel A. T. M.

2013-01-01

Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance

DNA biosensor for detection of Salmonella typhi from blood sample of typhoid fever patient using gold electrode modified by self-assembled monolayers of thiols

Science.gov (United States)

Suryapratiwi, Windha Novita; Paat, Vlagia Indira; Gaffar, Shabarni; Hartati, Yeni Wahyuni

2017-05-01

Electrochemical biosensors are currently being developed in order to handle various clinical problems in diagnosing infectious diseases caused by pathogenic bacteria, or viruses. On this research, voltammetric DNA biosensor using gold electrode modified by thiols with self-assembled monolayers had been developed to detect a certain sequence of Salmonella typhi DNA from blood sample of typhoid fever patient. Thiol groups of cysteamines (Cys) and aldehyde groups from glutaraldehydes (Glu) were used as a link to increase the performance of gold electrode in detecting guanine oxidation signal of hybridized S. typhi DNA and ssDNA probe. Standard calibration method was used to determine analytical parameters from the measurements. The result shown that, the detection of S. typhi DNA from blood sample of typhoid fever patient can be carried out by voltammetry using gold electrode modified by self-assembled monolayers of thiols. A characteristic oxidation potential of guanine using Au/Cys/Gluwas obtained at +0.17 until +0.20 V. Limit of detection and limit of quantification from this measurements were 1.91μg mL-1 and 6.35 μg mL-1. The concentration of complement DNA from sample was 6.96 μg mL-1.

Mechanistic Basis for the Bypass of a Bulky DNA Adduct Catalyzed by a Y-Family DNA Polymerase

Science.gov (United States)

Vyas, Rajan; Efthimiopoulos, Georgia; Tokarsky, E. John; Malik, Chanchal K.; Basu, Ashis K.; Suo, Zucai

2015-01-01

1-Nitropyrene (1-NP), an environmental pollutant, induces DNA damage in vivo and is considered to be carcinogenic. The DNA adducts formed by the 1-NP metabolites stall replicative DNA polymerases but are presumably bypassed by error-prone Y-family DNA polymerases at the expense of replication fidelity and efficiency in vivo. Our running start assays confirmed that a site-specifically placed 8-(deoxyguanosin-N2-yl)-1-aminopyrene (dG1,8), one of the DNA adducts derived from 1-NP, can be bypassed by Sulfolobus solfataricus DNA polymerase IV (Dpo4), although this representative Y-family enzyme was paused strongly by the lesion. Pre-steady-state kinetic assays were employed to determine the low nucleotide incorporation fidelity and establish a minimal kinetic mechanism for the dG1,8 bypass by Dpo4. To reveal a structural basis for dCTP incorporation opposite dG1,8, we solved the crystal structures of the complexes of Dpo4 and DNA containing a templating dG1,8 lesion in the absence or presence of dCTP. The Dpo4·DNA-dG1,8 binary structure shows that the aminopyrene moiety of the lesion stacks against the primer/template junction pair, while its dG moiety projected into the cleft between the Finger and Little Finger domains of Dpo4. In the Dpo4·DNA-dG1,8·dCTP ternary structure, the aminopyrene moiety of the dG1,8 lesion, is sandwiched between the nascent and junction base pairs, while its base is present in the major groove. Moreover, dCTP forms a Watson–Crick base pair with dG, two nucleotides upstream from the dG1,8 site, creating a complex for “-2” frameshift mutation. Mechanistically, these crystal structures provide additional insight into the aforementioned minimal kinetic mechanism. PMID:26327169

S1-sensitive sites in DNA after γ-irradiation

International Nuclear Information System (INIS)

Martin-Bertram, H.

1981-01-01

DNA from γ-irradiated T 1 bacteriophages was analyzed for 'single-stranded' sites by cleavage with S1 nuclease from Aspergillus oryzae as lesion probe. The ratio of 'S1-sensitive sites' to the amount of radiation-induced single-strand breaks was about one. Presumably these 'denatured' sites were associated with single-strand breaks. The subsequent check for the persistence of 'single-stranded' sites within the DNA molecule by thermokinetics demonstrated a strong affinity of the nuclease to its substrate, the single-stranded lesion, and a perfect excision. It is assumed that the direct absorption of radiation energy in the DNA gives rise to the formation of such bulky lesions. (Auth.)

Misrepair of overlapping daughter strand gaps as a possible mechanism for UV induced mutagenesis in uvr strains of Escherichia coli: a general model for induced mutagenesis by misrepair (SOS repair) of closely spaced DNA lesions

International Nuclear Information System (INIS)

Sedgwick, S.G.

1976-01-01

It has been previously reported that an inducible form of post-replication repair appeared to be required for UV induced mutagenesis in an uvrA strain of Escherichia coli. It is shown here that the numbers of daughter strand gaps requiring inducible repair were similar to the numbers calculated to be overlapping one another in opposite daughter chromosomes. An estimation of survival with no repair of these gaps resembled the survival predicted with mutagenesis. It is thus proposed that inducible post-replication repair causes mutagenesis by the repair of overlapping daughter strand gaps. A general model for induced mutagenesis is presented. It is proposed that (a) some DNA lesions introduced by any DNA damaging agent may be close enough to interfere with constitutive repair replication of each other, (b) these lesions induce a repair system (SOS repair) which involves the recA + . lexA + and polC + genes (c) repair, and noncomitant mutagenesis occurs during repair replication by the insertion of mismatched bases oppposite the noncoding DNA lesions

Dichromatic laser radiation effects on DNA of Escherichia coli and plasmids

Science.gov (United States)

Martins, W. A.; Polignano, G. A. C.; Guimarães, O. R.; Geller, M.; Paoli, F.; Fonseca, A. S.

2015-04-01

Dichromatic and consecutive laser radiations have attracted increased attention for clinical applications as offering new tools for the treatment of dysfunctional tissues in situations where monochromatic radiation is not effective. This work evaluated the survival, filamentation and morphology of Escherichia coli cells, and the induction of DNA lesions, in plasmid DNA exposed to low-intensity consecutive dichromatic laser radiation. Exponential and stationary wild type and formamidopyrimidine DNA glycosylase/MutM protein deficient E. coli cultures were exposed to consecutive low-intensity dichromatic laser radiation (infrared laser immediately after red laser) to study the survival, filamentation and morphology of bacterial cells. Plasmid DNA samples were exposed to dichromatic radiation to study DNA lesions by electrophoretic profile. Dichromatic laser radiation affects the survival, filamentation and morphology of E. coli cultures depending on the growth phase and the functional repair mechanism of oxidizing lesions in DNA, but does not induce single/double strands breaks or alkali-labile DNA lesions. Results show that low-intensity consecutive dichromatic laser radiation induces biological effects that differ from those induced by monochromatic laser radiation, suggesting that other therapeutic effects could be obtained using dichromatic radiation.

Transgenerational inheritance of modified DNA methylation patterns and enhanced tolerance induced by heavy metal stress in rice (Oryza sativa L.).

Science.gov (United States)

Ou, Xiufang; Zhang, Yunhong; Xu, Chunming; Lin, Xiuyun; Zang, Qi; Zhuang, Tingting; Jiang, Lili; von Wettstein, Diter; Liu, Bao

2012-01-01

DNA methylation is sensitive and responsive to stressful environmental conditions. Nonetheless, the extent to which condition-induced somatic methylation modifications can impose transgenerational effects remains to be fully understood. Even less is known about the biological relevance of the induced epigenetic changes for potentially altered well-being of the organismal progenies regarding adaptation to the specific condition their progenitors experienced. We analyzed DNA methylation pattern by gel-blotting at genomic loci representing transposable elements and protein-coding genes in leaf-tissue of heavy metal-treated rice (Oryza sativa) plants (S0), and its three successive organismal generations. We assessed expression of putative genes involved in establishing and/or maintaining DNA methylation patterns by reverse transcription (RT)-PCR. We measured growth of the stressed plants and their unstressed progenies vs. the control plants. We found (1) relative to control, DNA methylation patterns were modified in leaf-tissue of the immediately treated plants, and the modifications were exclusively confined to CHG hypomethylation; (2) the CHG-demethylated states were heritable via both maternal and paternal germline, albeit often accompanying further hypomethylation; (3) altered expression of genes encoding for DNA methyltransferases, DNA glycosylase and SWI/SNF chromatin remodeling factor (DDM1) were induced by the stress; (4) progenies of the stressed plants exhibited enhanced tolerance to the same stress their progenitor experienced, and this transgenerational inheritance of the effect of condition accompanying heritability of modified methylation patterns. Our findings suggest that stressful environmental condition can produce transgenerational epigenetic modifications. Progenies of stressed plants may develop enhanced adaptability to the condition, and this acquired trait is inheritable and accord with transmission of the epigenetic modifications. We suggest

Quantitation of DNA adducts by stable isotope dilution mass spectrometry

Science.gov (United States)

Tretyakova, Natalia; Goggin, Melissa; Janis, Gregory

2012-01-01

Exposure to endogenous and exogenous chemicals can lead to the formation of structurally modified DNA bases (DNA adducts). If not repaired, these nucleobase lesions can cause polymerase errors during DNA replication, leading to heritable mutations potentially contributing to the development of cancer. Due to their critical role in cancer initiation, DNA adducts represent mechanism-based biomarkers of carcinogen exposure, and their quantitation is particularly useful for cancer risk assessment. DNA adducts are also valuable in mechanistic studies linking tumorigenic effects of environmental and industrial carcinogens to specific electrophilic species generated from their metabolism. While multiple experimental methodologies have been developed for DNA adduct analysis in biological samples – including immunoassay, HPLC, and 32P-postlabeling – isotope dilution high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) generally has superior selectivity, sensitivity, accuracy, and reproducibility. As typical DNA adducts concentrations in biological samples are between 0.01 – 10 adducts per 108 normal nucleotides, ultrasensitive HPLC-ESI-MS/MS methodologies are required for their analysis. Recent developments in analytical separations and biological mass spectrometry – especially nanoflow HPLC, nanospray ionization MS, chip-MS, and high resolution MS – have pushed the limits of analytical HPLC-ESI-MS/MS methodologies for DNA adducts, allowing researchers to accurately measure their concentrations in biological samples from patients treated with DNA alkylating drugs and in populations exposed to carcinogens from urban air, drinking water, cooked food, alcohol, and cigarette smoke. PMID:22827593

Understanding DNA Under Oxidative Stress and Sensitization: The Role of Molecular Modeling

Directory of Open Access Journals (Sweden)

Antonio eMonari

2015-07-01

Full Text Available DNA is constantly exposed to damaging threats coming from oxidative stress, i.e. from the presence of free radicals and reactive oxygen species. Sensitization from exogenous and endogenous compounds that strongly enhance the frequency of light-induced lesions also plays an important role. The experimental determination of DNA lesions, though a difficult subject, is somehow well established and allows to elucidate even extremely rare DNA lesions. In parallel, molecular modeling has become fundamental to clearly understand the fine mechanisms related to DNA defects induction. Indeed, it offers an unprecedented possibility to get access to an atomistic or even electronic resolution. Ab initio molecular dynamics may also describe the time-evolution of the molecular system and its reactivity. Yet the modeling of DNA (photo-reactions does necessitate elaborate multi-scale methodologies to tackle a damage induction reactivity that takes place in a complex environment. The double-stranded DNA environment is first characterized by a very high flexibility, that dynamical effects are to be taken into account, but also a strongly inhomogeneous electrostatic embedding. Additionally, one aims at capturing more subtle effects, such as the sequence selectivity which is of critical important for DNA damage. The structure and dynamics of the DNA/sensitizers complexes, as well as the photo-induced electron- and energy-transfer phenomena taking place upon sensitization, should be carefully modeled. Finally the factors inducing different repair ratios for different lesions should also be rationalized.In this review we will critically analyze the different computational strategies used to model DNA lesions. A clear picture of the complex interplay between reactivity and structural factors will be sketched. The use of proper multi-scale modeling leads to the in-depth comprehension of DNA lesions mechanism and also to the rational design of new chemo-therapeutic agents.

A single-strand specific lesion drives MMS-induced hyper-mutability at a double-strand break in yeast.

Science.gov (United States)

Yang, Yong; Gordenin, Dmitry A; Resnick, Michael A

2010-08-05

Localized hyper-mutability (LHM) can be important in evolution, immunity, and genetic diseases. We previously reported that single-strand DNA (ssDNA) can be an important source of damage-induced LHM in yeast. Here, we establish that the generation of LHM by methyl methanesulfonate (MMS) during repair of a chromosomal double-strand break (DSB) can result in over 0.2 mutations/kb, which is approximately 20,000-fold higher than the MMS-induced mutation density without a DSB. The MMS-induced mutations associated with DSB repair were primarily due to substitutions via translesion DNA synthesis at damaged cytosines, even though there are nearly 10 times more MMS-induced lesions at other bases. Based on this mutation bias, the promutagenic lesion dominating LHM is likely 3-methylcytosine, which is single-strand specific. Thus, the dramatic increase in mutagenesis at a DSB is concluded to result primarily from the generation of non-repairable lesions in ssDNA associated with DSB repair along with efficient induction of highly mutagenic ssDNA-specific lesions. These findings with MMS-induced LHM have broad biological implications for unrepaired damage generated in ssDNA and possibly ssRNA. Published by Elsevier B.V.

Immunological detection and quantification of DNA components structurally modified by alkylating carcinogens, mutagens and chemotherapeutic agents

International Nuclear Information System (INIS)

Rajewsky, M.F.

1983-01-01

The detection and quantification of defined reaction products of chemical mutagens and carcinogens (and of many cancer chemotherapeutic agents) with DNA require highly sensitive analytical techniques. The exceptional capability of immunoglobulins to recognize subtle alterations of molecular structure (especially when monoclonal antibodies are used to maximize specificity), outstanding sensitivity of immunoanalysis by high-affinity antibodies, and the fact that radioactively-labelled agents are not required suggest the utility of a radioimmunoassay to recognize and quantitate alkylated DNA products. We have recently developed a set of high-affinity monoclonal antibodies (secreted by mouse x mouse as well as by rat x rat hybridomas; antibody affinity constants, 10 9 to > 10 10 lmol) specifically directed against several DNA alkylation products with possible relevance in relation to both mutagenesis and malignant transformation of mammalian cells. These alkylation products include 0 6 -N-butyldeoxyguanosine, and 0 4 -ethyldeoxythymidine. When used in a radioimmunassay, an antibody specific for 0 6 -ethyldeoxyguanosine, for example, will detect this product at an 0 6 -ethyldeoxyguanosine/deoxyguanosine molar ratio of approx. 3 x 10 -7 in a hydrolysate of 100 ug of DNA. The limit of detection can be lowered further if the respective alkyldeoxynucleosides are separated by HPLC from the DNA hydrolysate prior to the RIA. The anti-alkyldeoxynucleoside monoclonal antibodies can also be used to visualize, by immunostaining and fluorescence microscopy combined with electronic image intensification, specific alkylation products in the nuclear DNA of individual cells, and to localize structurally modified bases in double-stranded DNA molecules by transmission electron microscopy

Nuclear magnetic resonance solution structure of an N(2)-guanine DNA adduct derived from the potent tumorigen dibenzo[a,l]pyrene: intercalation from the minor groove with ruptured Watson-Crick base pairing.

Science.gov (United States)

Tang, Yijin; Liu, Zhi; Ding, Shuang; Lin, Chin H; Cai, Yuqin; Rodriguez, Fabian A; Sayer, Jane M; Jerina, Donald M; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E

2012-12-04

The most potent tumorigen identified among the polycyclic aromatic hydrocarbons (PAH) is the nonplanar fjord region dibenzo[a,l]pyrene (DB[a,l]P). It is metabolically activated in vivo through the widely studied diol epoxide (DE) pathway to form covalent adducts with DNA bases, predominantly guanine and adenine. The (+)-11S,12R,13R,14S DE enantiomer forms adducts via its C14 position with the exocyclic amino group of guanine. Here, we present the first nuclear magnetic resonance solution structure of a DB[a,l]P-derived adduct, the 14R-(+)-trans-anti-DB[a,l]P-N(2)-dG (DB[a,l]P-dG) lesion in double-stranded DNA. In contrast to the stereochemically identical benzo[a]pyrene-derived N(2)-dG adduct (B[a]P-dG) in which the B[a]P rings reside in the B-DNA minor groove on the 3'-side of the modifed deoxyguanosine, in the DB[a,l]P-derived adduct the DB[a,l]P rings intercalate into the duplex on the 3'-side of the modified base from the sterically crowded minor groove. Watson-Crick base pairing of the modified guanine with the partner cytosine is broken, but these bases retain some stacking with the bulky DB[a,l]P ring system. This new theme in PAH DE-DNA adduct conformation differs from (1) the classical intercalation motif in which Watson-Crick base pairing is intact at the lesion site and (2) the base-displaced intercalation motif in which the damaged base and its partner are extruded from the helix. The structural considerations that lead to the intercalated conformation of the DB[a,l]P-dG lesion in contrast to the minor groove alignment of the B[a]P-dG adduct, and the implications of the DB[a,l]P-dG conformational motif for the recognition of such DNA lesions by the human nucleotide excision repair apparatus, are discussed.

Alkylation damage in DNA and RNA--repair mechanisms and medical significance

DEFF Research Database (Denmark)

Drabløs, Finn; Feyzi, Emadoldin; Aas, Per Arne

2004-01-01

Alkylation lesions in DNA and RNA result from endogenous compounds, environmental agents and alkylating drugs. Simple methylating agents, e.g. methylnitrosourea, tobacco-specific nitrosamines and drugs like temozolomide or streptozotocin, form adducts at N- and O-atoms in DNA bases. These lesions...... are mainly repaired by direct base repair, base excision repair, and to some extent by nucleotide excision repair (NER). The identified carcinogenicity of O(6)-methylguanine (O(6)-meG) is largely caused by its miscoding properties. Mutations from this lesion are prevented by O(6)-alkylG-DNA alkyltransferase......, inactivation of the MMR system in an AGT-defective background causes resistance to the killing effects of O(6)-alkylating agents, but not to the mutagenic effect. Bifunctional alkylating agents, such as chlorambucil or carmustine (BCNU), are commonly used anti-cancer drugs. DNA lesions caused by these agents...

Base damage within single-strand DNA underlies in vivo hypermutability induced by a ubiquitous environmental agent.

Directory of Open Access Journals (Sweden)

Kin Chan

Full Text Available Chromosomal DNA must be in single-strand form for important transactions such as replication, transcription, and recombination to occur. The single-strand DNA (ssDNA is more prone to damage than double-strand DNA (dsDNA, due to greater exposure of chemically reactive moieties in the nitrogenous bases. Thus, there can be agents that damage regions of ssDNA in vivo while being inert toward dsDNA. To assess the potential hazard posed by such agents, we devised an ssDNA-specific mutagenesis reporter system in budding yeast. The reporter strains bear the cdc13-1 temperature-sensitive mutation, such that shifting to 37°C results in telomere uncapping and ensuing 5' to 3' enzymatic resection. This exposes the reporter region, containing three closely-spaced reporter genes, as a long 3' ssDNA overhang. We validated the ability of the system to detect mutagenic damage within ssDNA by expressing a modified human single-strand specific cytosine deaminase, APOBEC3G. APOBEC3G induced a high density of substitutions at cytosines in the ssDNA overhang strand, resulting in frequent, simultaneous inactivation of two reporter genes. We then examined the mutagenicity of sulfites, a class of reactive sulfur oxides to which humans are exposed frequently via respiration and food intake. Sulfites, at a concentration similar to that found in some foods, induced a high density of mutations, almost always as substitutions at cytosines in the ssDNA overhang strand, resulting in simultaneous inactivation of at least two reporter genes. Furthermore, sulfites formed a long-lived adducted 2'-deoxyuracil intermediate in DNA that was resistant to excision by uracil-DNA N-glycosylase. This intermediate was bypassed by error-prone translesion DNA synthesis, frequently involving Pol ζ, during repair synthesis. Our results suggest that sulfite-induced lesions in DNA can be particularly deleterious, since cells might not possess the means to repair or bypass such lesions

Transition-state destabilization reveals how human DNA polymerase β proceeds across the chemically unstable lesion N7-methylguanine

Science.gov (United States)

Ouzon-Shubeita, Hala; Lee, Seongmin

2014-01-01

N7-Methyl-2′-deoxyguanosine (m7dG) is the predominant lesion formed by methylating agents. A systematic investigation on the effect of m7dG on DNA replication has been difficult due to the chemical instability of m7dG. To gain insights into the m7dG effect, we employed a 2′-fluorine-mediated transition-state destabilzation strategy. Specifically, we determined kinetic parameters for dCTP insertion opposite a chemically stable m7dG analogue, 2′-fluoro-m7dG (Fm7dG), by human DNA polymerase β (polβ) and solved three X-ray structures of polβ in complex with the templating Fm7dG paired with incoming dCTP or dTTP analogues. The kinetic studies reveal that the templating Fm7dG slows polβ catalysis ∼300-fold, suggesting that m7dG in genomic DNA may impede replication by some DNA polymerases. The structural analysis reveals that Fm7dG forms a canonical Watson–Crick base pair with dCTP, but metal ion coordination is suboptimal for catalysis in the polβ-Fm7dG:dCTP complex, which partially explains the slow insertion of dCTP opposite Fm7dG by polβ. In addition, the polβ-Fm7dG:dTTP structure shows open protein conformations and staggered base pair conformations, indicating that N7-methylation of dG does not promote a promutagenic replication. Overall, the first systematic studies on the effect of m7dG on DNA replication reveal that polβ catalysis across m7dG is slow, yet highly accurate. PMID:24966350

Can a Modified Bosniak Classification System Risk Stratify Pediatric Cystic Renal Masses?

Science.gov (United States)

Saltzman, Amanda F; Carrasco, Alonso; Colvin, Alexandra N; Meyers, Mariana L; Cost, Nicholas G

2018-03-20

We characterize and apply the modified Bosniak classification system to a cohort of children with cystic renal lesions and known surgical pathology. We identified all patients at our institution with cystic renal masses who also underwent surgery for these lesions. Patients without available preoperative imaging or pathology were excluded. All radiological imaging was independently reviewed by a pediatric radiologist blinded to pathological findings. Imaging characteristics (size, border, septations, calcifications, solid components, vascularity) were recorded from the most recent preoperative ultrasounds and computerized tomograms. The modified Bosniak classification system was applied to these scans and then correlated with final pathology. A total of 22 patients met study criteria. Median age at surgery was 6.1 years (range 11 months to 16.8 years). Of the patients 12 (54.5%) underwent open nephrectomy, 6 (27.3%) open partial nephrectomy, 2 (9.1%) laparoscopic cyst decortication, 1 (4.5%) open renal biopsy and 1 (4.5%) laparoscopic partial nephrectomy. Final pathology was benign in 9 cases (41%), intermediate in 6 (27%) and malignant in 7 (32%). All malignant lesions were modified Bosniak class 4, all intermediate lesions were modified class 3 or 4 and 8 of 9 benign lesions (89%) were modified class 1 or 2. Cystic renal lesions in children with a modified Bosniak class of 1 or 2 were most often benign, while class 3 or 4 lesions warranted surgical excision since more than 90% of masses harbored intermediate or malignant pathology. The modified Bosniak classification system appears to allow for a reasonable clinical risk stratification of pediatric cystic renal masses. Copyright © 2018 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Push back to respond better: regulatory inhibition of the DNA double-strand break response.

Science.gov (United States)

Panier, Stephanie; Durocher, Daniel

2013-10-01

Single DNA lesions such as DNA double-strand breaks (DSBs) can cause cell death or trigger genome rearrangements that have oncogenic potential, and so the pathways that mend and signal DNA damage must be highly sensitive but, at the same time, selective and reversible. When initiated, boundaries must be set to restrict the DSB response to the site of the lesion. The integration of positive and, crucially, negative control points involving post-translational modifications such as phosphorylation, ubiquitylation and acetylation is key for building fast, effective responses to DNA damage and for mitigating the impact of DNA lesions on genome integrity.

DNA enrichment approaches to identify unauthorized genetically modified organisms (GMOs).

Science.gov (United States)

Arulandhu, Alfred J; van Dijk, Jeroen P; Dobnik, David; Holst-Jensen, Arne; Shi, Jianxin; Zel, Jana; Kok, Esther J

2016-07-01

With the increased global production of different genetically modified (GM) plant varieties, chances increase that unauthorized GM organisms (UGMOs) may enter the food chain. At the same time, the detection of UGMOs is a challenging task because of the limited sequence information that will generally be available. PCR-based methods are available to detect and quantify known UGMOs in specific cases. If this approach is not feasible, DNA enrichment of the unknown adjacent sequences of known GMO elements is one way to detect the presence of UGMOs in a food or feed product. These enrichment approaches are also known as chromosome walking or gene walking (GW). In recent years, enrichment approaches have been coupled with next generation sequencing (NGS) analysis and implemented in, amongst others, the medical and microbiological fields. The present review will provide an overview of these approaches and an evaluation of their applicability in the identification of UGMOs in complex food or feed samples.

2,3,7,8-Tetrachlorodibenzo-p-dioxin modulates estradiol-induced aldehydic DNA lesions in human breast cancer cells through alteration of CYP1A1 and CYP1B1 expression.

Science.gov (United States)

Chen, Shou-Tung; Chen, Dar-Ren; Fang, Ju-Pin; Lin, Po-Hsiung

2015-05-01

Many genes responsible for the bioactivation of endogenous estrogen to reactive quinonoid metabolites, including cytochrome P450 (CYP) 1A1, 1A2, and 1B1, are well-known target genes of the aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The purpose of this research was to investigate the roles of TCDD-mediated altered gene expression in the induction of aldehydic DNA lesions (ADLs) by 17β-estradiol (E2) in human MDA-MB-231 and MCF-7 breast cancer cells. We demonstrated that increases in the number of oxidant-mediated ADLs, including abasic sites and aldehydic base/sugar lesions, were detected in MDA-MB-231 cells exposed to E2. The DNA-damaging effects of E2 in MDA-MB-231 cells were prevented by pretreatment of cells with TCDD. In contrast, we did not observe statistically significant increases in the number of ADLs in MCF-7 cells exposed to E2. However, with TCDD pretreatment, an approximately twofold increase in the number of ADLs was detected in MCF-7 cells exposed to E2. TCDD pretreatment induces disparity in the disposition of E2 to reactive quinonoid metabolites and the subsequent formation of oxidative DNA lesions through alteration of CYP1A1 and CYP1B1 expression in human breast cancer cells.

A “four-ferrocene” modified stem-loop structure as a probe for sensitive detection and single-base mismatch discrimination of DNA

International Nuclear Information System (INIS)

Chatelain, Grégory; Ripert, Micaël; Farre, Carole; Ansanay-Alex, Salomé; Chaix, Carole

2012-01-01

We report the use of a four-ferrocene modified oligonucleotide as a probe for DNA detection with a gold electrode microsystem. This oligonucleotide is synthesized by automated solid-phase synthesis with four successive ferrocene moieties at the 5′-end and a C6-thiol modifier group at the 3′-end. The grafting of this 4Fc-DNA probe on a gold electrode microsystem results in the appearance of the ferrocene redox couple in cyclic voltammetry. The probe sequence is a stem-loop structure that folds efficiently on the electrode, thus optimizing electron transfer. Such architecture serves as sensor for DNA detection which is based on hybridization. The resulting disposable voltammetric sensor allowed direct, reagentless DNA detection in a small volume (20 μL). Electrochemical response upon hybridization with complementary short sequence (30-base length) and long sequence (50-base length) strands was observed by differential pulse voltammetry. Current variations were compared. The longer the sequence, the greater the decrease in current. The system's detection limit was estimated at 3.5 pM (0.07 fmol in 20 μL) with the 50-base length target and provided a dynamic detection range between 3.5 pM and 5 nM. Single mismatch detection showed a good level of sensitivity. The system was regenerated twice with no significant loss of Fc signal. Finally, 1 pM sensitivity was reached with a long chain analog of DNA PCR products of Influenza virus.

Inhibition of DNA glycosylases via small molecule purine analogs.

Directory of Open Access Journals (Sweden)

Aaron C Jacobs

Full Text Available Following the formation of oxidatively-induced DNA damage, several DNA glycosylases are required to initiate repair of the base lesions that are formed. Recently, NEIL1 and other DNA glycosylases, including OGG1 and NTH1 were identified as potential targets in combination chemotherapeutic strategies. The potential therapeutic benefit for the inhibition of DNA glycosylases was validated by demonstrating synthetic lethality with drugs that are commonly used to limit DNA replication through dNTP pool depletion via inhibition of thymidylate synthetase and dihydrofolate reductase. Additionally, NEIL1-associated synthetic lethality has been achieved in combination with Fanconi anemia, group G. As a prelude to the development of strategies to exploit the potential benefits of DNA glycosylase inhibition, it was necessary to develop a reliable high-throughput screening protocol for this class of enzymes. Using NEIL1 as the proof-of-principle glycosylase, a fluorescence-based assay was developed that utilizes incision of site-specifically modified oligodeoxynucleotides to detect enzymatic activity. This assay was miniaturized to a 1536-well format and used to screen small molecule libraries for inhibitors of the combined glycosylase/AP lyase activities. Among the top hits of these screens were several purine analogs, whose postulated presence in the active site of NEIL1 was consistent with the paradigm of NEIL1 recognition and excision of damaged purines. Although a subset of these small molecules could inhibit other DNA glycosylases that excise oxidatively-induced DNA adducts, they could not inhibit a pyrimidine dimer-specific glycosylase.

DNA hybridization on membrane-modified carbon electrodes

Czech Academy of Sciences Publication Activity Database

Kouřilová, Alena; Babkina, S. S.; Cahová, Kateřina; Havran, Luděk; Jelen, František; Paleček, Emil; Fojta, Miroslav

2005-01-01

Roč. 38, - (2005), s. 2493-2507 ISSN 0003-2719 R&D Projects: GA MPO(CZ) 1H-PK/42; GA AV ČR(CZ) IAA4004402; GA AV ČR(CZ) IBS5004355 Institutional research plan: CEZ:AV0Z50040507 Keywords : DNA hybridization * electrochemical DNA sensor * nitrocellulose membrane Subject RIV: BO - Biophysics Impact factor: 1.036, year: 2005

Cellular responses to environmental DNA damage

Energy Technology Data Exchange (ETDEWEB)

1994-08-01

This volume contains the proceedings of the conference entitled Cellular Responses to Environmental DNA Damage held in Banff,Alberta December 1--6, 1991. The conference addresses various aspects of DNA repair in sessions titled DNA repair; Basic Mechanisms; Lesions; Systems; Inducible Responses; Mutagenesis; Human Population Response Heterogeneity; Intragenomic DNA Repair Heterogeneity; DNA Repair Gene Cloning; Aging; Human Genetic Disease; and Carcinogenesis. Individual papers are represented as abstracts of about one page in length.

Repair of endogenous and ionizing radiation-induced DNA damages: mechanisms and biological functions

International Nuclear Information System (INIS)

Boiteux, S.

2002-01-01

The cellular DNA is continuously exposed to endogenous and exogenous stress. Oxidative stress due to cellular metabolism is the major cause of endogenous DNA damage. On the other hand, ionizing radiation (IR) is an important exogenous stress. Both induce similar DNA damages: damaged bases, abasic sites and strand breakage. Most of these lesions are lethal and/or mutagenic. The survival of the cell is managed by efficient and accurate DNA repair mechanisms that remove lesions before their replication or transcription. DNA repair pathways involved in the removal of IR-induced lesions are briefly described. Base excision repair (BER) is mostly involved in the removal of base damage, abasic sites and single strand breaks. In contrast, DNA double strand breaks are mostly repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). How DNA repair pathways prevent cancer process is also discussed. (author)

DNA damage induced by the direct effect of He ion particles

International Nuclear Information System (INIS)

Urushibara, A.; Shikazono, N.; Watanabe, R.; Fujii, K.; O'Neill, P.; Yokoya, A.

2006-01-01

We present here evidence showing that the yields of DNA lesions induced by He 2+ ions strongly depend on Linear energy transfer (LET). In this study, hydrated plasmid DNA was irradiated with He 2+ ions with LET values of 19, 63 and 95 keVμm -1 . The yields of prompt single-strand breaks (SSBs) are very similar at the varying LET values, whereas the yields of prompt double-strand breaks (DSBs) increase with increasing LET. Further, base lesions were revealed as additional strand breaks by post-irradiation treatment of the DNA with endonuclease III (Nth) and formamido-pyrimidine-DNA glycosylase (Fpg). The reduction in the yield of these enzymatically induced SSBs and DSBs becomes significant as the LET increases. These results suggest that the clustering of DNA lesions becomes more probable in regions of high LET. (authors)

Possible role(s) of nuclear matrix and DNA loop organization in fixation or repair of DNA double-strand breaks

International Nuclear Information System (INIS)

Malyapa, R.S.; Wright, W.D.; Roti Roti, J.L.

1995-01-01

DNA double-strand breaks produced by ionizing radiation are considered to be a critical radiation-induced lesion responsible, in part, for cell killing. However, the manner in which structures within the nucleus involving DNA organization contribute to the balance between fixation or repair of these critical lesions remains largely obscure. The repair process requires both functional enzymes and substrate availability, i.e., access to and orientation of damage sites. Therefore, the ability to repair damaged DNA could be influenced not only by DNA integrity but also by the spatial organization of DNA. Therefore, the authors investigated the possibility that radiation-induced DNA damage differentially affects DNA supercoiling ability in cells of differing radiosensitivities using radioresistant and radiosensitive mutants of different origins. This study was also designed to determine if differences in the composition of the nuclear matrix exist between cell lines of each origin. Results from these studies indicate that differences in the composition of the nuclear matrix proteins and DNA stability might be related to intrinsic radiation resistance

Preparation of carboxyl group-modified palladium nanoparticles in an aqueous solution and their conjugation with DNA

Science.gov (United States)

Wang, Zhifei; Li, Hongying; Zhen, Shuang; He, Nongyue

2012-05-01

The use of nanomaterials in biomolecular labeling and their corresponding detection has been attracting much attention, recently. There are currently very few studies on palladium nanoparticles (Pd NPs) due to their lack of appropriate surface functionalities for conjugation with DNA. In this paper, we thus firstly present an approach to prepare carboxyl group-modified Pd NPs (with an average size of 6 nm) by the use of 11-mercaptoundecanoic acid (MUDA) as a stabilizer in the aqueous solution. The effect of the various reducing reaction conditions on the morphology of the Pd NPs was investigated. The particles were further characterized by TEM, UV-vis, FT-IR and XPS techniques. DNA was finally covalently conjugated to the surface of the Pd NPs through the activation of the carboxyl group, which was confirmed by agarose gel electrophoresis and fluorescence analysis. The resulting Pd NPs-DNA conjugates show high single base pair mismatch discrimination capabilities. This work therefore sets a good foundation for further applications of Pd NPs in bio-analytical research.

Roles of Aag, Alkbh2, and Alkbh3 in the Repair of Carboxymethylated and Ethylated Thymidine Lesions.

Science.gov (United States)

You, Changjun; Wang, Pengcheng; Nay, Stephanie L; Wang, Jianshuang; Dai, Xiaoxia; O'Connor, Timothy R; Wang, Yinsheng

2016-05-20

Environmental and endogenous genotoxic agents can result in a variety of alkylated and carboxymethylated DNA lesions, including N3-ethylthymidine (N3-EtdT), O(2)-EtdT, and O(4)-EtdT as well as N3-carboxymethylthymidine (N3-CMdT) and O(4)-CMdT. By using nonreplicative double-stranded vectors harboring a site-specifically incorporated DNA lesion, we assessed the potential roles of alkyladenine DNA glycosylase (Aag); alkylation repair protein B homologue 2 (Alkbh2); or Alkbh3 in modulating the effects of N3-EtdT, O(2)-EtdT, O(4)-EtdT, N3-CMdT, or O(4)-CMdT on DNA transcription in mammalian cells. We found that the depletion of Aag did not significantly change the transcriptional inhibitory or mutagenic properties of all five examined lesions, suggesting a negligible role of Aag in the repair of these DNA adducts in mammalian cells. In addition, our results revealed that N3-EtdT, but not other lesions, could be repaired by Alkbh2 and Alkbh3 in mammalian cells. Furthermore, we demonstrated the direct reversal of N3-EtdT by purified human Alkbh2 protein in vitro. These findings provided important new insights into the repair of the carboxymethylated and alkylated thymidine lesions in mammalian cells.

Causes and consequences of plant radio-resistance. Formation of DNA basis lesions and self-repairing activity of one of them, the 8-oxo-7,8-dihydro-guanine in Arabidopsis thaliana

International Nuclear Information System (INIS)

Dany, A.L.

2001-01-01

In this research thesis, the author first explains how and why DNA is injured when it is submitted to an oxidizing stress, and describes precisely the formation and the biological consequences of lesions of DNA bases, the 8-oxo-7,8-dihydro-guanine (8-oxoGua). She describes the repairing activities of the oxidized DNA, and more particularly the repairing of 8-oxoGua, in prokaryotes as well as in yeast, mammals and plants. Methodologies used are described, together with the repair activities of the 8-oxo-7,8-dihydro-guanine following a biochemical type approach and a molecular biology approach

Recurring DNA copy number gain at chromosome 9p13 plays a role in the activation of multiple candidate oncogenes in progressing oral premalignant lesions

International Nuclear Information System (INIS)

Towle, Rebecca; Tsui, Ivy F L; Zhu, Yuqi; MacLellan, Sara; Poh, Catherine F; Garnis, Cathie

2014-01-01

Genomic alteration at chromosome 9p has been previously reported as a frequent and critical event in oral premalignancy. While this alteration is typically reported as a loss driven by selection for CDKN2A deactivation (at 9p21.3), we detect a recurrent DNA copy number gain of ∼2.49 Mbp at chromosome 9p13 in oral premalignant lesions (OPLs) that later progressed to invasive lesions. This recurrent alteration event has been validated using fluorescence in situ hybridization in an independent set of OPLs. Analysis of publicly available gene expression datasets aided in identifying three oncogene candidates that may have driven selection for DNA copy number increases in this region (VCP, DCTN3, and STOML2). We performed in vitro silencing and activation experiments for each of these genes in oral cancer cell lines and found that each gene is independently capable of upregulating proliferation and anchorage-independent growth. We next analyzed the activity of each of these genes in biopsies of varying histological grades that were obtained from a diseased oral tissue field in a single patient, finding further molecular evidence of parallel activation of VCP, DCTN3, and STOML2 during progression from normal healthy tissue to invasive oral carcinoma. Our results support the conclusion that DNA gain at 9p13 is important to the earliest stages of oral tumorigenesis and that this alteration event likely contributes to the activation of multiple oncogene candidates capable of governing oral cancer phenotypes

Proton density fat fraction (PDFF) MRI for differentiation of benign and malignant vertebral lesions.

Science.gov (United States)

Schmeel, Frederic Carsten; Luetkens, Julian Alexander; Wagenhäuser, Peter Johannes; Meier-Schroers, Michael; Kuetting, Daniel Lloyd; Feißt, Andreas; Gieseke, Jürgen; Schmeel, Leonard Christopher; Träber, Frank; Schild, Hans Heinz; Kukuk, Guido Matthias

2018-06-01

To investigate whether proton density fat fraction (PDFF) measurements using a six-echo modified Dixon sequence can help to differentiate between benign and malignant vertebral bone marrow lesions. Sixty-six patients were prospectively enrolled in our study. In addition to conventional MRI at 3.0-Tesla including at least sagittal T2-weighted/spectral attenuated inversion recovery and T1-weighted sequences, all patients underwent a sagittal six-echo modified Dixon sequence of the spine. The mean PDFF was calculated using regions of interest and compared between vertebral lesions. A cut-off value of 6.40% in PDFF was determined by receiver operating characteristic curves and used to differentiate between malignant (benign and malignant vertebral lesions with a high diagnostic accuracy. • Establishing a diagnosis of indeterminate vertebral lesions is a common clinical problem • Benign bone marrow processes may mimic the signal alterations observed in malignancy • PDFF differentiates between benign and malignant lesions with a high diagnostic accuracy • PDFF of non-neoplastic vertebral lesions is significantly higher than that of malignancy • PDFF from six-echo modified Dixon may help avoid potentially harmful bone biopsy.

Differential recruitment of DNA Ligase I and III to DNA repair sites

Science.gov (United States)

Mortusewicz, Oliver; Rothbauer, Ulrich; Cardoso, M. Cristina; Leonhardt, Heinrich

2006-01-01

DNA ligation is an essential step in DNA replication, repair and recombination. Mammalian cells contain three DNA Ligases that are not interchangeable although they use the same catalytic reaction mechanism. To compare the recruitment of the three eukaryotic DNA Ligases to repair sites in vivo we introduced DNA lesions in human cells by laser microirradiation. Time lapse microscopy of fluorescently tagged proteins showed that DNA Ligase III accumulated at microirradiated sites before DNA Ligase I, whereas we could detect only a faint accumulation of DNA Ligase IV. Recruitment of DNA Ligase I and III to repair sites was cell cycle independent. Mutational analysis and binding studies revealed that DNA Ligase I was recruited to DNA repair sites by interaction with PCNA while DNA Ligase III was recruited via its BRCT domain mediated interaction with XRCC1. Selective recruitment of specialized DNA Ligases may have evolved to accommodate the particular requirements of different repair pathways and may thus enhance efficiency of DNA repair. PMID:16855289

Bisphenol a promotes cell survival following oxidative DNA damage in mouse fibroblasts.

Directory of Open Access Journals (Sweden)

Natalie R Gassman

Full Text Available Bisphenol A (BPA is a biologically active industrial chemical used in production of consumer products. BPA has become a target of intense public scrutiny following concerns about its association with human diseases such as obesity, diabetes, reproductive disorders, and cancer. Recent studies link BPA with the generation of reactive oxygen species, and base excision repair (BER is responsible for removing oxidatively induced DNA lesions. Yet, the relationship between BPA and BER has yet to be examined. Further, the ubiquitous nature of BPA allows continuous exposure of the human genome concurrent with the normal endogenous and exogenous insults to the genome, and this co-exposure may impact the DNA damage response and repair. To determine the effect of BPA exposure on base excision repair of oxidatively induced DNA damage, cells compromised in double-strand break repair were treated with BPA alone or co-exposed with either potassium bromate (KBrO3 or laser irradiation as oxidative damaging agents. In experiments with KBrO3, co-treatment with BPA partially reversed the KBrO3-induced cytotoxicity observed in these cells, and this was coincident with an increase in guanine base lesions in genomic DNA. The improvement in cell survival and the increase in oxidatively induced DNA base lesions were reminiscent of previous results with alkyl adenine DNA glycosylase-deficient cells, suggesting that BPA may prevent initiation of repair of oxidized base lesions. With laser irradiation-induced DNA damage, treatment with BPA suppressed DNA repair as revealed by several indicators. These results are consistent with the hypothesis that BPA can induce a suppression of oxidized base lesion DNA repair by the base excision repair pathway.

Cleavage of phosphorothioated DNA and methylated DNA by the type IV restriction endonuclease ScoMcrA.

Directory of Open Access Journals (Sweden)

Guang Liu

2010-12-01

Full Text Available Many taxonomically diverse prokaryotes enzymatically modify their DNA by replacing a non-bridging oxygen with a sulfur atom at specific sequences. The biological implications of this DNA S-modification (phosphorothioation were unknown. We observed that simultaneous expression of the dndA-E gene cluster from Streptomyces lividans 66, which is responsible for the DNA S-modification, and the putative Streptomyces coelicolor A(32 Type IV methyl-dependent restriction endonuclease ScoA3McrA (Sco4631 leads to cell death in the same host. A His-tagged derivative of ScoA3McrA cleaved S-modified DNA and also Dcm-methylated DNA in vitro near the respective modification sites. Double-strand cleavage occurred 16-28 nucleotides away from the phosphorothioate links. DNase I footprinting demonstrated binding of ScoA3McrA to the Dcm methylation site, but no clear binding could be detected at the S-modified site under cleavage conditions. This is the first report of in vitro endonuclease activity of a McrA homologue and also the first demonstration of an enzyme that specifically cleaves S-modified DNA.

Processing of free radical damaged DNA bases

International Nuclear Information System (INIS)

Wallace, S.

2003-01-01

Free radicals produced during the radiolysis of water gives rise to a plethora of DNA damages including single strand breaks, sites of base loss and a wide variety of purine and pyrimidine base lesions. All these damages are processed in cells by base excision repair. The oxidative DNA glycosylases which catalyze the first step in the removal of a base damage during base excision repair evolved primarily to protect the cells from the deleterious mutagenic effects of single free radical-induced DNA lesions arising during oxidative metabolism. This is evidenced by the high spontaneous mutation rate in bacterial mutants lacking the oxidative DNA glycosylases. However, when a low LET photon transverses the DNA molecule, a burst of free radicals is produced during the radiolysis of water that leads to the formation of clustered damages in the DNA molecule, that are recognized by the oxidative DNA glycosylases. When substrates containing two closely opposed sugar damages or base and sugar damages are incubated with the oxidative DNA glycosylases in vitro, one strand is readily incised by the lyase activity of the DNA glycosylase. Whether or not the second strand is incised depends on the distance between the strand break resulting from the incised first strand and the remaining DNA lesion on the other strand. If the lesions are more than two or three base pairs apart, the second strand is readily cleaved by the DNA glycosylase, giving rise to a double strand break. Even if the entire base excision repair system is reconstituted in vitro, whether or not a double strand break ensues depends solely upon the ability of the DNA glycosylase to cleave the second strand. These data predicted that cells deficient in the oxidative DNA glycosylases would be radioresistant while those that overproduce an oxidative DNA glycosylase would be radiosensitive. This prediction was indeed borne in Escherichia coli that is, mutants lacking the oxidative DNA glycosylases are radioresistant

Selective base excision repair of DNA damage by the non-base-flipping DNA glycosylase AlkC

Energy Technology Data Exchange (ETDEWEB)

Shi, Rongxin; Mullins, Elwood A.; Shen, Xing; #8208; Xing; Lay, Kori T.; Yuen, Philip K.; David, Sheila S.; Rokas, Antonis; Eichman, Brandt F. (UCD); (Vanderbilt)

2017-10-20

DNA glycosylases preserve genome integrity and define the specificity of the base excision repair pathway for discreet, detrimental modifications, and thus, the mechanisms by which glycosylases locate DNA damage are of particular interest. Bacterial AlkC and AlkD are specific for cationic alkylated nucleobases and have a distinctive HEAT-like repeat (HLR) fold. AlkD uses a unique non-base-flipping mechanism that enables excision of bulky lesions more commonly associated with nucleotide excision repair. In contrast, AlkC has a much narrower specificity for small lesions, principally N3-methyladenine (3mA). Here, we describe how AlkC selects for and excises 3mA using a non-base-flipping strategy distinct from that of AlkD. A crystal structure resembling a catalytic intermediate complex shows how AlkC uses unique HLR and immunoglobulin-like domains to induce a sharp kink in the DNA, exposing the damaged nucleobase to active site residues that project into the DNA. This active site can accommodate and excise N3-methylcytosine (3mC) and N1-methyladenine (1mA), which are also repaired by AlkB-catalyzed oxidative demethylation, providing a potential alternative mechanism for repair of these lesions in bacteria.

Multiplex electrochemical DNA platform for femtomolar-level quantification of genetically modified soybean.

Science.gov (United States)

Manzanares-Palenzuela, C Lorena; de-los-Santos-Álvarez, Noemí; Lobo-Castañón, María Jesús; López-Ruiz, Beatriz

2015-06-15

Current EU regulations on the mandatory labeling of genetically modified organisms (GMOs) with a minimum content of 0.9% would benefit from the availability of reliable and rapid methods to detect and quantify DNA sequences specific for GMOs. Different genosensors have been developed to this aim, mainly intended for GMO screening. A remaining challenge, however, is the development of genosensing platforms for GMO quantification, which should be expressed as the number of event-specific DNA sequences per taxon-specific sequences. Here we report a simple and sensitive multiplexed electrochemical approach for the quantification of Roundup-Ready Soybean (RRS). Two DNA sequences, taxon (lectin) and event-specific (RR), are targeted via hybridization onto magnetic beads. Both sequences are simultaneously detected by performing the immobilization, hybridization and labeling steps in a single tube and parallel electrochemical readout. Hybridization is performed in a sandwich format using signaling probes labeled with fluorescein isothiocyanate (FITC) or digoxigenin (Dig), followed by dual enzymatic labeling using Fab fragments of anti-Dig and anti-FITC conjugated to peroxidase or alkaline phosphatase, respectively. Electrochemical measurement of the enzyme activity is finally performed on screen-printed carbon electrodes. The assay gave a linear range of 2-250 pM for both targets, with LOD values of 650 fM (160 amol) and 190 fM (50 amol) for the event-specific and the taxon-specific targets, respectively. Results indicate that the method could be applied for GMO quantification below the European labeling threshold level (0.9%), offering a general approach for the rapid quantification of specific GMO events in foods. Copyright © 2015 Elsevier B.V. All rights reserved.

PNA-PEG modified silicon platforms as functional bio-interfaces for applications in DNA microarrays and biosensors.

Science.gov (United States)

Cattani-Scholz, Anna; Pedone, Daniel; Blobner, Florian; Abstreiter, Gerhard; Schwartz, Jeffrey; Tornow, Marc; Andruzzi, Luisa

2009-03-09

The synthesis and characterization of two types of silicon-based biofunctional interfaces are reported; each interface bonds a dense layer of poly(ethylene glycol) (PEG(n)) and peptide nucleic acid (PNA) probes. Phosphonate self-assembled monolayers were derivatized with PNA using a maleimido-terminated PEG(45). Similarly, siloxane monolayers were functionalized with PNA using a maleimido-terminated PEG(45) spacer and were subsequently modified with a shorter methoxy-terminated PEG(12) ("back-filling"). The long PEG(45) spacer was used to distance the PNA probe from the surface and to minimize undesirable nonspecific adsorption of DNA analyte. The short PEG(12) "back-filler" was used to provide additional passivation of the surface against nonspecific DNA adsorption. X-ray photoelectron spectroscopic (XPS) analysis near the C 1s and N 1s ionization edges was done to characterize chemical groups formed in the near-surface region, which confirmed binding of PEG and PNA to the phosphonate and silane films. XPS also indicated that additional PEG chains were tethered to the surface during the back-filling process. Fluorescence hybridization experiments were carried out with complementary and noncDNA strands; both phosphonate and siloxane biofunctional surfaces were effective for hybridization of cDNA strands and significantly reduced nonspecific adsorption of the analyte. Spatial patterns were prepared by polydimethylsiloxane (PDMS) micromolding on the PNA-functionalized surfaces; selective hybridization of fluorescently labeled DNA was shown at the PNA functionalized regions, and physisorption at the probe-less PEG-functionalized regions was dramatically reduced. These results show that PNA-PEG derivatized phosphonate monolayers hold promise for the smooth integration of device surface chemistry with semiconductor technology for the fabrication of DNA biosensors. In addition, our results confirm that PNA-PEG derivatized self-assembled carboxyalkylsiloxane films are

Assessment of DNA degradation induced by thermal and UV radiation processing: implications for quantification of genetically modified organisms.

Science.gov (United States)

Ballari, Rajashekhar V; Martin, Asha

2013-12-01

DNA quality is an important parameter for the detection and quantification of genetically modified organisms (GMO's) using the polymerase chain reaction (PCR). Food processing leads to degradation of DNA, which may impair GMO detection and quantification. This study evaluated the effect of various processing treatments such as heating, baking, microwaving, autoclaving and ultraviolet (UV) irradiation on the relative transgenic content of MON 810 maize using pRSETMON-02, a dual target plasmid as a model system. Amongst all the processing treatments examined, autoclaving and UV irradiation resulted in the least recovery of the transgenic (CaMV 35S promoter) and taxon-specific (zein) target DNA sequences. Although a profound impact on DNA degradation was seen during the processing, DNA could still be reliably quantified by Real-time PCR. The measured mean DNA copy number ratios of the processed samples were in agreement with the expected values. Our study confirms the premise that the final analytical value assigned to a particular sample is independent of the degree of DNA degradation since the transgenic and the taxon-specific target sequences possessing approximately similar lengths degrade in parallel. The results of our study demonstrate that food processing does not alter the relative quantification of the transgenic content provided the quantitative assays target shorter amplicons and the difference in the amplicon size between the transgenic and taxon-specific genes is minimal. Copyright © 2013 Elsevier Ltd. All rights reserved.

Damages induced in lambda phage DNA by enzyme-generated triplet acetone

International Nuclear Information System (INIS)

Menck, C.F.; Cabral Neto, J.B.; Gomes, R.A.; Faljoni-Alario, A.

1985-01-01

Exposure of lambda phage to triplet acetone, generated during the aerobic oxidation of isobutanal by peroxidase, leads to genome lesions. The majority of these lesions are detected as DNA single-strand breaks only in alkaline conditions, so true breaks were not observed. Also, no sites sensitive to UV-endonuclease from Micrococcus luteus were found in DNA from treated phage. The participation of triplet acetone in the generation of such DNA damage is discussed. (Author) [pt

Pnc1p-mediated nicotinamide clearance modifies the epigenetic properties of rDNA silencing in Saccharomyces cerevisiae.

Science.gov (United States)

McClure, Julie M; Gallo, Christopher M; Smith, Daniel L; Matecic, Mirela; Hontz, Robert D; Buck, Stephen W; Racette, Frances G; Smith, Jeffrey S

2008-10-01

The histone deacetylase activity of Sir2p is dependent on NAD(+) and inhibited by nicotinamide (NAM). As a result, Sir2p-regulated processes in Saccharomyces cerevisiae such as silencing and replicative aging are susceptible to alterations in cellular NAD(+) and NAM levels. We have determined that high concentrations of NAM in the growth medium elevate the intracellular NAD(+) concentration through a mechanism that is partially dependent on NPT1, an important gene in the Preiss-Handler NAD(+) salvage pathway. Overexpression of the nicotinamidase, Pnc1p, prevents inhibition of Sir2p by the excess NAM while maintaining the elevated NAD(+) concentration. This growth condition alters the epigenetics of rDNA silencing, such that repression of a URA3 reporter gene located at the rDNA induces growth on media that either lacks uracil or contains 5-fluoroorotic acid (5-FOA), an unusual dual phenotype that is reminiscent of telomeric silencing (TPE) of URA3. Despite the similarities to TPE, the modified rDNA silencing phenotype does not require the SIR complex. Instead, it retains key characteristics of typical rDNA silencing, including RENT and Pol I dependence, as well as a requirement for the Preiss-Handler NAD(+) salvage pathway. Exogenous nicotinamide can therefore have negative or positive impacts on rDNA silencing, depending on the PNC1 expression level.

DNA damage and mutagenesis of lambda phage induced by gamma-rays

International Nuclear Information System (INIS)

Bertram, Heidi

1988-01-01

Lambda phage DNA was gamma irradiated in aqueous solution and strand breakage determined. Twice as much minor structural damage per lethal hit was found in this DNA compared with DNA from irradiated phage suspensions. The in vitro irradiated DNA was repackaged into infectious particles. Induction of mutations in the cI or cII cistron was scored using SOS-induced host cells. In vitro prepared particles were found to have second-order kinetics for mutagenesis induced by gamma rays indicating two pre-mutational events were necessary to produce a mutation, but bacteria-free phage suspensions ('lys-phage') showed single hit kinetics for mutagenesis after irradiation. Increase in the mutation rate in the phage particles was mainly due to minor lesions, i.e. ssb, als and unidentified base damage. In lys-phage, mutagenesis might be enhanced by clustered DNA damage - configuration not existing in pack-phage. Loss of infectivity was analysed in comparison with structural damage. All lesions contributed to biological inactivation. Minor lesions were tolerated by lambda phage to a limited extent. Major lesions (e.g. dsb) contributed most to infectivity loss and were considered lethal events. (U.K.)

Detection, characterization and measure of a new radiation-induced damage in isolated and cellular DNA; Detection, caracterisation et mesure d'un nouveau dommage radio-induit de l'ADN isole et cellulaire

Energy Technology Data Exchange (ETDEWEB)

Regulus, P

2006-10-15

Deoxyribonucleic acid (DNA) contains the genetic information and chemical injury to this macromolecule may have severe biological consequences. We report here the detection of 4 new radiation-induced DNA lesions by using a high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) approach. For that purpose, the characteristic fragmentation of most 2'-deoxy-ribo nucleosides, the loss of 116 Da corresponding to the loss of the 2-deoxyribose moiety, was used in the so-called neutral loss mode of the HPLC-MS/MS. One of the newly detected lesions, named dCyd341 because it is a 2'-deoxycytidine modification exhibiting a molecular weight of 341 Da, was also detected in cellular DNA. Characterization of this modified nucleoside was performed using NMR and exact mass determination of the product obtained by chemical synthesis. A mechanism of formation was then proposed, in which the first event is the H-abstraction at the C4 position of a 2-deoxyribose moiety. Then, the sugar modification produced exhibits a reactive aldehyde that, through reaction with a vicinal cytosine base, gives rise to dCyd341. dCyd341 could be considered as a complex damage since its formation involves a DNA strand break and a cross-link between a damaged sugar residue and a vicinal cytosine base located most probably on the complementary DNA strand. In addition to its characterization, preliminary biological studies revealed that cells are able to remove the lesion from DNA. Repair studies have revealed the ability of cells to excise the lesion. Identification of the repair systems involved could represent an interesting challenge. (author)

Human papillomavirus in oral lesions Virus papiloma humano en lesiones orales

Directory of Open Access Journals (Sweden)

Joaquín V. Gónzalez

2007-08-01

Full Text Available Growing evidence suggests a role for human papillomavirus (HPV in oral cancer; however its involvement is still controversial. This study evaluates the frequency of HPV DNA in a variety of oral lesions in patients from Argentina. A total of 77 oral tissue samples from 66 patients were selected (cases; the clinical-histopathological diagnoses corresponded to: 11 HPV- associated benign lesions, 8 non-HPV associated benign lesions, 33 premalignant lesions and 25 cancers. Sixty exfoliated cell samples from normal oral mucosa were used as controls. HPV detection and typing were performed by polymerase chain reaction (PCR using primers MY09, 11, combined with RFLP or alternatively PCR using primers GP5+, 6+ combined with dot blot hybridization. HPV was detected in 91.0% of HPV- associated benign lesions, 14.3% of non-HPV associated benign lesions, 51.5% of preneoplasias and 60.0% of cancers. No control sample tested HPV positive. In benign HPV- associated lesions, 30.0% of HPV positive samples harbored high-risk types, while in preneoplastic lesions the value rose to 59.9%. In cancer lesions, HPV detection in verrucous carcinoma was 88.9% and in squamous cell carcinoma 43.8%, with high-risk type rates of 75.5% and 85.6%, respectively. The high HPV frequency detected in preneoplastic and neoplastic lesions supports an HPV etiological role in at least a subset of oral cancers.Crecientes evidencias sugieren que el virus Papiloma humano (HPV tiene un rol en el cáncer oral; sin embargo su participación es todavía controvertida. Este estudio evalúa la frecuencia de ADN de HPV en una variedad de lesiones orales de pacientes de Argentina. Se seleccionaron 77 muestras de tejido oral de 66 pacientes (casos; el diagnóstico histo-patológico correspondió a: 11 lesiones benignas asociadas a HPV, 8 lesiones benignas no asociadas a HPV, 33 lesiones premalignas y 25 cánceres. Como controles se usaron 60 muestras de células exfoliadas de mucosa oral normal. La

Repair of DNA damage in Deinococcus radiodurans

International Nuclear Information System (INIS)

Evans, D.M.

1984-01-01

The repair of DNA lesions in Deinococcus radiodurans was examined with particular reference to DNA excision repair of ultraviolet light (UV) induced pyrimidine dimers. The characteristics of excision repair via UV endonucleases α and β in vivo varied with respect to (a) the substrate range of the enzymes, (b) the rate of repair of DNA damage (c) the requirement for a protein synthesised in response to DNA damage to attenuate exonuclease action at repairing regions. UV endonuclease α is postulated to incise DNA in a different manner from UV endonuclease β thus defining the method of subsequent repair. Several DNA damage specific endonuclease activities independent of α and β are described. Mutations of the uvsA, uvsF and uvsG genes resulted in an increase in single-strand breaks in response to DNA damage producing uncontrolled DNA degradation. Evidence is presented that these genes have a role in limiting the access of UV endonuclease β to DNA lesions. uvsF and uvsG are also shown to be linked to the mtoA gene. Mutation of uvsH and reo-1 produces further distinct phenotypes which are discussed. An overall model of excision repair of DNA damage in Deinococcus radiodurans is presented. (author)

Quantification of intrahepatic total HBV DNA in liver biopsies of HBV-infected patients by a modified version of COBAS® Ampliprep/COBAS®TaqMan HBV test v2.0.

Science.gov (United States)

Salpini, Romina; Piermatteo, Lorenzo; Gill, Upkar; Battisti, Arianna; Stazi, Francesca; Guenci, Tania; Giannella, Sara; Serafini, Valentina; Kennedy, Patrick T F; Perno, Carlo Federico; Svicher, Valentina; Ciotti, Marco

2017-08-01

Intrahepatic total HBV DNA (it-HBV DNA) level might reflect the size of virus reservoir and correlate with the histological status of the liver. To quantitate it-HBV DNA in a series of 70 liver biopsies obtained from hepatitis B chronic patients, a modified version of the COBAS ® Ampliprep/COBAS ® TaqMan HBV test v2.0 was used for this purpose. The linearity and reproducibility of the modified protocol was tested by quantifying serial dilutions of a full-length HBV containing plasmid and it-HBV DNA from a reference patient. A good linear trend between the expected values and those generated by the assay was observed at different concentrations of both plasmid and reference patient (R 2  = 0.994 and 0.962, respectively). Differences between the values obtained in two independent runs were ≤0.3 log IU for the plasmid and ≤0.6 log IU/mg for the reference patient, showing a high inter-run reproducibility. In the 70 liver biopsies, it-HBV DNA level ranged from 1.4 to 5.4 log IU/mg, with a good linearity and reproducibility between the values obtained in two runs [R 2  = 0.981; median (IQR) difference of it-HBV DNA 0.05 (0.02-0.09) IU/mg]. The modified COBAS ® Ampliprep/COBAS ® TaqMan HBV test v2.0 allows an accurate quantitation of it-HBV DNA. Its determination may have prognostic value and may be a useful tool for the new therapeutic strategies aimed at eradicating the HBV infection.

DNA degradation in genetically modified rice with Cry1Ab by food processing methods: implications for the quantification of genetically modified organisms.

Science.gov (United States)

Xing, Fuguo; Zhang, Wei; Selvaraj, Jonathan Nimal; Liu, Yang

2015-05-01

Food processing methods contribute to DNA degradation, thereby affecting genetically modified organism detection and quantification. This study evaluated the effect of food processing methods on the relative transgenic content of genetically modified rice with Cry1Ab. In steamed rice and rice noodles, the levels of Cry1Ab were ⩾ 100% and <83%, respectively. Frying and baking in rice crackers contributed to a reduction in Pubi and Cry1Ab, while microwaving caused a decrease in Pubi and an increase in Cry1Ab. The processing methods of sweet rice wine had the most severe degradation effects on Pubi and Cry1Ab. In steamed rice and rice noodles, Cry1Ab was the most stable, followed by SPS and Pubi. However, in rice crackers and sweet rice wine, SPS was the most stable, followed by Cry1Ab and Pubi. Therefore, Cry1Ab is a better representative of transgenic components than is Pubi because the levels of Cry1Ab were less affected compared to Pubi. Copyright © 2014 Elsevier Ltd. All rights reserved.

DNA methylation in metabolic disorders

DEFF Research Database (Denmark)

Barres, Romain; Zierath, Juleen R

2011-01-01

DNA methylation is a major epigenetic modification that controls gene expression in physiologic and pathologic states. Metabolic diseases such as diabetes and obesity are associated with profound alterations in gene expression that are caused by genetic and environmental factors. Recent reports...... have provided evidence that environmental factors at all ages could modify DNA methylation in somatic tissues, which suggests that DNA methylation is a more dynamic process than previously appreciated. Because of the importance of lifestyle factors in metabolic disorders, DNA methylation provides...... a mechanism by which environmental factors, including diet and exercise, can modify genetic predisposition to disease. This article considers the current evidence that defines a role for DNA methylation in metabolic disorders....

TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism.

Science.gov (United States)

Harley, Margaret E; Murina, Olga; Leitch, Andrea; Higgs, Martin R; Bicknell, Louise S; Yigit, Gökhan; Blackford, Andrew N; Zlatanou, Anastasia; Mackenzie, Karen J; Reddy, Kaalak; Halachev, Mihail; McGlasson, Sarah; Reijns, Martin A M; Fluteau, Adeline; Martin, Carol-Anne; Sabbioneda, Simone; Elcioglu, Nursel H; Altmüller, Janine; Thiele, Holger; Greenhalgh, Lynn; Chessa, Luciana; Maghnie, Mohamad; Salim, Mahmoud; Bober, Michael B; Nürnberg, Peter; Jackson, Stephen P; Hurles, Matthew E; Wollnik, Bernd; Stewart, Grant S; Jackson, Andrew P

2016-01-01

DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism. We establish that TRAIP relocalizes to sites of DNA damage, where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to ultraviolet (UV) irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.

Mannan-Modified PLGA Nanoparticles for Targeted Gene Delivery

Directory of Open Access Journals (Sweden)

Fansheng Kong

2012-01-01

Full Text Available The studies of targeted gene delivery nanocarriers have gained increasing attention during the past decades. In this study, mannan modified DNA loaded bioadhesive PLGA nanoparticles (MAN-DNA-NPs were investigated for targeted gene delivery to the Kupffer cells (KCs. Bioadhesive PLGA nanoparticles were prepared and subsequently bound with pEGFP. Following the coupling of the mannan-based PE-grafted ligands (MAN-PE with the DNA-NPs, the MAN-DNA-NPs were delivered intravenously to rats. The transfection efficiency was determined from the isolated KCs and flow cytometry was applied for the quantitation of gene expression after 48 h post transfection. The size of the MAN-DNA-NPs was found to be around 190 nm and the Zeta potential was determined to be −15.46mV. The pEGFP binding capacity of MAN-DNA-NPs was (88.9±5.8% and the in vitro release profiles of the MAN-DNA-NPs follow the Higuchi model. When compared with non-modified DNA-NPs and Lipofectamine 2000-DNA, MAN-DNA-NPs produced the highest gene expressions, especially in vivo. The in vivo data from flow cytometry analysis showed that MAN-DNA-NPs displayed a remarkably higher transfection efficiency (39% than non-modified DNA-NPs (25% and Lipofectamine 2000-DNA (23% in KCs. The results illustrate that MAN-DNA-NPs have the ability to target liver KCs and could function as promising active targeting drug delivery vectors.

DNA extraction techniques compared for accurate detection of genetically modified organisms (GMOs) in maize food and feed products.

Science.gov (United States)

Turkec, Aydin; Kazan, Hande; Karacanli, Burçin; Lucas, Stuart J

2015-08-01

In this paper, DNA extraction methods have been evaluated to detect the presence of genetically modified organisms (GMOs) in maize food and feed products commercialised in Turkey. All the extraction methods tested performed well for the majority of maize foods and feed products analysed. However, the highest DNA content was achieved by the Wizard, Genespin or the CTAB method, all of which produced optimal DNA yield and purity for different maize food and feed products. The samples were then screened for the presence of GM elements, along with certified reference materials. Of the food and feed samples, 8 % tested positive for the presence of one GM element (NOS terminator), of which half (4 % of the total) also contained a second element (the Cauliflower Mosaic Virus 35S promoter). The results obtained herein clearly demonstrate the presence of GM maize in the Turkish market, and that the Foodproof GMO Screening Kit provides reliable screening of maize food and feed products.

Neocarzinostatin as a probe for DNA protection activity--molecular interaction with caffeine.

Science.gov (United States)

Chin, Der-Hang; Li, Huang-Hsien; Kuo, Hsiu-Maan; Chao, Pei-Dawn Lee; Liu, Chia-Wen

2012-04-01

Neocarzinostatin (NCS), a potent mutagen and carcinogen, consists of an enediyne prodrug and a protein carrier. It has a unique double role in that it intercalates into DNA and imposes radical-mediated damage after thiol activation. Here we employed NCS as a probe to examine the DNA-protection capability of caffeine, one of common dietary phytochemicals with potential cancer-chemopreventive activity. NCS at the nanomolar concentration range could induce significant single- and double-strand lesions in DNA, but up to 75 ± 5% of such lesions were found to be efficiently inhibited by caffeine. The percentage of inhibition was caffeine-concentration dependent, but was not sensitive to the DNA-lesion types. The well-characterized activation reactions of NCS allowed us to explore the effect of caffeine on the enediyne-generated radicals. Postactivation analyses by chromatographic and mass spectroscopic methods identified a caffeine-quenched enediyne-radical adduct, but the yield was too small to fully account for the large inhibition effect on DNA lesions. The affinity between NCS chromophore and DNA was characterized by a fluorescence-based kinetic method. The drug-DNA intercalation was hampered by caffeine, and the caffeine-induced increases in DNA-drug dissociation constant was caffeine-concentration dependent, suggesting importance of binding affinity in the protection mechanism. Caffeine has been shown to be both an effective free radical scavenger and an intercalation inhibitor. Our results demonstrated that caffeine ingeniously protected DNA against the enediyne-induced damages mainly by inhibiting DNA intercalation beforehand. The direct scavenging of the DNA-bound NCS free radicals by caffeine played only a minor role. Copyright © 2011 Wiley Periodicals, Inc.

Voxel-based lesion-symptom mapping of stroke lesions underlying somatosensory deficits

Directory of Open Access Journals (Sweden)

Sarah Meyer

2016-01-01

Full Text Available The aim of this study was to investigate the relationship between stroke lesion location and the resulting somatosensory deficit. We studied exteroceptive and proprioceptive somatosensory symptoms and stroke lesions in 38 patients with first-ever acute stroke. The Erasmus modified Nottingham Sensory Assessment was used to clinically evaluate somatosensory functioning in the arm and hand within the first week after stroke onset. Additionally, more objective measures such as the perceptual threshold of touch and somatosensory evoked potentials were recorded. Non-parametric voxel-based lesion-symptom mapping was performed to investigate lesion contribution to different somatosensory deficits in the upper limb. Additionally, structural connectivity of brain areas that demonstrated the strongest association with somatosensory symptoms was determined, using probabilistic fiber tracking based on diffusion tensor imaging data from a healthy age-matched sample. Voxels with a significant association to somatosensory deficits were clustered in two core brain regions: the central parietal white matter, also referred to as the sensory component of the superior thalamic radiation, and the parietal operculum close to the insular cortex, representing the secondary somatosensory cortex. Our objective recordings confirmed findings from clinical assessments. Probabilistic tracking connected the first region to thalamus, internal capsule, brain stem, postcentral gyrus, cerebellum, and frontal pathways, while the second region demonstrated structural connections to thalamus, insular and primary somatosensory cortex. This study reveals that stroke lesions in the sensory fibers of the superior thalamocortical radiation and the parietal operculum are significantly associated with multiple exteroceptive and proprioceptive deficits in the arm and hand.

Voxel-based lesion-symptom mapping of stroke lesions underlying somatosensory deficits.

Science.gov (United States)

Meyer, Sarah; Kessner, Simon S; Cheng, Bastian; Bönstrup, Marlene; Schulz, Robert; Hummel, Friedhelm C; De Bruyn, Nele; Peeters, Andre; Van Pesch, Vincent; Duprez, Thierry; Sunaert, Stefan; Schrooten, Maarten; Feys, Hilde; Gerloff, Christian; Thomalla, Götz; Thijs, Vincent; Verheyden, Geert

2016-01-01

The aim of this study was to investigate the relationship between stroke lesion location and the resulting somatosensory deficit. We studied exteroceptive and proprioceptive somatosensory symptoms and stroke lesions in 38 patients with first-ever acute stroke. The Erasmus modified Nottingham Sensory Assessment was used to clinically evaluate somatosensory functioning in the arm and hand within the first week after stroke onset. Additionally, more objective measures such as the perceptual threshold of touch and somatosensory evoked potentials were recorded. Non-parametric voxel-based lesion-symptom mapping was performed to investigate lesion contribution to different somatosensory deficits in the upper limb. Additionally, structural connectivity of brain areas that demonstrated the strongest association with somatosensory symptoms was determined, using probabilistic fiber tracking based on diffusion tensor imaging data from a healthy age-matched sample. Voxels with a significant association to somatosensory deficits were clustered in two core brain regions: the central parietal white matter, also referred to as the sensory component of the superior thalamic radiation, and the parietal operculum close to the insular cortex, representing the secondary somatosensory cortex. Our objective recordings confirmed findings from clinical assessments. Probabilistic tracking connected the first region to thalamus, internal capsule, brain stem, postcentral gyrus, cerebellum, and frontal pathways, while the second region demonstrated structural connections to thalamus, insular and primary somatosensory cortex. This study reveals that stroke lesions in the sensory fibers of the superior thalamocortical radiation and the parietal operculum are significantly associated with multiple exteroceptive and proprioceptive deficits in the arm and hand.

Delayed repair of radiation induced clustered DNA damage: Friend or foe?

International Nuclear Information System (INIS)

Eccles, Laura J.; O'Neill, Peter; Lomax, Martine E.

2011-01-01

A signature of ionizing radiation exposure is the induction of DNA clustered damaged sites, defined as two or more lesions within one to two helical turns of DNA by passage of a single radiation track. Clustered damage is made up of double strand breaks (DSB) with associated base lesions or abasic (AP) sites, and non-DSB clusters comprised of base lesions, AP sites and single strand breaks. This review will concentrate on the experimental findings of the processing of non-DSB clustered damaged sites. It has been shown that non-DSB clustered damaged sites compromise the base excision repair pathway leading to the lifetime extension of the lesions within the cluster, compared to isolated lesions, thus the likelihood that the lesions persist to replication and induce mutation is increased. In addition certain non-DSB clustered damaged sites are processed within the cell to form additional DSB. The use of E. coli to demonstrate that clustering of DNA lesions is the major cause of the detrimental consequences of ionizing radiation is also discussed. The delayed repair of non-DSB clustered damaged sites in humans can be seen as a 'friend', leading to cell killing in tumour cells or as a 'foe', resulting in the formation of mutations and genetic instability in normal tissue.

Principles of DNA architectonics: design of DNA-based nanoobjects

International Nuclear Information System (INIS)

Vinogradova, O A; Pyshnyi, D V

2012-01-01

The methods of preparation of monomeric DNA blocks that serve as key building units for the construction of complex DNA objects are described. Examples are given of the formation of DNA blocks based on native and modified oligonucleotide components using hydrogen bonding and nucleic acid-specific types of bonding and also some affinity interactions with RNA, proteins, ligands. The static discrete and periodic two- and three-dimensional DNA objects reported to date are described systematically. Methods used to prove the structures of DNA objects and the prospects for practical application of nanostructures based on DNA and its analogues in biology, medicine and biophysics are considered. The bibliography includes 195 references.

The DinB•RecA complex of Escherichia coli mediates an efficient and high-fidelity response to ubiquitous alkylation lesions.

Science.gov (United States)

Cafarelli, Tiziana M; Rands, Thomas J; Godoy, Veronica G

2014-03-01

Alkylation DNA lesions are ubiquitous, and result from normal cellular metabolism as well as from treatment with methylating agents and chemotherapeutics. DNA damage tolerance by translesion synthesis DNA polymerases has an important role in cellular resistance to alkylating agents. However, it is not yet known whether Escherichia coli (E. coli) DNA Pol IV (DinB) alkylation lesion bypass efficiency and fidelity in vitro are similar to those inferred by genetic analyses. We hypothesized that DinB-mediated bypass of 3-deaza-3-methyladenine, a stable analog of 3-methyladenine, the primary replication fork-stalling alkylation lesion, would be of high fidelity. We performed here the first kinetic analyses of E. coli DinB•RecA binary complexes. Whether alone or in a binary complex, DinB inserted the correct deoxyribonucleoside triphosphate (dNTP) opposite either lesion-containing or undamaged template; the incorporation of other dNTPs was largely inefficient. DinB prefers undamaged DNA, but the DinB•RecA binary complex increases its catalytic efficiency on lesion-containing template, perhaps as part of a regulatory mechanism to better respond to alkylation damage. Notably, we find that a DinB derivative with enhanced affinity for RecA, either alone or in a binary complex, is less efficient and has a lower fidelity than DinB or DinB•RecA. This finding contrasts our previous genetic analyses. Therefore, mutagenesis resulting from alkylation lesions is likely limited in cells by the activity of DinB•RecA. These two highly conserved proteins play an important role in maintaining genomic stability when cells are faced with ubiquitous DNA damage. Kinetic analyses are important to gain insights into the mechanism(s) regulating TLS DNA polymerases. Copyright © 2013 Wiley Periodicals, Inc.

[Examination of processed vegetable foods for the presence of common DNA sequences of genetically modified tomatoes].

Science.gov (United States)

Kitagawa, Mamiko; Nakamura, Kosuke; Kondo, Kazunari; Ubukata, Shoji; Akiyama, Hiroshi

2014-01-01

The contamination of processed vegetable foods with genetically modified tomatoes was investigated by the use of qualitative PCR methods to detect the cauliflower mosaic virus 35S promoter (P35S) and the kanamycin resistance gene (NPTII). DNA fragments of P35S and NPTII were detected in vegetable juice samples, possibly due to contamination with the genomes of cauliflower mosaic virus infecting juice ingredients of Brassica species and soil bacteria, respectively. Therefore, to detect the transformation construct sequences of GM tomatoes, primer pairs were designed for qualitative PCR to specifically detect the border region between P35S and NPTII, and the border region between nopaline synthase gene promoter and NPTII. No amplification of the targeted sequences was observed using genomic DNA purified from the juice ingredients. The developed qualitative PCR method is considered to be a reliable tool to check contamination of products with GM tomatoes.

Fanconi anemia (cross)linked to DNA repair.

Science.gov (United States)

Niedernhofer, Laura J; Lalai, Astrid S; Hoeijmakers, Jan H J

2005-12-29

Fanconi anemia is characterized by hypersensitivity to DNA interstrand crosslinks (ICLs) and susceptibility to tumor formation. Despite the identification of numerous Fanconi anemia (FANC) genes, the mechanism by which proteins encoded by these genes protect a cell from DNA interstrand crosslinks remains unclear. The recent discovery of two DNA helicases that, when defective, cause Fanconi anemia tips the balance in favor of the direct involvement of the FANC proteins in DNA repair and the bypass of DNA lesions.

Inhibition of DNA replication by ultraviolet light

International Nuclear Information System (INIS)

Edenberg, H.J.

1976-01-01

DNA replication in ultraviolet-irradiated HeLa cells was studied by two different techniques: measurements of the kinetics of semiconservative DNA synthesis, and DNA fiber autoradiography. In examining the kinetics of semiconservative DNA synthesis, density label was used to avoid measuring the incorporation due to repair replication. The extent of inhibition varied with time. After doses of less than 10 J/m 2 the rate was initially depressed but later showed some recovery. After higher doses, a constant, low rate of synthesis was seen for at least the initial 6 h. An analysis of these data indicated that the inhibition of DNA synthesis could be explained by replication forks halting at pyrimidine dimers. DNA fiber autoradiography was used to further characterize replication after ultraviolet irradiation. The average length of labeled segments in irradiated cells increased in the time immediately after irradiation, and then leveled off. This is the predicted pattern if DNA synthesis in each replicon continued at its previous rate until a lesion is reached, and then halted. The frequency of lesions that block synthesis is approximately the same as the frequency of pyrimidine dimers

Quantitative Analysis of the Mutagenic Potential of 1-Aminopyrene-DNA Adduct Bypass Catalyzed by Y-Family DNA Polymerases

Science.gov (United States)

Sherrer, Shanen M.; Taggart, David J.; Pack, Lindsey R.; Malik, Chanchal K.; Basu, Ashis K.; Suo, Zucai

2012-01-01

N- (deoxyguanosin-8-yl)-1-aminopyrene (dGAP) is the predominant nitro polyaromatic hydrocarbon product generated from the air pollutant 1-nitropyrene reacting with DNA. Previous studies have shown that dGAP induces genetic mutations in bacterial and mammalian cells. One potential source of these mutations is the error-prone bypass of dGAP lesions catalyzed by the low-fidelity Y-family DNA polymerases. To provide a comparative analysis of the mutagenic potential of the translesion DNA synthesis (TLS) of dGAP, we employed short oligonucleotide sequencing assays (SOSAs) with the model Y-family DNA polymerase from Sulfolobus solfataricus, DNA Polymerase IV (Dpo4), and the human Y-family DNA polymerases eta (hPolη), kappa (hPolκ), and iota (hPolι). Relative to undamaged DNA, all four enzymes generated far more mutations (base deletions, insertions, and substitutions) with a DNA template containing a site-specifically placed dGAP. Opposite dGAP and at an immediate downstream template position, the most frequent mutations made by the three human enzymes were base deletions and the most frequent base substitutions were dAs for all enzymes. Based on the SOSA data, Dpo4 was the least error-prone Y-family DNA polymerase among the four enzymes during the TLS of dGAP. Among the three human Y-family enzymes, hPolκ made the fewest mutations at all template positions except opposite the lesion site. hPolκ was significantly less error-prone than hPolι and hPolη during the extension of dGAP bypass products. Interestingly, the most frequent mutations created by hPolι at all template positions were base deletions. Although hRev1, the fourth human Y-family enzyme, could not extend dGAP bypass products in our standing start assays, it preferentially incorporated dCTP opposite the bulky lesion. Collectively, these mutagenic profiles suggest that hPolkk and hRev1 are the most suitable human Y-family DNA polymerases to perform TLS of dGAP in humans. PMID:22917544

TTV and HPV co-infection in cervical smears of patients with cervical lesions

Directory of Open Access Journals (Sweden)

Tachezy Ruth

2009-07-01

Full Text Available Abstract Background The female lower genital tract is a gateway for pathogens entering the host through the mucous membrane. One of the prevalent human viruses is Torque teno virus (TTV. The major reported routes of TTV transmission are fecal-oral and parenteral. Furthermore, other modes of transmission, e.g. sexual contact, are suggested. To investigate the sexual route of TTV transmission, cervical smears of healthy women and those with cervical lesions were screened for the presence of TTV DNA. Methods TTV DNA was studied in cervical smears of 95 patients with cervical lesions and 55 healthy women. Paired serum samples were available from 55 and 42 women, respectively. All healthy women had normal cytology while 44 patients had histologically confirmed low-grade lesion (LGL and 51 high-grade lesion (HGL. TTV DNA was detected with primers specific for the non-coding region. In 40 paired cervical smears and serum samples, the phylogenetic group of TTV isolates was determined. The presence of HPV DNA in cervical smears was detected by means of PCR with MY09/11 primers. Results The prevalence of TTV DNA in cervical smears of healthy women was 52.7% and was comparable with that in paired serum samples (50%. Symptomatic women had significantly higher prevalence of TTV DNA in cervical smears (74.7% than healthy controls. The TTV DNA prevalence in patient serum samples was 51%. The phylogenetic groups of TTV serum isolates were concordant with those of TTV from cervical smears of the same subjects. In cervical smears, a wider variety of TTV isolates was found. The viral loads in cervical smears were 10 to 1000 times as high as in sera. The HPV-positive study subjects had significantly higher TTV DNA prevalence than HPV negatives. The prevalence of TTV was not associated with disease severity. Conclusion High prevalence of TTV in cervical smears suggests that sexual transmission is another mode of expansion of TTV infection among the population. The

Multiple effects of fluorescent light on repair of ultraviolet-induced DNA lesions in cultured goldfish cells

International Nuclear Information System (INIS)

Uchida, Nobuhiro; Mitani, Hiroshi; Shima, Akihiro

1995-01-01

It is known that fluorescent light illumination prior to UV irradiation (FL preillumination) of cultured fish cells increases photorepair (PR) ability. In the present study, it was found that FL preillumination also enhanced UV resistance of logarithmically growing cells in the dark. This enhancement of UV resistance differs from induction of PR because it was not suppressed by cyclohexamide (CH) and it occurred immediately after FL preillumination. The effects of FL preillumination on repair of UV-induced DNA lesions in the dark were examined by an endonuclease-sensitive site assay to measure the repair of cyclobutyl pyrimidine dimers, and by enzyme-linked immunosorbent assay to quantitate the repair of (6-4) photoproducts. It was found that excision repair ability for (6-4) photoproducts in the genome overall was increased by FL preillumination. Moreover, a decrease in (6-4) photoproducts by FL illumination immediately after UV irradiation of the cells was found, the decrement being enhanced by FL preillumination with or without CH. (author)

Multiple effects of fluorescent light on repair of ultraviolet-induced DNA lesions in cultured goldfish cells

Energy Technology Data Exchange (ETDEWEB)

Uchida, Nobuhiro; Mitani, Hiroshi; Shima, Akihiro [Tokyo Univ. (Japan). Lab. of Radiation Biology

1995-01-01

It is known that fluorescent light illumination prior to UV irradiation (FL preillumination) of cultured fish cells increases photorepair (PR) ability. In the present study, it was found that FL preillumination also enhanced UV resistance of logarithmically growing cells in the dark. This enhancement of UV resistance differs from induction of PR because it was not suppressed by cyclohexamide (CH) and it occurred immediately after FL preillumination. The effects of FL preillumination on repair of UV-induced DNA lesions in the dark were examined by an endonuclease-sensitive site assay to measure the repair of cyclobutyl pyrimidine dimers, and by enzyme-linked immunosorbent assay to quantitate the repair of (6-4) photoproducts. It was found that excision repair ability for (6-4) photoproducts in the genome overall was increased by FL preillumination. Moreover, a decrease in (6-4) photoproducts by FL illumination immediately after UV irradiation of the cells was found, the decrement being enhanced by FL preillumination with or without CH. (author).

Characterization of DNA antigens from immune complexes deposited in the skin of patients with systemic lupus erythematosus

Institute of Scientific and Technical Information of China (English)

曾凡钦; 尹若菲; 谭国珍; 郭庆; 许德清

2004-01-01

Background Skin lesions are common manifestations in systemic lupus erythematosus (SLE). It is still unknown what the definite pathogenesis of skin involvement was and whether DNA participated in it. Our study was designed to explore the pathogenetic role and nature of nuclear antigen (DNA) deposited in the skin lesions of patients with SLE.Methods Thirty skin samples from patients with SLE and 2 normal skin samples were studied. Extracellular DNA was evaluated by indirect immunofluorescence methods. The deposited immune complexes were extracted by cryoprecipitation, and DNA was then isolated with phenol and chloroform. DNA fragment sizes were detected by agarose gel electrophoresis. Finally, 8 different probes were used to analyze the origin of these DNA molecules using Dot hybridization.Results Extracellular DNA staining was found only in skin lesions, mainly those located in the basement membrane zone, vascular wall, and hair follicle wall. Normal skin and non-lesion SLE skin showed no fluorescence at locations outside the nuclei. There were no differences in the rate and intensity of extracellular DNA staining when comparing active phase to remission phase patients. No relationship was found between extracellular DNA and circulating anti-dsDNA antibodies. Deposited DNA fragments clustered into four bands of somewhat discrete sizes: 20 000 bp, 1300 bp, 800-900 bp, 100-200 bp. Small sized fragments (100-200 bp) were positively correlated with disease activity (P<0.05, r=0.407). Dot hybridization showed significant homology of the various extracellular DNA fragments examined with human genomic DNA, but not with DNA from the microorganisms and viruses we examined. There were also homologies between DNA samples from different individuals.Conclusions DNA and its immune complexes may contribute to the pathogenesis of skin lesions in SLE. These DNA molecules range in size from 100 bp to 20 kb and may be endogenous in origin.

Coordinating repair of oxidative DNA damage with transcription and replication

International Nuclear Information System (INIS)

Cooper, P.K.

2003-01-01

Transcription-coupled repair (TCR) preferentially removes DNA lesions from template strands of active genes. Defects in TCR, which acts both on lesions removed by nucleotide excision repair (NER) and on oxidative lesions removed by base excision repair (BER), underlie the fatal developmental disorder Cockayne syndrome. Although its detailed mechanism remains unknown, TCR involves recognition of a stalled RNA polymerase (RNAP), removal or remodeling of RNAP to allow access to the lesion, and recruitment of repair enzymes. At a minimum, these early steps require a non-enzymatic function of the multifunctional repair protein XPG, the CSB protein with ATP-dependent chromatin remodeling activity, and the TFIIH complex (including the XPB and XPD helicases) that is also required for basal transcription initiation and NER. XPG exists in the cell in a complex with TFIIH, and in vitro evidence has suggested that it interacts with CSB. To address the mechanism of TCR, we are characterizing protein-DNA and protein-protein interactions of XPG. We show that XPG preferentially binds to double-stranded DNA containing bubbles resembling in size the unpaired regions associated with transcription. Two distinct domains of XPG are required for the observed strong binding specificity and stability. XPG both interacts directly with CSB and synergistically binds with it to bubble DNA, and it strongly stimulates the bubble DNA-dependent ATPase activity of CSB. Significantly for TCR, XPG also interacts directly with RNAP II, binds both the protein and nucleic acid components (the R-loop) of a stalled RNA polymerase, and forms a ternary complex with CSB and the stalled RNAP. These results are consistent with the model that XPG and CSB jointly interact with the DNA/chromatin structure in the vicinity of the stalled transcriptional apparatus and with the transcriptional machinery itself to remodel the chromatin and either move or remodel the blocked RNA polymerase to expose the lesion

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

Science.gov (United States)

Ceylan, Deniz; Tuna, Gamze; Kirkali, Güldal; Tunca, Zeliha; Can, Güneş; Arat, Hidayet Ece; Kant, Melis; Dizdaroglu, Miral; Özerdem, Ayşegül

2018-05-01

Oxidatively-induced DNA damage has previously been associated with bipolar disorder. More recently, impairments in DNA repair mechanisms have also been reported. We aimed to investigate oxidatively-induced DNA lesions and expression of DNA glycosylases involved in base excision repair in euthymic patients with bipolar disorder compared to healthy individuals. DNA base lesions including both base and nucleoside modifications were measured using gas chromatography-tandem mass spectrometry and liquid chromatography-tandem mass spectrometry with isotope-dilution in DNA samples isolated from leukocytes of euthymic patients with bipolar disorder (n = 32) and healthy individuals (n = 51). The expression of DNA repair enzymes OGG1 and NEIL1 were measured using quantitative real-time polymerase chain reaction. The levels of malondialdehyde were measured using high performance liquid chromatography. Seven DNA base lesions in DNA of leukocytes of patients and healthy individuals were identified and quantified. Three of them had significantly elevated levels in bipolar patients when compared to healthy individuals. No elevation of lipid peroxidation marker malondialdehyde was observed. The level of OGG1 expression was significantly reduced in bipolar patients compared to healthy individuals, whereas the two groups exhibited similar levels of NEIL1 expression. Our results suggest that oxidatively-induced DNA damage occurs and base excision repair capacity may be decreased in bipolar patients when compared to healthy individuals. Measurement of oxidatively-induced DNA base lesions and the expression of DNA repair enzymes may be of great importance for large scale basic research and clinical studies of bipolar disorder. Copyright © 2018 Elsevier B.V. All rights reserved.

Inhibition of HIV-1 reverse transcriptase-catalyzed synthesis by intercalated DNA Benzo[a]Pyrene 7,8-Dihydrodiol-9,10-Epoxide adducts.

Directory of Open Access Journals (Sweden)

Parvathi Chary

Full Text Available To aid in the characterization of the relationship of structure and function for human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT, this investigation utilized DNAs containing benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE-modified primers and templates as a probe of the architecture of this complex. BPDE lesions that differed in their stereochemistry around the C10 position were covalently linked to N (6-adenine and positioned in either the primer or template strand of a duplex template-primer. HIV-1 RT exhibited a stereoisomer-specific and strand-specific difference in replication when the BPDE-lesion was placed in the template versus the primer strand. When the C10 R-BPDE adduct was positioned in the primer strand in duplex DNA, 5 nucleotides from the 3΄ end of the primer terminus, HIV-1 RT could not fully replicate the template, producing truncated products; this block to further synthesis did not affect rates of dissociation or DNA binding affinity. Additionally, when the adducts were in the same relative position, but located in the template strand, similar truncated products were observed with both the C10 R and C10 S BPDE adducts. These data suggest that the presence of covalently-linked intercalative DNA adducts distant from the active site can lead to termination of DNA synthesis catalyzed by HIV-1 RT.

Metatranscriptomics reveals overall active bacterial composition in caries lesions

Directory of Open Access Journals (Sweden)

Aurea Simón-Soro

2014-10-01

Full Text Available Background: Identifying the microbial species in caries lesions is instrumental to determine the etiology of dental caries. However, a significant proportion of bacteria in carious lesions have not been cultured, and the use of molecular methods has been limited to DNA-based approaches, which detect both active and inactive or dead microorganisms. Objective: To identify the RNA-based, metabolically active bacterial composition of caries lesions at different stages of disease progression in order to provide a list of potential etiological agents of tooth decay. Design: Non-cavitated enamel caries lesions (n=15 and dentin caries lesions samples (n=12 were collected from 13 individuals. RNA was extracted and cDNA was constructed, which was used to amplify the 16S rRNA gene. The resulting 780 bp polymerase chain reaction products were pyrosequenced using Titanium-plus chemistry, and the sequences obtained were used to determine the bacterial composition. Results: A mean of 4,900 sequences of the 16S rRNA gene with an average read length of 661 bp was obtained per sample, giving a comprehensive view of the active bacterial communities in caries lesions. Estimates of bacterial diversity indicate that the microbiota of cavities is highly complex, each sample containing between 70 and 400 metabolically active species. The composition of these bacterial consortia varied among individuals and between caries lesions of the same individuals. In addition, enamel and dentin lesions had a different bacterial makeup. Lactobacilli were found almost exclusively in dentin cavities. Streptococci accounted for 40% of the total active community in enamel caries, and 20% in dentin caries. However, Streptococcus mutans represented only 0.02–0.73% of the total bacterial community. Conclusions: The data indicate that the etiology of dental caries is tissue dependent and that the disease has a clear polymicrobial origin. The low proportion of mutans streptococci

Conformational Effects of UV Light on DNA Origami.

Science.gov (United States)

Chen, Haorong; Li, Ruixin; Li, Shiming; Andréasson, Joakim; Choi, Jong Hyun

2017-02-01

The responses of DNA origami conformation to UV radiation of different wavelengths and doses are investigated. Short- and medium-wavelength UV light can cause photo-lesions in DNA origami. At moderate doses, the lesions do not cause any visible defects in the origami, nor do they significantly affect the hybridization capability. Instead, they help relieve the internal stress in the origami structure and restore it to the designed conformation. At high doses, staple dissociation increases which causes structural disintegration. Long-wavelength UV does not show any effect on origami conformation by itself. We show that this UV range can be used in conjunction with photoactive molecules for photo-reconfiguration, while avoiding any damage to the DNA structures.

Rhodium metalloinsertor binding generates a lesion with selective cytotoxicity for mismatch repair-deficient cells.

Science.gov (United States)

Bailis, Julie M; Weidmann, Alyson G; Mariano, Natalie F; Barton, Jacqueline K

2017-07-03

The DNA mismatch repair (MMR) pathway recognizes and repairs errors in base pairing and acts to maintain genome stability. Cancers that have lost MMR function are common and comprise an important clinical subtype that is resistant to many standard of care chemotherapeutics such as cisplatin. We have identified a family of rhodium metalloinsertors that bind DNA mismatches with high specificity and are preferentially cytotoxic to MMR-deficient cells. Here, we characterize the cellular mechanism of action of the most potent and selective complex in this family, [Rh(chrysi)(phen)(PPO)] 2+ (Rh-PPO). We find that Rh-PPO binding induces a lesion that triggers the DNA damage response (DDR). DDR activation results in cell-cycle blockade and inhibition of DNA replication and transcription. Significantly, the lesion induced by Rh-PPO is not repaired in MMR-deficient cells, resulting in selective cytotoxicity. The Rh-PPO mechanism is reminiscent of DNA repair enzymes that displace mismatched bases, and is differentiated from other DNA-targeted chemotherapeutics such as cisplatin by its potency, cellular mechanism, and selectivity for MMR-deficient cells.

Sensitive DNA impedance biosensor for detection of cancer, chronic lymphocytic leukemia, based on gold nanoparticles/gold modified electrode

International Nuclear Information System (INIS)

Ensafi, Ali A.; Taei, M.; Rahmani, H.R.; Khayamian, T.

2011-01-01

Highlights: → Chronic lymphocytic leukemia causes an increase in the number of white blood cells. → We introduced a highly sensitive biosensor for the detection of chronic lymphocytic leukemia. → A suitable 25-mer ssDNA probe was immobilized on the surface of the gold nanoparticles. → We used electrochemical impedance spectroscopy as a suitable tool for the detection. → Detection of chronic lymphocytic leukemia in blood sample was checked using the sensor. - Abstract: A simple and sensitive DNA impedance sensor was prepared for the detection of chronic lymphocytic leukemia. The DNA electrochemical biosensor is worked based on the electrochemical impedance spectroscopic (EIS) detection of the sequence-specific DNA related to chronic lymphocytic leukemia. The ssDNA probe was immobilized on the surface of the gold nanoparticles. Compared to the bare gold electrode, the gold nanoparticles-modified electrode could improve the density of the probe DNA attachment and hence the sensitivity of the DNA sensor greatly. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy were performed in a solution containing 1.0 mmol L -1 K 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ] and 50 mmol L -1 phosphate buffer saline pH 6.87 plus 50 mmol L -1 KCl. In the CV studied, the potential was cycled from 0.0 to +0.65 V with a scan rate of 50 mV s -1 . Using EIS, the difference of the electron transfer resistance (ΔR et ) was linear with the logarithm of the complementary oligonucleotides sequence concentrations in the range of 7.0 x 10 -12 -2.0 x 10 -7 mol L -1 , with a detection limit of 1.0 x 10 -12 mol L -1 . In addition, the DNA sensor showed a good reproducibility and stability during repeated regeneration and hybridization cycles.

Transcriptome Analysis Reveals Markers of Aberrantly Activated Innate Immunity in Vitiligo Lesional and Non-Lesional Skin

Science.gov (United States)

Huang, Yuanshen; Wang, Yang; Yu, Jie; Gao, Min; Levings, Megan; Wei, Shencai; Zhang, Shengquan; Xu, Aie; Su, Mingwan; Dutz, Jan; Zhang, Xuejun; Zhou, Youwen

2012-01-01

Background Vitiligo is characterized by the death of melanocytes in the skin. This is associated with the presence of T cell infiltrates in the lesional borders. However, at present, there is no detailed and systematic characterization on whether additional cellular or molecular changes are present inside vitiligo lesions. Further, it is unknown if the normal appearing non-lesional skin of vitiligo patients is in fact normal. The purpose of this study is to systematically characterize the molecular and cellular characteristics of the lesional and non-lesional skin of vitiligo patients. Methods and Materials Paired lesional and non-lesional skin biopsies from twenty-three vitiligo patients and normal skin biopsies from sixteen healthy volunteers were obtained with informed consent. The following aspects were analyzed: (1) transcriptome changes present in vitiligo skin using DNA microarrays and qRT-PCR; (2) abnormal cellular infiltrates in vitiligo skin explant cultures using flow cytometry; and (3) distribution of the abnormal cellular infiltrates in vitiligo skin using immunofluorescence microscopy. Results Compared with normal skin, vitiligo lesional skin contained 17 genes (mostly melanocyte-specific genes) whose expression was decreased or absent. In contrast, the relative expression of 13 genes was up-regulated. The up-regulated genes point to aberrant activity of the innate immune system, especially natural killer cells in vitiligo. Strikingly, the markers of heightened innate immune responses were also found to be up-regulated in the non-lesional skin of vitiligo patients. Conclusions and Clinical Implications As the first systematic transcriptome characterization of the skin in vitiligo patients, this study revealed previously unknown molecular markers that strongly suggest aberrant innate immune activation in the microenvironment of vitiligo skin. Since these changes involve both lesional and non-lesional skin, our results suggest that therapies targeting

Modifying action of DNA synthesis precursors on Aspergillus nidulans conidium irradiated by ultraviolet and X-rays

International Nuclear Information System (INIS)

Muronets, E.M.; Kameneva, S.V.

1975-01-01

Modification of inactivation action of radiation on conidia Aspergillus nidulans, UVS + and UVS strains, by desoxynucleosides, purine and pyrimidine bases is shown. The modification manifested in increased conidia survival is revealed when the precursor of DNA synthesis is added to the suspension before exposure to ultraviolet or X-rays. In the case of postradiation application of the substance no modification is observed. The modifying effect of different precursors becomes equally apparent with equimolar solutions and increases at higher concentration of the latter. An increase in thymidine endogenic pool in the exposed conidia does not affect their survival. When conidia are exposed to ultraviolet rays through a thymidine filter the survival rate increases to the same extent as in the case when they are exposed to irradiation in thymidine solution. The authors suggest that modification of the inactivating radiation action by DNA precursors at exposure of conidia Aspergillus nidulans is caused by the radioprotective effect of precursors not related to reparation [ru

Pre-steady-state fluorescence analysis of damaged DNA transfer from human DNA glycosylases to AP endonuclease APE1.

Science.gov (United States)

Kuznetsova, Alexandra A; Kuznetsov, Nikita A; Ishchenko, Alexander A; Saparbaev, Murat K; Fedorova, Olga S

2014-10-01

DNA glycosylases remove the modified, damaged or mismatched bases from the DNA by hydrolyzing the N-glycosidic bonds. Some enzymes can further catalyze the incision of a resulting abasic (apurinic/apyrimidinic, AP) site through β- or β,δ-elimination mechanisms. In most cases, the incision reaction of the AP-site is catalyzed by special enzymes called AP-endonucleases. Here, we report the kinetic analysis of the mechanisms of modified DNA transfer from some DNA glycosylases to the AP endonuclease, APE1. The modified DNA contained the tetrahydrofurane residue (F), the analogue of the AP-site. DNA glycosylases AAG, OGG1, NEIL1, MBD4(cat) and UNG from different structural superfamilies were used. We found that all DNA glycosylases may utilise direct protein-protein interactions in the transient ternary complex for the transfer of the AP-containing DNA strand to APE1. We hypothesize a fast "flip-flop" exchange mechanism of damaged and undamaged DNA strands within this complex for monofunctional DNA glycosylases like MBD4(cat), AAG and UNG. Bifunctional DNA glycosylase NEIL1 creates tightly specific complex with DNA containing F-site thereby efficiently competing with APE1. Whereas APE1 fast displaces other bifunctional DNA glycosylase OGG1 on F-site thereby induces its shifts to undamaged DNA regions. Kinetic analysis of the transfer of DNA between human DNA glycosylases and APE1 allows us to elucidate the critical step in the base excision repair pathway. Copyright © 2014 Elsevier B.V. All rights reserved.

DNA-mediated gene transfer into ataxia-telangiectasia cells

International Nuclear Information System (INIS)

Crescenzi, M.; Pulciani, S.; Carbonari, M.; Tedesco, L.; Russo, G.; Gaetano, C.; Fiorilli, M.

1986-01-01

The complete description of the genetic lesion(s) underlying the AT mutation might, therefore, highlight not only a DNA-repair pathwa, but also an important aspect of the physiology of lymphocytes. DNA-mediated gene transfer into eukaryotic cells has proved a powerful tool for the molecular cloning of certain mammalian genes. The possibility to clone a given gene using this technology depends, basically, on the availability of a selectable marker associated with the expression of the transfected gene in the recipient cell. Recently, a human DNA repair gene has been cloned in CHO mutant cells by taking advantage of the increased resistance to ultraviolet radiation of the transformants. As a preliminary step toward the molecular cloning of the AT gene(s), the authors have attempted to confer radioresistance to AT cells by transfection with normal human DNA

The cyclopurine deoxynucleosides: DNA repair, biological effects, mechanistic insights, and unanswered questions.

Science.gov (United States)

Brooks, Philip J

2017-06-01

Patients with the genetic disease xeroderma pigmentosum (XP) who lack the capacity to carry out nucleotides excision repair (NER) have a dramatically elevated risk of skin cancer on sun exposed areas of the body. NER is the DNA repair mechanism responsible for the removal of DNA lesions resulting from ultraviolet light. In addition, a subset of XP patients develop a progressive neurodegenerative disease, referred to as XP neurologic disease, which is thought to be the result of accumulation of endogenous DNA lesions that are repaired by NER but not other repair pathways. The 8,5-cyclopurine deoxynucleotides (cyPu) have emerged as leading candidates for such lesions, in that they result from the reaction of the hydroxyl radical with DNA, are strong blocks to transcription in human cells, and are repaired by NER but not base excision repair. Here I present a focused perspective on progress into understating the repair and biological effects of these lesions. In doing so, I emphasize the role of Tomas Lindahl and his laboratory in stimulating cyPu research. I also include a critical evaluation of the evidence supporting a role for cyPu lesions in XP neurologic disease, with a focus on outstanding questions, and conceptual and technologic challenges. Copyright © 2017. Published by Elsevier Inc.

A model system for DNA repair studies

International Nuclear Information System (INIS)

Lange, C.S.; Perlmutter, E.

1984-01-01

The search for the ''lethal lesion:'' which would yield a molecular explanation of biological survival curves led to attempts to correlate unrepaired DNA lesions with loss of reproductive integrity. Such studies have shown the crucial importance of DNA repair systems. The unrepaired DSB has been sought for such correlation, but in such study the DNA was too large, polydisperse, and/or structurally complex to permit precise measurement of break induction and repair. Therefore, an analog of higher order systems but with a genome of readily measurable size, is needed. Bacteriophage T4 is such an analog. Both its biological (PFU) and molecular (DNA) survival curves are exponentials. Its aerobic /sub PFU/D/sub 37///sub DNA/D/sub 37/ ratio, (410 +- 4.5Gy/540 +- 25 Gy) indicates that 76 +- 4% of lethality at low multiplicity infection (moi 1) the survival is greater than can be explained if the assumption of no parental DSB repair were valid. Both T4 and its host have DSB repair systems which can be studied by the infectious center method. Results of such studies are discussed

Electrochemical detection of avian influenza virus H5N1 gene sequence using a DNA aptamer immobilized onto a hybrid nanomaterial-modified electrode

International Nuclear Information System (INIS)

Liu Xianggang; Cheng Ziqiang; Fan Hai; Ai Shiyun; Han Ruixia

2011-01-01

Highlights: → A sensitive electrochemical biosensor for the detection of gene sequence was developed. → The biosensor was assembled by MWNT, polypyrrole nanowires and gold nanoparticles. → The hybrid nanomaterials could provide a porous structure with good properties. → The biosensor has highly selectivity and sensitivity. → The design strategy is expected to have extensive applications in other biosensors - Abstract: A sensitive electrochemical method for the detection of avian influenza virus (AIV) H5N1 gene sequence using a DNA aptamer immobilized onto a hybrid nanomaterial-modified electrode was developed. To enhance the selectivity and sensitivity, the modified electrode was assembled with multi-wall carbon nanotubes (MWNT), polypyrrole nanowires (PPNWs) and gold nanoparticles (GNPs). This electrode offered a porous structure with a large effective surface area, highly electrocatalytic activities and electronic conductivity. Therefore, the amount of DNA aptamer immobilized onto the electrode was increased while the accessibility of the detection target was maintained. The biosensor is based on the hybridization and preferred orientation of a DNA aptamer immobilized onto a modified electrode surface with its target (H5N1 specific sequence) present in solution. It is selective for the H5N1 specific sequence, and the signal of the indicator was approximately linear to log(concentration) of the H5N1 specific sequence from 5.0 x 10 -12 to 1.0 x 10 -9 M (R = 0.9863) with a detection limit of 4.3 x 10 -13 M. These studies showed that the new hybrid nanomaterial (MWNT/PPNWs/GNPs) and the DNA aptamer could be used to fabricate an electrochemical biosensor for gene sequence detection. Furthermore, this design strategy is expected to have extensive applications in other biosensors.

Electrochemical detection of avian influenza virus H5N1 gene sequence using a DNA aptamer immobilized onto a hybrid nanomaterial-modified electrode

Energy Technology Data Exchange (ETDEWEB)

Liu Xianggang [College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, Shandong (China); Cheng Ziqiang, E-mail: czqsd@126.com [College of Animal Science and Technology, Shandong Agricultural University, Taian 271018, Shandong (China); Fan Hai [College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, Shandong (China); Ai Shiyun, E-mail: ashy@sdau.edu.cn [College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, Shandong (China); Han Ruixia [College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, Shandong (China)

2011-07-15

Highlights: > A sensitive electrochemical biosensor for the detection of gene sequence was developed. > The biosensor was assembled by MWNT, polypyrrole nanowires and gold nanoparticles. > The hybrid nanomaterials could provide a porous structure with good properties. > The biosensor has highly selectivity and sensitivity. > The design strategy is expected to have extensive applications in other biosensors - Abstract: A sensitive electrochemical method for the detection of avian influenza virus (AIV) H5N1 gene sequence using a DNA aptamer immobilized onto a hybrid nanomaterial-modified electrode was developed. To enhance the selectivity and sensitivity, the modified electrode was assembled with multi-wall carbon nanotubes (MWNT), polypyrrole nanowires (PPNWs) and gold nanoparticles (GNPs). This electrode offered a porous structure with a large effective surface area, highly electrocatalytic activities and electronic conductivity. Therefore, the amount of DNA aptamer immobilized onto the electrode was increased while the accessibility of the detection target was maintained. The biosensor is based on the hybridization and preferred orientation of a DNA aptamer immobilized onto a modified electrode surface with its target (H5N1 specific sequence) present in solution. It is selective for the H5N1 specific sequence, and the signal of the indicator was approximately linear to log(concentration) of the H5N1 specific sequence from 5.0 x 10{sup -12} to 1.0 x 10{sup -9} M (R = 0.9863) with a detection limit of 4.3 x 10{sup -13} M. These studies showed that the new hybrid nanomaterial (MWNT/PPNWs/GNPs) and the DNA aptamer could be used to fabricate an electrochemical biosensor for gene sequence detection. Furthermore, this design strategy is expected to have extensive applications in other biosensors.

Amperometric biosensor for hydrogen peroxide based on Hemoglobin/DNA/Poly-2,6-pyridinediamine modified gold electrode

International Nuclear Information System (INIS)

Tong Zhongqiang; Yuan Ruo; Chai Yaqin; Chen Shihong; Xie Yi

2007-01-01

An amperometric biosensor for hydrogen peroxide (H 2 O 2 ) was fabricated based on immobilization of hemoglobin (Hb) on DNA/Poly-2,6-pyridinediamine (PPD) modified Au electrode. PPD thin films were firstly electro-deposited on Au electrode surface which provide a template to attach negatively charged DNA molecules by electrostatic attraction. The adsorbed DNA network provides a good microenvironment for the immobilization of biomolecules and promotes electron transfer between the immobilized Hb and the electrode surface. The fabrication process of the biosensor was characterized by electrochemical impedance spectroscopy. Experimental conditions influencing the biosensor performance such as pH, potential and temperature were assessed and optimized. The proposed biosensor displayed a good electrocatalytic response to the reduction of H 2 O 2 , its linear range is 1.7 μM to 3 mM with a detection limit of 1.0 μM based on the signal-to-noise ratio of 3 (S/N = 3) under the optimized conditions. The Michaelis-Menten constant K m app of Hb immobilized on the electrode surface was found to be 0.8 mM. The biosensor shows high sensitivity and stability. Importantly, this deposition methodology could be further developed for the immobilization of other proteins and biocompounds

Delayed repair of radiation induced clustered DNA damage: Friend or foe?

Science.gov (United States)

Eccles, Laura J.; O’Neill, Peter; Lomax, Martine E.

2011-01-01

A signature of ionizing radiation exposure is the induction of DNA clustered damaged sites, defined as two or more lesions within one to two helical turns of DNA by passage of a single radiation track. Clustered damage is made up of double strand breaks (DSB) with associated base lesions or abasic (AP) sites, and non-DSB clusters comprised of base lesions, AP sites and single strand breaks. This review will concentrate on the experimental findings of the processing of non-DSB clustered damaged sites. It has been shown that non-DSB clustered damaged sites compromise the base excision repair pathway leading to the lifetime extension of the lesions within the cluster, compared to isolated lesions, thus the likelihood that the lesions persist to replication and induce mutation is increased. In addition certain non-DSB clustered damaged sites are processed within the cell to form additional DSB. The use of E. coli to demonstrate that clustering of DNA lesions is the major cause of the detrimental consequences of ionizing radiation is also discussed. The delayed repair of non-DSB clustered damaged sites in humans can be seen as a “friend”, leading to cell killing in tumour cells or as a “foe”, resulting in the formation of mutations and genetic instability in normal tissue. PMID:21130102

Recruitment of DNA methyltransferase I to DNA repair sites

Science.gov (United States)

Mortusewicz, Oliver; Schermelleh, Lothar; Walter, Joachim; Cardoso, M. Cristina; Leonhardt, Heinrich

2005-01-01

In mammalian cells, the replication of genetic and epigenetic information is directly coupled; however, little is known about the maintenance of epigenetic information in DNA repair. Using a laser microirradiation system to introduce DNA lesions at defined subnuclear sites, we tested whether the major DNA methyltransferase (Dnmt1) or one of the two de novo methyltransferases (Dnmt3a, Dnmt3b) are recruited to sites of DNA repair in vivo. Time lapse microscopy of microirradiated mammalian cells expressing GFP-tagged Dnmt1, Dnmt3a, or Dnmt3b1 together with red fluorescent protein-tagged proliferating cell nuclear antigen (PCNA) revealed that Dnmt1 and PCNA accumulate at DNA damage sites as early as 1 min after irradiation in S and non-S phase cells, whereas recruitment of Dnmt3a and Dnmt3b was not observed. Deletion analysis showed that Dnmt1 recruitment was mediated by the PCNA-binding domain. These data point to a direct role of Dnmt1 in the restoration of epigenetic information during DNA repair. PMID:15956212

DNA repair and radiation sensitivity in mammalian cells

International Nuclear Information System (INIS)

Chen, D.J.C.; Stackhouse, M.; Chen, D.S.

1993-01-01

Ionizing radiation induces various types of damage in mammalian cells including DNA single-strand breaks, DNA double-strand breaks (DSB), DNA-protein cross links, and altered DNA bases. Although human cells can repair many of these lesions there is little detailed knowledge of the nature of the genes and the encoded enzymes that control these repair processes. We report here on the cellular and genetic analyses of DNA double-strand break repair deficient mammalian cells. It has been well established that the DNA double-strand break is one of the major lesions induced by ionizing radiation. Utilizing rodent repair-deficient mutant, we have shown that the genes responsible for DNA double-strand break repair are also responsible for the cellular expression of radiation sensitivity. The molecular genetic analysis of DSB repair in rodent/human hybrid cells indicate that at least 6 different genes in mammalian cells are responsible for the repair of radiation-induced DNA double-strand breaks. Mapping and the prospect of cloning of human radiation repair genes are reviewed. Understanding the molecular and genetic basis of radiation sensitivity and DNA repair in man will provide a rational foundation to predict the individual risk associated with radiation exposure and to prevent radiation-induced genetic damage in the human population

Epigenetic reprogramming of pericentromeric satellite DNA in premalignant and malignant lesions

DEFF Research Database (Denmark)

Brückmann, Nadine Heidi; Pedersen, Christina Bøg; Ditzel, Henrik Jørn

2018-01-01

on pericentromeric satellites in primary melanocytes. This suggests that polycomb bodies form in cancer cells with global DNA demethylation to control the stability of pericentromeric satellite DNA. These results reveal a novel epigenetic perturbation specific to premalignant and malignant cells thatmaybe used...... as an early diagnostic marker for detection of precancerous changes and a new therapeutic entry point. Implications: Pericentromeric satellite DNA is epigenetically reprogrammed into polycomb bodies as a premalignant event with implications for transcriptional activity and genomic stability. Mol Cancer Res...

DNA damage in plant herbarium tissue.

Science.gov (United States)

Staats, Martijn; Cuenca, Argelia; Richardson, James E; Vrielink-van Ginkel, Ria; Petersen, Gitte; Seberg, Ole; Bakker, Freek T

2011-01-01

Dried plant herbarium specimens are potentially a valuable source of DNA. Efforts to obtain genetic information from this source are often hindered by an inability to obtain amplifiable DNA as herbarium DNA is typically highly degraded. DNA post-mortem damage may not only reduce the number of amplifiable template molecules, but may also lead to the generation of erroneous sequence information. A qualitative and quantitative assessment of DNA post-mortem damage is essential to determine the accuracy of molecular data from herbarium specimens. In this study we present an assessment of DNA damage as miscoding lesions in herbarium specimens using 454-sequencing of amplicons derived from plastid, mitochondrial, and nuclear DNA. In addition, we assess DNA degradation as a result of strand breaks and other types of polymerase non-bypassable damage by quantitative real-time PCR. Comparing four pairs of fresh and herbarium specimens of the same individuals we quantitatively assess post-mortem DNA damage, directly after specimen preparation, as well as after long-term herbarium storage. After specimen preparation we estimate the proportion of gene copy numbers of plastid, mitochondrial, and nuclear DNA to be 2.4-3.8% of fresh control DNA and 1.0-1.3% after long-term herbarium storage, indicating that nearly all DNA damage occurs on specimen preparation. In addition, there is no evidence of preferential degradation of organelle versus nuclear genomes. Increased levels of C→T/G→A transitions were observed in old herbarium plastid DNA, representing 21.8% of observed miscoding lesions. We interpret this type of post-mortem DNA damage-derived modification to have arisen from the hydrolytic deamination of cytosine during long-term herbarium storage. Our results suggest that reliable sequence data can be obtained from herbarium specimens.

Modified protocol for genomic DNA extraction from newly plucked feathers of lophura leucomelana hamiltoni (Galliformes) for genetic studies and its endo-restriction analysis

International Nuclear Information System (INIS)

Andleeb, S.; Shamim, S.; Minhas, R.A.

2012-01-01

A rapid and accurate protocol was used first time to isolate the high-quality genomic DNA from newly plucked feathers of Lophura leucomelana. Two different lysis protocols were used depending on the feather size and it was observed that 55 deg. C for 3 to 4 days showed better results of feathers lysis as compared with the 37 deg. C for overnight with gentle shaking. Purification of genomic DNA was also performed with phenol: chloroform: isoamyl alcohol and 100% absolute ethanol precipitation methods. By using this protocol, a significant amount of high-quality genomic DNA was obtained and the purity of DNA was analyzed through endo-restriction analysis. Genomic DNA isolated with this modified method will be used for Southern blotting and also in several polymerase chain reaction systems devoted to sex determination and paternity testing and the evolutionary relationships among the other pheasants. (author)

Comparison of the diagnostic value of cervical cytology and HPV HR DNA testing for the diagnosis of low-grade and high-grade squamous intraepithelial lesions across different age groups.

Science.gov (United States)

Paluszkiewicz, Aleksandra; Pruski, Dominik; Iwaniec, Kinga; Kędzia, Witold

2017-01-01

To assess the diagnostic value of cervical cytology and HPV HR DNA testing for the diagnosis of low grade and high-grade squamous intraepithelial lesions across different age groups. The study included 1103 patients, age 25-70 years. All patients underwent in-depth diagnostic tests following either an abnormal Pap test result or a clinically suspicious cervical lesion. In all women the following examinations were performed: a molecular test detecting 14 high-risk types of HPV, a colposcopy examination, as well as directed-biopsy of the cervix. The studied population was subdivided into four age groups. It was observed that the percentage of high grade squamous intraepithelial lesions (HSIL) and cancers increased with women's age. Sensitivity of both methods for detecting high-grade squamous intraepithelial lesions was highest for women aged 40-49 years. Sensitivity values of HPV testing was higher than that of cervical cytology among women under age 50. Specificity of HPV testing increased significantly with age of women and was several fold higher across all age groups than the specificity of cervical cytology.

Association between selected oral pathogens and gastric precancerous lesions.

Directory of Open Access Journals (Sweden)

Christian R Salazar

Full Text Available We examined whether colonization of selected oral pathogens is associated with gastric precancerous lesions in a cross-sectional study. A total of 119 participants were included, of which 37 were cases of chronic atrophic gastritis, intestinal metaplasia, or dysplasia. An oral examination was performed to measure periodontal indices. Plaque and saliva samples were tested with real-time quantitative PCR for DNA levels of pathogens related to periodontal disease (Porphyromonas gingivalis, Tannerella forsythensis, Treponema denticola, Actinobacillus actinomycetemcomitans and dental caries (Streptococcus mutans and S. sobrinus. There were no consistent associations between DNA levels of selected bacterial species and gastric precancerous lesions, although an elevated but non-significant odds ratio (OR for gastric precancerous lesions was observed in relation to increasing colonization of A. actinomycetemcomitans (OR = 1.36 for one standard deviation increase, 95% Confidence Interval = 0.87-2.12, P. gingivalis (OR = 1.12, 0.67-1.88 and T. denticola (OR = 1.34, 0.83-2.12 measured in plaque. To assess the influence of specific long-term infection, stratified analyses by levels of periodontal indices were conducted. A. actinomycetemcomitans was significantly associated with gastric precancerous lesions (OR = 2.51, 1.13-5.56 among those with ≥ median of percent tooth sites with PD ≥ 3 mm, compared with no association among those below the median (OR = 0.86, 0.43-1.72. A significantly stronger relationship was observed between the cumulative bacterial burden score of periodontal disease-related pathogens and gastric precancerous lesions among those with higher versus lower levels of periodontal disease indices (p-values for interactions: 0.03-0.06. Among individuals with periodontal disease, high levels of colonization of periodontal pathogens are associated with an increased risk of gastric precancerous lesions.

LET-effects in DNA

International Nuclear Information System (INIS)

Kraft, G.; Taucher-Scholz, G.; Heilmann, J.

1994-11-01

In this contribution, an introductory view on the physical properties of ions is given and the cellular response to high LET radiation is summarized. Then the measurements of strand break induction of DNA in solution and in intracellular DNA are reported and compared to cell survival. The possibility of changes in the quality of the lesions is discussed and finally the present status of model calculations in comparison to the experiments is given. (orig./HSI)

Learning tasks as a possible treatment for DNA lesions induced by oxidative stress in hippocampal neurons

Institute of Scientific and Technical Information of China (English)

DragoCrneci; Radu Silaghi-Dumitrescu

2013-01-01

Reactive oxygen species have been implicated in conditions ranging from cardiovascular dysfunc-tion, arthritis, cancer, to aging and age-related disorders. The organism developed several path-ways to counteract these effects, with base excision repair being responsible for repairing one of the major base lesions (8-oxoG) in al organisms. Epidemiological evidence suggests that cognitive stimulation makes the brain more resilient to damage or degeneration. Recent studies have linked enriched environment to reduction of oxidative stressin neurons of mice with Alzheimer’s dis-ease-like disease, but given its complexity it is not clear what specific aspect of enriched environ-ment has therapeutic effects. Studies from molecular biology have shown that the protein p300, which is a transcription co-activator required for consolidation of memories during specific learning tasks, is at the same time involved in DNA replication and repair, playing a central role in the long-patch pathway of base excision repair. Based on the evidence, we propose that learning tasks such as novel object recognition could be tested as possible methods of base excision repair faci-litation, hence inducing DNA repair in the hippocampal neurons. If this method proves to be effective, it could be the start for designing similar tasks for humans, as a behavioral therapeutic complement to the classical drug-based therapy in treating neurodegenerative disorders. This review presents the current status of therapeutic methods used in treating neurodegenerative diseases induced by reactive oxygen species and proposes a new approach based on existing data.

Radiation induced degradation of DNA in photodynamic therapy of cancer

International Nuclear Information System (INIS)

Ion, Rodica; Scarlat, F.; Niculescu, V.I.R.; Scarlat, Fl.; Gunaydin, Keriman

2001-01-01

DNA is a critical cellular target for oxidative processes induced by physical and chemical stresses. It is known that the direct effect of ionizing radiation on DNA results mainly in base ionization and may lead to mutation, carcinogenesis and cell death. The degradation of DNA induced by laser and ionizing radiation (electron and photon beam) is analyzed in this paper. The ionizing radiation degradation of DNA is a radical process. A series of lesions among the major base degradation product has been measured in isolated DNA exposed to gamma radiation in aerated aqueous solution. Degradation can be accounted for by the formation of hydroxyl radicals upon radiolysis of water (indirect effect). The production of DNA damage by ionizing radiation involves two mechanisms, direct and indirect effects. Direct effect leads to ionization and excitation of DNA molecules, while indirect effect is due to the interaction of reactive species, in particular of OH radicals produced by water radiolysis, with targets in DNA. The relative contribution of the two mechanisms in damaging DNA depends on the type of radiation. Single strand breaks and base damage seem to be mainly produced by the attack of hydroxyl radicals on DNA, whereas double strand breaks result predominantly of direct energy deposition. The four bases are degraded in high yield. Direct effect has been mimicked by photo-induced electron abstraction from the bases producing their radical cation. The base damage may also occur from the formation of radical cation of purine and pyrimidine components. When DNA is irradiated in solution, single strand breaks are mainly due to the abstraction of an H atom from the 4 ' position of 2 ' -deoxyribose by the attack of OH radicals produced by water radiolysis. Quantification of the modified bases showed the guanine is the preferential target. Ionizing radiation induces several types of DNA modifications, including chain breaks, DNA-protein cross-links, oxidized DNA bases

Oxidative DNA damage of peripheral blood polymorphonuclear leukocytes, selectively induced by chronic arsenic exposure, is associated with extent of arsenic-related skin lesions

International Nuclear Information System (INIS)

Pei, Qiuling; Ma, Ning; Zhang, Jing; Xu, Wenchao; Li, Yong; Ma, Zhifeng; Li, Yunyun; Tian, Fengjie; Zhang, Wenping; Mu, Jinjun; Li, Yuanfei; Wang, Dongxing; Liu, Haifang; Yang, Mimi; Ma, Caifeng; Yun, Fen

2013-01-01

�� 8-OHdG staining of PMN nuclei was paralleled by increased debris of cells. ► Oxidative DNA damage of PMNs is associated with arsenic-related skin lesions.

Spectroscopic approaches to study DNA damage induced in genome exposed to ionizing radiation and its enzymatic repair

International Nuclear Information System (INIS)

Yokoya, Akinari; Fujii, Kentaro; Oka, Toshitaka; Watanabe, Ritsuko

2012-01-01

Recent progress on spectroscopic study on physicochemical process of DNA damage induction will be reported. It has been predicted by computer track simulation studies that complex DNA damage, so called clustered DNA damage sites, is produced along the tack particularly of high Linear Energy Transfer (LET) ions. The clustered DNA damage, consisting of two or more isolated lesions such as single strand breaks or nucleobase lesions, is thought to compromise DNA repair enzymes. We have revealed that the nucleobase lesions produced by He 2+ ion impact to simple model DNA (plasmid) are hardly processed by base excision repair enzymes (E. coli DNA glycosylases). Using the third generation synchrotron radiation facility (SPring-8), we have studied unpaired electron species or desorbed ions as intermediates of DNA damage using an EPR apparatus or mass spectrometer installed in the soft X-ray beamline in SPring-8. These aspects are compared with the yields of final products of single- and double-strand breaks and base lesions revealed biochemical techniques. Models of complex DNA damage induction will be proposed considering various modification factors of the damage induction, ionization of valence and inner-shell electrons, OH radicals, hydration layer and the impact of secondary electrons. (author)

Novel DNA lesions generated by the interaction between therapeutic thiopurines and UVA light.

Science.gov (United States)

Zhang, Xiaohong; Jeffs, Graham; Ren, Xiaolin; O'Donovan, Peter; Montaner, Beatriz; Perrett, Conal M; Karran, Peter; Xu, Yao-Zhong

2007-03-01

The therapeutic effect of the thiopurines, 6-thioguanine (6-TG), 6-mercaptopurine, and its prodrug azathioprine, depends on the incorporation of 6-TG into cellular DNA. Unlike normal DNA bases, 6-TG absorbs UVA radiation, and UVA-mediated photochemical damage of DNA 6-TG has potentially harmful side effects. When free 6-TG is UVA irradiated in solution in the presence of molecular oxygen, reactive oxygen species are generated and 6-TG is oxidized to guanine-6-sulfonate (G(SO3)) and guanine-6-thioguanine in reactions involving singlet oxygen. This conversion is prevented by antioxidants, including the dietary vitamin ascorbate. DNA G(SO3) is also the major photoproduct of 6-TG in DNA and it can be selectively introduced into DNA or oligonucleotides in vitro by mild chemical oxidation. Thermal stability measurements indicate that G(SO3) does not form stable base pairs with any of the normal DNA bases in duplex oligonucleotides and is a powerful block for elongation by Klenow DNA polymerase in primer extension experiments. In cultured human cells, DNA damage produced by 6-TG and UVA treatment is associated with replication inhibition and provokes a p53-dependent DNA damage response.

Regulation of rDNA stability by sumoylation

DEFF Research Database (Denmark)

Eckert-Boulet, Nadine; Lisby, Michael

2009-01-01

Repair of DNA lesions by homologous recombination relies on the copying of genetic information from an intact homologous sequence. However, many eukaryotic genomes contain repetitive sequences such as the ribosomal gene locus (rDNA), which poses a risk for illegitimate recombination. Therefore, t......6 complex and sumoylation of Rad52, which directs DNA double-strand breaks in the rDNA to relocalize from within the nucleolus to the nucleoplasm before association with the recombination machinery. The relocalization before repair is important for maintaining rDNA stability. The focus...

Mitochondrial respiratory modifiers confer survival advantage by facilitating DNA repair in cancer cells

International Nuclear Information System (INIS)

Chauhan, Ankit; Khanna, Suchit; Singh, Saurabh; Rai, Yogesh; Soni, Ravi; Kalra, Namita; Dwarakanath, B.S.; Bhatt, Anant Narayan

2014-01-01

High rate of aerobic glycolysis (Warburg effect), one of the primary hallmarks of cancer cells, acquired during the multistep development of tumors is also responsible for therapeutic resistance. Underlying this hallmark is the compromised respiratory metabolism that contributes to the acquisition of the glycolytic phenotype for sustained ATP production and cell proliferation. Nevertheless, the exact mechanisms underlying the glycolysis-linked radio-resistance in cancer cells remain elusive. In this study, we transiently elevated glycolysis by treating human cell lines (HEK293, BMG-1 and OCT-1) with mitochondrial respiratory modifiers (MRMs) viz. 2,4-dinitrophenol, Photosan-3, and Methylene blue to examine if transient stimulation of glycolysis before irradiation using MRMs is sufficient to confer radioresistance. Treatment with MRMs led to a significant (two-fold) increase in glucose consumption and lactate production together with a robust increase in the protein levels of two key regulators of glucose metabolism, i.e. GLUT-1 and HK-II. MRMs also enhanced the clonogenic survival and facilitated DNA repair by activating both non-homologous end joining (NHEJ) and homologous recombination (HR) pathways of DNA double strand break repair leading to reduction in radiation-induced cytogenetic damage (micronuclei formation) in these cells. Inhibition of glucose uptake by inhibitors like 2-deoxy-D-glucose (2-DG), 3-bromo pyruvate (3-BP) and fasentin under conditions of stimulated glycolysis not only reversed the effect but also sensitized the cells to radiation more profoundly. The inhibition of glycolysis using 2-DG also reduced the levels of Ku 70 (NHEJ) and Rad-51 (HR) proteins. Thus, our results suggest that enhanced glycolysis in cancer cells may confer radio-resistance and offers survival advantage partly by enhancing the repair of DNA damage. (author)

Critical points of DNA quantification by real-time PCR--effects of DNA extraction method and sample matrix on quantification of genetically modified organisms.

Science.gov (United States)

Cankar, Katarina; Stebih, Dejan; Dreo, Tanja; Zel, Jana; Gruden, Kristina

2006-08-14

Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was chosen as the primary criterion by which to

UV stimulation of DNA-mediated transformation of human cells

International Nuclear Information System (INIS)

van Duin, M.; Westerveld, A.; Hoeijmakers, J.H.

1985-01-01

Irradiation of dominant marker DNA with UV light (150 to 1,000 J/m2) was found to stimulate the transformation of human cells by this marker from two- to more than fourfold. This phenomenon is also displayed by xeroderma pigmentosum cells, which are deficient in the excision repair of UV-induced pyrimidine dimers in the DNA. Also, exposure to UV of the transfected (xeroderma pigmentosum) cells enhanced the transfection efficiency. Removal of the pyrimidine dimers from the DNA by photoreactivating enzyme before transfection completely abolished the stimulatory effect, indicating that dimer lesions are mainly responsible for the observed enhancement. A similar stimulation of the transformation efficiency is exerted by 2-acetoxy-2-acetylaminofluorene modification of the DNA. These findings suggest that lesions which are targets for the excision repair pathway induce the increase in transformation frequency. The stimulation was found to be independent of sequence homology between the irradiated DNA and the host chromosomal DNA. Therefore, the increase of the transformation frequency is not caused by a mechanism inducing homologous recombination between these two DNAs. UV treatment of DNA before transfection did not have a significant effect on the amount of DNA integrated into the xeroderma pigmentosum genome

The yield, processing, and biological consequences of clustered DNA damage induced by ionizing radiation

International Nuclear Information System (INIS)

Shikazono, Naoya; Noguchi, Miho; Fujii, Kentaro; Urushibara, Ayumi; Yokoya, Akinari

2009-01-01

After living cells are exposed to ionizing radiation, a variety of chemical modifications of DNA are induced either directly by ionization of DNA or indirectly through interactions with water-derived radicals. The DNA lesions include single strand breaks (SSB), base lesions, sugar damage, and apurinic/apyrimidinic sites (AP sites). Clustered DNA damage, which is defined as two or more of such lesions within one to two helical turns of DNA induced by a single radiation track, is considered to be a unique feature of ionizing radiation. A double strand break (DSB) is a type of clustered DNA damage, in which single strand breaks are formed on opposite strands in close proximity. Formation and repair of DSBs have been studied in great detail over the years as they have been linked to important biological endpoints, such as cell death, loss of genetic material, chromosome aberration. Although non-DSB clustered DNA damage has received less attention, there is growing evidence of its biological significance. This review focuses on the current understanding of (1) the yield of non-DSB clustered damage induced by ionizing radiation (2) the processing, and (3) biological consequences of non-DSB clustered DNA damage. (author)

Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints

DEFF Research Database (Denmark)

Bartkova, Jirina; Rezaei, Nousin; Liontos, Michalis

2006-01-01

Recent studies have indicated the existence of tumorigenesis barriers that slow or inhibit the progression of preneoplastic lesions to neoplasia. One such barrier involves DNA replication stress, which leads to activation of the DNA damage checkpoint and thereby to apoptosis or cell cycle arrest...... and senescence markers cosegregate closely. Thus, senescence in human preneoplastic lesions is a manifestation of oncogene-induced DNA replication stress and, together with apoptosis, provides a barrier to malignant progression....

Structure of human DNA polymerase iota and the mechanism of DNA synthesis.

Science.gov (United States)

Makarova, A V; Kulbachinskiy, A V

2012-06-01

Cellular DNA polymerases belong to several families and carry out different functions. Highly accurate replicative DNA polymerases play the major role in cell genome replication. A number of new specialized DNA polymerases were discovered at the turn of XX-XXI centuries and have been intensively studied during the last decade. Due to the special structure of the active site, these enzymes efficiently perform synthesis on damaged DNA but are characterized by low fidelity. Human DNA polymerase iota (Pol ι) belongs to the Y-family of specialized DNA polymerases and is one of the most error-prone enzymes involved in DNA synthesis. In contrast to other DNA polymerases, Pol ι is able to use noncanonical Hoogsteen interactions for nucleotide base pairing. This allows it to incorporate nucleotides opposite various lesions in the DNA template that impair Watson-Crick interactions. Based on the data of X-ray structural analysis of Pol ι in complexes with various DNA templates and dNTP substrates, we consider the structural peculiarities of the Pol ι active site and discuss possible mechanisms that ensure the unique behavior of the enzyme on damaged and undamaged DNA.

DNA-cell conjugates

Science.gov (United States)

Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn; Mathies, Richard; Chandra, Ravi; Douglas, Erik; Twite, Amy; Toriello, Nicholas; Onoe, Hiroaki

2016-05-03

The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

DNA-cell conjugates

Science.gov (United States)

Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn; Mathies, Richard; Chandra, Ravi; Douglas, Erik; Twite, Amy; Toriello, Nicholas; Onoe, Hiroaki

2018-05-15

The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

Role of Acute Lesion Topography in Initial Ischemic Stroke Severity and Long-Term Functional Outcomes.

Science.gov (United States)

Wu, Ona; Cloonan, Lisa; Mocking, Steven J T; Bouts, Mark J R J; Copen, William A; Cougo-Pinto, Pedro T; Fitzpatrick, Kaitlin; Kanakis, Allison; Schaefer, Pamela W; Rosand, Jonathan; Furie, Karen L; Rost, Natalia S

2015-09-01

Acute infarct volume, often proposed as a biomarker for evaluating novel interventions for acute ischemic stroke, correlates only moderately with traditional clinical end points, such as the modified Rankin Scale. We hypothesized that the topography of acute stroke lesions on diffusion-weighted magnetic resonance imaging may provide further information with regard to presenting stroke severity and long-term functional outcomes. Data from a prospective stroke repository were limited to acute ischemic stroke subjects with magnetic resonance imaging completed within 48 hours from last known well, admission NIH Stroke Scale (NIHSS), and 3-to-6 months modified Rankin Scale scores. Using voxel-based lesion symptom mapping techniques, including age, sex, and diffusion-weighted magnetic resonance imaging lesion volume as covariates, statistical maps were calculated to determine the significance of lesion location for clinical outcome and admission stroke severity. Four hundred ninety subjects were analyzed. Acute stroke lesions in the left hemisphere were associated with more severe NIHSS at admission and poor modified Rankin Scale at 3 to 6 months. Specifically, injury to white matter (corona radiata, internal and external capsules, superior longitudinal fasciculus, and uncinate fasciculus), postcentral gyrus, putamen, and operculum were implicated in poor modified Rankin Scale. More severe NIHSS involved these regions, as well as the amygdala, caudate, pallidum, inferior frontal gyrus, insula, and precentral gyrus. Acute lesion topography provides important insights into anatomic correlates of admission stroke severity and poststroke outcomes. Future models that account for infarct location in addition to diffusion-weighted magnetic resonance imaging volume may improve stroke outcome prediction and identify patients likely to benefit from aggressive acute intervention and personalized rehabilitation strategies. © 2015 American Heart Association, Inc.

Identification of multiple mRNA and DNA sequences from small tissue samples isolated by laser-assisted microdissection.

Science.gov (United States)

Bernsen, M R; Dijkman, H B; de Vries, E; Figdor, C G; Ruiter, D J; Adema, G J; van Muijen, G N

1998-10-01

Molecular analysis of small tissue samples has become increasingly important in biomedical studies. Using a laser dissection microscope and modified nucleic acid isolation protocols, we demonstrate that multiple mRNA as well as DNA sequences can be identified from a single-cell sample. In addition, we show that the specificity of procurement of tissue samples is not compromised by smear contamination resulting from scraping of the microtome knife during sectioning of lesions. The procedures described herein thus allow for efficient RT-PCR or PCR analysis of multiple nucleic acid sequences from small tissue samples obtained by laser-assisted microdissection.

Reactivation of DNA replication of the parvovirus MVM in UV preirradiated mouse cells

International Nuclear Information System (INIS)

Vos, J.M.; Rommelaere, Jean

1982-01-01

The parvovirus Minute-Virus-of-Mice (MVM) was used to probe the DNA replication activities expressed by mouse fibroblasts. This system allowed us to study quantitatively the effect of UV-induced DNA lesions on the progression of DNA replication in vivo. MVM was UV-irradiated prior to infection. Pyrimidine dimers induced in the viral genome account for the reduced level of intracellular viral DNA synthesis, assuming that most of these lesions block viral DNA replication in unirradiated cells. The inhibition of damaged MVM DNA synthesis is less severe if the host cells themselves are irradiated prior to virus infection. This stimulation of viral DNA replication in pretreated cells might account for the UV-enhanced viral reactivation phenomenon, i.e. the increased survival of nuclear-replicating viruses propagated in cells preexposed to various genotoxic agents [fr

Reactivation of DNA replication of the parvovirus MVM in UV preirradiated mouse cells

Energy Technology Data Exchange (ETDEWEB)

Vos, J.M.; Rommelaere, J. (Universite Libre de Bruxelles, Rhode-St-Genese (Belgium))

1982-07-01

The parvovirus Minute-Virus-of-Mice (MVM) was used to probe the DNA replication activities expressed by mouse fibroblasts. This system allowed us to study quantitatively the effect of UV-induced DNA lesions on the progression of DNA replication in vivo. MVM was UV-irradiated prior to infection. Pyrimidine dimers induced in the viral genome account for the reduced level of intracellular viral DNA synthesis, assuming that most of these lesions block viral DNA replication in unirradiated cells. The inhibition of damaged MVM DNA synthesis is less severe if the host cells themselves are irradiated prior to virus infection. This stimulation of viral DNA replication in pretreated cells might account for the UV-enhanced viral reactivation phenomenon, i.e. the increased survival of nuclear-replicating viruses propagated in cells preexposed to various genotoxic agents.

PREVALENCE OF HUMAN PAPILLOMAVIRUS GENOTYPES IN LOW AND HIGH GRADE SQUAMOUS INTRAEPITHELIAL LESIONS AT CERVICAL TISSUE

OpenAIRE

Prasetyo, Rizki Eko; Mastutik, Gondo; Mustokoweni, Sjahjenny

2017-01-01

HPV infection is known to cause cervical cancer. This study aimed to identify the variant of HPV genotypes of cervical precancerous lesions from low grade squamous intraepithelial lesion  (LSIL) and high grade squamous intraepithelial lesion (HSIL). This was an explorative study using formalin fix paraffin embedded (FFPE) from cervical precancerous lesions at Dr. Soetomo Hospital, Surabaya. DNA was extracted from FFPE and hybridized for HPV genotyping using Ampliquality HPV Type Express kit (...

Ionizing radiation sensitivity of DNA polymerase lambda-deficient cells.

NARCIS (Netherlands)

Vermeulen, C.; Bertocci, B.; Begg, A.C.; Vens, C.

2007-01-01

Ionizing radiation induces a diverse spectrum of DNA lesions, including strand breaks and oxidized bases. In mammalian cells, ionizing radiation-induced lesions are targets of non-homologous end joining, homologous recombination, and base excision repair. In vitro assays show a potential involvement

Detection of human cytomegalovirus and Epstein-Barr Virus in symptomatic and asymptomatic apical periodontitis lesions by real-time PCR.

Science.gov (United States)

Ozbek, Selcuk-M; Ozbek, Ahmet; Yavuz, Muhammed-Selim

2013-09-01

Recent studies have investigated the occurrence of human cytomegalovirus and Epstein-Barr Virus in samples from apical periodontitis lesions and a role in the pathogenesis of this disease has been suggested. Because genotype distribution and seroprevalence of EBV and HCMV differ among populations, it is important to determine the presence of these viruses in endodontic periapical lesions of different populations. The aims of this study were to determine the presence of HCMV and EBV DNAs in samples from Turkish patients with symptomatic and asymptomatic apical periodontitis lesions using real-time polymerase chain reaction method and to evaluate their presence in both symptomatic and asymptomatic apical periodontitis lesions. Periapical samples were collected from 12 asymptomatic and 16 symptomatic periapical lesions in conjunction with apicectomy. HCMV and EBV DNAs were identified in the samples by real-time PCR. The chi-squared test with Yates's correction or the Fisher's exact test was used to analyse the significance of differences. HCMV DNA was detected in 10 of the 16 (62.5%) symptomatic and in five of the 12 (41.7 %) asymptomatic periapical study lesions. The EBV DNA was identified in seven of the 16 (43.7 %) symptomatic and three of the 12 (25 %) asymptomatic periapical lesions. The difference in occurrence of HCMV and EBV DNA between symptomatic and asymptomatic periapical lesions was not statistically significant. (All comparisons have p > 0.05). Our findings suggest that HCMV and EBV is a frequent inhabitant of both symptomatic and asymptomatic apical periodontitis lesions of endodontic origin in Turkish population.

Genetic screening of spinal muscular atrophy using a real-time modified COP-PCR technique with dried blood-spot DNA.

Science.gov (United States)

Ar Rochmah, Mawaddah; Harahap, Nur Imma Fatimah; Niba, Emma Tabe Eko; Nakanishi, Kenta; Awano, Hiroyuki; Morioka, Ichiro; Iijima, Kazumoto; Saito, Toshio; Saito, Kayoko; Lai, Poh San; Takeshima, Yasuhiro; Takeuchi, Atsuko; Bouike, Yoshihiro; Okamoto, Maya; Nishio, Hisahide; Shinohara, Masakazu

2017-10-01

Spinal muscular atrophy (SMA) is a common neuromuscular disorder caused by mutations in SMN1. More than 95% of SMA patients carry homozygous SMN1 deletion. SMA is the leading genetic cause of infant death, and has been considered an incurable disease. However, a recent clinical trial with an antisense oligonucleotide drug has shown encouraging clinical efficacy. Thus, early and accurate detection of SMN1 deletion may improve prognosis of many infantile SMA patients. A total of 88 DNA samples (37 SMA patients, 12 carriers and 39 controls) from dried blood spots (DBS) on filter paper were analyzed. All participants had previously been screened for SMN genes by PCR restriction fragment length polymorphism (PCR-RFLP) using DNA extracted from freshly collected blood. DNA was extracted from DBS that had been stored at room temperature (20-25°C) for 1week to 5years. To ensure sufficient quality and quantity of DNA samples, target sequences were pre-amplified by conventional PCR. Real-time modified competitive oligonucleotide priming-PCR (mCOP-PCR) with the pre-amplified PCR products was performed for the gene-specific amplification of SMN1 and SMN2 exon 7. Compared with PCR-RFLP using DNA from freshly collected blood, results from real-time mCOP-PCR using DBS-DNA for detection of SMN1 exon 7 deletion showed a sensitivity of 1.00 (CI [0.87, 1.00])] and specificity of 1.00 (CI [0.90, 1.00]), respectively. We combined DNA extraction from DBS on filter paper, pre-amplification of target DNA, and real-time mCOP-PCR to specifically detect SMN1 and SMN2 genes, thereby establishing a rapid, accurate, and high-throughput system for detecting SMN1-deletion with practical applications for newborn screening. Copyright © 2017 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

PARP-1: Friend or Foe of DNA Damage and Repair in Tumorigenesis?

Energy Technology Data Exchange (ETDEWEB)

Swindall, Amanda F.; Stanley, Jennifer A. [Department of Radiation Oncology Comprehensive Cancer Center, University of Alabama at Birmingham School of Medicine, 176F HSROC Suite 2232B, 1700 6th Avenue South, Birmingham, AL 35249 (United States); Yang, Eddy S., E-mail: eyang@uab.edu [Department of Radiation Oncology Comprehensive Cancer Center, University of Alabama at Birmingham School of Medicine, 176F HSROC Suite 2232B, 1700 6th Avenue South, Birmingham, AL 35249 (United States); Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL 35249 (United States); Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL 35249 (United States)

2013-07-26

Oxidative stress induced by reactive oxygen species can result in DNA damage within cells and subsequently increase risk for carcinogenesis. This may be averted by repair of DNA damage through the base or nucleotide excision repair (BER/NER) pathways. PARP, a BER protein, is known for its role in DNA-repair. However, multiple lesions can occur within a small range of DNA, known as oxidative clustered DNA lesions (OCDLs), which are difficult to repair and may lead to the more severe DNA double-strand break (DSB). Inefficient DSB repair can then result in increased mutagenesis and neoplastic transformation. OCDLs occur more frequently within a variety of tumor tissues. Interestingly, PARP is highly expressed in several human cancers. Additionally, chronic inflammation may contribute to tumorigenesis through ROS-induced DNA damage. Furthermore, PARP can modulate inflammation through interaction with NFκB and regulating the expression of inflammatory signaling molecules. Thus, the upregulation of PARP may present a double-edged sword. PARP is needed to repair ROS-induced DNA lesions, but PARP expression may lead to increased inflammation via upregulation of NFκB signaling. Here, we discuss the role of PARP in the repair of oxidative damage versus the formation of OCDLs and speculate on the feasibility of PARP inhibition for the treatment and prevention of cancers by exploiting its role in inflammation.

PARP-1: Friend or Foe of DNA Damage and Repair in Tumorigenesis?

International Nuclear Information System (INIS)

Swindall, Amanda F.; Stanley, Jennifer A.; Yang, Eddy S.

2013-01-01

Oxidative stress induced by reactive oxygen species can result in DNA damage within cells and subsequently increase risk for carcinogenesis. This may be averted by repair of DNA damage through the base or nucleotide excision repair (BER/NER) pathways. PARP, a BER protein, is known for its role in DNA-repair. However, multiple lesions can occur within a small range of DNA, known as oxidative clustered DNA lesions (OCDLs), which are difficult to repair and may lead to the more severe DNA double-strand break (DSB). Inefficient DSB repair can then result in increased mutagenesis and neoplastic transformation. OCDLs occur more frequently within a variety of tumor tissues. Interestingly, PARP is highly expressed in several human cancers. Additionally, chronic inflammation may contribute to tumorigenesis through ROS-induced DNA damage. Furthermore, PARP can modulate inflammation through interaction with NFκB and regulating the expression of inflammatory signaling molecules. Thus, the upregulation of PARP may present a double-edged sword. PARP is needed to repair ROS-induced DNA lesions, but PARP expression may lead to increased inflammation via upregulation of NFκB signaling. Here, we discuss the role of PARP in the repair of oxidative damage versus the formation of OCDLs and speculate on the feasibility of PARP inhibition for the treatment and prevention of cancers by exploiting its role in inflammation

Human macrophage scavenger receptors: Primary structure, expression, and localization in atherosclerotic lesions

International Nuclear Information System (INIS)

Matsumoto, Akiyo; Itakura, Hiroshige; Kodama, Tatsuhiko; Naito, Makoto; Takahashi, Kiyoshi; Ikemoto, Shinji; Asaoka, Hitoshi; Hayakawa, Ikuho; Kanamori, Hiroshi; Takaku, Fumimaro; Aburatani, Hiroyuki; Suzuki, Hiroshi; Kobari, Yukage; Miyai, Tatsuya; Cohen, E.H.; Wydro, R.; Housman, D.E.

1990-01-01

Two types of cDNAs for human macrophage scavenger receptors were cloned from a cDNA library derived from the phorbol ester-treated human monocytic cell line THP-1. The type I and type II human scavenger receptors encoded by these cDNAs are homologous (73% and 71% amino acid identity) to their previously characterized bovine counterparts and consist of six domains: cytoplasmic (I), membrane-spanning (II), spacer (III), α-helical coiled-coil (IV), collagen-like (V), and a type-specific C-terminal (VI). The receptor gene is located on human chromosome 8. The human receptors expressed in CHO-K1 cells mediated endocytosis of modified low density lipoproteins. Two mRNAs, 4.0 and 3.2 kilobases, have been detected in human liver, placenta, and brain. Immunohistochemical studies using an anti-peptide antibody which recognizes human scavenger receptors indicated the presence of the scavenger receptors in the macrophages of lipid-rich atherosclerotic lesions, suggesting the involvement of scavenger receptors in atherogenesis

The effect of modulators of radiation-induced G2 arrest on the repair of radiation-induced DNA damage detectable by neutral filter elution

International Nuclear Information System (INIS)

Rowley, R.; Kort, L.

1988-01-01

The influence of cycloheximide (50 μg/ml), caffeine (5 mM) and cordycepin (0.15 mM) on the repair of the damage detectable in DNA by neutral filter elution was determined. Chinese hamster ovary cells (CHO) were irradiated with X-ray doses of 20, 60 and 100 Gy then allowed to repair without drug treatment or in the presence of each drug for intervals up to 6 h. DNA damage repair proceeded in two phases. The fast component of the repair process (t 1/2 approx. 7 min) was not modified by drug treatment; the slow component (t 1/2 170 min) was unaffected by cycloheximide or cordycepin, but appeared to be inhibited by caffeine. It was concluded that: (a) the lesion which results in radiation-induced G 2 arrest is not the lesion which is detectable by neutral filter elution, and (b) the influence of caffeine on dsb repair is specific to caffeine and is not mediated by a reduction in the duration of G 2 arrest. (author)

Modulation of mutagen-induced biological effects by inhibitors of DNA repair

International Nuclear Information System (INIS)

Natarajan, A.T.; Mullenders, L.F.H.; Zwanenburg, T.S.B.

1986-01-01

When lesions are induced in the DNA by mutagenic agents, they are subjected to cellular repair. Unrepaired and misrepaired lesions lead to biological effects, such as cell killing, point mutations and chromosomal alterations (aberrations and sister chromatid exchanges - SCEs). It is very difficult to directly correlate any particular type of lesion to a specific biological effect. However, in specific cases, this has been done. For example, short wave UV induced biological effects (cell killing, chromosomal alterations) result predominantly from induced cyclobutane dimers and by photoreactivation experiments, one can demonstrate that with the removal of dimers all types biological effects are diminished. In cases where many types of lesions are considered responsible for the observed biological effects other strategies have been employed to identify the possible lesion. The frequencies of induced chromosomal alterations and point mutations increase with the dose of the mutagen employed and an inhibition of DNA repair following treatment with the mutagen. Prevention of the cells from dividing following mutagen treatment allows them to repair premutational damage, thus reducing the biological effects induced. By comprehensive studies involving quantification of primary DNA lesions, their repair and biological effects will enable us to understand to some extent the complex processes involved in the manifestation of specific biological effects that follow the treatment of cells with mutagenic carcinogens

PET/MR imaging of bone lesions - implications for PET quantification from imperfect attenuation correction

International Nuclear Information System (INIS)

Samarin, Andrei; Burger, Cyrill; Crook, David W.; Burger, Irene A.; Schmid, Daniel T.; Schulthess, Gustav K. von; Kuhn, Felix P.; Wollenweber, Scott D.

2012-01-01

Accurate attenuation correction (AC) is essential for quantitative analysis of PET tracer distribution. In MR, the lack of cortical bone signal makes bone segmentation difficult and may require implementation of special sequences. The purpose of this study was to evaluate the need for accurate bone segmentation in MR-based AC for whole-body PET/MR imaging. In 22 patients undergoing sequential PET/CT and 3-T MR imaging, modified CT AC maps were produced by replacing pixels with values of >100 HU, representing mostly bone structures, by pixels with a constant value of 36 HU corresponding to soft tissue, thereby simulating current MR-derived AC maps. A total of 141 FDG-positive osseous lesions and 50 soft-tissue lesions adjacent to bones were evaluated. The mean standardized uptake value (SUVmean) was measured in each lesion in PET images reconstructed once using the standard AC maps and once using the modified AC maps. Subsequently, the errors in lesion tracer uptake for the modified PET images were calculated using the standard PET image as a reference. Substitution of bone by soft tissue values in AC maps resulted in an underestimation of tracer uptake in osseous and soft tissue lesions adjacent to bones of 11.2 ± 5.4 % (range 1.5-30.8 %) and 3.2 ± 1.7 % (range 0.2-4 %), respectively. Analysis of the spine and pelvic osseous lesions revealed a substantial dependence of the error on lesion composition. For predominantly sclerotic spine lesions, the mean underestimation was 15.9 ± 3.4 % (range 9.9-23.5 %) and for osteolytic spine lesions, 7.2 ± 1.7 % (range 4.9-9.3 %), respectively. CT data simulating treating bone as soft tissue as is currently done in MR maps for PET AC leads to a substantial underestimation of tracer uptake in bone lesions and depends on lesion composition, the largest error being seen in sclerotic lesions. Therefore, depiction of cortical bone and other calcified areas in MR AC maps is necessary for accurate quantification of tracer uptake

CLINICAL-EVALUATION OF A MODIFIED ELISA, USING PHOTOBIOTINYLATED DNA, FOR THE DETECTION OF ANTI-DNA ANTIBODIES

NARCIS (Netherlands)