Meta-analysis of genome-wide association studies identifies 8 novel loci involved in shape variation of human head hair

Science.gov (United States)

Liu, Fan; Chen, Yan; Zhu, Gu; Hysi, Pirro G; Wu, Sijie; Adhikari, Kaustubh; Breslin, Krystal; Pośpiech, Ewelina; Hamer, Merel A; Peng, Fuduan; Muralidharan, Charanya; Acuna-Alonzo, Victor; Canizales-Quinteros, Samuel; Bedoya, Gabriel; Gallo, Carla; Poletti, Giovanni; Rothhammer, Francisco; Bortolini, Maria Catira; Gonzalez-Jose, Rolando; Zeng, Changqing; Xu, Shuhua; Jin, Li; Uitterlinden, André G; Ikram, M Arfan; van Duijn, Cornelia M; Nijsten, Tamar; Walsh, Susan; Branicki, Wojciech; Wang, Sijia; Ruiz-Linares, Andrés; Spector, Timothy D; Martin, Nicholas G; Medland, Sarah E; Kayser, Manfred

2018-01-01

Abstract Shape variation of human head hair shows striking variation within and between human populations, while its genetic basis is far from being understood. We performed a series of genome-wide association studies (GWASs) and replication studies in a total of 28 964 subjects from 9 cohorts from multiple geographic origins. A meta-analysis of three European GWASs identified 8 novel loci (1p36.23 ERRFI1/SLC45A1, 1p36.22 PEX14, 1p36.13 PADI3, 2p13.3 TGFA, 11p14.1 LGR4, 12q13.13 HOXC13, 17q21.2 KRTAP, and 20q13.33 PTK6), and confirmed 4 previously known ones (1q21.3 TCHH/TCHHL1/LCE3E, 2q35 WNT10A, 4q21.21 FRAS1, and 10p14 LINC00708/GATA3), all showing genome-wide significant association with hair shape (P < 5e-8). All except one (1p36.22 PEX14) were replicated with nominal significance in at least one of the 6 additional cohorts of European, Native American and East Asian origins. Three additional previously known genes (EDAR, OFCC1, and PRSS53) were confirmed at the nominal significance level. A multivariable regression model revealed that 14 SNPs from different genes significantly and independently contribute to hair shape variation, reaching a cross-validated AUC value of 0.66 (95% CI: 0.62–0.70) and an AUC value of 0.64 in an independent validation cohort, providing an improved accuracy compared with a previous model. Prediction outcomes of 2504 individuals from a multiethnic sample were largely consistent with general knowledge on the global distribution of hair shape variation. Our study thus delivers target genes and DNA variants for future functional studies to further evaluate the molecular basis of hair shape in humans. PMID:29220522

Meta-analysis of genome-wide association studies identifies 8 novel loci involved in shape variation of human head hair.

Science.gov (United States)

Liu, Fan; Chen, Yan; Zhu, Gu; Hysi, Pirro G; Wu, Sijie; Adhikari, Kaustubh; Breslin, Krystal; Pospiech, Ewelina; Hamer, Merel A; Peng, Fuduan; Muralidharan, Charanya; Acuna-Alonzo, Victor; Canizales-Quinteros, Samuel; Bedoya, Gabriel; Gallo, Carla; Poletti, Giovanni; Rothhammer, Francisco; Bortolini, Maria Catira; Gonzalez-Jose, Rolando; Zeng, Changqing; Xu, Shuhua; Jin, Li; Uitterlinden, André G; Ikram, M Arfan; van Duijn, Cornelia M; Nijsten, Tamar; Walsh, Susan; Branicki, Wojciech; Wang, Sijia; Ruiz-Linares, Andrés; Spector, Timothy D; Martin, Nicholas G; Medland, Sarah E; Kayser, Manfred

2018-02-01

Shape variation of human head hair shows striking variation within and between human populations, while its genetic basis is far from being understood. We performed a series of genome-wide association studies (GWASs) and replication studies in a total of 28 964 subjects from 9 cohorts from multiple geographic origins. A meta-analysis of three European GWASs identified 8 novel loci (1p36.23 ERRFI1/SLC45A1, 1p36.22 PEX14, 1p36.13 PADI3, 2p13.3 TGFA, 11p14.1 LGR4, 12q13.13 HOXC13, 17q21.2 KRTAP, and 20q13.33 PTK6), and confirmed 4 previously known ones (1q21.3 TCHH/TCHHL1/LCE3E, 2q35 WNT10A, 4q21.21 FRAS1, and 10p14 LINC00708/GATA3), all showing genome-wide significant association with hair shape (P 5e-8). All except one (1p36.22 PEX14) were replicated with nominal significance in at least one of the 6 additional cohorts of European, Native American and East Asian origins. Three additional previously known genes (EDAR, OFCC1, and PRSS53) were confirmed at the nominal significance level. A multivariable regression model revealed that 14 SNPs from different genes significantly and independently contribute to hair shape variation, reaching a cross-validated AUC value of 0.66 (95% CI: 0.62-0.70) and an AUC value of 0.64 in an independent validation cohort, providing an improved accuracy compared with a previous model. Prediction outcomes of 2504 individuals from a multiethnic sample were largely consistent with general knowledge on the global distribution of hair shape variation. Our study thus delivers target genes and DNA variants for future functional studies to further evaluate the molecular basis of hair shape in humans. © The Author(s) 2017. Published by Oxford University Press.

Transcriptome Profile Analysis of Mechanisms of Black and White Plumage Determination in Black-Bone Chicken

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Shigang Yu

2018-05-01

Full Text Available Background/Aims: Melanin is a major and ubiquitous component of plumage colouration, and patterns of melanin pigmentation in birds are extremely varied. However, the molecular mechanism of pigmentation in avian plumage is still largely unknown. Methods: To elucidate the molecular mechanisms involved in the formation of black and white plumage, this study takes advantage of high-throughput sequencing technology to compare differences in the transcriptome between black and white chicken feather bulbs. In total, we constructed six cDNA libraries from black (Group B and white (Group W feather bulbs in the dorsal plumage of Muchuan black-boned chickens. Results: A comparison between Groups B and W revealed 61 differentially expressed genes, with 47 displaying higher, and 14 displaying lower, levels of expression in white feather bulbs. Our results revealed a set of candidate genes and two potential metabolic pathways involved in black-bone chicken plumage melanogenesis. These include four homeobox genes (HOXB9, HOXC8, HOXA9, and HOXC 9, two glutathione (GSH metabolism-related genes (CHAC1 and GPX3, and the transforming growth factor beta (TGF-β signalling pathway. Two known genes, TYR and MITF, were also shown to play a role in melanin formation. Conclusion: our data provide a valuable resource for discovering genes important in plumage melanin formation and will help further elucidate the molecular mechanisms for black and white plumage.

The Role of 8-Oxoguanine DNA Glycosylase-1 in Inflammation

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Xueqing Ba

2014-09-01

Full Text Available Many, if not all, environmental pollutants/chemicals and infectious agents increase intracellular levels of reactive oxygen species (ROS at the site of exposure. ROS not only function as intracellular signaling entities, but also induce damage to cellular molecules including DNA. Among the several dozen ROS-induced DNA base lesions generated in the genome, 8-oxo-7,8-dihydroguanine (8-oxoG is one of the most abundant because of guanine’s lowest redox potential among DNA bases. In mammalian cells, 8-oxoG is repaired by the 8-oxoguanine DNA glycosylase-1 (OGG1-initiated DNA base excision repair pathway (OGG1–BER. Accumulation of 8-oxoG in DNA has traditionally been associated with mutagenesis, as well as various human diseases and aging processes, while the free 8-oxoG base in body fluids is one of the best biomarkers of ongoing pathophysiological processes. In this review, we discuss the biological significance of the 8-oxoG base and particularly the role of OGG1–BER in the activation of small GTPases and changes in gene expression, including those that regulate pro-inflammatory chemokines/cytokines and cause inflammation.

New approaches to detect 8-hydroxyguanine in γ-irradiated cellular DNA

International Nuclear Information System (INIS)

Mei, Nan; Tamae, Kazuyoshi; Hirano, Takeshi; Kasai, Hiroshi; Kunugita, Naoki

2003-01-01

This report describes an assay to detect 8-hydroxydeoxyguanosine 5'-monophosphate (8-OH-dGMP) in cellular DNA by modification of enzyme treatment after DNA extraction, using a high-performance liquid chromatography system equipped with an electrochemical detector (HPLC-ECD). This modification greatly reduces the measured background level of 8-hydroxyguanine (8-OH-Gua) in DNA, and improves the HPLC-ECD sensitivity to measure oxidative DNA damage. The 8-OH-Gua value in the DNA was expressed by the ratio of 8-OH-dGMP to deoxycytidine 5'-monophosphate (dCMP). Background level of 8-OH-Gua in DNA under our conditions was several times lower than that by a previous method. The human lung carcinoma cells (A549) were exposed to γ-rays of 20-100 Gy. A dose-dependent increase in oxidative DNA damage of 8-OH-Gua was observed. Furthermore, using commercial FITC-kit of an immunohistochemical type procedure, 8-OH-Gua was clearly detected in A549 cells and the fluorescence intensity of cells with oxidative DNA damage increased with the doses of γ-irradiation. Using an 8-OH-Gua repair activity assay, we also found that γ-rays decreased the repair enzyme activity. We conclude that the 8-OH-Gua level in human cellular DNA increases partly by the generation of reactive oxygen species (ROS) and partly by the inhibition of repair activity for 8-OH-Gua. (author)

Aging and defense against generation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in DNA

DEFF Research Database (Denmark)

Mikkelsen, Lone; Bialkowski, Karol; Risom, Lotte

2009-01-01

damaged DNA and the activity of the DNA repair system and 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxodGTPase) activity in liver and lung tissue from mice at 10-100 weeks of age. The level of 8-oxodG increased with age, whereas the level of formamidopyrimidine DNA...... glycosylase sites was unaltered. The enzyme activity toward single oxygen-induced DNA damage and mRNA expression levels of Ercc1, Neil1, and Ogg1 remained unaltered with age. However, the 8-oxodGTPase activity in the liver was 18% (95% CI: 0.2-37%) lower in mice at 25 and 50 weeks than in 10-week-old mice....... The 10- and 100-week-old mice had similar 8-oxodGTPase activity. In contrast, the mRNA expression of Nudt1 was statistically unaltered that likely resulted from higher variation of measurements. The accumulation of 8-oxodG with age is not a direct consequence of decreased enzyme activity toward singlet...

Involvement of DNA polymerase δ in DNA repair synthesis in human fibroblasts at late times after ultraviolet irradiation

International Nuclear Information System (INIS)

Dresler, S.L.; Gowans, B.J.; Robinson-Hill, R.M.; Hunting, D.J.

1988-01-01

DNA repair synthesis following UV irradiation of confluent human fibroblasts has a biphasic time course with an early phase of rapid nucleotide incorporation and a late phase of much slower nucleotide incorporation. The biphasic nature of this curve suggests that two distinct DNA repair systems may be operative. Previous studies have specifically implicated DNA polymerase δ as the enzyme involved in DNA repair synthesis occurring immediately after UV damage. In this paper, the authors describe studies of DNA polymerase involvement in DNA repair synthesis in confluent human fibroblasts at late times after UV irradiation. Late UV-induced DNA repair synthesis in both intact and permeable cells was found to be inhibited by aphidicolin, indicating the involvement of one of the aphidicolin-sensitive DNA polymerases, α or δ. In permeable cells, the process was further analyzed by using the nucleotide analogue (butylphenyl)-2'-deoxyguanosine 5'-triphosphate, which inhibits DNA polymerase α several hundred times more strongly than it inhibits DNA polymerase δ. The (butylphenyl)-2'-deoxyguanosine 5'-triphosphate inhibition curve for late UV-induced repair synthesis was very similar to that for polymerase δ. It appears that repair synthesis at late time after UV irradiation, like repair synthesis at early times, is mediated by DNA polymerase δ

Cul8/Rtt101 Forms a Variety of Protein Complexes That Regulate DNA Damage Response and Transcriptional Silencing*

OpenAIRE

Mimura, Satoru; Yamaguchi, Tsuyoshi; Ishii, Satoru; Noro, Emiko; Katsura, Tomoya; Obuse, Chikashi; Kamura, Takumi

2010-01-01

The budding yeast, Saccharomyces cerevisiae, has three cullin proteins, which act as platforms for Cullin-based E3 ubiquitin ligases. Genetic evidence indicates that Cul8, together with Mms1, Mms22, and Esc4, is involved in the repair of DNA damage that can occur during DNA replication. Cul8 is thought to form a complex with these proteins, but the composition and the function of Cul8-based E3 ubiquitin ligases remain largely uncharacterized. Herein, we report a comprehensive biochemical anal...

DNA repair in human cells: Methods for the determination of calmodulin involvement

International Nuclear Information System (INIS)

Charp, P.A.

1987-01-01

Exposure of DNA to either physical or chemical agents can result in the formation of a number of different lesions which must be repaired enzymatically in order for DNA to carry on normal replication and transcription. In most cases, the enzymes involved in this repair of damaged DNA include endonucleases, exonucleases, glycosylases, polymerases, and ligases. Each group of enzymes is involved in precise steps in DNA repair. Exposure to physical agents such as ultraviolet light (UV) at a wavelength of 254 nm is repaired by two distinct and different mechanisms. One mode of enzymatic repair of pyrimidine dimers is accomplished in situ by photoreactivation of UV-induced pyrimidine dimers by photoreactivating light. The second mode of enzymatic repair is the excision repair of pyrimidine dimers involving several different enzymes including endonuclease, exonuclease, and DNA ligase. A summary of the sequence of enzymatic steps involved is shown. It has been observed that specific drugs which bind to and alter the action of calmodulin in cells block DNA synthesis. This suggests that calmodulin may play a role both in normal DNA replication and repair. Others using an indirect method measuring the degree of DNA nucleoid sedimentation, showed that the specific anti-calmodulin agent W-13 slowed the rate of DNA repair. Others showed that DNA synthesis in T51B rat liver cells could be blocked with the addition of either chlorpromazine or trifluoperazine

Enzymes involved in organellar DNA replication in photosynthetic eukaryotes.

Science.gov (United States)

Moriyama, Takashi; Sato, Naoki

2014-01-01

Plastids and mitochondria possess their own genomes. Although the replication mechanisms of these organellar genomes remain unclear in photosynthetic eukaryotes, several organelle-localized enzymes related to genome replication, including DNA polymerase, DNA primase, DNA helicase, DNA topoisomerase, single-stranded DNA maintenance protein, DNA ligase, primer removal enzyme, and several DNA recombination-related enzymes, have been identified. In the reference Eudicot plant Arabidopsis thaliana, the replication-related enzymes of plastids and mitochondria are similar because many of them are dual targeted to both organelles, whereas in the red alga Cyanidioschyzon merolae, plastids and mitochondria contain different replication machinery components. The enzymes involved in organellar genome replication in green plants and red algae were derived from different origins, including proteobacterial, cyanobacterial, and eukaryotic lineages. In the present review, we summarize the available data for enzymes related to organellar genome replication in green plants and red algae. In addition, based on the type and distribution of replication enzymes in photosynthetic eukaryotes, we discuss the transitional history of replication enzymes in the organelles of plants.

Sequence context effects on 8-methoxypsoralen photobinding to defined DNA fragments

International Nuclear Information System (INIS)

Sage, E.; Moustacchi, E.

1987-01-01

The photoreaction of 8-methoxypsoralen (8-MOP) with DNA fragments of defined sequence was studied. The authors took advantage of the blockage by bulky adducts of the 3'-5'-exonuclease activity associated with the T4 DNA polymerase. The action of the exonuclease is stopped by biadducts as well as by monoadducts. The termination products were analyzed on sequencing gels. A strong sequence specificity was observed in the DNA photobinding of 8-MOP. The exonuclease terminates its digestion near thymine residues, mainly at potentially cross-linkable sites. There is an increasing reactivity of thymine residues in the order T < TT << TTT in a GC environment. For thymine residues in cross-linkable sites, the reactivity follows the order AT << TA ∼ TAT << ATA < ATAT < ATATAA. Repeated A-T sequences are hot spots for the photochemical reaction of 8-MOP with DNA. Both monoadducts and interstrand cross-links are formed preferentially in 5'-TpA sites. The results highlight the role of the sequence and consequently of the conformation around a potential site in the photobinding of 8-MOP to DNA

Triphlorethol-A from Ecklonia cava Up-Regulates the Oxidant Sensitive 8-Oxoguanine DNA Glycosylase 1

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Ki Cheon Kim

2014-10-01

Full Text Available This study investigated the protective mechanisms of triphlorethol-A, isolated from Ecklonia cava, against oxidative stress-induced DNA base damage, especially 8-oxoguanine (8-oxoG, in Chinese hamster lung fibroblast V79-4 cells. 8-Oxoguanine DNA glycosylase-1 (OGG1 plays an important role in the removal of 8-oxoG during the cellular response to DNA base damage. Triphlorethol-A significantly decreased the levels of 8-oxoG induced by H2O2, and this correlated with increases in OGG1 mRNA and OGG1 protein levels. Furthermore, siOGG1-transfected cell attenuated the protective effect of triphlorethol-A against H2O2 treatment. Nuclear factor erythroid 2–related factor 2 (Nrf2 is a transcription factor for OGG1, and Nrf2 combines with small Maf proteins in the nucleus to bind to antioxidant response elements (ARE in the upstream promoter region of the OGG1 gene. Triphlorethol-A restored the expression of nuclear Nrf2, small Maf protein, and the Nrf2-Maf complex, all of which were reduced by oxidative stress. Furthermore, triphlorethol-A increased Nrf2 binding to ARE sequences and the resulting OGG1 promoter activity, both of which were also reduced by oxidative stress. The levels of the phosphorylated forms of Akt kinase, downstream of phosphatidylinositol 3-kinase (PI3K, and Erk, which are regulators of OGG1, were sharply decreased by oxidative stress, but these decreases were prevented by triphlorethol-A. Specific PI3K, Akt, and Erk inhibitors abolished the cytoprotective effects of triphlorethol-A, suggesting that OGG1 induction by triphlorethol-A involves the PI3K/Akt and Erk pathways. Taken together, these data indicate that by activating the DNA repair system, triphlorethol-A exerts protective effects against DNA base damage induced by oxidative stress.

DnaA protein DNA-binding domain binds to Hda protein to promote inter-AAA+ domain interaction involved in regulatory inactivation of DnaA.

Science.gov (United States)

Keyamura, Kenji; Katayama, Tsutomu

2011-08-19

Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis.

DnaA Protein DNA-binding Domain Binds to Hda Protein to Promote Inter-AAA+ Domain Interaction Involved in Regulatory Inactivation of DnaA*

Science.gov (United States)

Keyamura, Kenji; Katayama, Tsutomu

2011-01-01

Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis. PMID:21708944

Decreased uv mutagenesis in cdc8, a DNA replication mutant of Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Prakash, L.; Hinkle, D.; Prakash, S.

1978-01-01

A DNA replication mutant of yeast, cdc8, was found to decrease uv-induced reversion of lys2-1, arg4-17, tryl and ural. This effect was observed with all three alleles of cdc8 tested. Survival curves obtained following uv irradiation in cdc8 rad double mutants show that cdc8 is epistatic to rad6, as well as to rad1; cdc8 rad51 double mutants seem to be more sensitive than the single mutants. Since uv-induced reversion in cdc8 rad1 and cdc8 rad51 double mutants is like that of the cdc8 single mutants, we conclude that CDC8 plays a direct role in error-prone repair. To test whether CDC8 codes for a DNA polymerase, we have purified both DNA polymerase I and DNA polymerase II from cdc8 and CDC+ cells. The purified DNA polymerases from cdc8 were no more heat labile than those from CDC+, suggesting that CDC8 is not a structural gene for either enzyme

Islet expression of the DNA repair enzyme 8-oxoguanosine DNA glycosylase (Ogg1 in human type 2 diabetes

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Yoon Kun-Ho

2002-04-01

Full Text Available Abstract Background It has become increasingly clear that β-cell failure plays a critical role in the pathogenesis of type 2 diabetes. Free-radical mediated β-cell damage has been intensively studied in type 1 diabetes, but not in human type 2 diabetes. Therefore, we studied the protein expression of the DNA repair enzyme Ogg1 in pancreases from type 2 diabetics. Ogg1 was studied because it is the major enzyme involved in repairing 7,8-dihydro-8-oxoguanosine DNA adducts, a lesion previously observed in a rat model of type 2 diabetes. Moreover, in a gene expression screen, Ogg1 was over-expressed in islets from a human type 2 diabetic. Methods Immunofluorescent staining of Ogg1 was performed on pancreatic specimens from healthy controls and patients with diabetes for 2–23 years. The intensity and islet area stained for Ogg1 was evaluated by semi-quantitative scoring. Results Both the intensity and the area of islet Ogg1 staining were significantly increased in islets from the type 2 diabetic subjects compared to the healthy controls. A correlation between increased Ogg1 fluorescent staining intensity and duration of diabetes was also found. Most of the staining observed was cytoplasmic, suggesting that mitochondrial Ogg1 accounts primarily for the increased Ogg1 expression. Conclusion We conclude that oxidative stress related DNA damage may be a novel important factor in the pathogenesis of human type 2 diabetes. An increase of Ogg1 in islet cell mitochondria is consistent with a model in which hyperglycemia and consequent increased β-cell oxidative metabolism lead to DNA damage and the induction of Ogg1 expression.

Identification of genes involved in DNA replication of the Autographa californica baculovirus

NARCIS (Netherlands)

Kool, M.; Ahrens, C. H.; Goldbach, R. W.; Rohrmann, G. F.; Vlak, J. M.

1994-01-01

By use of a transient replication assay, nine genes involved in DNA replication were identified in the genome of the Autographa californica baculovirus. Six genes encoding helicase, DNA polymerase, IE-1, LEF-1, LEF-2, and LEF-3 are essential for DNA replication while three genes encoding P35, IE-2,

Involvement of DNA mismatch repair in the maintenance of heterochromatic DNA stability in Saccharomyces cerevisiae.

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Basanta K Dahal

2017-10-01

Full Text Available Heterochromatin contains a significant part of nuclear DNA. Little is known about the mechanisms that govern heterochromatic DNA stability. We show here that in the yeast Saccharomyces cerevisiae (i DNA mismatch repair (MMR is required for the maintenance of heterochromatic DNA stability, (ii MutLα (Mlh1-Pms1 heterodimer, MutSα (Msh2-Msh6 heterodimer, MutSβ (Msh2-Msh3 heterodimer, and Exo1 are involved in MMR at heterochromatin, (iii Exo1-independent MMR at heterochromatin frequently leads to the formation of Pol ζ-dependent mutations, (iv MMR cooperates with the proofreading activity of Pol ε and the histone acetyltransferase Rtt109 in the maintenance of heterochromatic DNA stability, (v repair of base-base mismatches at heterochromatin is less efficient than repair of base-base mismatches at euchromatin, and (vi the efficiency of repair of 1-nt insertion/deletion loops at heterochromatin is similar to the efficiency of repair of 1-nt insertion/deletion loops at euchromatin.

Genome-Wide DNA Methylation Profiles of Phlegm-Dampness Constitution

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Haiqiang Yao

2018-03-01

Full Text Available Background/Aims: Metabolic diseases are leading health concerns in today’s global society. In traditional Chinese medicine (TCM, one body type studied is the phlegm-dampness constitution (PC, which predisposes individuals to complex metabolic disorders. Genomic studies have revealed the potential metabolic disorders and the molecular features of PC. The role of epigenetics in the regulation of PC, however, is unknown. Methods: We analyzed a genome-wide DNA methylation in 12 volunteers using Illumina Infinium Human Methylation450 BeadChip on peripheral blood mononuclear cells (PBMCs. Eight volunteers had PC and 4 had balanced constitutions. Results: Methylation data indicated a genome-scale hyper-methylation pattern in PC. We located 288 differentially methylated probes (DMPs. A total of 256 genes were mapped, and some of these were metabolic-related. SQSTM1, DLGAP2 and DAB1 indicated diabetes mellitus; HOXC4 and SMPD3, obesity; and GRWD1 and ATP10A, insulin resistance. According to Ingenuity Pathway Analysis (IPA, differentially methylated genes were abundant in multiple metabolic pathways. Conclusion: Our results suggest the potential risk for metabolic disorders in individuals with PC. We also explain the clinical characteristics of PC with DNA methylation features.

Altered DNA methylation: a secondary mechanism involved in carcinogenesis.

Science.gov (United States)

Goodman, Jay I; Watson, Rebecca E

2002-01-01

This review focuses on the role that DNA methylation plays in the regulation of normal and aberrant gene expression and on how, in a hypothesis-driven fashion, altered DNA methylation may be viewed as a secondary mechanism involved in carcinogenesis. Research aimed at discerning the mechanisms by which chemicals can transform normal cells into frank carcinomas has both theoretical and practical implications. Through an increased understanding of the mechanisms by which chemicals affect the carcinogenic process, we learn more about basic biology while, at the same time, providing the type of information required to make more rational safety assessment decisions concerning their actual potential to cause cancer under particular conditions of exposure. One key question is: does the mechanism of action of the chemical in question involve a secondary mechanism and, if so, what dose may be below its threshold?

Phosphorylation of human INO80 is involved in DNA damage tolerance

International Nuclear Information System (INIS)

Kato, Dai; Waki, Mayumi; Umezawa, Masaki; Aoki, Yuka; Utsugi, Takahiko; Ohtsu, Masaya; Murakami, Yasufumi

2012-01-01

Highlights: ► Depletion of hINO80 significantly reduced PCNA ubiquitination. ► Depletion of hINO80 significantly reduced nuclear dots intensity of RAD18 after UV irradiation. ► Western blot analyses showed phosphorylated hINO80 C-terminus. ► Overexpression of phosphorylation mutant hINO80 reduced PCNA ubiquitination. -- Abstract: Double strand breaks (DSBs) are the most serious type of DNA damage. DSBs can be generated directly by exposure to ionizing radiation or indirectly by replication fork collapse. The DNA damage tolerance pathway, which is conserved from bacteria to humans, prevents this collapse by overcoming replication blockages. The INO80 chromatin remodeling complex plays an important role in the DNA damage response. The yeast INO80 complex participates in the DNA damage tolerance pathway. The mechanisms regulating yINO80 complex are not fully understood, but yeast INO80 complex are necessary for efficient proliferating cell nuclear antigen (PCNA) ubiquitination and for recruitment of Rad18 to replication forks. In contrast, the function of the mammalian INO80 complex in DNA damage tolerance is less clear. Here, we show that human INO80 was necessary for PCNA ubiquitination and recruitment of Rad18 to DNA damage sites. Moreover, the C-terminal region of human INO80 was phosphorylated, and overexpression of a phosphorylation-deficient mutant of human INO80 resulted in decreased ubiquitination of PCNA during DNA replication. These results suggest that the human INO80 complex, like the yeast complex, was involved in the DNA damage tolerance pathway and that phosphorylation of human INO80 was involved in the DNA damage tolerance pathway. These findings provide new insights into the DNA damage tolerance pathway in mammalian cells.

Functional characterization of 8-oxoguanine DNA glycosylase of Trypanosoma cruzi.

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Carolina Furtado

Full Text Available The oxidative lesion 8-oxoguanine (8-oxoG is removed during base excision repair by the 8-oxoguanine DNA glycosylase 1 (Ogg1. This lesion can erroneously pair with adenine, and the excision of this damaged base by Ogg1 enables the insertion of a guanine and prevents DNA mutation. In this report, we identified and characterized Ogg1 from the protozoan parasite Trypanosoma cruzi (TcOgg1, the causative agent of Chagas disease. Like most living organisms, T. cruzi is susceptible to oxidative stress, hence DNA repair is essential for its survival and improvement of infection. We verified that the TcOGG1 gene encodes an 8-oxoG DNA glycosylase by complementing an Ogg1-defective Saccharomyces cerevisiae strain. Heterologous expression of TcOGG1 reestablished the mutation frequency of the yeast mutant ogg1(-/- (CD138 to wild type levels. We also demonstrate that the overexpression of TcOGG1 increases T. cruzi sensitivity to hydrogen peroxide (H(2O(2. Analysis of DNA lesions using quantitative PCR suggests that the increased susceptibility to H(2O(2 of TcOGG1-overexpressor could be a consequence of uncoupled BER in abasic sites and/or strand breaks generated after TcOgg1 removes 8-oxoG, which are not rapidly repaired by the subsequent BER enzymes. This hypothesis is supported by the observation that TcOGG1-overexpressors have reduced levels of 8-oxoG both in the nucleus and in the parasite mitochondrion. The localization of TcOgg1 was examined in parasite transfected with a TcOgg1-GFP fusion, which confirmed that this enzyme is in both organelles. Taken together, our data indicate that T. cruzi has a functional Ogg1 ortholog that participates in nuclear and mitochondrial BER.

ZTF-8 interacts with the 9-1-1 complex and is required for DNA damage response and double-strand break repair in the C. elegans germline.

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Hyun-Min Kim

2014-10-01

Full Text Available Germline mutations in DNA repair genes are linked to tumor progression. Furthermore, failure in either activating a DNA damage checkpoint or repairing programmed meiotic double-strand breaks (DSBs can impair chromosome segregation. Therefore, understanding the molecular basis for DNA damage response (DDR and DSB repair (DSBR within the germline is highly important. Here we define ZTF-8, a previously uncharacterized protein conserved from worms to humans, as a novel factor involved in the repair of both mitotic and meiotic DSBs as well as in meiotic DNA damage checkpoint activation in the C. elegans germline. ztf-8 mutants exhibit specific sensitivity to γ-irradiation and hydroxyurea, mitotic nuclear arrest at S-phase accompanied by activation of the ATL-1 and CHK-1 DNA damage checkpoint kinases, as well as accumulation of both mitotic and meiotic recombination intermediates, indicating that ZTF-8 functions in DSBR. However, impaired meiotic DSBR progression partially fails to trigger the CEP-1/p53-dependent DNA damage checkpoint in late pachytene, also supporting a role for ZTF-8 in meiotic DDR. ZTF-8 partially co-localizes with the 9-1-1 DDR complex and interacts with MRT-2/Rad1, a component of this complex. The human RHINO protein rescues the phenotypes observed in ztf-8 mutants, suggesting functional conservation across species. We propose that ZTF-8 is involved in promoting repair at stalled replication forks and meiotic DSBs by transducing DNA damage checkpoint signaling via the 9-1-1 pathway. Our findings define a conserved function for ZTF-8/RHINO in promoting genomic stability in the germline.

Distinct DNA repair pathways involving RecA and nonhomologous end joining in Mycobacterium smegmatis.

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Korycka-Machala, Malgorzata; Brzostek, Anna; Rozalska, Sylwia; Rumijowska-Galewicz, Anna; Dziedzic, Renata; Bowater, Richard; Dziadek, Jaroslaw

2006-05-01

Mycobacterium smegmatis was used to study the relationship between DNA repair processes involving RecA and nonhomologous end joining (NHEJ). The effect of gene deletions in recA and/or in two genes involved in NHEJ (ku and ligD) was tested on the ability of bacteria to join breaks in plasmids transformed into them and in their response to chemicals that damage DNA. The results provide in vivo evidence that only NHEJ is required for the repair of noncompatible DNA ends. By contrast, the response of mycobacteria to mitomycin C preferentially involved a RecA-dependent pathway.

Biochemical studies of DNA strand break repair and molecular characterization of mei-41, a gene involved in DNA break repair

International Nuclear Information System (INIS)

Oliveri, D.R.

1989-01-01

The ability to repair X-irradiation induced single-strand DNA breaks was examined in mutagen-sensitive mutants of Drosophila melanogaster. This analysis demonstrated that examined stocks possess a normal capacity to repair X-ray induced single-strand breaks. One of the mutants in this study, mei-41, has been shown to be involved in a number of DNA metabolizing functions. A molecular characterization of this mutant is presented. A cDNA hybridizing to genomic DNA both proximal and distal to a P element inducing a mei-41 mutation was isolated from both embryonic and adult female recombinant lambda phage libraries. A 2.2 kilobase embryonic cDNA clone was sequenced; the sequence of an open reading frame was identified which would predict a protein of 384 amino acids with a molecular weight of 43,132 daltons. An examination of homologies to sequences in protein and nucleic acid data bases revealed no sequences with significant homology to mei-41, however, two potential Zinc-finger domains were identified. Analysis of RNA hybridizing to the embryonic cDNA demonstrated the existence of a major 2.2 kilobase transcript expressed primarily in embryos and adult flies. An examination of the transcription of this gene in mei-41 mutants revealed significant variation from wild-type, an indication that the embryonic cDNA does represent a mei-41 transcript. Expression in tissues from adult animals demonstrated that the 2.2 kilobase RNA is expressed primarily in reproductive tissues. A 3.8kb transcript is the major species of RNA in the adult head and thorax. Evidence is presented which implies that expression of the mei-41 gene is strongly induced by exposure of certain cells to mutagens

DNA glycosylases involved in base excision repair may be associated with cancer risk in BRCA1 and BRCA2 mutation carriers.

Directory of Open Access Journals (Sweden)

Ana Osorio

2014-04-01

Full Text Available Single Nucleotide Polymorphisms (SNPs in genes involved in the DNA Base Excision Repair (BER pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of the components of the BER pathway, PARP1 (poly ADP ribose polymerase, and both BRCA1 and BRCA2. In the present study, we have performed a comprehensive analysis of 18 genes involved in BER using a tagging SNP approach in a large series of BRCA1 and BRCA2 mutation carriers. 144 SNPs were analyzed in a two stage study involving 23,463 carriers from the CIMBA consortium (the Consortium of Investigators of Modifiers of BRCA1 and BRCA2. Eleven SNPs showed evidence of association with breast and/or ovarian cancer at p<0.05 in the combined analysis. Four of the five genes for which strongest evidence of association was observed were DNA glycosylases. The strongest evidence was for rs1466785 in the NEIL2 (endonuclease VIII-like 2 gene (HR: 1.09, 95% CI (1.03-1.16, p = 2.7 × 10(-3 for association with breast cancer risk in BRCA2 mutation carriers, and rs2304277 in the OGG1 (8-guanine DNA glycosylase gene, with ovarian cancer risk in BRCA1 mutation carriers (HR: 1.12 95%CI: 1.03-1.21, p = 4.8 × 10(-3. DNA glycosylases involved in the first steps of the BER pathway may be associated with cancer risk in BRCA1/2 mutation carriers and should be more comprehensively studied.

The effect of DNA replication on mutation of the Saccharomyces cerevisiae CDC8 gene.

Science.gov (United States)

Zaborowska, D; Zuk, J

1990-04-01

Incubation in YPD medium under permissive conditions when DNA replication is going on, strongly stimulates the induction of cdc+ colonies of UV-irradiated cells of yeast strains HB23 (cdc8-1/cdc8-3), HB26 (cdc8-3/cdc8-3) and HB7 (cdc8-1/cdc8-1). Inhibition of DNA replication by hydroxyurea, araCMP, cycloheximide or caffeine or else by incubation in phosphate buffer pH 7.0, abolishes this stimulation. Thus the replication of DNA is strongly correlated with the high induction of cdc+ colonies by UV irradiation. It is postulated that these UV-induced cdc+ colonies arise as the result infidelity in DNA replication.

Distinct DNA repair pathways involving RecA and nonhomologous end joining in Mycobacterium smegmatis.

OpenAIRE

Korycka-Machala, M; Brzostek, A; Rozalska, S; Rumijowska-Galewicz, A; Dziedzic, R; Bowater, R; Dziadek, J

2006-01-01

Mycobacterium smegmatis was used to study the relationship between DNA repair processes involving RecA and nonhomologous end joining (NHEJ). The effect of gene deletions in recA and/or in two genes involved in NHEJ (ku and ligD) was tested on the ability of bacteria to join breaks in plasmids transformed into them and in their response to chemicals that damage DNA. The results provide in vivo evidence that only NHEJ is required for the repair of noncompatible DNA ends. By contrast, the respon...

The Bipolar Filaments Formed by Herpes Simplex Virus Type 1 SSB/Recombination Protein (ICP8) Suggest a Mechanism for DNA Annealing

Energy Technology Data Exchange (ETDEWEB)

Makhov, A.M.; Simon, M.; Sen, A.; Yu, X.; Griffith, J. D.; Egelman, E. H.

2009-02-20

Herpes simplex virus type 1 encodes a multifunctional protein, ICP8, which serves both as a single-strand binding protein and as a recombinase, catalyzing reactions involved in replication and recombination of the viral genome. In the presence of divalent ions and at low temperature, previous electron microscopic studies showed that ICP8 will form long left-handed helical filaments. Here, electron microscopic image reconstruction reveals that the filaments are bipolar, with an asymmetric unit containing two subunits of ICP8 that constitute a symmetrical dimer. This organization of the filament has been confirmed using scanning transmission electron microscopy. The pitch of the filaments is {approx} 250 {angstrom}, with {approx} 6.2 dimers per turn. Docking of a crystal structure of ICP8 into the reconstructed filament shows that the C-terminal domain of ICP8, attached to the body of the subunit by a flexible linker containing {approx} 10 residues, is packed into a pocket in the body of a neighboring subunit in the crystal in a similar manner as in the filament. However, the interactions between the large N-terminal domains are quite different in the filament from that observed in the crystal. A previously proposed model for ICP8 binding single-stranded DNA (ssDNA), based upon the crystal structure, leads to a model for a continuous strand of ssDNA near the filament axis. The bipolar nature of the ICP8 filaments means that a second strand of ssDNA would be running through this filament in the opposite orientation, and this provides a potential mechanism for how ICP8 anneals complementary ssDNA into double-stranded DNA, where each strand runs in opposite directions.

Structure of a preternary complex involving a prokaryotic NHEJ DNA polymerase.

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Brissett, Nigel C; Martin, Maria J; Pitcher, Robert S; Bianchi, Julie; Juarez, Raquel; Green, Andrew J; Fox, Gavin C; Blanco, Luis; Doherty, Aidan J

2011-01-21

In many prokaryotes, a specific DNA primase/polymerase (PolDom) is required for nonhomologous end joining (NHEJ) repair of DNA double-strand breaks (DSBs). Here, we report the crystal structure of a catalytically active conformation of Mycobacterium tuberculosis PolDom, consisting of a polymerase bound to a DNA end with a 3' overhang, two metal ions, and an incoming nucleotide but, significantly, lacking a primer strand. This structure represents a polymerase:DNA complex in a preternary intermediate state. This polymerase complex occurs in solution, stabilizing the enzyme on DNA ends and promoting nucleotide extension of short incoming termini. We also demonstrate that the invariant Arg(220), contained in a conserved loop (loop 2), plays an essential role in catalysis by regulating binding of a second metal ion in the active site. We propose that this NHEJ intermediate facilitates extension reactions involving critically short or noncomplementary DNA ends, thus promoting break repair and minimizing sequence loss during DSB repair. Copyright © 2011 Elsevier Inc. All rights reserved.

DNA damage tolerance pathway involving DNA polymerase ι and the tumor suppressor p53 regulates DNA replication fork progression.

Science.gov (United States)

Hampp, Stephanie; Kiessling, Tina; Buechle, Kerstin; Mansilla, Sabrina F; Thomale, Jürgen; Rall, Melanie; Ahn, Jinwoo; Pospiech, Helmut; Gottifredi, Vanesa; Wiesmüller, Lisa

2016-07-26

DNA damage tolerance facilitates the progression of replication forks that have encountered obstacles on the template strands. It involves either translesion DNA synthesis initiated by proliferating cell nuclear antigen monoubiquitination or less well-characterized fork reversal and template switch mechanisms. Herein, we characterize a novel tolerance pathway requiring the tumor suppressor p53, the translesion polymerase ι (POLι), the ubiquitin ligase Rad5-related helicase-like transcription factor (HLTF), and the SWI/SNF catalytic subunit (SNF2) translocase zinc finger ran-binding domain containing 3 (ZRANB3). This novel p53 activity is lost in the exonuclease-deficient but transcriptionally active p53(H115N) mutant. Wild-type p53, but not p53(H115N), associates with POLι in vivo. Strikingly, the concerted action of p53 and POLι decelerates nascent DNA elongation and promotes HLTF/ZRANB3-dependent recombination during unperturbed DNA replication. Particularly after cross-linker-induced replication stress, p53 and POLι also act together to promote meiotic recombination enzyme 11 (MRE11)-dependent accumulation of (phospho-)replication protein A (RPA)-coated ssDNA. These results implicate a direct role of p53 in the processing of replication forks encountering obstacles on the template strand. Our findings define an unprecedented function of p53 and POLι in the DNA damage response to endogenous or exogenous replication stress.

Trans-Lesion DNA Polymerases May Be Involved in Yeast Meiosis

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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

Chromosomal Alterations and Gene Expression Changes Associated with the Progression of Leukoplakia to Advanced Gingivobuccal Cancer

Directory of Open Access Journals (Sweden)

Priyanka G. Bhosale

2017-06-01

Full Text Available We present an integrative genome-wide analysis that can be used to predict the risk of progression from leukoplakia to oral squamous cell carcinoma (OSCC arising in the gingivobuccal complex (GBC. We find that the genomic and transcriptomic profiles of leukoplakia resemble those observed in later stages of OSCC and that several changes are associated with this progression, including amplification of 8q24.3, deletion of 8p23.2, and dysregulation of DERL3, EIF5A2, ECT2, HOXC9, HOXC13, MAL, MFAP5 and NELL2. Comparing copy number profiles of primary tumors with and without lymph-node metastasis, we identify alterations associated with metastasis, including amplifications of 3p26.3, 8q24.21, 11q22.1, 11q22.3 and deletion of 8p23.2. Integrative analysis reveals several biomarkers that have never or rarely been reported in previous OSCC studies, including amplifications of 1p36.33 (attributable to MXRA8, 3q26.31 (EIF5A2, 9p24.1 (CD274, and 12q13.2 (HOXC9 and HOXC13. Additionally, we find that amplifications of 1p36.33 and 11q22.1 are strongly correlated with poor clinical outcome. Overall, our findings delineate genomic changes that can be used in treatment management for patients with potentially malignant leukoplakia and OSCC patients with higher risk of lymph-node metastasis.

Increased urinary excretion of 8-oxo-2'-deoxyguanosine, a biomarker of oxidative DNA damage, in urban bus drivers

DEFF Research Database (Denmark)

Loft, S; Poulsen, H E; Vistisen, K

1999-01-01

Oxidative damage to DNA could be involved in the increased risk of cancer associated with exposure to polluted urban air, which contains a number of oxidants. CYP1A2 is induced by and metabolizes polyaromatic hydrocarbons (PAH) and aromatic amines and could modify effects of exposure to ambient air...... pollution. Similarly, DNA repair may be influenced by occupational and other exposures as well as modify the effect of DNA damaging agents. As part of a large investigation of the genotoxic burden to diesel exposed workers in transport sectors we studied oxidative DNA damage in 57 non-smoking bus drivers...... from the greater Copenhagen area. The drivers were studied on a workday and on a day off work. Comparisons were made between drivers from the central (n=30) and rural/suburban (n=27) areas of Copenhagen. The rate of oxidative DNA damage was estimated from 24 h urinary excretion of 8-oxo-2...

Kaposi's sarcoma-associated herpesvirus-like DNA sequences (KSHV/HHV-8) in oral AIDS-Kaposi's sarcoma: a PCR and clinicopathologic study.

Science.gov (United States)

Flaitz, C M; Jin, Y T; Hicks, M J; Nichols, C M; Wang, Y W; Su, I J

1997-02-01

Recently, a new human herpesvirus (KSHV/HHV-8) has been identified in classic, transplant, endemic, and AIDS Kaposi's sarcoma that may be involved in the pathogenesis of Kaposi's sarcoma. The purpose of this study was to evaluate oral AIDS-Kaposi's sarcoma for detection of KSHV/HHV-8 DNA. DNA extracted from 54 oral AIDS-Kaposi's sarcoma lesions (47 initial, 7 postvinblastine treated), 5 non-Kaposi's sarcoma HIV-positive lesions, and 3 non-Kaposi's sarcoma HIV-negative lesions was evaluated by polymerase chain reaction (KS330(233bp)amplicon) for KSHV/HHV-8. The AIDS-Kaposi's sarcoma study population consisted of 52 patients (51:1, men:woman; 92% men having sex with men, 8% heterosexual; mean age, 38 years; mean, CD4 59/mm3) Opportunistic infections occurred in 88% (candidiasis, 65%; Pneumocystis carinii pneumonia, 31%; nonoral Kaposi's sarcoma, 25%; mycobacterium avium-intracellulare (MAI), 16%; cytomegalovirus, 14%; herpes simplex virus, 14%). Sexually transmitted diseases occurred in 73% (gonorrhea, 37%; syphilis, 23%; condyloma, 22%; HSV, 16%). Most frequent lesion sites were palate (74%) and gingiva (17%). Most common lesion types were purple nodular (48%) and macular (42%). Histopathologic subtypes were nodular (71%), plaque (27%), and patch (2%). Polymerase chain reaction analysis detected KSHV/HHV-8 DNA in 53 of 54 AIDS-Kaposi's sarcoma lesions (47 of 47 initial, 6 of 7 postvinblastine treatment). KSHV/HHV-8 DNA was not detected in non-Kaposi's sarcoma lesions in HIV-positive or HIV-negative persons. KSHV/HHV-8 DNA sequence is present in a high proportion of oral AIDS-Kaposi's sarcoma lesions. Whether KSHV/HHV-8 is an etiologic agent or a cofactor in the development of this vascular neoplasm is uncertain and remains to be proven. Polymerase chain reaction analysis for KSHV/HHV-8 DNA sequence detection may be helpful in identifying Kaposi's sarcoma in early vascular proliferations, when the characteristic histopathologic features are not present.

Miscoding and mutagenic properties of 8-oxoguanine and abasic sites: Ubiquitous lesions in damaged DNA

International Nuclear Information System (INIS)

Grollman, A.P.; Takeshita, Masaru

1995-01-01

More than twenty oxidatively-damaged bases, including 8-oxoguanine, have been found to occur in genomic DNA. Some of these lesions block DNA replication and are potentially lethal; others generate mutations which can initiate carcinogenesis and promote cellular aging. In this report, the authors focus attention on the mutagenicity and repair of 8-oxoguanine. Kasai and Nishimura's discovery that hydroxyl radicals react with guanine residues in DNA to form 8-oxoguanine and the development of sensitive methods for the detection and quantitation of this modified base led to the observation that approximately 1 in 10 5 guanine residues in mammalian DNA are oxidized at the C-8 position. DNA containing 8-oxoguanine and synthetic analogs of the abasic site have been used to investigate the miscoding and mutagenic potential of these ubiquitous lesions. Studies in the laboratory were facilitated by the development of solid state synthetic methods by which these lesions could be introduced at defined positions in DNA. In this paper, the authors review studies in which 8-oxoguanine and abasic sites have been used in model systems to explore various early events in the replication of selectively damaged DNA

Menadione-induced DNA fragmentation without 8-oxo-2'-deoxyguanosine formation in isolated rat hepatocytes

DEFF Research Database (Denmark)

Fischer-Nielsen, A; Corcoran, G B; Poulsen, H E

1995-01-01

Menadione (2-methyl-1,4-naphthoquinone) induces oxidative stress in cells causing perturbations in the cytoplasm as well as nicking of DNA. The mechanisms by which DNA damage occurs are still unclear, but a widely discussed issue is whether menadione-generated reactive oxygen species (ROS) directly...... damage DNA. In the present study, we measured the effect of menadione on formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG), an index of oxidative DNA base modifications, and on DNA fragmentation. Isolated hepatocytes from phenobarbital-pretreated rats were exposed to menadione, 25-400 micro......M, for 15, 90 or 180 min with or without prior depletion of reduced glutathione (GSH) by diethyl maleate. Menadione caused profound GSH depletion and internucleosomal DNA fragmentation, which was demonstrated by a prominent fragmentation ladder on agarose gel electrophoresis. We found no oxidative...

In vitro study of DNA Adduct 8-OHdG Formation by using Bisphenol A in Calf Thymus DNA and 2’-Deoxyguanosine

Science.gov (United States)

Budiawan; Cahaya Dani, Intan; Bakri, Ridla; Handayani, Sri; Ratna Dewi, Evi

2018-01-01

The in vitro study of DNA Adduct 8-OHdG Formation due to BisphenolA (BPA) as xenobiotics has been conducted by using calf thymus DNA and 2’deoxyguanosine. The method of study was conducted by incubating calf thymus DNA and 2’dG with compounds trigger to radicals in the variation of pH (7.4 and 8.4), temperature (37°C and 60°C), and BPA concentrations (2 ppm and 10 ppm). To represent the work of CYP 450 enzyme in metabolic process of xenobiotics in the body and the effect of metal presence to the formation of radicals that can lead to 8-OHdG formation, we used iron(II) solution and also fenton reagent (Fe(II) and H2O2). The DNA used has 1.8 purity ratio (checked at λ260/λ280 by using Spectrophotometry UV-Vis). The results by using HPLC method showed that BPA could interact with DNA and DNA base (represent as calf thymus and 2’dG) and potentially induced 8-OHdG formation. The presence of iron(II) metal and Fenton reagent also induced the higher 8-OHdG formation. The higher of pH, temperature and concentrations also lead to 8-OHdG formation (ranger between 4 - 70 ppb).

Effect of DNA polymerase inhibitors on DNA repair in intact and permeable human fibroblasts: Evidence that DNA polymerases δ and β are involved in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine

International Nuclear Information System (INIS)

Hammond, R.A.; Miller, M.R.; McClung, J.K.

1990-01-01

The involvement of DNA polymerases α, β, and δ in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was investigated in human fibroblasts (HF). The effects of anti-(DNA polymerase α) monoclonal antibody, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), dideoxythymidine triphosphate (ddTTP), and aphidicolin on MNNG-induced DNA repair synthesis were investigated to dissect the roles of the different DNA polymerases. A subcellular system (permeable cells), in which DNA repair synthesis and DNA replication were differentiated by CsCl gradient centrifugation of BrdUMP density-labeled DNA, was used to examine the effects of the polymerase inhibitors. Another approach investigated the effects of several of these inhibitors of MNNG-induced DNA repair synthesis in intact cells by measuring the amount of [ 3 H]thymidine incorporated into repair DNA as determined by autoradiography and quantitation with an automated video image analysis system. In permeable cells, MNNG-induced DNA repair synthesis was inhibited 56% by 50 μg of aphidicolin/mL, 6% by 10 μM BuPdGTP, 13% by anti-(DNA polymerse α) monoclonal antibodies, and 29% by ddTTP. In intact cells, MNNG-induced DNA repair synthesis was inhibited 57% by 50 μg of aphidicolin/mL and was not significantly inhibited by microinjecting anti-(DNA polymerase α) antibodies into HF nuclei. These results indicate that both DNA polymerase δ and β are involved in repairing DNA damage caused by MNNG

8-oxo-7,8-dihydroguanine level - the DNA oxidative stress marker - recognized by fluorescence image analysis in sporadic uterine adenocarcinomas in women.

Science.gov (United States)

Postawski, Krzysztof; Przadka-Rabaniuk, Dorota; Piersiak, Tomasz

2013-01-01

In the case of carcinogenesis in human endometrium no information exists on tissue concentration of 8-oxo-7,8-dihydroguanine, the DNA oxidative stress marker This was the main reason to undertake the investigation of this DNA modification in human uterine estrogen-dependent tissue cancers. In order to estimate the level of oxidative damage, 8-oxo-7,8-dihydroguanine was determined directly in cells of tissue microscope slides using OxyDNA Assay Kit, Fluorometric. Cells were investigated under confocal microscope. Images of individual cells were captured by computer-interfaced digital photography and analyzed for fluorescence intensities (continuous inverted 8-bit gray-scale = 0 [black]-255 [white]). Fluorescence scores were calculated for each of 13 normal endometrial samples and 31 uterine adenocarcinoma specimens. Finally the level of the oxidative stress marker was also analyzed according to histological and clinical features of the neoplasms. The obtained data revealed that: 8-oxo-7,8-dihydroguanine levels were higher in uterine adenocarcinomas than in normal endometrial samples (48,32 vs. 38,64; p<0,001); in contrast to normal endometrium there was no correlation between age and DNA oxidative modification content in uterine cancer; highest mean fluorescence intensity was recognized in G2 endometrial adenocarcinomas; level of 8-oxo-7,8-dihydroguanine does not depend on Body Mass Index (BMI) and cancer uterine wall infiltration or tumor FIGO stage. Our study indicates that accumulation of the oxidized DNA base may contribute to the development of endometrial neoplasia, however oxidative DNA damage does not seem to increase with tumor progression.

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.

Isolation of proteins involved in the replication of adenoviral DNA in vitro

International Nuclear Information System (INIS)

Lichy, J.H.; Nagata, K.; Friefeld, B.R.; Enomoto, T.; Field, J.; Guggenheimer, R.A.; Ikeda, J.E.; Horwitz, M.S.; Hurwitz, J.

1983-01-01

The simple mechanism of replication of adenoviral DNA has made adenovirus an especially useful model system for studies of eukaryotic replication mechanisms. The availability of this in vitro system that replicates exogenously added Ad DNA-pro has made it possible to characterize the factors involved in replication. The results presented in this paper summarize our further fractionation of the in vitro system. First, the properties of two factors purified from the uninfected nuclear extract are described. Second, the separation of the pTP/Ad Pol complex into subunits and the properties of the isolated subunits are presented. The 140K protein is shown to possess the Ad DNA polymerase activity. The results suggest that the only DNA polymerase required for adenoviral DNA replication in vitro is the 140K Ad DNA polymerase and that this enzyme is probably a viral gene product. 50 references, 10 figures, 3 tables

Opposing roles of RNF8/RNF168 and deubiquitinating enzymes in ubiquitination-dependent DNA double-strand break response signaling and DNA-repair pathway choice

International Nuclear Information System (INIS)

Nakada, Shinichiro

2016-01-01

The E3 ubiquitin ligases ring finger protein (RNF) 8 and RNF168 transduce the DNA double-strand break (DSB) response (DDR) signal by ubiquitinating DSB sites. The depletion of RNF8 or RNF168 suppresses the accumulation of DNA-repair regulating factors such as 53BP1 and RAP80 at DSB sites, suggesting roles for RNF8- and RNF168-mediated ubiquitination in DSB repair. This mini-review provides a brief overview of the RNF8- and RNF168-dependent DDR-signaling and DNA-repair pathways. The choice of DNA-repair pathway when RNF8- and RNF168-mediated ubiquitination-dependent DDR signaling is negatively regulated by deubiquitinating enzymes (DUBs) is reviewed to clarify how the opposing roles of RNF8/RNF168 and DUBs regulate ubiquitination-dependent DDR signaling and the choice of DNA-repair pathway

Differential DNA Methylation in Relation to Age and Health Risks of Obesity

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María Luisa Mansego

2015-07-01

Full Text Available The aim of this study was to evaluate whether genome-wide levels of DNA methylation are associated with age and the health risks of obesity (HRO; defined according to BMI categories as “Low HRO” (overweight and class 1 obesity versus “High HRO” (class 2 and class 3 obesity. Anthropometric measurements were assessed in a subsample of 48 volunteers from the Metabolic Syndrome Reduction in Navarra (RESMENA study and 24 women from another independent study, Effects of Lipoic Acid and Eicosapentaenoic Acid in Human Obesity (OBEPALIP study. In the pooled population; the methylation levels of 55 CpG sites were significantly associated with age after Benjamini-Hochberg correction. In addition, DNA methylation of three CpG sites located in ELOVL2; HOXC4 and PI4KB were further negatively associated with their mRNA levels. Although no differentially methylated CpG sites were identified in relation to HRO after multiple testing correction; several nominally significant CpG sites were identified in genes related to insulin signaling; energy and lipid metabolism. Moreover, statistically significant associations between BMI or mRNA levels and two HRO-related CpG sites located in GPR133 and ITGB5 are reported. As a conclusion, these findings from two Spanish cohorts add knowledge about the important role of DNA methylation in the age-related regulation of gene expression. In addition; a relevant influence of age on DNA methylation in white blood cells was found, as well as, on a trend level, novel associations between DNA methylation and obesity.

Nucleolar exit of RNF8 and BRCA1 in response to DNA damage

International Nuclear Information System (INIS)

Guerra-Rebollo, Marta; Mateo, Francesca; Franke, Kristin; Huen, Michael S.Y.; Lopitz-Otsoa, Fernando; Rodríguez, Manuel S.; Plans, Vanessa; Thomson, Timothy M.

2012-01-01

The induction of DNA double-strand breaks (DSBs) elicits a plethora of responses that redirect many cellular functions to the vital task of repairing the injury, collectively known as the DNA damage response (DDR). We have found that, in the absence of DNA damage, the DSB repair factors RNF8 and BRCA1 are associated with the nucleolus. Shortly after exposure of cells to γ-radiation, RNF8 and BRCA1 translocated from the nucleolus to damage foci, a traffic that was reverted several hours after the damage. RNF8 interacted through its FHA domain with the ribosomal protein RPSA, and knockdown of RPSA caused a depletion of nucleolar RNF8 and BRCA1, suggesting that the interaction of RNF8 with RPSA is critical for the nucleolar localization of these DDR factors. Knockdown of RPSA or RNF8 impaired bulk protein translation, as did γ-irradiation, the latter being partially countered by overexpression of exogenous RNF8. Our results suggest that RNF8 and BRCA1 are anchored to the nucleolus through reversible interactions with RPSA and that, in addition to its known functions in DDR, RNF8 may play a role in protein synthesis, possibly linking the nucleolar exit of this factor to the attenuation of protein synthesis in response to DNA damage. -- Highlights: ► RNF8 and BRCA1 are associated with the nucleolus of undamaged cells. ► Upon γ-radiation, RNF8 and BRCA1 are translocated from the nucleolus to damage foci. ► The ribosomal protein RPSA anchors RNF8 to the nucleolus. ► RNF8 may play previously unsuspected roles in protein synthesis.

Dietary Berries and Ellagic Acid Prevent Oxidative DNA Damage and Modulate Expression of DNA Repair Genes

Directory of Open Access Journals (Sweden)

Ramesh C. Gupta

2008-03-01

Full Text Available DNA damage is a pre-requisite for the initiation of cancer and agents that reduce this damage are useful in cancer prevention. In this study, we evaluated the ability of whole berries and berry phytochemical, ellagic acid to reduce endogenous oxidative DNA damage. Ellagic acid was selected based on > 95% inhibition of 8-oxodeoxyguosine (8-oxodG and other unidentified oxidative DNA adducts induced by 4-hydroxy-17B;-estradiol and CuCl2 in vitro. Inhibition of the latter occurred at lower concentrations (10 u(microM than that for 8-oxodG (100 u(microM. In the in vivo study, female CD-1 mice (n=6 were fed either a control diet or diet supplemented with ellagic acid (400 ppm and dehydrated berries (5% w/w with varying ellagic acid contents -- blueberry (low, strawberry (medium and red raspberry (high, for 3 weeks. Blueberry and strawberry diets showed moderate reductions in endogenous DNA adducts (25%. However, both red raspberry and ellagic acid diets showed a significant reduction of 59% (p < 0.001 and 48% (p < 0.01, respectively. Both diets also resulted in a 3-8 fold over-expression of genes involved in DNA repair such as xeroderma pigmentosum group A complementing protein (XPA, DNA excision repair protein (ERCC5 and DNA ligase III (DNL3. These results suggest that red raspberry and ellagic acid reduce endogenous oxidative DNA damage by mechanisms which may involve increase in DNA repair.

Evaluation of DNA damage in patients with arsenic poisoning: urinary 8-hydroxydeoxyguanine

International Nuclear Information System (INIS)

Yamauchi, Hiroshi; Aminaka, Yoshito; Yoshida, Katsumi; Sun Guifan; Pi Jingbo; Waalkes, Michael P.

2004-01-01

The relationship between arsenic exposure and DNA damage in patients with acute or chronic arsenic poisoning was analyzed. Urinary 8-hydroxydeoxyguanine (8-OHdG) concentrations were measured as an indication of oxidative DNA damage. A remarkable increase in 8-OHdG in the urine was observed in 60% of 52 patients with acute arsenic poisoning from the accidental oral intake of the arsenic trioxide. This was two- to threefold higher than levels in normal healthy subjects (n = 248). There was a clear relationship between arsenic concentrations in urine after acute poisoning and elevated levels of 8-OHdG. Levels of urinary 8-OHdG returned to normal within 180 days after the acute arsenic poisoning event. In patients chronically poisoned by the consumption of well water with elevated levels of arsenate [As(V)], elevated 8-OHdG concentrations in urine were also observed. A significant correlation between the 8-OHdG levels and arsenic levels in the urine was observed in 82 patients with chronic poisoning. Thus, evidence of oxidative DNA damage occurred in acute arsenic poisoning by arsenite [As(III)] and in chronic arsenic poisoning by As(V). In chronic poisoning patients provided low-arsenic drinking water, evidence of DNA damage subsided between 9 months and 1 year after the high levels of arsenic intake were reduced. The initial level of arsenic exposure appeared to dictate the length of this recovery period. These data indicate that some aspects of chronic and acute arsenic poisoning may be reversible with the cessation of exposure. This knowledge may contribute to our understanding of the risk elevation from arsenic carcinogenesis and perhaps be used in a prospective fashion to assess individual risk

Dideoxynucleoside triphosphate-sensitive DNA polymerase from rice is involved in base excision repair and immunologically similar to mammalian DNA pol beta.

Science.gov (United States)

Sarkar, Sailendra Nath; Bakshi, Sankar; Mokkapati, Sanath K; Roy, Sujit; Sengupta, Dibyendu N

2004-07-16

A single polypeptide with ddNTP-sensitive DNA polymerase activity was purified to near homogeneity from the shoot tips of rice seedlings and analysis of the preparations by SDS-PAGE followed by silver staining showed a polypeptide of 67 kDa size. The DNA polymerase activity was found to be inhibitory by ddNTP in both in vitro DNA polymerase activity assay and activity gel analysis. Aphidicolin, an inhibitor of other types of DNA polymerases, had no effect on plant enzyme. The 67 kDa rice DNA polymerase was found to be recognized by the polyclonal antibody (purified IgG) made against rat DNA polymerase beta (pol beta) both in solution and also on Western blot. The recognition was found to be very specific as the activity of Klenow enzyme was unaffected by the antibody. The ability of rice nuclear extract to correct G:U mismatch of oligo-duplex was observed when oligo-duplex with 32P-labeled lower strand containing U (at 22nd position) was used as substrate. Differential appearance of bands at 21-mer, 22-mer, and 51-mer position in presence of dCTP was visible only with G:U mismatch oligo-duplex, but not with G:C oligo-duplex. While ddCTP or polyclonal antibody against rat-DNA pol beta inhibits base excision repair (BER), aphidicolin had no effect. These results for the first time clearly demonstrate the ability of rice nuclear extract to run BER and the involvement of ddNTP-sensitive pol beta type DNA polymerase. Immunological similarity of the ddNTP-sensitive DNA polymerase beta of rice and rat and its involvement in BER revealed the conservation of structure and function of ddNTP-sensitive DNA pol beta in plant and animal.

Nucleolar exit of RNF8 and BRCA1 in response to DNA damage

Energy Technology Data Exchange (ETDEWEB)

Guerra-Rebollo, Marta; Mateo, Francesca; Franke, Kristin [Department of Cell Biology, Molecular Biology Institute of Barcelona (IBMB), CSIC, Barcelona Science Park, Helix Building, Baldiri Reixac 15-21, 08028 Barcelona (Spain); Huen, Michael S.Y. [Department of Anatomy, Centre for Cancer Research, The University of Hong Kong, L1, Laboratory Block, 21 Sassoon Road, Hong Kong Special Administrative Region (Hong Kong); Lopitz-Otsoa, Fernando; Rodriguez, Manuel S. [Proteomics Unit, CIC bioGUNE CIBERehd, ProteoRed, Technology Park of Bizkaia, Building 801A, 48160 Derio (Spain); Plans, Vanessa [Department of Cell Biology, Molecular Biology Institute of Barcelona (IBMB), CSIC, Barcelona Science Park, Helix Building, Baldiri Reixac 15-21, 08028 Barcelona (Spain); Thomson, Timothy M., E-mail: titbmc@ibmb.csic.es [Department of Cell Biology, Molecular Biology Institute of Barcelona (IBMB), CSIC, Barcelona Science Park, Helix Building, Baldiri Reixac 15-21, 08028 Barcelona (Spain)

2012-11-01

The induction of DNA double-strand breaks (DSBs) elicits a plethora of responses that redirect many cellular functions to the vital task of repairing the injury, collectively known as the DNA damage response (DDR). We have found that, in the absence of DNA damage, the DSB repair factors RNF8 and BRCA1 are associated with the nucleolus. Shortly after exposure of cells to {gamma}-radiation, RNF8 and BRCA1 translocated from the nucleolus to damage foci, a traffic that was reverted several hours after the damage. RNF8 interacted through its FHA domain with the ribosomal protein RPSA, and knockdown of RPSA caused a depletion of nucleolar RNF8 and BRCA1, suggesting that the interaction of RNF8 with RPSA is critical for the nucleolar localization of these DDR factors. Knockdown of RPSA or RNF8 impaired bulk protein translation, as did {gamma}-irradiation, the latter being partially countered by overexpression of exogenous RNF8. Our results suggest that RNF8 and BRCA1 are anchored to the nucleolus through reversible interactions with RPSA and that, in addition to its known functions in DDR, RNF8 may play a role in protein synthesis, possibly linking the nucleolar exit of this factor to the attenuation of protein synthesis in response to DNA damage. -- Highlights: Black-Right-Pointing-Pointer RNF8 and BRCA1 are associated with the nucleolus of undamaged cells. Black-Right-Pointing-Pointer Upon {gamma}-radiation, RNF8 and BRCA1 are translocated from the nucleolus to damage foci. Black-Right-Pointing-Pointer The ribosomal protein RPSA anchors RNF8 to the nucleolus. Black-Right-Pointing-Pointer RNF8 may play previously unsuspected roles in protein synthesis.

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.

Protozoan ALKBH8 Oxygenases Display both DNA Repair and tRNA Modification Activities

DEFF Research Database (Denmark)

Zdżalik, Daria; Vågbø, Cathrine B; Kirpekar, Finn

2014-01-01

The ALKBH family of Fe(II) and 2-oxoglutarate dependent oxygenases comprises enzymes that display sequence homology to AlkB from E. coli, a DNA repair enzyme that uses an oxidative mechanism to dealkylate methyl and etheno adducts on the nucleobases. Humans have nine different ALKBH proteins, ALKBH......1-8 and FTO. Mammalian and plant ALKBH8 are tRNA hydroxylases targeting 5-methoxycarbonylmethyl-modified uridine (mcm5U) at the wobble position of tRNAGly(UCC). In contrast, the genomes of some bacteria encode a protein with strong sequence homology to ALKBH8, and robust DNA repair activity...... was previously demonstrated for one such protein. To further explore this apparent functional duality of the ALKBH8 proteins, we have here enzymatically characterized a panel of such proteins, originating from bacteria, protozoa and mimivirus. All the enzymes showed DNA repair activity in vitro, but...

Interstrand DNA crosslinking by 4,5',8-trimethylpsoralen plus monochromatic ultraviolet light

International Nuclear Information System (INIS)

Cohen, L.F.; Ewig, R.A.G.; Kohn, K.W.; Glaubiger, D.

1980-01-01

DNA crosslinking by 4,5',8-trimethylpsoralen plus monochromatic ultraviolet light of wavelength 365 nm was studied in mouse L1210 leukemia cells. DNA breaks and crosslinking were evaluated by alkaline elution of DNA from poly(vinyl chloride) filters. Trimethylpsoralen plus 365 nm light produced DNA crosslinks but not breaks. The kinetics of crosslinging were linear with respect to concentration and second-order with respect to light exposure time. The latter finding supports the proposed two photon mechanism for the formation of diadducts. In contrast to DNA crosslinking agents such as nitrogen mustard, nitrosoureas and platinums, trimethylpsoralen crosslinks were resistant to proteolytic digestion. Thus, trimethylpsoralen plus 365 nm light produced interstrand crosslinks, as proposed for a bifunctional agent binding to bases on opposite DNA strands. (Auth.)

The study of DNA adduct 8-hydroxy-2‧deoxyguanosine (8-OHdG) formation of butylated hydroxyanisole (BHA) and its metabolite ter-butyl hydroquinone (TBHQ) through in vitro reaction with Calf Thymus DNA and 2‧deoxyguanosine

Science.gov (United States)

Budiawan; Purwaningsih, S. S.; Cahaya, D. I.

2017-04-01

Butylated Hydroxyanisole (BHA) and its metabolite Tert-Butyl Hydroquinone (TBHQ) are synthetic antioxidants, commonly used as food and beverage preservatives. Although WHO declared their safety, the use of these preservatives are still controversial because some studies showed that BHA induced proliferative effects in animal testing and TBHQ is considered as carcinogenic and causes DNA cleavage. This study is aimed to analyze the interaction between Calf Thymus DNA with BHA and TBHQ which are mediated with Copper (II) Chloride. The result of the study in spectrophotometric showed there was bathochromic shift as much as 2-3 nm in DNA treated with TBHQ. The next analysis used HPLC method in stationary phase of ODS, mobile phase of 10mM Natrium Hydrogen Phosphate Buffer and Methanol (85 : 15) for DNA adduct formation, 8-Hydroxy-2-Deoxyguanosine (8-OHDG) as biomarker of risk cancer. The resultof the study showed the formation of DNA adduct 8-OHDG in the interaction between DNA and 20-500 ppm of TBHQ. The 8-OHdG formation was greatly increased by the higher concentration of TBHQ. The relative amount of 8 OHDG which formed was reached 946/105 deoxyguanosine in DNA bases. Confirmation test by LCMS/MS was characterized with the detection of mother ion peak (m/z 284); fragment ion peaks at m/z 167.9, and 139.9; at retention time 3.52 min. Meanwhile the interaction between DNA and 50-250 ppm BHA did not induce 8-OHDG.

The Crystal Structure of PF-8, the DNA Polymerase Accessory Subunit from Kaposi's Sarcoma-Associated Herpesvirus

Energy Technology Data Exchange (ETDEWEB)

Baltz, Jennifer L.; Filman, David J.; Ciustea, Mihai; Silverman, Janice Elaine Y.; Lautenschlager, Catherine L.; Coen, Donald M.; Ricciardi, Robert P.; Hogle, James M.; (UPENN)

2009-12-01

Kaposi's sarcoma-associated herpesvirus is an emerging pathogen whose mechanism of replication is poorly understood. PF-8, the presumed processivity factor of Kaposi's sarcoma-associated herpesvirus DNA polymerase, acts in combination with the catalytic subunit, Pol-8, to synthesize viral DNA. We have solved the crystal structure of residues 1 to 304 of PF-8 at a resolution of 2.8 {angstrom}. This structure reveals that each monomer of PF-8 shares a fold common to processivity factors. Like human cytomegalovirus UL44, PF-8 forms a head-to-head dimer in the form of a C clamp, with its concave face containing a number of basic residues that are predicted to be important for DNA binding. However, there are several differences with related proteins, especially in loops that extend from each monomer into the center of the C clamp and in the loops that connect the two subdomains of each protein, which may be important for determining PF-8's mode of binding to DNA and to Pol-8. Using the crystal structures of PF-8, the herpes simplex virus catalytic subunit, and RB69 bacteriophage DNA polymerase in complex with DNA and initial experiments testing the effects of inhibition of PF-8-stimulated DNA synthesis by peptides derived from Pol-8, we suggest a model for how PF-8 might form a ternary complex with Pol-8 and DNA. The structure and the model suggest interesting similarities and differences in how PF-8 functions relative to structurally similar proteins.

Identification of Bicarbonate as a Trigger and Genes Involved with Extracellular DNA Export in Mycobacterial Biofilms

Directory of Open Access Journals (Sweden)

Sasha J. Rose

2016-12-01

Full Text Available Extracellular DNA (eDNA is an integral biofilm matrix component of numerous pathogens, including nontuberculous mycobacteria (NTM. Cell lysis is the source of eDNA in certain bacteria, but the source of eDNA remains unidentified for NTM, as well as for other eDNA-containing bacterial species. In this study, conditions affecting eDNA export were examined, and genes involved with the eDNA export mechanism were identified. After a method for monitoring eDNA in real time in undisturbed biofilms was established, different conditions affecting eDNA were investigated. Bicarbonate positively influenced eDNA export in a pH-independent manner in Mycobacterium avium, M. abscessus, and M. chelonae. The surface-exposed proteome of M. avium in eDNA-containing biofilms revealed abundant carbonic anhydrases. Chemical inhibition of carbonic anhydrases with ethoxzolamide significantly reduced eDNA export. An unbiased transposon mutant library screen for eDNA export in M. avium identified many severely eDNA-attenuated mutants, including one not expressing a unique FtsK/SpoIIIE-like DNA-transporting pore, two with inactivation of carbonic anhydrases, and nine with inactivation of genes belonging to a unique genomic region, as well as numerous mutants involved in metabolism and energy production. Complementation of nine mutants that included the FtsK/SpoIIIE and carbonic anhydrase significantly restored eDNA export. Interestingly, several attenuated eDNA mutants have mutations in genes encoding proteins that were found with the surface proteomics, and many more mutations are localized in operons potentially encoding surface proteins. Collectively, our data strengthen the evidence of eDNA export being an active mechanism that is activated by the bacterium responding to bicarbonate.

Involvement of specialized DNA polymerases Pol II, Pol IV and DnaE2 in DNA replication in the absence of Pol I in Pseudomonas putida

International Nuclear Information System (INIS)

Sidorenko, Julia; Jatsenko, Tatjana; Saumaa, Signe; Teras, Riho; Tark-Dame, Mariliis; Horak, Rita; Kivisaar, Maia

2011-01-01

The majority of bacteria possess a different set of specialized DNA polymerases than those identified in the most common model organism Escherichia coli. Here, we have studied the ability of specialized DNA polymerases to substitute Pol I in DNA replication in Pseudomonas putida. Our results revealed that P. putida Pol I-deficient cells have severe growth defects in LB medium, which is accompanied by filamentous cell morphology. However, growth of Pol I-deficient bacteria on solid rich medium can be restored by reduction of reactive oxygen species in cells. Also, mutants with improved growth emerge rapidly. Similarly to the initial Pol I-deficient P. putida, its adapted derivatives express a moderate mutator phenotype, which indicates that DNA replication carried out in the absence of Pol I is erroneous both in the original Pol I-deficient bacteria and the adapted derivatives. Analysis of the spectra of spontaneous Rif r mutations in P. putida strains lacking different DNA polymerases revealed that the presence of specialized DNA polymerases Pol II and Pol IV influences the frequency of certain base substitutions in Pol I-proficient and Pol I-deficient backgrounds in opposite ways. Involvement of another specialized DNA polymerase DnaE2 in DNA replication in Pol I-deficient bacteria is stimulated by UV irradiation of bacteria, implying that DnaE2-provided translesion synthesis partially substitutes the absence of Pol I in cells containing heavily damaged DNA.

DNA crosslinking and cell survival in human lymphoid cells treated with 8-methoxypsoralen and long wavelength ultraviolet radiation

Energy Technology Data Exchange (ETDEWEB)

Cohen, L F; Glaubiger, D L [National Cancer Inst., Bethesda, MD (USA). Pediatric Oncology Branch; Kraemer, K H; Waters, H L [National Cancer Inst., Bethesda, MD (USA). Lab. of Molecular Carcinogenesis; Kohn, K W [National Cancer Inst., Bethesda, MD (USA). Lab. of Molecular Pharmacology

1981-01-01

8-Methoxypsoralen (8-MOP) when irradiated with long wavelength ultraviolet radiation (UV-A) inhibits DNA synthesis in lymphocytes in vitro and in vivo. 8-MOP binds reversibly to DNA in the dark; when exposed to UV-A, covalent monoadducts and cross-links are formed with the DNA. The present study correlates the cytotoxic effects of 8-MOP plus UV-A with DNA crosslinking. E-B virus transformed human lymphoblastoid cells were suspended in a colorless salt solution containing 8-MOP and exposed to UV-A from fluorescent lamps filtered to remove radiation below 320 nm (22.5 J/m/sup 2/-sec). Cells were then returned to complete medium and assayed for survival (by daily counts of viable cells and by cloning in microtiter wells) and for DNA crosslinking by alkaline elution. 8-MOP alone or UV-A alone resulted in minimal to no alterations in survivial or in DNA crosslinking. DNA crosslinking was found to be linearly dependent on 8-MOP concentration (in the range of 0.01-1.0 ..mu..g/ml) for 3 different UV-A doses (3000-15000 J/m/sup 2/). The surviving fraction declined exponentially as a function of the relative number of DNA crosslinks.

DNA gains at 8q23.2

DEFF Research Database (Denmark)

da Silva Veiga, Luciana Caricati; Bérgamo, Nádia Aparecida; dos Reis, Patrícia Pintor

2003-01-01

Gains or amplifications involving chromosome arm 8q are one of the most recurrent chromosomal alterations in head and neck tumors. To characterize previously reported gains, we performed fluorescence in situ hybridization (FISH) using the sequences BAC RP1179E1 and 8-centromere PMJ 128 as probes....

DNA supercoiling in Escherichia coli is under tight and subtle homeostatic control, involving gene-expression and metabolic regulation of both topoisomerase I and DNA gyrase

DEFF Research Database (Denmark)

Snoep, J.L.; van der Weijden, C.C.; Andersen, H.W.

2002-01-01

DNA of prokaryotes is in a nonequilibrium. structural state, characterized as 'active' DNA supercoiling. Alterations in this state affect many life processes and a homeostatic control of DNA supercoiling has been suggested [Menzel, R. & Gellert. M. (1983) Cell 34, 105-113]. We here report on a ne...... of the nonequilibrium DNA structure in wild-type Escherichia coli is almost complete and subtle (i.e. involving at least three regulatory mechanisms)....

CD8 Memory Cells Develop Unique DNA Repair Mechanisms Favoring Productive Division.

Science.gov (United States)

Galgano, Alessia; Barinov, Aleksandr; Vasseur, Florence; de Villartay, Jean-Pierre; Rocha, Benedita

2015-01-01

Immune responses are efficient because the rare antigen-specific naïve cells are able to proliferate extensively and accumulate upon antigen stimulation. Moreover, differentiation into memory cells actually increases T cell accumulation, indicating improved productive division in secondary immune responses. These properties raise an important paradox: how T cells may survive the DNA lesions necessarily induced during their extensive division without undergoing transformation. We here present the first data addressing the DNA damage responses (DDRs) of CD8 T cells in vivo during exponential expansion in primary and secondary responses in mice. We show that during exponential division CD8 T cells engage unique DDRs, which are not present in other exponentially dividing cells, in T lymphocytes after UV or X irradiation or in non-metastatic tumor cells. While in other cell types a single DDR pathway is affected, all DDR pathways and cell cycle checkpoints are affected in dividing CD8 T cells. All DDR pathways collapse in secondary responses in the absence of CD4 help. CD8 T cells are driven to compulsive suicidal divisions preventing the propagation of DNA lesions. In contrast, in the presence of CD4 help all the DDR pathways are up regulated, resembling those present in metastatic tumors. However, this up regulation is present only during the expansion phase; i.e., their dependence on antigen stimulation prevents CD8 transformation. These results explain how CD8 T cells maintain genome integrity in spite of their extensive division, and highlight the fundamental role of DDRs in the efficiency of CD8 immune responses.

The involvement of DNA repair genes in the hypoxia-dependent NLCQ-1 (NSC 709257) toxicity and its synergistic interaction with cisplatin or melphalan

International Nuclear Information System (INIS)

Papadopoulou, M.V.; Xue, C.-J.; Bloomer, W.D.

2003-01-01

4-[3-(2-Nitro-1-imidazolyl)-propylamino]-7-chloro-quinoline hydrochloride (NLCQ-1) is a weakly DNA-intercalating hypoxia selective cytotoxin, which synergistically enhances the antitumor effect of several chemotherapeutic agents or radiation against mouse tumors or human xenografts. Synergy with melphalan (L-PAM) or cisplatin (cisPt) requires hypoxic pre-exposure of cells to NLCQ-1 or, in mice, administration of NLCQ-1 about 1 h before L-PAM or cisPt. This suggests that NLCQ-1 may cause DNA lesions upon reductive metabolism. To indirectly identify such lesions, rodent cell lines defective in specific DNA repair genes (EM9 and UV41) and their repair-proficient parental AA8, were exposed to NLCQ-1 alone and in combination with L-PAM or cisPt under hypoxic/aerobic conditions and appropriate routes, and assessed for clonogenicity. Selected comparisons with tirapazamine (TPZ) were also performed. DNA ssbs were identified by using the alkaline comet assay. Synergism was assessed by isobologramic analysis. EM9, which lack the functional XRCC1 gene and are unable to efficiently repair DNA ssbs, were 3.7x and 4.5x more sensitive to NLCQ-1 and TPZ, respectively, than the parental AA8 cells. Similarly, UV41, which are defective in the ERCC4/XPF gene and thus, hypersensitive to DNA cross-linking agents, were 4.1x more sensitive than AA8 cells to NLCQ-1. Equitoxic concentrations of NLCQ-1 and TPZ gave similar numbers of ssbs in AA8 and EM9 cells exposed to each compound for 1 h under hypoxic conditions. In combination with L-PAM or cisPt, synergy was observed in AA8 but not in EM9 or UV41 cells, with either NLCQ-1 or TPZ. These results suggest that NLCQ-1 is involved in the formation of DNA ssbs and interstrand crosslinks, with the latter being most likely responsible for NLCQ-1 hypoxic toxicity. The synergistic interaction of NLCQ-1 with L-PAM or cisPt is probably due to an enhancement in the L-PAM/cisPt-induced DNA interstrand crosslinks, possibly as a result of an inhibited

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...

The blood DNA virome in 8,000 humans.

Directory of Open Access Journals (Sweden)

Ahmed Moustafa

2017-03-01

Full Text Available The characterization of the blood virome is important for the safety of blood-derived transfusion products, and for the identification of emerging pathogens. We explored non-human sequence data from whole-genome sequencing of blood from 8,240 individuals, none of whom were ascertained for any infectious disease. Viral sequences were extracted from the pool of sequence reads that did not map to the human reference genome. Analyses sifted through close to 1 Petabyte of sequence data and performed 0.5 trillion similarity searches. With a lower bound for identification of 2 viral genomes/100,000 cells, we mapped sequences to 94 different viruses, including sequences from 19 human DNA viruses, proviruses and RNA viruses (herpesviruses, anelloviruses, papillomaviruses, three polyomaviruses, adenovirus, HIV, HTLV, hepatitis B, hepatitis C, parvovirus B19, and influenza virus in 42% of the study participants. Of possible relevance to transfusion medicine, we identified Merkel cell polyomavirus in 49 individuals, papillomavirus in blood of 13 individuals, parvovirus B19 in 6 individuals, and the presence of herpesvirus 8 in 3 individuals. The presence of DNA sequences from two RNA viruses was unexpected: Hepatitis C virus is revealing of an integration event, while the influenza virus sequence resulted from immunization with a DNA vaccine. Age, sex and ancestry contributed significantly to the prevalence of infection. The remaining 75 viruses mostly reflect extensive contamination of commercial reagents and from the environment. These technical problems represent a major challenge for the identification of novel human pathogens. Increasing availability of human whole-genome sequences will contribute substantial amounts of data on the composition of the normal and pathogenic human blood virome. Distinguishing contaminants from real human viruses is challenging.

Molecular cloning and functional expression of a human cDNA encoding the antimutator enzyme 8-hydroxyguanine-DNA glycosylase

Science.gov (United States)

Roldán-Arjona, Teresa; Wei, Ying-Fei; Carter, Kenneth C.; Klungland, Arne; Anselmino, Catherine; Wang, Rui-Ping; Augustus, Meena; Lindahl, Tomas

1997-01-01

The major mutagenic base lesion in DNA caused by exposure to reactive oxygen species is 8-hydroxyguanine (8-oxo-7,8-dihydroguanine). In bacteria and Saccharomyces cerevisiae, this damaged base is excised by a DNA glycosylase with an associated lyase activity for chain cleavage. We have cloned, sequenced, and expressed a human cDNA with partial sequence homology to the relevant yeast gene. The encoded 47-kDa human enzyme releases free 8-hydroxyguanine from oxidized DNA and introduces a chain break in a double-stranded oligonucleotide specifically at an 8-hydroxyguanine residue base paired with cytosine. Expression of the human protein in a DNA repair-deficient E. coli mutM mutY strain partly suppresses its spontaneous mutator phenotype. The gene encoding the human enzyme maps to chromosome 3p25. These results show that human cells have an enzyme that can initiate base excision repair at mutagenic DNA lesions caused by active oxygen. PMID:9223306

Involvement of the yeast DNA polymerase delta in DNA repair in vivo

Energy Technology Data Exchange (ETDEWEB)

Giot, L. [State University of New York at Stony Brook, Stony Brook, NY. (United States); Chanet, R.; Simon, M.; Facca, C.; Faye, G.

1997-08-15

The POL3 encoded catalytic subunit of DNA polymerase delta possesses a highly conserved C-terminal cysteine-rich domain in Saccharomyces cerevisiae. Mutations in some of its cysteine codons display a lethal phenotype, which demonstrates an essential function of this domain. The thermosensitive mutant pol3-13, in which a serine replaces a cysteine of this domain, exhibits a range of defects in DNA repair, such as hypersensitivity to different DNA-damaging agents and deficiency for induced mutagenesis and for recombination. These phenotypes are observed at 24 degrees, a temperature at which DNA replication is almost normal; this differentiates the functions of POL3 in DNA repair and DNA replication. Since spontaneous mutagenesis and spontaneous recombination are efficient in pol3-13, we propose that POL3 plays an important role in DNA repair after irradiation, particularly in the error-prone and recombinational pathways. Extragenic suppressors of pol3-13 are allelic to sdp5-1, previously identified as an extragenic suppressor of pol3-11. SDP5, which is identical to HYS2, encodes a protein homologous to the p50 subunit of bovine and human DNA polymerase delta. SDP5 is most probably the p55 subunit of Pol delta of S. cerevisiae and seems to be associated with the catalytic subunit for both DNA replication and DNA repair. (author)

Identification of activated enhancers and linked transcription factors in breast, prostate, and kidney tumors by tracing enhancer networks using epigenetic traits.

Science.gov (United States)

Rhie, Suhn Kyong; Guo, Yu; Tak, Yu Gyoung; Yao, Lijing; Shen, Hui; Coetzee, Gerhard A; Laird, Peter W; Farnham, Peggy J

2016-01-01

Although technological advances now allow increased tumor profiling, a detailed understanding of the mechanisms leading to the development of different cancers remains elusive. Our approach toward understanding the molecular events that lead to cancer is to characterize changes in transcriptional regulatory networks between normal and tumor tissue. Because enhancer activity is thought to be critical in regulating cell fate decisions, we have focused our studies on distal regulatory elements and transcription factors that bind to these elements. Using DNA methylation data, we identified more than 25,000 enhancers that are differentially activated in breast, prostate, and kidney tumor tissues, as compared to normal tissues. We then developed an analytical approach called Tracing Enhancer Networks using Epigenetic Traits that correlates DNA methylation levels at enhancers with gene expression to identify more than 800,000 genome-wide links from enhancers to genes and from genes to enhancers. We found more than 1200 transcription factors to be involved in these tumor-specific enhancer networks. We further characterized several transcription factors linked to a large number of enhancers in each tumor type, including GATA3 in non-basal breast tumors, HOXC6 and DLX1 in prostate tumors, and ZNF395 in kidney tumors. We showed that HOXC6 and DLX1 are associated with different clusters of prostate tumor-specific enhancers and confer distinct transcriptomic changes upon knockdown in C42B prostate cancer cells. We also discovered de novo motifs enriched in enhancers linked to ZNF395 in kidney tumors. Our studies characterized tumor-specific enhancers and revealed key transcription factors involved in enhancer networks for specific tumor types and subgroups. Our findings, which include a large set of identified enhancers and transcription factors linked to those enhancers in breast, prostate, and kidney cancers, will facilitate understanding of enhancer networks and mechanisms

Mycobacterium smegmatis PafBC is involved in regulation of DNA damage response.

Science.gov (United States)

Fudrini Olivencia, Begonia; Müller, Andreas U; Roschitzki, Bernd; Burger, Sibylle; Weber-Ban, Eilika; Imkamp, Frank

2017-10-25

Two genes, pafB and pafC, are organized in an operon with the Pup-ligase gene pafA, which is part of the Pup-proteasome system (PPS) present in mycobacteria and other actinobacteria. The PPS is crucial for Mycobacterium tuberculosis resistance towards reactive nitrogen intermediates (RNI). However, pafB and pafC apparently play only a minor role in RNI resistance. To characterize their function, we generated a pafBC deletion in Mycobacterium smegmatis (Msm). Proteome analysis of the mutant strain revealed decreased cellular levels of various proteins involved in DNA damage repair, including recombinase A (RecA). In agreement with this finding, Msm ΔpafBC displayed increased sensitivity to DNA damaging agents. In mycobacteria two pathways regulate DNA repair genes: the LexA/RecA-dependent SOS response and a predominant pathway that controls gene expression via a LexA/RecA-independent promoter, termed P1. PafB and PafC feature winged helix-turn-helix DNA binding motifs and we demonstrate that together they form a stable heterodimer in vitro, implying a function as a heterodimeric transcriptional regulator. Indeed, P1-driven transcription of recA was decreased in Msm ΔpafBC under standard conditions and induction of recA expression upon DNA damage was strongly impaired. Taken together, our data indicate an important regulatory function of PafBC in the mycobacterial DNA damage response.

Double-stranded DNA-dependent ATPase Irc3p is directly involved in mitochondrial genome maintenance.

Science.gov (United States)

Sedman, Tiina; Gaidutšik, Ilja; Villemson, Karin; Hou, YingJian; Sedman, Juhan

2014-12-01

Nucleic acid-dependent ATPases are involved in nearly all aspects of DNA and RNA metabolism. Previous studies have described a number of mitochondrial helicases. However, double-stranded DNA-dependent ATPases, including translocases or enzymes remodeling DNA-protein complexes, have not been identified in mitochondria of the yeast Saccharomyces cerevisae. Here, we demonstrate that Irc3p is a mitochondrial double-stranded DNA-dependent ATPase of the Superfamily II. In contrast to the other mitochondrial Superfamily II enzymes Mss116p, Suv3p and Mrh4p, which are RNA helicases, Irc3p has a direct role in mitochondrial DNA (mtDNA) maintenance. Specific Irc3p-dependent mtDNA metabolic intermediates can be detected, including high levels of double-stranded DNA breaks that accumulate in irc3Δ mutants. irc3Δ-related topology changes in rho- mtDNA can be reversed by the deletion of mitochondrial RNA polymerase RPO41, suggesting that Irc3p counterbalances adverse effects of transcription on mitochondrial genome stability. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Possible mechanism of psoralen phototoxicity not involving direct interaction with DNA

International Nuclear Information System (INIS)

Laskin, J.D.; Lee, E.; Yurkow, E.J.; Laskin, D.L.; Gallo, M.A.

1985-01-01

Psoralens in combination with ultraviolet light (UVA; 320-400 nm) are used in the photochemical treatment of a variety of skin diseases including vitiligo, a skin depigmentational disorder, and psoriasis, a disease of accelerated epidermal cell proliferation. Although it is generally assumed that the major site of action of the psoralens is DNA, the authors have obtained evidence that another site may be the primary target for these compounds. They have identified specific, saturable, high-affinity binding sites for 8-methoxypsoralen on HeLa cells and have detected specific binding of 8-methoxypsoralen to four other human cell lines and five mouse cell lines. In HeLa cells, specific binding is reversible and independent of the ability of the compound to intercalate into DNA. In addition, binding sites become covalently modified by the psoralen after UVA exposure. Specific binding of 8-[methyoxy- 3 H]methoxypsoralen constitutes 79% of the label bound to the cells. Scatchard analysis indicated two classes of psoralen binding sites. Based on these findings, the authors hypothesize that specific binding sites for psoralens on mammalian cells mediate, at least in part, psoralen-induced phototoxicity

Larger yield of cyclobutane dimers than 8-oxo-7,8-dihydroguanine in the DNA of UVA-irradiated human skin cells

International Nuclear Information System (INIS)

Courdavault, Sophie; Baudouin, Caroline; Charveron, Marie; Favier, Alain; Cadet, Jean; Douki, Thierry

2004-01-01

Exposure to solar ultraviolet light is the major cause of most skin cancers. While the genotoxic properties of UVB radiation are now well understood, the DNA damaging processes triggered by less energetic but more abundant UVA photons remain to be elucidated. Evidence has been provided for the induction of oxidative lesions to cellular DNA including strand breaks and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). Formation of cyclobutane pyrimidine dimers (CPDs) has also been reported, mostly in rodent cells. In order to gain insights into the relevance of the latter photoproducts in UVA-mutagenesis of human skin, we quantified the level of 8-oxodGuo and CPDs within primary cultures of normal fibroblasts and keratinocytes using specific chromatographic assays. The yield of formation of CPDs was found to be higher than that of 8-oxodGuo in both cell types. In addition, CPDs were mostly TT derivatives, and neither (6-4) photoproducts nor Dewar valence isomers were detected. These observations are reminiscent of results obtained in rodent cells and suggest that a photosensitized triplet energy transfer occurs and that this reaction is more efficient than photooxidation of DNA components. The predominant formation of CPDs with respect to oxidative damage within normal human skin cells exposed to UVA radiation should be taken into account in photoprotection strategies

Larger yield of cyclobutane dimers than 8-oxo-7,8-dihydroguanine in the DNA of UVA-irradiated human skin cells

Energy Technology Data Exchange (ETDEWEB)

Courdavault, Sophie [Laboratoire Lesions des Acides Nucleiques, Service de Chimie Inorganique et Biologique, CEA/DSM/Departement de Recherche Fondamentale sur la Matiere Condensee, CEA-Grenoble, 17, avenue des Martyrs, 38054 Grenoble Cedex 9 (France); Baudouin, Caroline [Institut de Recherche Pierre Fabre, Laboratoire de Biologie Cellulaire, Toulouse (France); Charveron, Marie [Institut de Recherche Pierre Fabre, Laboratoire de Biologie Cellulaire, Toulouse (France); Favier, Alain [Laboratoire Lesions des Acides Nucleiques, Service de Chimie Inorganique et Biologique, CEA/DSM/Departement de Recherche Fondamentale sur la Matiere Condensee, CEA-Grenoble, 17, avenue des Martyrs, 38054 Grenoble Cedex 9 (France); Cadet, Jean [Laboratoire Lesions des Acides Nucleiques, Service de Chimie Inorganique et Biologique, CEA/DSM/Departement de Recherche Fondamentale sur la Matiere Condensee, CEA-Grenoble, 17, avenue des Martyrs, 38054 Grenoble Cedex 9 (France); Douki, Thierry [Laboratoire Lesions des Acides Nucleiques, Service de Chimie Inorganique et Biologique, CEA/DSM/Departement de Recherche Fondamentale sur la Matiere Condensee, CEA-Grenoble, 17, avenue des Martyrs, 38054 Grenoble Cedex 9 (France)]. E-mail: tdouki@cea.fr

2004-11-22

Exposure to solar ultraviolet light is the major cause of most skin cancers. While the genotoxic properties of UVB radiation are now well understood, the DNA damaging processes triggered by less energetic but more abundant UVA photons remain to be elucidated. Evidence has been provided for the induction of oxidative lesions to cellular DNA including strand breaks and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). Formation of cyclobutane pyrimidine dimers (CPDs) has also been reported, mostly in rodent cells. In order to gain insights into the relevance of the latter photoproducts in UVA-mutagenesis of human skin, we quantified the level of 8-oxodGuo and CPDs within primary cultures of normal fibroblasts and keratinocytes using specific chromatographic assays. The yield of formation of CPDs was found to be higher than that of 8-oxodGuo in both cell types. In addition, CPDs were mostly TT derivatives, and neither (6-4) photoproducts nor Dewar valence isomers were detected. These observations are reminiscent of results obtained in rodent cells and suggest that a photosensitized triplet energy transfer occurs and that this reaction is more efficient than photooxidation of DNA components. The predominant formation of CPDs with respect to oxidative damage within normal human skin cells exposed to UVA radiation should be taken into account in photoprotection strategies.0.

Mechanisms involved in repairing the lesions induced in pBR 322 by PUVA treatment (8-Methoxypsoralen + ultraviolet A light)

International Nuclear Information System (INIS)

Bauluz, C.

1988-01-01

This work deals with the genotoxic effects derived from damaging pBR322 DNA through PUVA treatment (8-Methoxypsoralen plusUVA light), both with respect to the lethality and mutagenicity of the lesions produced by the treatment. The mechanisms involved in the repair of the plasmid lesions have been investigated by transforming several strains of E. coli differing in their DNA-repair capacities. The frequency, distribution and type of mutations occurring in a restriction fragment of the damaged plasmid were determined in order to establish the mutagenic features of the PUVA treatment. Damages produced bY PUVA habe a strong lethal effect on plasmid survival; however, partial recovery is possible through some of the bacterial DNA repair pathways, namely Excision repair, SOS-repair and a third mechanism which appears to be independent from the analised genes and is detected at high density of lesions per plasmid molecule. PUVA treatment produces a high increase in plasmid mutagenesis; however, the contribution of such an increase to the whole plasmid survival is negligible. Only punctual mutations were detected and consisted mainly in base-pair substitutions. Some mutation-prone regions were sound inside the investigated DNA fragment, a though their existence is more likely to be related with the structure acquired by the damaged DNA than with the type of damaging agent. (Author)

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

A novel human AP endonuclease with conserved zinc-finger-like motifs involved in DNA strand break responses

OpenAIRE

Kanno, Shin-ichiro; Kuzuoka, Hiroyuki; Sasao, Shigeru; Hong, Zehui; Lan, Li; Nakajima, Satoshi; Yasui, Akira

2007-01-01

DNA damage causes genome instability and cell death, but many of the cellular responses to DNA damage still remain elusive. We here report a human protein, PALF (PNK and APTX-like FHA protein), with an FHA (forkhead-associated) domain and novel zinc-finger-like CYR (cysteine–tyrosine–arginine) motifs that are involved in responses to DNA damage. We found that the CYR motif is widely distributed among DNA repair proteins of higher eukaryotes, and that PALF, as well as a Drosophila protein with...

Kin3 protein, a NIMA-related kinase of Saccharomyces cerevisiae, is involved in DNA adduct damage response.

Science.gov (United States)

Moura, Dinara J; Castilhos, Bruna; Immich, Bruna F; Cañedo, Andrés D; Henriques, João A P; Lenz, Guido; Saffi, Jenifer

2010-06-01

Kin3 is a nonessential serine/threonine protein kinase of the budding yeast Saccharomyces cerevisiae with unknown cellular role. It is an ortholog of the Aspergillus nidulans protein kinase NIMA (Never-In Mitosis, gene A), which is involved in the regulation of G2/M phase progression, DNA damage response and mitosis. The aim of this study was to determine whether Kin3 is required for proper checkpoint activation and DNA repair. Here we show that KIN3 gene deficient cells present sensitivity and fail to arrest properly at G2/M-phase checkpoint in response to the DNA damage inducing agents MMS, cisplatin, doxorubicin and nitrogen mustard, suggesting that Kin3 can be involved in DNA strand breaks recognition or signaling. In addition, there is an increase in KIN3 gene expression in response to the mutagenic treatment, which was confirmed by the increase of Kin3 protein. We also showed that co-treatment with caffeine induces a slight increase in the susceptibility to genotoxic agents in kin3 cells and abolishes KIN3 gene expression in wild-type strain, suggesting that Kin3p can play a role in Tel1/Mec1-dependent pathway activation induced after genotoxic stress. These data provide the first evidence of the involvement of S. cerevisiae Kin3 in the DNA damage response.

Involvement of DNA topoisomerase I in transcription of human ribosomal RNA genes

International Nuclear Information System (INIS)

Zhang, H.; Wang, J.C.; Liu, L.F.

1988-01-01

Treatment of HeLa cells with a DNA topoisomerase I-specific inhibitor, camptothecin, results in rapid cessation of the synthesis of the 45S rRNA precursor. The inhibition of rRNA synthesis is reversible following drug removal and correlates with the presence of camptothecin-trapped topoisomerase I-DNA abortive complexes, which can be detected as topoisomerase I-linked DNA breaks upon lysis with sodium dodecyl sulfate. These breaks were found to be concentrated within the transcribed region of human rRNA genes. No such sites can be detected in the inactive human rRNA genes in mouse-human hybrid cells, suggesting a preferential association of topoisomerase I with actively transcribed genes. The distribution of RNA polymerase molecules along the transcription unit of human rRNA genes in camptothecin-treated HeLa cells, as assayed by nuclear run-on transcription, shows a graded decrease of the RNA polymerase density toward the 3' end of the transcription unit; the density is minimally affected near the 5' start of the transcription unit. These results suggest that DNA topoisomerase I is normally involved in the elongation step of transcription, especially when the transcripts are long, and that camptothecin interferes with this role

DNA Polymerases ImuC and DinB Are Involved in DNA Alkylation Damage Tolerance in Pseudomonas aeruginosa and Pseudomonas putida.

Science.gov (United States)

Jatsenko, Tatjana; Sidorenko, Julia; Saumaa, Signe; Kivisaar, Maia

2017-01-01

Translesion DNA synthesis (TLS), facilitated by low-fidelity polymerases, is an important DNA damage tolerance mechanism. Here, we investigated the role and biological function of TLS polymerase ImuC (former DnaE2), generally present in bacteria lacking DNA polymerase V, and TLS polymerase DinB in response to DNA alkylation damage in Pseudomonas aeruginosa and P. putida. We found that TLS DNA polymerases ImuC and DinB ensured a protective role against N- and O-methylation induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in both P. aeruginosa and P. putida. DinB also appeared to be important for the survival of P. aeruginosa and rapidly growing P. putida cells in the presence of methyl methanesulfonate (MMS). The role of ImuC in protection against MMS-induced damage was uncovered under DinB-deficient conditions. Apart from this, both ImuC and DinB were critical for the survival of bacteria with impaired base excision repair (BER) functions upon alkylation damage, lacking DNA glycosylases AlkA and/or Tag. Here, the increased sensitivity of imuCdinB double deficient strains in comparison to single mutants suggested that the specificity of alkylated DNA lesion bypass of DinB and ImuC might also be different. Moreover, our results demonstrated that mutagenesis induced by MMS in pseudomonads was largely ImuC-dependent. Unexpectedly, we discovered that the growth temperature of bacteria affected the efficiency of DinB and ImuC in ensuring cell survival upon alkylation damage. Taken together, the results of our study disclosed the involvement of ImuC in DNA alkylation damage tolerance, especially at low temperatures, and its possible contribution to the adaptation of pseudomonads upon DNA alkylation damage via increased mutagenesis.

DNA methylation in the human cerebral cortex is dynamically regulated throughout the life span and involves differentiated neurons.

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Kimberly D Siegmund

Full Text Available The role of DNA cytosine methylation, an epigenetic regulator of chromatin structure and function, during normal and pathological brain development and aging remains unclear. Here, we examined by MethyLight PCR the DNA methylation status at 50 loci, encompassing primarily 5' CpG islands of genes related to CNS growth and development, in temporal neocortex of 125 subjects ranging in age from 17 weeks of gestation to 104 years old. Two psychiatric disease cohorts--defined by chronic neurodegeneration (Alzheimer's or lack thereof (schizophrenia--were included. A robust and progressive rise in DNA methylation levels across the lifespan was observed for 8/50 loci (GABRA2, GAD1, HOXA1, NEUROD1, NEUROD2, PGR, STK11, SYK typically in conjunction with declining levels of the corresponding mRNAs. Another 16 loci were defined by a sharp rise in DNA methylation levels within the first few months or years after birth. Disease-associated changes were limited to 2/50 loci in the Alzheimer's cohort, which appeared to reflect an acceleration of the age-related change in normal brain. Additionally, methylation studies on sorted nuclei provided evidence for bidirectional methylation events in cortical neurons during the transition from childhood to advanced age, as reflected by significant increases at 3, and a decrease at 1 of 10 loci. Furthermore, the DNMT3a de novo DNA methyl-transferase was expressed across all ages, including a subset of neurons residing in layers III and V of the mature cortex. Therefore, DNA methylation is dynamically regulated in the human cerebral cortex throughout the lifespan, involves differentiated neurons, and affects a substantial portion of genes predominantly by an age-related increase.

Involvement of DNA gyrase in replication and transcription of bacteriophage T7 DNA

International Nuclear Information System (INIS)

De Wyngaert, M.A.; Hinkle, D.C.

1979-01-01

Growth of bacteriophage T7 is inhibited by the antibiotic coumermycin A 1 , an inhibitor of the Escherichia coli DNA gyrase. Since growth of the phage is insensitive to the antibiotic in strains containing a coumermycin-resistent DNA gyrase, this enzyme appears to be required for phage growth. We have investigated the effect of coumermycin on the kinetics of DNA, RNA, and protein synthesis during T7 infection. DNA synthesis is completely inhibited by the antibiotic. In addition, coumermycin significantly inhibits transcription of late but not early genes. Thus, E. coli DNA gyrase may play an important role in transcription as well as in replication of T7 DNA

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

44 CFR 5.8 - Records involved in litigation or other judicial process.

Science.gov (United States)

2010-10-01

... litigation or other judicial process. 5.8 Section 5.8 Emergency Management and Assistance FEDERAL EMERGENCY... Provisions § 5.8 Records involved in litigation or other judicial process. Where there is reason to believe that any records requested may be involved in litigation or other judicial process in which the United...

Some new methyl-8-methoxypsoralens: synthesis, photobinding to DNA, photobiological properties and molecular modelling.

Science.gov (United States)

Gia, O; Anselmo, A; Pozzan, A; Antonello, C; Magno, S M; Uriarte, E

1997-01-01

The tricyclic structure of known natural photochemotherapeutic drugs such as 8-methoxypsoralen and 5-methoxypsoralen is often taken as a model in the search of new photosensitizer agents with less phototoxic and mutagenic effects. This paper describes the synthesis, characterization, photobinding to DNA, photobiological properties and computational chemistry of some 8-methoxypsoralen derivatives bearing two or three methyl groups at the key positions of the two photoactive double bonds. Results showed that photoreactivity and photobiological behaviour depend on the pattern of methyl substitutions. Antiproliferative activity in cell lines shows good correlation with DNA interaction data.

Implication of the E. coli K12 uvrA and recA genes in the repair of 8-methoxypsoralen-induced mono adducts and crosslinks on plasmid DNA; Implicacion de los genes uvrA de E. coli K12 en la reparacion de monoaductos y entrecruzamien tos inducidos en DNA plasmidico por 8-metoxipso raleno mas luz ultravioleta A

Energy Technology Data Exchange (ETDEWEB)

Paramio, J M; Bauluz, C; Vidania, R de

1986-07-01

Genotoxicity of psoralen damages on plasmid DNA has been studied. pBR322 DNA was randomly modified with several concentrations of 8-methoxypsoralen plus 365 nm-UV light. After transformation into E. coli strains (wild-type, uvrA and recA) plasmid survival and mutagenesis were analyzed. To study the influence of the SOS response on plasmid recovery, preirradiation of the cells was performed. In absence of cell preirradiation, crosslinks were not repaired in any strain. Mono adducts were also lethal but in part removed by the excision-repair pathway. Preirradiation of the cells significantly. increased plasmid recovery in recA+ celia. In uvrA- only the mutagenic pathway seemed to be involved in the repair of the damaged DNA. Wild type strain showed the highest increase in plasmid survival, involving the repair of mono adducts and some fraction of crosslinks mainly through an error-free repair pathway. This suggests an enhancement of the excision repair promoted by the induction of SOS functions. (Author) 32 refs.

Regulation of DNA methylation on EEF1D and RPL8 expression in cattle.

Science.gov (United States)

Liu, Xuan; Yang, Jie; Zhang, Qin; Jiang, Li

2017-10-01

Dynamic changes to the epigenome play a critical role in a variety of biology processes and complex traits. Many important candidate genes have been identified through our previous genome wide association study (GWAS) on milk production traits in dairy cattle. However, the underlying mechanism of candidate genes have not yet been clearly understood. In this study, we analyzed the methylation variation of the candidate genes, EEF1D and RPL8, which were identified to be strongly associated with milk production traits in dairy cattle in our previous studies, and its effect on protein and mRNA expression. We compared DNA methylation profiles and gene expression levels of EEF1D and RPL8 in five different tissues (heart, liver, mammary gland, ovary and muscle) of three cows. Both genes showed the highest expression level in mammary gland. For RPL8, there was no difference in the DNA methylation pattern in the five tissues, suggesting no effect of DNA methylation on gene expression. For EEF1D, the DNA methylation levels of its first CpG island differed in the five tissues and were negatively correlated with the gene expression levels. To further investigate the function of DNA methylation on the expression of EEF1D, we collected blood samples of three cows at early stage of lactation and in dry period and analyzed its expression and the methylation status of the first CpG island in blood. As a result, the mRNA expression of EEF1D in the dry period was higher than that at the early stage of lactation, while the DNA methylation level in the dry period was lower than that at the early stage of lactation. Our result suggests that the DNA methylation of EEF1D plays an important role in the spatial and temporal regulation of its expression and possibly have an effect on the milk production traits.

Increased levels of mitochondrial DNA copy number in patients with vitiligo.

Science.gov (United States)

Vaseghi, H; Houshmand, M; Jadali, Z

2017-10-01

Oxidative stress is known to be involved in the pathogenesis of autoimmune diseases such as vitiligo. Evidence suggests that the human mitochondrial DNA copy number (mtDNAcn) is vulnerable to damage mediated by oxidative stress. The purpose of this study was to examine and compare peripheral blood mtDNAcn and oxidative DNA damage byproducts (8-hydroxy-2-deoxyguanosine; 8-OHdG) in patients with vitiligo and healthy controls (HCs). The relative mtDNAcn and the oxidative damage (formation of 8-OHdG in mtDNA) of each sample were determined by real-time quantitative PCR. Blood samples were obtained from 56 patients with vitiligo and 46 HCs. The mean mtDNAcn and the degree of mtDNA damage were higher in patients with vitiligo than in HCs. These data suggest that increase in mtDNAcn and oxidative DNA damage may be involved in the pathogenesis of vitiligo. © 2017 British Association of Dermatologists.

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

An EG-VEGF-dependent decrease in homeobox gene NKX3.1 contributes to cytotrophoblast dysfunction: a possible mechanism in human fetal growth restriction.

Science.gov (United States)

Murthi, P; Brouillet, S; Pratt, A; Borg, Aj; Kalionis, B; Goffin, F; Tsatsaris, V; Munaut, C; Feige, Jj; Benharouga, M; Fournier, T; Alfaidy, N

2015-07-21

Idiopathic fetal growth restriction (FGR) is frequently associated with placental insufficiency. Previous reports have provided evidence that EG-VEGF (endocrine gland derived-vascular endothelial growth factor), a placental secreted protein, is expressed during the first trimester of pregnancy, controls both trophoblast proliferation and invasion, and its increased expression is associated with human FGR. In this study, we hypothesise that EG-VEGF-dependent change in placental homeobox gene expressions contribute to trophoblast dysfunction in idiopathic FGR. The changes in EG-VEGF-dependent homeobox gene expressions were determined using a Homeobox gene cDNA array on placental explants of 8-12 weeks' gestation after stimulation with EG-VEGF in vitro for 24 hours. The Homeobox gene array identified a >5-fold increase in HOXA9, HOXC8, HOXC10, HOXD1, HOXD8, HOXD9 and HOXD11, while NKX 3.1 showed a >2 fold-decrease in mRNA expression compared to untreated controls. Homeobox gene NKX3.1 was selected as a candidate because it is a downstream target of EG-VEGF and its expression and functional role are largely unknown in control and idiopathic FGR-affected placentae. Real-time PCR and immunoblotting showed a significant decrease in NKX3.1 mRNA and protein levels, respectively, in placentae from FGR compared to control pregnancies. Gene inactivation in vitro using short-interference RNA specific for NKX3.1 demonstrated an increase in BeWo cell differentiation and a decrease in HTR8-SVneo proliferation. We conclude that the decreased expression of homeobox gene NKX3.1 down-stream of EG-VEGF may contribute to the trophoblast dysfunction associated with idiopathic FGR pregnancies.

Chromosomal location of the human gene for DNA polymerase β

International Nuclear Information System (INIS)

McBride, O.W.; Zmudzka, B.Z.; Wilson, S.H.

1987-01-01

Inhibition studies indicate that DNA polymerase β has a synthetic role in DNA repair after exposure of mammalian cells to some types of DNA-damaging agents. The primary structure of the enzyme is highly conserved in vertebrates, and nearly full-length cDNAs for the enzyme were recently cloned from mammalian cDNA libraries. Southern blot analysis of DNA from a panel of human-rodent somatic cell hybrids, using portions of the cDNA as probe, indicates that the gene for human DNA polymerase β is single copy and located on the short arm or proximal long arm of chromosome 8 (8pter-8q22). A restriction fragment length polymorphism (RFLP) was detected in normal individuals by using a probe from the 5' end of the cDNA, and this RFLP probably is due to an insertion or duplication of DNA in 20-25% of the population. This restriction site can be used as one marker for chromosome 8 genetic linkage studies and for family studies of traits potentially involving this DNA repair gene

DNA repair is responsible for the presence of oxidatively damaged DNA lesions in urine

International Nuclear Information System (INIS)

Cooke, Marcus S.; Evans, Mark D.; Dove, Rosamund; Rozalski, Rafal; Gackowski, Daniel; Siomek, Agnieszka; Lunec, Joseph; Olinski, Ryszard

2005-01-01

The repair of oxidatively damaged DNA is integral to the maintenance of genomic stability, and hence prevention of a wide variety of pathological conditions, such as aging, cancer and cardiovascular disease. The ability to non-invasively assess DNA repair may provide information regarding repair pathways, variability in repair capacity, and susceptibility to disease. The development of assays to measure urinary DNA lesions offered this potential, although it rapidly became clear that possible contribution from diet and cell turnover may influence urinary lesion levels. Whilst early studies attempted to address these issues, up until now, much of the data appears conflicting. However, recent work from our laboratories, in which human volunteers were fed highly oxidatively modified 15 N-labelled DNA demonstrates that diet does not appear to contribute to urinary levels of 8-hydroxyguanine and 7,8-dihydro-8-oxo-2'-deoxyguanosine. Furthermore, we propose that a number of literature reports form an argument against a contribution from cell death. Indeed we, and others, have presented evidence, which strongly suggests the involvement of cell death to be minimal. Taken together, these data would appear to rule out various confounding factors, leaving DNA repair pathways as the principal source of urinary purine, if not DNA, lesions enabling such measurements to be used as indicators of repair

Dumbbell DNA-templated CuNPs as a nano-fluorescent probe for detection of enzymes involved in ligase-mediated DNA repair.

Science.gov (United States)

Qing, Taiping; He, Xiaoxiao; He, Dinggeng; Ye, Xiaosheng; Shangguan, Jingfang; Liu, Jinquan; Yuan, Baoyin; Wang, Kemin

2017-08-15

DNA repair processes are responsible for maintaining genome stability. Ligase and polynucleotide kinase (PNK) have important roles in ligase-mediated DNA repair. The development of analytical methods to monitor these enzymes involved in DNA repair pathways is of great interest in biochemistry and biotechnology. In this work, we reported a new strategy for label-free monitoring PNK and ligase activity by using dumbbell-shaped DNA templated copper nanoparticles (CuNPs). In the presence of PNK and ligase, the dumbbell-shaped DNA probe (DP) was locked and could resist the digestion of exonucleases and then served as an efficient template for synthesizing fluorescent CuNPs. However, in the absence of ligase or PNK, the nicked DP could be digested by exonucleases and failed to template fluorescent CuNPs. Therefore, the fluorescence changes of CuNPs could be used to evaluate these enzymes activity. Under the optimal conditions, highly sensitive detection of ligase activity of about 1U/mL and PNK activity down to 0.05U/mL is achieved. To challenge the practical application capability of this strategy, the detection of analyte in dilute cells extracts was also investigated and showed similar linear relationships. In addition to ligase and PNK, this sensing strategy was also extended to the detection of phosphatase, which illustrates the versatility of this strategy. Copyright © 2017 Elsevier B.V. All rights reserved.

Implication of the E. coli K12 uvrA and recA genes in the repair of 8-methoxypsoralen-induced mono adducts and crosslinks on plasmid DNA

International Nuclear Information System (INIS)

Paramio, J.M.; Bauluz, C.; Vidania, R. de

1986-01-01

Genotoxicity of psoralen damages on plasmid DNA has been studied. pBR322 DNA was randomly modified with several concentrations of 8-methoxypsoralen plus 365 nm-UV light. After transformation into E. coli strains (wild-type, uvrA and recA) plasmid survival and mutagenesis were analyzed. To study the influence of the SOS response on plasmid recovery, preirradiation of the cells was performed. In absence of cell preirradiation, crosslinks were not repaired in any strain. Mono adducts were also lethal but in part removed by the excision-repair pathway. Preirradiation of the cells significantly. increased plasmid recovery in recA+ celia. In uvrA- only the mutagenic pathway seemed to be involved in the repair of the damaged DNA. Wild type strain showed the highest increase in plasmid survival, involving the repair of mono adducts and some fraction of crosslinks mainly through an error-free repair pathway. This suggests an enhancement of the excision repair promoted by the induction of SOS functions. (Author) 32 refs

Episodic weakness due to mitochondrial DNA MT-ATP6/8 mutations.

Science.gov (United States)

Auré, Karine; Dubourg, Odile; Jardel, Claude; Clarysse, Lucie; Sternberg, Damien; Fournier, Emmanuel; Laforêt, Pascal; Streichenberger, Nathalie; Petiot, Philippe; Gervais-Bernard, Hélène; Vial, Christophe; Bedat-Millet, Anne-Laure; Drouin-Garraud, Valérie; Bouillaud, Frédéric; Vandier, Christophe; Fontaine, Bertrand; Lombès, Anne

2013-11-19

To report that homoplasmic deleterious mutations in the mitochondrial DNA MT-ATP6/8 genes may be responsible for acute episodes of limb weakness mimicking periodic paralysis due to channelopathies and dramatically responding to acetazolamide. Mitochondrial DNA sequencing and restriction PCR, oxidative phosphorylation functional assays, reactive oxygen species metabolism, and patch-clamp technique in cultured skin fibroblasts. Occurrence of a typical MELAS (mitochondrial encephalopathy with lactic acidosis and stroke-like episodes) syndrome in a single member of a large pedigree with episodic weakness associated with a later-onset distal motor neuropathy led to the disclosure of 2 deleterious mitochondrial DNA mutations. The MT-ATP6 m.9185T>C p.Leu220Pro mutation, previously associated with Leigh syndrome, was present in all family members, while the MT-TL1 m.3271T>C mutation, a known cause of MELAS syndrome, was observed in the sole patient with MELAS presentation. Significant defect of complexes V and I as well as oxidative stress were observed in both primary fibroblasts and cybrid cells with 100% m.9185T>C mutation. Permanent plasma membrane depolarization and altered permeability to K(+) in fibroblasts provided a link with the paralysis episodes. Screening of 9 patients, based on their clinical phenotype, identified 4 patients with similar deleterious MT-ATP6 mutations (twice m.9185T>C and once m.9176T>C or m.8893T>C). A fifth patient presented with an original potentially deleterious MT-ATP8 mutation (m.8403T>C). All mutations were associated with almost-normal complex V activity but significant oxidative stress and permanent plasma membrane depolarization. Homoplasmic mutations in the MT-ATP6/8 genes may cause episodic weakness responding to acetazolamide treatment.

Multiple and variable NHEJ-like genes are involved in resistance to DNA damage in Streptomyces ambofaciens

Directory of Open Access Journals (Sweden)

Grégory Hoff

2016-11-01

Full Text Available Non homologous end-joining (NHEJ is a double strand break (DSB repair pathway which does not require any homologous template and can ligate two DNA ends together. The basic bacterial NHEJ machinery involves two partners: the Ku protein, a DNA end binding protein for DSB recognition and the multifunctional LigD protein composed a ligase, a nuclease and a polymerase domain, for end processing and ligation of the broken ends. In silico analyses performed in the 38 sequenced genomes of Streptomyces species revealed the existence of a large panel of NHEJ-like genes. Indeed, ku genes or ligD domain homologues are scattered throughout the genome in multiple copies and can be distinguished in two categories: the core NHEJ gene set constituted of conserved loci and the variable NHEJ gene set constituted of NHEJ-like genes present in only a part of the species. In Streptomyces ambofaciens ATCC 23877, not only the deletion of core genes but also that of variable genes led to an increased sensitivity to DNA damage induced by electron beam irradiation. Multiple mutants of ku, ligase or polymerase encoding genes showed an aggravated phenotype compared to single mutants. Biochemical assays revealed the ability of Ku-like proteins to protect and to stimulate ligation of DNA ends. RT-qPCR and GFP fusion experiments suggested that ku-like genes show a growth phase dependent expression profile consistent with their involvement in DNA repair during spores formation and/or germination.

Physical association of pyrimidine dimer DNA glycosylase and apurinic/apyrimidinic DNA endonuclease essential for repair of ultraviolet-damaged DNA

International Nuclear Information System (INIS)

Nakabeppu, Y.; Sekiguchi, M.

1981-01-01

T4 endonuclease, which is involved in repair of uv-damaged DNA, has been purified to apparent physical homogeneity. Incubation of uv-irradiated poly(dA).poly(dT) with the purified enzyme preparations resulted in production of alkali-labile apyrimidinic sites, followed by formation of nicks in the polymer. By performing a limited reaction with T4 endonuclease V at pH 8.5, irradiated polymer was converted to an intermediate form that carried a large number of alkali-labile sites but only a few nicks. The intermediate was used as substrate for the assay of apurinic/apyrimidinic DNA endonuclease activity. The two activities, a pyrimidine dimer DNA glycosylase and an apurinic/apyrimidinic DNA endonuclease, were copurified and found in enzyme preparations that contained only a 16,000-dalton polypeptide. These results strongly suggested that a DNA glycosylase specific for pyrimidine dimers and an apurinic/apyrimidinic DNA endonuclease reside in a single polypeptide chain coded by the denV gene of bacteriophage T4

TbPIF5 is a Trypanosoma brucei mitochondrial DNA helicase involved in processing of minicircle Okazaki fragments.

Directory of Open Access Journals (Sweden)

Beiyu Liu

2009-09-01

Full Text Available Trypanosoma brucei's mitochondrial genome, kinetoplast DNA (kDNA, is a giant network of catenated DNA rings. The network consists of a few thousand 1 kb minicircles and several dozen 23 kb maxicircles. Here we report that TbPIF5, one of T. brucei's six mitochondrial proteins related to Saccharomyces cerevisiae mitochondrial DNA helicase ScPIF1, is involved in minicircle lagging strand synthesis. Like its yeast homolog, TbPIF5 is a 5' to 3' DNA helicase. Together with other enzymes thought to be involved in Okazaki fragment processing, TbPIF5 localizes in vivo to the antipodal sites flanking the kDNA. Minicircles in wild type cells replicate unidirectionally as theta-structures and are unusual in that Okazaki fragments are not joined until after the progeny minicircles have segregated. We now report that overexpression of TbPIF5 causes premature removal of RNA primers and joining of Okazaki fragments on theta structures. Further elongation of the lagging strand is blocked, but the leading strand is completed and the minicircle progeny, one with a truncated H strand (ranging from 0.1 to 1 kb, are segregated. The minicircles with a truncated H strand electrophorese on an agarose gel as a smear. This replication defect is associated with kinetoplast shrinkage and eventual slowing of cell growth. We propose that TbPIF5 unwinds RNA primers after lagging strand synthesis, thus facilitating processing of Okazaki fragments.

Involvement of the DNA mismatch repair system in cisplatin sensitivity of testicular germ cell tumours

DEFF Research Database (Denmark)

Rudolph, Christiane; Melau, Cecilie; Nielsen, John E.

2017-01-01

in the majority of tumours, although the underlying mechanism largely remains to be elucidated. The aim of this study was to investigate the role of the DNA mismatch repair (MMR) system in the cisplatin sensitivity of TGCT. MethodsThe expression pattern of key MMR proteins, including MSH2, MSH6, MLH1 and PMS2...... proteins, in particular MSH2 and MLH1, which are involved in the recognition of cisplatin adducts and in activation of the DNA damage response pathway to initiate apoptosis....

Integral parametrization of the Kinetics of Crosslink production in plasmid DNA as a function of 8-methoxypsoralen concentration

Energy Technology Data Exchange (ETDEWEB)

Vidania, R. de; Paramio, J. M.; Bauluz, C.

1986-07-01

In this paper we present results of crosslink production in pBR322 DNA along a wide range of 8-methoxypsoralen (8-MOP) concentration. Experimental data were obtained as DNA renaturation percentages, from the shift in hyperchromicity after a temperature-dependent denaturation-renaturation process. the experimental results showed a three-stage profile when represented as a function of the natural logarithms of 8-MOP concentration. an integral parametrization which allows a simultaneous fit of the three observed stages is presented here. the theoretical values of crosslink production determined from the fit are useful to asses the genotoxicity of psoralen-induced crosslinks in plasmid DNA. (Author) 24 refs.

Integral parametrization of the Kinetics of Crosslink production in plasmid DNA as a function of 8-methoxypsoralen concentration

International Nuclear Information System (INIS)

Vidania, R. de; Paramio, J. M.; Bauluz, C.

1986-01-01

In this paper we present results of crosslink production in pBR322 DNA along a wide range of 8-methoxypsoralen (8-MOP) concentration. Experimental data were obtained as DNA renaturation percentages, from the shift in hyperchromicity after a temperature-dependent denaturation-renaturation process. the experimental results showed a three-stage profile when represented as a function of the natural logarithms of 8-MOP concentration. an integral parametrization which allows a simultaneous fit of the three observed stages is presented here. the theoretical values of crosslink production determined from the fit are useful to asses the genotoxicity of psoralen-induced crosslinks in plasmid DNA. (Author) 24 refs

Action of some drugs on enzymes involved in DNA-repair and semiconservative DNA-synthesis

International Nuclear Information System (INIS)

Wawra, E.; Klein, W.; Kocsis, F.; Weniger, P.

1975-07-01

Different antirheumatic and cytostatic drugs had been tested by measurement of the thymidine incorporation into DNA of spleen cells under conditions, under which either DNA-synthesis or repair after gamma- or UV-irradiation takes place. There are substances, which inhibit either only the semiconservative DNA-synthesis (vinblastine, isonicotinic acid hydracide) or only DNA-repair after gamma-irradiation (mixture of penicillin-G and procaine-penicillin-G) or both (cyclophosphamide, phenylbutazone, procarbazine, nalidixic acid). Vincristine shows no effect on the thymidine incorporation in DNA, but by density gradient centrifugation it has been found that it influences the ligase reaction. Two DNA polymerases had been isolated from spleen cells, one of the low molecular and one of the high molecular weight type. The influences of the described drugs on these enzymes and on a deoxyribonuclease I from beef pancreas have been tested in ''in vitro'' systems. In all cases, it has been found that there is no effect or only a very small one, compared with the action of well known inhibitors as e.g. ethidium bromide and p-chloromercuribenzoate, and this cannot be responsible for the suppressions found in DNA-repair and semiconservative DNA-synthesis. (author)

Are glutathione S transferases involved in DNA damage signalling? Interactions with DNA damage and repair revealed from molecular epidemiology studies

International Nuclear Information System (INIS)

Dusinska, Maria; Staruchova, Marta; Horska, Alexandra; Smolkova, Bozena; Collins, Andrew; Bonassi, Stefano; Volkovova, Katarina

2012-01-01

Glutathione S-transferases (GSTs) are members of a multigene family of isoenzymes that are important in the control of oxidative stress and in phase II metabolism. Acting non-enzymically, GSTs can modulate signalling pathways of cell proliferation, cell differentiation and apoptosis. Using a molecular epidemiology approach, we have investigated a potential involvement of GSTs in DNA damage processing, specifically the modulation of DNA repair in a group of 388 healthy adult volunteers; 239 with at least 5 years of occupational exposure to asbestos, stone wool or glass fibre, and 149 reference subjects. We measured DNA damage in lymphocytes using the comet assay (alkaline single cell gel electrophoresis): strand breaks (SBs) and alkali-labile sites, oxidised pyrimidines with endonuclease III, and oxidised purines with formamidopyrimidine DNA glycosylase. We also measured GST activity in erythrocytes, and the capacity for base excision repair (BER) in a lymphocyte extract. Polymorphisms in genes encoding three GST isoenzymes were determined, namely deletion of GSTM1 and GSTT1 and single nucleotide polymorphism Ile105Val in GSTP1. Consumption of vegetables and wine correlated negatively with DNA damage and modulated BER. GST activity correlated with oxidised bases and with BER capacity, and differed depending on polymorphisms in GSTP1, GSTT1 and GSTM1. A significantly lower BER rate was associated with the homozygous GSTT1 deletion in all asbestos site subjects and in the corresponding reference group. Multifactorial analysis revealed effects of sex and exposure in GSTP1 Ile/Val heterozygotes but not in Ile/Ile homozygotes. These variants affected also SBs levels, mainly by interactions of GSTP1 genotype with exposure, with sex, and with smoking habit; and by an interaction between sex and smoking. Our results show that GST polymorphisms and GST activity can apparently influence DNA stability and repair of oxidised bases, suggesting a potential new role for these

Are glutathione S transferases involved in DNA damage signalling? Interactions with DNA damage and repair revealed from molecular epidemiology studies

Energy Technology Data Exchange (ETDEWEB)

Dusinska, Maria, E-mail: Maria.DUSINSKA@nilu.no [CEE-Health Effects Group, NILU - Norwegian Institute for Air Research, Kjeller (Norway); Staruchova, Marta; Horska, Alexandra [Department of Experimental and Applied Genetics, Slovak Medical University, Bratislava (Slovakia); Smolkova, Bozena [Laboratory of Cancer Genetics, Cancer Research Institute of the Slovak Academy of Sciences, Bratislava (Slovakia); Collins, Andrew [Department of Nutrition, Faculty of Medicine, University of Oslo (Norway); Bonassi, Stefano [Unit of Clinical and Molecular Epidemiology, IRCCS San Raffaele Pisana, Rome (Italy); Volkovova, Katarina [Department of Experimental and Applied Genetics, Slovak Medical University, Bratislava (Slovakia)

2012-08-01

Glutathione S-transferases (GSTs) are members of a multigene family of isoenzymes that are important in the control of oxidative stress and in phase II metabolism. Acting non-enzymically, GSTs can modulate signalling pathways of cell proliferation, cell differentiation and apoptosis. Using a molecular epidemiology approach, we have investigated a potential involvement of GSTs in DNA damage processing, specifically the modulation of DNA repair in a group of 388 healthy adult volunteers; 239 with at least 5 years of occupational exposure to asbestos, stone wool or glass fibre, and 149 reference subjects. We measured DNA damage in lymphocytes using the comet assay (alkaline single cell gel electrophoresis): strand breaks (SBs) and alkali-labile sites, oxidised pyrimidines with endonuclease III, and oxidised purines with formamidopyrimidine DNA glycosylase. We also measured GST activity in erythrocytes, and the capacity for base excision repair (BER) in a lymphocyte extract. Polymorphisms in genes encoding three GST isoenzymes were determined, namely deletion of GSTM1 and GSTT1 and single nucleotide polymorphism Ile105Val in GSTP1. Consumption of vegetables and wine correlated negatively with DNA damage and modulated BER. GST activity correlated with oxidised bases and with BER capacity, and differed depending on polymorphisms in GSTP1, GSTT1 and GSTM1. A significantly lower BER rate was associated with the homozygous GSTT1 deletion in all asbestos site subjects and in the corresponding reference group. Multifactorial analysis revealed effects of sex and exposure in GSTP1 Ile/Val heterozygotes but not in Ile/Ile homozygotes. These variants affected also SBs levels, mainly by interactions of GSTP1 genotype with exposure, with sex, and with smoking habit; and by an interaction between sex and smoking. Our results show that GST polymorphisms and GST activity can apparently influence DNA stability and repair of oxidised bases, suggesting a potential new role for these

Cytochrome P450 2C8 and flavin-containing monooxygenases are involved in the metabolism of tazarotenic acid in humans.

Science.gov (United States)

Attar, Mayssa; Dong, Dahai; Ling, Kah-Hiing John; Tang-Liu, Diane D-S

2003-04-01

Upon oral administration, tazarotene is rapidly converted to tazarotenic acid by esterases. The main circulating agent, tazarotenic acid is subsequently oxidized to the inactive sulfoxide metabolite. Therefore, alterations in the metabolic clearance of tazarotenic acid may have significant effects on its systemic exposure. The objective of this study was to identify the human liver microsomal enzymes responsible for the in vitro metabolism of tazarotenic acid. Tazarotenic acid was incubated with 1 mg/ml pooled human liver microsomes, in 100 mM potassium phosphate buffer (pH 7.4), at 37 degrees C, over a period of 30 min. The microsomal enzymes that may be involved in tazarotenic acid metabolism were identified through incubation with microsomes containing cDNA-expressed human microsomal isozymes. Chemical inhibition studies were then conducted to confirm the identity of the enzymes potentially involved in tazarotenic acid metabolism. Reversed-phase high performance liquid chromatography was used to quantify the sulfoxide metabolite, the major metabolite of tazarotenic acid. Upon incubation of tazarotenic acid with microsomes expressing CYP2C8, flavin-containing monooxygenase 1 (FMO1), or FMO3, marked formation of the sulfoxide metabolite was observed. The involvement of these isozymes in tazarotenic acid metabolism was further confirmed by inhibition of metabolite formation in pooled human liver microsomes by specific inhibitors of CYP2C8 or FMO. In conclusion, the in vitro metabolism of tazarotenic acid to its sulfoxide metabolite in human liver microsomes is mediated by CYP2C8 and FMO.

DHX9 helicase is involved in preventing genomic instability induced by alternatively structured DNA in human cells.

Science.gov (United States)

Jain, Aklank; Bacolla, Albino; Del Mundo, Imee M; Zhao, Junhua; Wang, Guliang; Vasquez, Karen M

2013-12-01

Sequences that have the capacity to adopt alternative (i.e. non-B) DNA structures in the human genome have been implicated in stimulating genomic instability. Previously, we found that a naturally occurring intra-molecular triplex (H-DNA) caused genetic instability in mammals largely in the form of DNA double-strand breaks. Thus, it is of interest to determine the mechanism(s) involved in processing H-DNA. Recently, we demonstrated that human DHX9 helicase preferentially unwinds inter-molecular triplex DNA in vitro. Herein, we used a mutation-reporter system containing H-DNA to examine the relevance of DHX9 activity on naturally occurring H-DNA structures in human cells. We found that H-DNA significantly increased mutagenesis in small-interfering siRNA-treated, DHX9-depleted cells, affecting mostly deletions. Moreover, DHX9 associated with H-DNA in the context of supercoiled plasmids. To further investigate the role of DHX9 in the recognition/processing of H-DNA, we performed binding assays in vitro and chromatin immunoprecipitation assays in U2OS cells. DHX9 recognized H-DNA, as evidenced by its binding to the H-DNA structure and enrichment at the H-DNA region compared with a control region in human cells. These composite data implicate DHX9 in processing H-DNA structures in vivo and support its role in the overall maintenance of genomic stability at sites of alternatively structured DNA.

Incomplete effector/memory differentiation of antigen-primed CD8+ T cells in gene gun DNA-vaccinated mice

DEFF Research Database (Denmark)

Bartholdy, Christina; Stryhn, Anette; Hansen, Nils Jacob Vest

2003-01-01

DNA vaccination is an efficient way to induce CD8+ T cell memory, but it is still unclear to what extent such memory responses afford protection in vivo. To study this, we induced CD8+ memory responses directed towards defined viral epitopes, using DNA vaccines encoding immunodominant MHC class I......-restricted epitopes of lymphocytic choriomeningitis virus covalently linked to beta2-microglobulin. This vaccine construct primed for a stronger recall response than did a more conventional minigene construct. Despite this, vaccinated mice were only protected against systemic infection whereas protection against...... sites. Thus, our DNA vaccine induces a long-lived memory CD8+ T cell population that provides efficient protection against high-dose systemic infection. However, viral replication in solid non-lymphoid organs is not curtailed sufficiently fast to prevent significant virus-induced inflammation. Our...

The F8H Glycosyltransferase is a Functional Paralog of FRA8 Involved in Glucuronoxylan Biosynthesis in Arabidopsis

Science.gov (United States)

The FRAGILE FIBER8 gene was previously shown to be required for the biosynthesis of the reducing end tetrasaccharide sequence of glucuronoxylan (GX) in Arabidopsis thaliana. Here, we demonstrate that F8H, a close homolog of FRA8, is a functional ortholog of FRA8 involved in GX bi...

DNA-binding protects p53 from interactions with cofactors involved in transcription-independent functions.

Science.gov (United States)

Lambrughi, Matteo; De Gioia, Luca; Gervasio, Francesco Luigi; Lindorff-Larsen, Kresten; Nussinov, Ruth; Urani, Chiara; Bruschi, Maurizio; Papaleo, Elena

2016-11-02

Binding-induced conformational changes of a protein at regions distant from the binding site may play crucial roles in protein function and regulation. The p53 tumour suppressor is an example of such an allosterically regulated protein. Little is known, however, about how DNA binding can affect distal sites for transcription factors. Furthermore, the molecular details of how a local perturbation is transmitted through a protein structure are generally elusive and occur on timescales hard to explore by simulations. Thus, we employed state-of-the-art enhanced sampling atomistic simulations to unveil DNA-induced effects on p53 structure and dynamics that modulate the recruitment of cofactors and the impact of phosphorylation at Ser215. We show that DNA interaction promotes a conformational change in a region 3 nm away from the DNA binding site. Specifically, binding to DNA increases the population of an occluded minor state at this distal site by more than 4-fold, whereas phosphorylation traps the protein in its major state. In the minor conformation, the interface of p53 that binds biological partners related to p53 transcription-independent functions is not accessible. Significantly, our study reveals a mechanism of DNA-mediated protection of p53 from interactions with partners involved in the p53 transcription-independent signalling. This also suggests that conformational dynamics is tightly related to p53 signalling. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

DNA ligase III is involved in a DNA-PK independent pathway of NHEJ in human cells

International Nuclear Information System (INIS)

Wang, H.; Perrault, A.R.; Qin, W.; Wang, H.; Iliakis, G.

2003-01-01

Full text: Double strand breaks (DSB) induced by ionizing radiation (IR) and other cytotoxic agents in the genome of higher eukaryotes are thought to be repaired either by homologous recombination repair (HRR), or non-homologous endjoining (NHEJ). We previously reported the operation of two components of NHEJ in vivo: a DNA-PK dependent component that operates with fast kinetics (D-NHEJ), and a DNA-PK independent component that acts as a backup (basic or B-NHEJ) and operates with kinetics an order of magnitude slower. To gain further insight into the mechanisms of B-NHEJ, we investigated DNA endjoining in extracts 180BR, a human cell line deficient in DNA ligase IV, using an in vitro plasmid-based DNA endjoining assay. An anti DNA ligase III antibody inhibited almost completely DNA endjoining activity in these extracts. On the other hand, an anti DNA ligase I antibody had no measurable effect in DNA endjoining activity. Immunodepletion of DNA ligase III from 180BR cell extracts abolished the DNA endjoining activity, which could be restored by addition of purified human DNA ligase IIIb. Full-length DNA ligase III bound to double stranded DNA and stimulated DNA endjoining in both intermolecular and intramolecular ligation. Furthermore, fractionation of HeLa cell extracts demonstrated the presence of an activity stimulating the function of DNA ligase III. Based on these observations we propose that DNA ligase III is the ligase operating in B-NHEJ

The role of the PHP domain associated with DNA polymerase X from Thermus thermophilus HB8 in base excision repair.

Science.gov (United States)

Nakane, Shuhei; Nakagawa, Noriko; Kuramitsu, Seiki; Masui, Ryoji

2012-11-01

Base excision repair (BER) is one of the most commonly used DNA repair pathways involved in genome stability. X-family DNA polymerases (PolXs) play critical roles in BER, especially in filling single-nucleotide gaps. In addition to a polymerase core domain, bacterial PolXs have a polymerase and histidinol phosphatase (PHP) domain with phosphoesterase activity which is also required for BER. However, the role of the PHP domain of PolX in bacterial BER remains unresolved. We found that the PHP domain of Thermus thermophilus HB8 PolX (ttPolX) functions as two types of phosphoesterase in BER, including a 3'-phosphatase and an apurinic/apyrimidinic (AP) endonuclease. Experiments using T. thermophilus HB8 cell lysates revealed that the majority of the 3'-phosphatase and AP endonuclease activities are attributable to the another phosphoesterase in T. thermophilus HB8, endonuclease IV (ttEndoIV). However, ttPolX possesses significant 3'-phosphatase activity in ΔttendoIV cell lysate, indicating possible complementation. Our experiments also reveal that there are only two enzymes that display the 3'-phosphatase activity in the T. thermophilus HB8 cell, ttPolX and ttEndoIV. Furthermore, phenotypic analysis of ΔttpolX, ΔttendoIV, and ΔttpolX/ΔttendoIV using hydrogen peroxide and sodium nitrite supports the hypothesis that ttPolX functions as a backup for ttEndoIV in BER. Copyright © 2012 Elsevier B.V. All rights reserved.

Aminothiols linked to quinoline and acridine chromophores efficiently decrease 7,8-dihydro-8-oxo-2'-deoxyguanosine formation in γ-irradiated DNA

International Nuclear Information System (INIS)

Laayoun, A.; Coulombeau, C.; Constant, J.F.; Lhomme, J.; Berger, M.; Cadet, J.

1994-01-01

In a search for more active radioprotective compounds, we have prepared and examined a series of model molecules in which the radioprotective β-aminothiol unit (free or derivatized as acetate or phosphorothioate) is tethered to the DNA-binding chromophores quinoline and acridine through links of variable length. The modifying activity of these 'hybrid' molecules was estimated by measuring the formation of 8-oxo-2'-deoxyguanosine (8-oxodGuo) in double-strand DNA upon exposure to γ-rays in oxygen-free solution in the presence of the drugs. We show that all hybrid molecules protect the guanine moiety from oxidation more efficiently than the parent β-aminothiol units. The degree of protection is the highest for the molecules in which the thiol is linked to the strong binding intercalator acridine through a long polyaminochain. (author)

The yeast Saccharomyces cerevisiae DNA polymerase IV: possible involvement in double strand break DNA repair.

OpenAIRE

Leem, S H; Ropp, P A; Sugino, A

1994-01-01

We identified and purified a new DNA polymerase (DNA polymerase IV), which is similar to mammalian DNA polymerase beta, from Saccharomyces cerevisiae and suggested that it is encoded by YCR14C (POLX) on chromosome III. Here, we provided a direct evidence that the purified DNA polymerase IV is indeed encoded by POLX. Strains harboring a pol4 deletion mutation exhibit neither mitotic growth defect nor a meiosis defect, suggesting that DNA polymerase IV participates in nonessential functions in ...

A polycomb group protein, PHF1, is involved in the response to DNA double-strand breaks in human cell

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Hong, Zehui; Jiang, Jie; Lan, Li; Nakajima, Satoshi; Kanno, Shin-ichiro; Koseki, Haruhiko; Yasui, Akira

2008-01-01

DNA double-strand breaks (DSBs) represent the most toxic DNA damage arisen from endogenous and exogenous genotoxic stresses and are known to be repaired by either homologous recombination or nonhomologous end-joining processes. Although many proteins have been identified to participate in either of the processes, the whole processes still remain elusive. Polycomb group (PcG) proteins are epigenetic chromatin modifiers involved in gene silencing, cancer development and the maintenance of embryonic and adult stem cells. By screening proteins responding to DNA damage using laser micro-irradiation, we found that PHF1, a human homolog of Drosophila polycomb-like, Pcl, protein, was recruited to DSBs immediately after irradiation and dissociated within 10 min. The accumulation at DSBs is Ku70/Ku80-dependent, and knockdown of PHF1 leads to X-ray sensitivity and increases the frequency of homologous recombination in HeLa cell. We found that PHF1 interacts physically with Ku70/Ku80, suggesting that PHF1 promotes nonhomologous end-joining processes. Furthermore, we found that PHF1 interacts with a number of proteins involved in DNA damage responses, RAD50, SMC1, DHX9 and p53, further suggesting that PHF1, besides the function in PcG, is involved in genome maintenance processes. PMID:18385154

Folate, colorectal cancer and the involvement of DNA methylation.

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Williams, Elizabeth A

2012-11-01

Diet is a major factor in the aetiology of colorectal cancer (CRC). Epidemiological evidence suggests that folate confers a modest protection against CRC risk. However, the relationship is complex, and evidence from human intervention trials and animal studies suggests that a high-dose of folic acid supplementation may enhance the risk of colorectal carcinogenesis in certain circumstances. The molecular mechanisms underlying the apparent dual modulatory effect of folate on colorectal carcinogenesis are not fully understood. Folate is central to C1 metabolism and is needed for both DNA synthesis and DNA methylation, providing plausible biological mechanisms through which folate could modulate cancer risk. Aberrant DNA methylation is an early event in colorectal carcinogenesis and is typically associated with the transcriptional silencing of tumour suppressor genes. Folate is required for the production of S-adenosyl methionine, which serves as a methyl donor for DNA methylation events; thereby folate availability is proposed to modulate DNA methylation status. The evidence for an effect of folate on DNA methylation in the human colon is limited, but a modulation of DNA methylation in response to folate has been demonstrated. More research is required to clarify the optimum intake of folate for CRC prevention and to elucidate the effect of folate availability on DNA methylation and the associated impact on CRC biology.

The role of hnRPUL1 involved in DNA damage response is related to PARP1.

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Zehui Hong

Full Text Available Heterogeneous nuclear ribonucleoprotein U-like 1 (hnRPUL1 -also known as adenovirus early region 1B-associated proteins 5 (E1B-AP5 - plays a role in RNA metabolism. Recently, hnRPUL1 has also been shown to be involved in DNA damage response, but the function of hnRPUL1 in response to DNA damage remains unclear. Here, we have demonstrated that hnRPUL1 is associated with PARP1 and recruited to DNA double-strand breaks (DSBs sites in a PARP1-mediated poly (ADP-ribosyl ation dependent manner. In turn, hnRPUL1 knockdown enhances the recruitment of PARP1 to DSBs sites. Specifically, we showed that hnRPUL1 is also implicated in the transcriptional regulation of PARP1 gene. Thus, we propose hnRPUL1 as a new component related to PARP1 in DNA damage response and repair.

UVA photoactivation of DNA containing halogenated thiopyrimidines induces cytotoxic DNA lesions

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Brem, Reto; Zhang, Xiaohui; Xu, Yao-Zhong; Karran, Peter

2015-01-01

Photochemotherapy, the combination of a photosensitiser and ultraviolet (UV) or visible light, is an effective treatment for skin conditions including cancer. The high mutagenicity and non-selectivity of photochemotherapy regimes warrants the development of alternative approaches. We demonstrate that the thiopyrimidine nucleosides 5-bromo-4-thiodeoxyuridine (SBrdU) and 5-iodo-4-thiodeoxyuridine (SIdU) are incorporated into the DNA of cultured human and mouse cells where they synergistically sensitise killing by low doses of UVA radiation. The DNA halothiopyrimidine/UVA combinations induce DNA interstrand crosslinks, DNA-protein crosslinks, DNA strand breaks, nucleobase damage and lesions that resemble UV-induced pyrimidine(6-4)pyrimidone photoproducts. These are potentially lethal DNA lesions and cells defective in their repair are hypersensitive to killing by SBrdU/UVA and SIdU/UVA. DNA SIdU and SBrdU generate lethal DNA photodamage by partially distinct mechanisms that reflect the different photolabilities of their C–I and C–Br bonds. Although singlet oxygen is involved in photolesion formation, DNA SBrdU and SIdU photoactivation does not detectably increase DNA 8-oxoguanine levels. The absence of significant collateral damage to normal guanine suggests that UVA activation of DNA SIdU or SBrdU might offer a strategy to target hyperproliferative skin conditions that avoids the extensive formation of a known mutagenic DNA lesion. PMID:25747491

An ameliorative protocol for the quantification of purine 5',8-cyclo-2'-deoxynucleosides in oxidized DNA

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Terzidis, Michael; Chatgilialoglu, Chryssostomos

2015-07-01

5',8-Cyclo-2'-deoxyadenosine (cdA) and 5',8-cyclo-2'-deoxyguanosine (cdG) are lesions resulting from hydroxyl radical (HO•) attack on the 5'H of the nucleoside sugar moiety and exist in both 5'R and 5'S diastereomeric forms. Increased levels of cdA and cdG are linked to Nucleotide Excision Repair mechanism deficiency and mutagenesis. Discrepancies in the damage measurements reported over recent years indicated the weakness of the actual protocols, in particular for ensuring the quantitative release of these lesions from the DNA sample and the appropriate method for their analysis. Herein we report the detailed revision leading to a cost-effective and efficient protocol for the DNA damage measurement, consisting of the nuclease benzonase and nuclease P1 enzymatic combination for DNA digestion followed by liquid chromatography isotope dilution tandem mass spectrometry analysis.

DNA transfer-a never ending story. A study on scenarios involving a second person as carrier.

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Helmus, Janine; Bajanowski, Thomas; Poetsch, Micaela

2016-01-01

The transfer of DNA directly from one item to another has been shown in many studies with elaborate discussions on the nature of the DNA donor as well as material and surface of the items or surrounding features. Every DNA transfer scenario one can imagine seems to be possible. This evokes more and more intricate scenarios proposed by lawyers or attorneys searching for an explanation of the DNA of a certain person on a distinct item with impact on a crime. At court, the forensic genetic scientist has to comment on the probability of these scenarios thus calling for extensive studies on such settings. Here, the possibility of an involvement of a second person as a carrier of the donor's DNA in a variety of different scenarios including three pairs of people and two kinds of items (textiles and plastic bags) was investigated. All transfer settings were executed with and without gloves on the carrier's hands. DNA left on the items was isolated and analyzed using the Powerplex® ESX17 kit. In 21 out of 180 samples, all alleles of the donor DNA could be obtained on the second item (12%), on eight samples, the donor's DNA was dominant compared to all other alleles (38% of samples with complete donor profile). Additionally, 51 samples displayed at least more than half of the donor's alleles (28%). The complete DNA profile of the carrier was found in 47 out of 180 samples (42 partial profiles). In summary, it could be shown that a transfer of donor DNA from epithelial cells through a carrier to a second item is possible, even if the carrier does not wear gloves.

Fluorometric detection of adenine in target DNA by exciplex formation with fluorescent 8-arylethynylated deoxyguanosine.

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Saito, Yoshio; Kugenuma, Kenji; Tanaka, Makiko; Suzuki, Azusa; Saito, Isao

2012-06-01

We demonstrated an intriguing method to discriminate adenine by incident appearance of an intense new emission via exciplex formation in hybridization of target DNA with newly designed fluorescent 8-arylethynylated deoxyguanosine derivatives. We described the synthesis of such highly electron donating fluorescent guanosine derivatives and their incorporation into DNA oligomers which may be used for the structural study and the fluorometric analysis of nucleic acids. Copyright © 2012 Elsevier Ltd. All rights reserved.

8-oxoguanine DNA glycosylase (OGG1 deficiency elicits coordinated changes in lipid and mitochondrial metabolism in muscle.

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Vladimir Vartanian

Full Text Available Oxidative stress resulting from endogenous and exogenous sources causes damage to cellular components, including genomic and mitochondrial DNA. Oxidative DNA damage is primarily repaired via the base excision repair pathway that is initiated by DNA glycosylases. 8-oxoguanine DNA glycosylase (OGG1 recognizes and cleaves oxidized and ring-fragmented purines, including 8-oxoguanine, the most commonly formed oxidative DNA lesion. Mice lacking the OGG1 gene product are prone to multiple features of the metabolic syndrome, including high-fat diet-induced obesity, hepatic steatosis, and insulin resistance. Here, we report that OGG1-deficient mice also display skeletal muscle pathologies, including increased muscle lipid deposition and alterations in genes regulating lipid uptake and mitochondrial fission in skeletal muscle. In addition, expression of genes of the TCA cycle and of carbohydrate and lipid metabolism are also significantly altered in muscle of OGG1-deficient mice. These tissue changes are accompanied by marked reductions in markers of muscle function in OGG1-deficient animals, including decreased grip strength and treadmill endurance. Collectively, these data indicate a role for skeletal muscle OGG1 in the maintenance of optimal tissue function.

Characterization of DNA polymerase X from Thermus thermophilus HB8 reveals the POLXc and PHP domains are both required for 3'-5' exonuclease activity.

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Nakane, Shuhei; Nakagawa, Noriko; Kuramitsu, Seiki; Masui, Ryoji

2009-04-01

The X-family DNA polymerases (PolXs) comprise a highly conserved DNA polymerase family found in all kingdoms. Mammalian PolXs are known to be involved in several DNA-processing pathways including repair, but the cellular functions of bacterial PolXs are less known. Many bacterial PolXs have a polymerase and histidinol phosphatase (PHP) domain at their C-termini in addition to a PolX core (POLXc) domain, and possess 3'-5' exonuclease activity. Although both domains are highly conserved in bacteria, their molecular functions, especially for a PHP domain, are unknown. We found Thermus thermophilus HB8 PolX (ttPolX) has Mg(2+)/Mn(2+)-dependent DNA/RNA polymerase, Mn(2+)-dependent 3'-5' exonuclease and DNA-binding activities. We identified the domains of ttPolX by limited proteolysis and characterized their biochemical activities. The POLXc domain was responsible for the polymerase and DNA-binding activities but exonuclease activity was not detected for either domain. However, the POLXc and PHP domains interacted with each other and a mixture of the two domains had Mn(2+)-dependent 3'-5' exonuclease activity. Moreover, site-directed mutagenesis revealed catalytically important residues in the PHP domain for the 3'-5' exonuclease activity. Our findings provide a molecular insight into the functional domain organization of bacterial PolXs, especially the requirement of the PHP domain for 3'-5' exonuclease activity.

The yeast Saccharomyces cerevisiae DNA polymerase IV: possible involvement in double strand break DNA repair.

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Leem, S H; Ropp, P A; Sugino, A

1994-08-11

We identified and purified a new DNA polymerase (DNA polymerase IV), which is similar to mammalian DNA polymerase beta, from Saccharomyces cerevisiae and suggested that it is encoded by YCR14C (POLX) on chromosome III. Here, we provided a direct evidence that the purified DNA polymerase IV is indeed encoded by POLX. Strains harboring a pol4 deletion mutation exhibit neither mitotic growth defect nor a meiosis defect, suggesting that DNA polymerase IV participates in nonessential functions in DNA metabolism. The deletion strains did not exhibit UV-sensitivity. However, they did show weak sensitivity to MMS-treatment and exhibited a hyper-recombination phenotype when intragenic recombination was measured during meiosis. Furthermore, MAT alpha pol4 delta segregants had a higher frequency of illegitimate mating with a MAT alpha tester strain than that of wild-type cells. These results suggest that DNA polymerase IV participates in a double-strand break repair pathway. A 3.2kb of the POL4 transcript was weakly expressed in mitotically growing cells. During meiosis, a 2.2 kb POL4 transcript was greatly induced, while the 3.2 kb transcript stayed at constant levels. This induction was delayed in a swi4 delta strain during meiosis, while no effect was observed in a swi6 delta strain.

Crystallization and preliminary X-ray analysis of a complex formed between the antibiotic simocyclinone D8 and the DNA breakage–reunion domain of Escherichia coli DNA gyrase

International Nuclear Information System (INIS)

Edwards, Marcus J.; Flatman, Ruth H.; Mitchenall, Lesley A.; Stevenson, Clare E. M.; Maxwell, Anthony; Lawson, David M.

2009-01-01

Crystals of a complex formed between the 59 kDa N-terminal fragment of the E. coli DNA gyrase A subunit and the antibiotic simocyclinone D8 were obtained and X-ray data were recorded to a resolution of 2.75 Å. Crystals of a complex formed between the 59 kDa N-terminal fragment of the Escherichia coli DNA gyrase A subunit (also known as the breakage–reunion domain) and the antibiotic simocyclinone D8 were grown by vapour diffusion. The complex crystallized with I-centred orthorhombic symmetry and X-ray data were recorded to a resolution of 2.75 Å from a single crystal at the synchrotron. DNA gyrase is an essential bacterial enzyme and thus represents an attractive target for drug development

The RTR Complex Partner RMI2 and the DNA Helicase RTEL1 Are Both Independently Involved in Preserving the Stability of 45S rDNA Repeats in Arabidopsis thaliana.

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Sarah Röhrig

2016-10-01

Full Text Available The stability of repetitive sequences in complex eukaryotic genomes is safeguarded by factors suppressing homologues recombination. Prominent in this is the role of the RTR complex. In plants, it consists of the RecQ helicase RECQ4A, the topoisomerase TOP3α and RMI1. Like mammals, but not yeast, plants harbor an additional complex partner, RMI2. Here, we demonstrate that, in Arabidopsis thaliana, RMI2 is involved in the repair of aberrant replication intermediates in root meristems as well as in intrastrand crosslink repair. In both instances, RMI2 is involved independently of the DNA helicase RTEL1. Surprisingly, simultaneous loss of RMI2 and RTEL1 leads to loss of male fertility. As both the RTR complex and RTEL1 are involved in suppression of homologous recombination (HR, we tested the efficiency of HR in the double mutant rmi2-2 rtel1-1 and found a synergistic enhancement (80-fold. Searching for natural target sequences we found that RTEL1 is required for stabilizing 45S rDNA repeats. In the double mutant with rmi2-2 the number of 45S rDNA repeats is further decreased sustaining independent roles of both factors in this process. Thus, loss of suppression of HR does not only lead to a destabilization of rDNA repeats but might be especially deleterious for tissues undergoing multiple cell divisions such as the male germline.

The RTR Complex Partner RMI2 and the DNA Helicase RTEL1 Are Both Independently Involved in Preserving the Stability of 45S rDNA Repeats in Arabidopsis thaliana.

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Röhrig, Sarah; Schröpfer, Susan; Knoll, Alexander; Puchta, Holger

2016-10-01

The stability of repetitive sequences in complex eukaryotic genomes is safeguarded by factors suppressing homologues recombination. Prominent in this is the role of the RTR complex. In plants, it consists of the RecQ helicase RECQ4A, the topoisomerase TOP3α and RMI1. Like mammals, but not yeast, plants harbor an additional complex partner, RMI2. Here, we demonstrate that, in Arabidopsis thaliana, RMI2 is involved in the repair of aberrant replication intermediates in root meristems as well as in intrastrand crosslink repair. In both instances, RMI2 is involved independently of the DNA helicase RTEL1. Surprisingly, simultaneous loss of RMI2 and RTEL1 leads to loss of male fertility. As both the RTR complex and RTEL1 are involved in suppression of homologous recombination (HR), we tested the efficiency of HR in the double mutant rmi2-2 rtel1-1 and found a synergistic enhancement (80-fold). Searching for natural target sequences we found that RTEL1 is required for stabilizing 45S rDNA repeats. In the double mutant with rmi2-2 the number of 45S rDNA repeats is further decreased sustaining independent roles of both factors in this process. Thus, loss of suppression of HR does not only lead to a destabilization of rDNA repeats but might be especially deleterious for tissues undergoing multiple cell divisions such as the male germline.

Male infertility is significantly associated with multiple deletions in an 8.7-kb segment of sperm mtDNA in Pakistan.

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Mughal, Irfan Afzal; Irfan, Asma; Jahan, Sarwat; Hameed, Abdul

2017-06-12

This study aimed to find a link between sperm mitochondrial DNA mutations and male infertility in Pakistan. DNA from semen samples was extracted and amplified by PCR using 7.8-kb deletion-specific primers. The PCR products were separated on agarose gel, visualized under UV-illumination, and then photographed. The results were genotyped and the data were analyzed using SPSS. Deletion analysis of the 8.7-kb fragment by long PCR revealed multiple deletions. The frequency of deletion was much higher in infertile groups as compared to the control group. Further, on comparison between different subtypes of infertile groups, the deletions were highest in the oligoasthenoteratozoospermia (OAT) group. The statistical analysis of case and control groups showed a significant association of the 8.7-kb deletion with human male infertile groups (P = 0.031), and particularly a very significant association with the OAT subgroup (P = 0.019). A significant association has been found between human male infertility and mtDNA deletions in an 8.7-kb segment of sperm mtDNA in a Pakistani population.

Vagus nerve is involved in the changes in body temperature induced by intragastric administration of 1,8-cineole via TRPM8 in mice.

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Urata, Tomomi; Mori, Noriyuki; Fukuwatari, Tsutomu

2017-05-22

Transient Receptor Potential Melastatin 8 (TRPM8) is a cold receptor activated by mild cold temperature (<28°C). TRPM8 expressed in cutaneous sensory nerves is involved in cold sensation and thermoregulation. TRPM8 mRNA is detected in various tissues, including the gastrointestinal mucosa, and in the vagal afferent nerve. The relationship between vagal afferent nerve-specific expression of TRPM8 and thermoregulation remains unclear. In this study, we aimed to investigate whether TRPM8 expression in the vagal afferent nerve is involved in autonomic thermoregulation. We found that intragastric administration of 1,8-cineole, a TRPM8 agonist, increased intrascapular brown adipose tissue and colonic temperatures, and M8-B-treatment (TRPM8 antagonist) inhibited these responses. Intravenous administration of 1,8-cineole also showed similar effects. In vagotomized mice, the responses induced by intragastric administration of 1,8-cineole were attenuated. These results suggest that TRPM8 expressed in tissues apart from cutaneous sensory nerves are involved in autonomic thermoregulation response. Copyright © 2017 Elsevier B.V. All rights reserved.

Stimulation of ribosomal RNA gene promoter by transcription factor Sp1 involves active DNA demethylation by Gadd45-NER pathway.

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Rajput, Pallavi; Pandey, Vijaya; Kumar, Vijay

2016-08-01

The well-studied Pol II transcription factor Sp1 has not been investigated for its regulatory role in rDNA transcription. Here, we show that Sp1 bound to specific sites on rDNA and localized into the nucleoli during the G1 phase of cell cycle to activate rDNA transcription. It facilitated the recruitment of Pol I pre-initiation complex and impeded the binding of nucleolar remodeling complex (NoRC) to rDNA resulting in the formation of euchromatin active state. More importantly, Sp1 also orchestrated the site-specific binding of Gadd45a-nucleotide excision repair (NER) complex resulting in active demethylation and transcriptional activation of rDNA. Interestingly, knockdown of Sp1 impaired rDNA transcription due to reduced engagement of the Gadd45a-NER complex and hypermethylation of rDNA. Thus, the present study unveils a novel role of Sp1 in rDNA transcription involving promoter demethylation. Copyright © 2016 Elsevier B.V. All rights reserved.

A PHF8 homolog in C. elegans promotes DNA repair via homologous recombination.

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Changrim Lee

Full Text Available PHF8 is a JmjC domain-containing histone demethylase, defects in which are associated with X-linked mental retardation. In this study, we examined the roles of two PHF8 homologs, JMJD-1.1 and JMJD-1.2, in the model organism C. elegans in response to DNA damage. A deletion mutation in either of the genes led to hypersensitivity to interstrand DNA crosslinks (ICLs, while only mutation of jmjd-1.1 resulted in hypersensitivity to double-strand DNA breaks (DSBs. In response to ICLs, JMJD-1.1 did not affect the focus formation of FCD-2, a homolog of FANCD2, a key protein in the Fanconi anemia pathway. However, the dynamic behavior of RPA-1 and RAD-51 was affected by the mutation: the accumulations of both proteins at ICLs appeared normal, but their subsequent disappearance was retarded, suggesting that later steps of homologous recombination were defective. Similar changes in the dynamic behavior of RPA-1 and RAD-51 were seen in response to DSBs, supporting a role of JMJD-1.1 in homologous recombination. Such a role was also supported by our finding that the hypersensitivity of jmjd-1.1 worms to ICLs was rescued by knockdown of lig-4, a homolog of Ligase 4 active in nonhomologous end-joining. The hypersensitivity of jmjd-1.1 worms to ICLs was increased by rad-54 knockdown, suggesting that JMJD-1.1 acts in parallel with RAD-54 in modulating chromatin structure. Indeed, the level of histone H3 Lys9 tri-methylation, a marker of heterochromatin, was higher in jmjd-1.1 cells than in wild-type cells. We conclude that the histone demethylase JMJD-1.1 influences homologous recombination either by relaxing heterochromatin structure or by indirectly regulating the expression of multiple genes affecting DNA repair.

OGG1 Involvement in High Glucose-Mediated Enhancement of Bupivacaine-Induced Oxidative DNA Damage in SH-SY5Y Cells

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Liu, Zhong-Jie; Zhao, Wei; Zhang, Qing-Guo; Li, Le; Lai, Lu-Ying; Jiang, Shan; Xu, Shi-Yuan

2015-01-01

Hyperglycemia can inhibit expression of the 8-oxoG-DNA glycosylase (OGG1) which is one of the key repair enzymes for DNA oxidative damage. The effect of hyperglycemia on OGG1 expression in response to local anesthetics-induced DNA damage is unknown. This study was designed to determine whether high glucose inhibits OGG1 expression and aggravates bupivacaine-induced DNA damage via reactive oxygen species (ROS). SH-SY5Y cells were cultured with or without 50 mM glucose for 8 days before they were treated with 1.5 mM bupivacaine for 24 h. OGG1 expression was measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. ROS was estimated using the redox-sensitive fluorescent dye DCFH-DA. DNA damage was investigated with immunostaining for 8-oxodG and comet assays. OGG1 expression was inhibited in cells exposed to high glucose with concomitant increase in ROS production and more severe DNA damage as compared to control culture conditions, and these changes were further exacerbated by bupivacaine. Treatment with the antioxidant N-acetyl-L-cysteine (NAC) prevented high glucose and bupivacaine mediated increase in ROS production and restored functional expression of OGG1, which lead to attenuated high glucose-mediated exacerbation of bupivacaine neurotoxicity. Our findings indicate that subjects with diabetes may experience more detrimental effects following bupivacaine use. PMID:26161242

OGG1 Involvement in High Glucose-Mediated Enhancement of Bupivacaine-Induced Oxidative DNA Damage in SH-SY5Y Cells

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Zhong-Jie Liu

2015-01-01

Full Text Available Hyperglycemia can inhibit expression of the 8-oxoG-DNA glycosylase (OGG1 which is one of the key repair enzymes for DNA oxidative damage. The effect of hyperglycemia on OGG1 expression in response to local anesthetics-induced DNA damage is unknown. This study was designed to determine whether high glucose inhibits OGG1 expression and aggravates bupivacaine-induced DNA damage via reactive oxygen species (ROS. SH-SY5Y cells were cultured with or without 50 mM glucose for 8 days before they were treated with 1.5 mM bupivacaine for 24 h. OGG1 expression was measured by quantitative real-time polymerase chain reaction (qRT-PCR and western blot. ROS was estimated using the redox-sensitive fluorescent dye DCFH-DA. DNA damage was investigated with immunostaining for 8-oxodG and comet assays. OGG1 expression was inhibited in cells exposed to high glucose with concomitant increase in ROS production and more severe DNA damage as compared to control culture conditions, and these changes were further exacerbated by bupivacaine. Treatment with the antioxidant N-acetyl-L-cysteine (NAC prevented high glucose and bupivacaine mediated increase in ROS production and restored functional expression of OGG1, which lead to attenuated high glucose-mediated exacerbation of bupivacaine neurotoxicity. Our findings indicate that subjects with diabetes may experience more detrimental effects following bupivacaine use.

A novel human AP endonuclease with conserved zinc-finger-like motifs involved in DNA strand break responses

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Kanno, Shin-ichiro; Kuzuoka, Hiroyuki; Sasao, Shigeru; Hong, Zehui; Lan, Li; Nakajima, Satoshi; Yasui, Akira

2007-01-01

DNA damage causes genome instability and cell death, but many of the cellular responses to DNA damage still remain elusive. We here report a human protein, PALF (PNK and APTX-like FHA protein), with an FHA (forkhead-associated) domain and novel zinc-finger-like CYR (cysteine–tyrosine–arginine) motifs that are involved in responses to DNA damage. We found that the CYR motif is widely distributed among DNA repair proteins of higher eukaryotes, and that PALF, as well as a Drosophila protein with tandem CYR motifs, has endo- and exonuclease activities against abasic site and other types of base damage. PALF accumulates rapidly at single-strand breaks in a poly(ADP-ribose) polymerase 1 (PARP1)-dependent manner in human cells. Indeed, PALF interacts directly with PARP1 and is required for its activation and for cellular resistance to methyl-methane sulfonate. PALF also interacts directly with KU86, LIGASEIV and phosphorylated XRCC4 proteins and possesses endo/exonuclease activity at protruding DNA ends. Various treatments that produce double-strand breaks induce formation of PALF foci, which fully coincide with γH2AX foci. Thus, PALF and the CYR motif may play important roles in DNA repair of higher eukaryotes. PMID:17396150

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

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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.

Public involvement in pharmacogenomics research: a national survey on patients' attitudes towards pharmacogenomics research and the willingness to donate DNA samples to a DNA bank in Japan.

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Kobayashi, Eriko; Sakurada, Tomoya; Ueda, Shiro; Satoh, Nobunori

2011-05-01

To assess the attitude of Japanese patients towards pharmacogenomics research and a DNA bank for identifying genomic markers associated with adverse drug reactions (ADRs) and their willingness to donate DNA samples, we conducted a survey of 550 male and female patients. The majority of the respondents showed a positive attitude towards pharmacogenomics research (87.6%) and a DNA bank (75.1%). The willingness to donate DNA samples when experiencing severe ADRs (55.8%) was higher than when taking medications (40.4%). Positive attitudes towards a DNA bank and organ donation were significantly associated with an increased willingness to donate. Though the level of positive attitude in the patient population was higher than that in the general public in our former study (81.0 and 70.4%, respectively), the level of the willingness of patients to donate was 40.4% when taking medications and 55.8% when experiencing severe ADRs which was lower than that of the general public in our former study (45.3 and 61.7%). The results suggested that the level of true willingness in the patient population was lower than that of the general public considering the fictitious situation presented to the public (to suppose that they were patients receiving medication). It is important to assess the willingness of patients who are true potential donors, not the general public.

Persistent Amplification of DNA Damage Signal Involved in Replicative Senescence of Normal Human Diploid Fibroblasts

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Masatoshi Suzuki

2012-01-01

Full Text Available Foci of phosphorylated histone H2AX and ATM are the surrogate markers of DNA double strand breaks. We previously reported that the residual foci increased their size after irradiation, which amplifies DNA damage signals. Here, we addressed whether amplification of DNA damage signal is involved in replicative senescence of normal human diploid fibroblasts. Large phosphorylated H2AX foci (>1.5 μm diameter were specifically detected in presenescent cells. The frequency of cells with large foci was well correlated with that of cells positive for senescence-associated β-galactosidase staining. Hypoxic cell culture condition extended replicative life span of normal human fibroblast, and we found that the formation of large foci delayed in those cells. Our immuno-FISH analysis revealed that large foci partially localized at telomeres in senescent cells. Importantly, large foci of phosphorylated H2AX were always colocalized with phosphorylated ATM foci. Furthermore, Ser15-phosphorylated p53 showed colocalization with the large foci. Since the treatment of senescent cells with phosphoinositide 3-kinase inhibitor, wortmannin, suppressed p53 phosphorylation, it is suggested that amplification of DNA damage signaling sustains persistent activation of ATM-p53 pathway, which is essential for replicative senescence.

Xeroderma Pigmentosum Group A Suppresses Mutagenesis Caused by Clustered Oxidative DNA Adducts in the Human Genome

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Sassa, Akira; Kamoshita, Nagisa; Kanemaru, Yuki; Honma, Masamitsu; Yasui, Manabu

2015-01-01

Clustered DNA damage is defined as multiple sites of DNA damage within one or two helical turns of the duplex DNA. This complex damage is often formed by exposure of the genome to ionizing radiation and is difficult to repair. The mutagenic potential and repair mechanisms of clustered DNA damage in human cells remain to be elucidated. In this study, we investigated the involvement of nucleotide excision repair (NER) in clustered oxidative DNA adducts. To identify the in vivo protective roles of NER, we established a human cell line lacking the NER gene xeroderma pigmentosum group A (XPA). XPA knockout (KO) cells were generated from TSCER122 cells derived from the human lymphoblastoid TK6 cell line. To analyze the mutagenic events in DNA adducts in vivo, we previously employed a system of tracing DNA adducts in the targeted mutagenesis (TATAM), in which DNA adducts were site-specifically introduced into intron 4 of thymidine kinase genes. Using the TATAM system, one or two tandem 7,8-dihydro-8-oxoguanine (8-oxoG) adducts were introduced into the genomes of TSCER122 or XPA KO cells. In XPA KO cells, the proportion of mutants induced by a single 8-oxoG (7.6%) was comparable with that in TSCER122 cells (8.1%). In contrast, the lack of XPA significantly enhanced the mutant proportion of tandem 8-oxoG in the transcribed strand (12%) compared with that in TSCER122 cells (7.4%) but not in the non-transcribed strand (12% and 11% in XPA KO and TSCER122 cells, respectively). By sequencing the tandem 8-oxoG-integrated loci in the transcribed strand, we found that the proportion of tandem mutations was markedly increased in XPA KO cells. These results indicate that NER is involved in repairing clustered DNA adducts in the transcribed strand in vivo. PMID:26559182

Xeroderma Pigmentosum Group A Suppresses Mutagenesis Caused by Clustered Oxidative DNA Adducts in the Human Genome.

Science.gov (United States)

Sassa, Akira; Kamoshita, Nagisa; Kanemaru, Yuki; Honma, Masamitsu; Yasui, Manabu

2015-01-01

Clustered DNA damage is defined as multiple sites of DNA damage within one or two helical turns of the duplex DNA. This complex damage is often formed by exposure of the genome to ionizing radiation and is difficult to repair. The mutagenic potential and repair mechanisms of clustered DNA damage in human cells remain to be elucidated. In this study, we investigated the involvement of nucleotide excision repair (NER) in clustered oxidative DNA adducts. To identify the in vivo protective roles of NER, we established a human cell line lacking the NER gene xeroderma pigmentosum group A (XPA). XPA knockout (KO) cells were generated from TSCER122 cells derived from the human lymphoblastoid TK6 cell line. To analyze the mutagenic events in DNA adducts in vivo, we previously employed a system of tracing DNA adducts in the targeted mutagenesis (TATAM), in which DNA adducts were site-specifically introduced into intron 4 of thymidine kinase genes. Using the TATAM system, one or two tandem 7,8-dihydro-8-oxoguanine (8-oxoG) adducts were introduced into the genomes of TSCER122 or XPA KO cells. In XPA KO cells, the proportion of mutants induced by a single 8-oxoG (7.6%) was comparable with that in TSCER122 cells (8.1%). In contrast, the lack of XPA significantly enhanced the mutant proportion of tandem 8-oxoG in the transcribed strand (12%) compared with that in TSCER122 cells (7.4%) but not in the non-transcribed strand (12% and 11% in XPA KO and TSCER122 cells, respectively). By sequencing the tandem 8-oxoG-integrated loci in the transcribed strand, we found that the proportion of tandem mutations was markedly increased in XPA KO cells. These results indicate that NER is involved in repairing clustered DNA adducts in the transcribed strand in vivo.

Involvement of DNA methylation in memory processing in the honey bee.

Science.gov (United States)

Lockett, Gabrielle A; Helliwell, Paul; Maleszka, Ryszard

2010-08-23

DNA methylation, an important and evolutionarily conserved epigenetic mechanism, is implicated in learning and memory processes in vertebrates, but its role in behaviour in invertebrates is unknown. We examined the role of DNA methylation in memory in the honey bee using an appetitive Pavlovian olfactory discrimination task, and by assessing the expression of DNA methyltransferase3, a key driver of epigenetic reprogramming. Here we report that DNA methyltransferase inhibition reduces acquisition retention and alters the extinction depending on treatment time, and DNA methyltransferase3 is upregulated after training. Our findings add to the understanding of epigenetic mechanisms in learning and memory, extending known roles of DNA methylation to appetitive and extinction memory, and for the first time implicate DNA methylation in memory in invertebrates.

Mitochondrial DNA alteration in obstructive sleep apnea.

Science.gov (United States)

Lacedonia, Donato; Carpagnano, Giovanna E; Crisetti, Elisabetta; Cotugno, Grazia; Palladino, Grazia P; Patricelli, Giulia; Sabato, Roberto; Foschino Barbaro, Maria P

2015-04-07

Obstructive Sleep Apnea (OSAS) is a disease associated with the increase of cardiovascular risk and it is characterized by repeated episodes of Intermittent Hypoxia (IH) which inducing oxidative stress and systemic inflammation. Mitochondria are cell organelles involved in the respiratory that have their own DNA (MtDNA). The aim of this study was to investigate if the increase of oxidative stress in OSAS patients can induce also MtDNA alterations. 46 OSAS patients (age 59.27 ± 11.38; BMI 30.84 ± 3.64; AHI 36.63 ± 24.18) were compared with 36 control subjects (age 54.42 ± 6.63; BMI 29.06 ± 4.7; AHI 3.8 ± 1.10). In blood cells Content of MtDNA and nuclear DNA (nDNA) was measured in OSAS patients by Real Time PCR. The ratio between MtDNA/nDNA was then calculated. Presence of oxidative stress was evaluated by levels of Reactive Oxygen Metabolites (ROMs), measured by diacron reactive oxygen metabolite test (d-ROM test). MtDNA/nDNA was higher in patients with OSAS than in the control group (150.94 ± 49.14 vs 128.96 ± 45.8; p = 0.04), the levels of ROMs were also higher in OSAS subjects (329.71 ± 70.17 vs 226 ± 36.76; p = 0.04) and they were positively correlated with MtDNA/nDNA (R = 0.5, p DNA damage induced by the increase of oxidative stress. Intermittent hypoxia seems to be the main mechanism which leads to this process.

A polycomb group protein, PHF1, is involved in the response to DNA double-strand breaks in human cell

OpenAIRE

Hong, Zehui; Jiang, Jie; Lan, Li; Nakajima, Satoshi; Kanno, Shin-ichiro; Koseki, Haruhiko; Yasui, Akira

2008-01-01

DNA double-strand breaks (DSBs) represent the most toxic DNA damage arisen from endogenous and exogenous genotoxic stresses and are known to be repaired by either homologous recombination or nonhomologous end-joining processes. Although many proteins have been identified to participate in either of the processes, the whole processes still remain elusive. Polycomb group (PcG) proteins are epigenetic chromatin modifiers involved in gene silencing, cancer development and the maintenance of embry...

DNA radio-induced tandem lesions: formation, introduction in oligonucleotides and repair

International Nuclear Information System (INIS)

Bourdat, Anne-Gaelle

2000-01-01

Cell killing induced by excited photosensitizers, ionizing radiation or radiomimetic drugs can not be only explained by the formation of single DNA lesions. Thus, multiply damaged sites, are likely to have harmful biological consequences. One example of tandem base damage induced by ".OH radical in X-irradiated aqueous solution of DNA oligomers is N-(2-deoxy-β-D-erythro-pentofuranosyl)-formyl-amine (dβF)/8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). In order to investigate the biological significance of such a tandem lesion, both 8-oxodGuo and dβF were introduced in synthetic oligonucleotides at vicinal positions using the solid phase phosphoramidite method with the 'Pac phosphoramidite' chemistry. The purity of the synthetic DNA fragments and the integrity of modified nucleosides was confirmed using different complementary techniques: HPLC, PAGE, ESI MS, MALDI-TOF MS and capillary electrophoresis. Using the above synthetic substrates, investigations were carried out in order to determine the substrate specificity and the excision mechanism of three glycosylases involved in the base excision repair pathway: endonuclease III, Fpg and yOggl. Both tandem lesions were substrates for the BER enzymes. However, the tandem lesion are not completely excised by the repair enzymes. The rates of excision as inferred from the determination of the ratios of Vm/Km Michaelis kinetics constants were not found to be significantly affected by the presence of the tandem lesions. MALDI-TOF mass spectrometry was used in order to gain insights into mechanistic aspects of oligonucleotide cleavage by the BER enzymes. During in vitro DNA synthesis by Taq DNA polymerase, Klenow fragment exo- and DNA polymerase β, tandem base damage were found to block the progression of the enzymes. Finally, the level of tandem base damage in the DNA exposed to γ-ray using the liquid chromatography coupled to electro-spray ionization tandem mass spectrometry was determined. Both dβF-8-oxodGuo and 8

Extensive 5.8S nrDNA polymorphism in Mammillaria (Cactaceae) with special reference to the identification of pseudogenic internal transcribed spacer regions.

Science.gov (United States)

Harpke, Doerte; Peterson, Angela

2008-05-01

The internal transcribed spacer (ITS) region (ITS1, 5.8S rDNA, ITS2) represents the most widely applied nuclear marker in eukaryotic phylogenetics. Although this region has been assumed to evolve in concert, the number of investigations revealing high degrees of intra-individual polymorphism connected with the presence of pseudogenes has risen. The 5.8S rDNA is the most important diagnostic marker for functionality of the ITS region. In Mammillaria, intra-individual 5.8S rDNA polymorphisms of up to 36% and up to nine different types have been found. Twenty-eight of 30 cloned genomic Mammillaria sequences were identified as putative pseudogenes. For the identification of pseudogenic ITS regions, in addition to formal tests based on substitution rates, we attempted to focus on functional features of the 5.8S rDNA (5.8S motif, secondary structure). The importance of functional data for the identification of pseudogenes is outlined and discussed. The identification of pseudogenes is essential, because they may cause erroneous phylogenies and taxonomic problems.

cDNA cloning and expression analysis of two distinct Sox8 genes in Paramisgurnus dabryanus (Cypriniformes).

Science.gov (United States)

Xia, Xiaohua; Zhao, Jie; Du, Qiyan; Chang, Zhongjie

2010-08-01

The Sox9 gene attracts a lot of attention because of its connection with gonadal development and differentiation. However, Sox8, belonging to the same subgroup SoxE, has rarely been studied. To investigate the function as well as the evolutionary origin of SOXE subgroup, we amplified the genomic DNA of Paramisgurnus dabryanu using a pair of degenerate primers. Using rapid amplification of the cDNA ends (RACE), it was discovered that P. dabryanu has two duplicates: Sox8a and Sox8b. Each has an intron of different length in the conserved HMG-box region. The overall sequence similarity of the deduced amino acid of PdSox8a and PdSox8b was 46.26%, and only two amino acids changed in the HMG-box. This is the first evidence showing that there are two distinct duplications of Sox8 genes in Cypriniformes. Southern blot analysis showed only one hybrid band, with lengths 7.4 or 9.2 kb. Both semi-quantitative RT-PCR and real-time quantitative PCR assay displayed that both PdSox8a and PdSox8b are downregulated during early embryonic development. In adult tissues, the two Sox8 genes expressed ubiquitously, and expression levels are particularly high in the gonads and brain. In gonads, both PdSox8a and PdSox8b are expressed at a higher level in the tesis than in the ovary. PdSox8a and PdSox8b may have functional overlaps and are essential for the neuronal development and differentiation of gonads.

Action spectra and chromophores for lethal photosensitization of Candida albicans by DNA monoadducts formed by 8-methoxypsoralen and monofunctional furocoumarins

International Nuclear Information System (INIS)

Baydoun, S.; Gibbs, N.K.; Young, A.R.

1992-01-01

The red-shift furocoumarin action spectra, compared with their absorption spectra, has been investigated. An action spectrum for 8-methoxypsoralen (8-Mop) monoadduct formation in the yeast Candida albicans has been determined. The yeast cells were initially exposed to sublethal doses of monochromatic UV A at different wavelengths. Monoadduct formation was monitored by growth inhibition induced, after washing out any unbound 8-Mop, by re-irradiation with a constant second (non-lethal) dose of 330 nm radiation. A comparison between this action spectrum and the absorption spectrum of the dark complex of 8-Mop and DNA was made. In addition, the action spectra of monoadduct formation of five monofunctional compounds including a coumarin derivative have been determined. These action spectra were compared with their respective DNA dark complex absorption spectra. In general, the peaks of the furocoumarin DNA dark complexes show a red-shift when compared with the free furocoumarin molecule and the action spectra show peaks which correspond with the peaks of the dark complexes. Such data indicate that the DNA dark complex is the chromophore for growth inhibition in yeast rather than the free furocoumarin. The similarity of the 8-Mop monoadduct formation spectrum and 8-Mop action spectra suggests that spectral dependence for the photobiological effects (including the red-shift) is dependent on monoadduct formation rather than, as previously suggested by several authors, crosslink formation. The action spectrum for the coumarin derivative 4-methyl N-ethylpyrrolo (3,2-g) coumarin (PCNEt) correlated well with the free molecule absorption spectrum rather than DNA dark complex indicating that the free molecule is the chromophore. This was supported by studies which showed that PCNEt photosensitization is oxygen dependent. (author). 38 refs., 3 tabs., 7 figs

Two Sets of Piwi Proteins Are Involved in Distinct sRNA Pathways Leading to Elimination of Germline-Specific DNA

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Dominique I. Furrer

2017-07-01

Full Text Available Piwi proteins and piRNAs protect eukaryotic germlines against the spread of transposons. During development in the ciliate Paramecium, two Piwi-dependent sRNA classes are involved in the elimination of transposons and transposon-derived DNA: scan RNAs (scnRNAs, associated with Ptiwi01 and Ptiwi09, and iesRNAs, whose binding partners we now identify as Ptiwi10 and Ptiwi11. scnRNAs derive from the maternal genome and initiate DNA elimination during development, whereas iesRNAs continue DNA targeting until the removal process is complete. Here, we show that scnRNAs and iesRNAs are processed by distinct Dicer-like proteins and bind Piwi proteins in a mutually exclusive manner, suggesting separate biogenesis pathways. We also demonstrate that the PTIWI10 gene is transcribed from the developing nucleus and that its transcription depends on prior DNA excision, suggesting a mechanism of gene expression control triggered by the removal of short DNA segments interrupting the gene.

Postradiation DNA repair in mammalian cells under the combined effect of hyperthermia and 8-bromocaffeine and actinomycin D

International Nuclear Information System (INIS)

Rezvaya, S.P.; Khanson, K.P.

1981-01-01

A study was made of the influence of postirradiation hyperthermia combined with chemical inhibitirors of DNA repa on rejoining the singlestranded DNA breaks induced by X-irradiation (50 Gy) of LL, cells. Separation of single- and double-stranded DNA fragments on a column with hydroxyapatite has revealed that elevation of the postradiation incubation temperature up to 41 deg C does not influence the degree of repair of single-stranded breaks. No repair is detected at 43 deg C. 8-Bromocaffeine and actinomycin combined with the elevated temperature (41 deg C) remove the inhibitory effect of the preparations on the postradiation repair of DNA [ru

Regulating DNA Self-assembly by DNA-Surface Interactions.

Science.gov (United States)

Liu, Longfei; Li, Yulin; Wang, Yong; Zheng, Jianwei; Mao, Chengde

2017-12-14

DNA self-assembly provides a powerful approach for preparation of nanostructures. It is often studied in bulk solution and involves only DNA-DNA interactions. When confined to surfaces, DNA-surface interactions become an additional, important factor to DNA self-assembly. However, the way in which DNA-surface interactions influence DNA self-assembly is not well studied. In this study, we showed that weak DNA-DNA interactions could be stabilized by DNA-surface interactions to allow large DNA nanostructures to form. In addition, the assembly can be conducted isothermally at room temperature in as little as 5 seconds. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Xanthorrhizol induced DNA fragmentation in HepG2 cells involving Bcl-2 family proteins

International Nuclear Information System (INIS)

Tee, Thiam-Tsui; Cheah, Yew-Hoong; Meenakshii, Nallappan; Mohd Sharom, Mohd Yusof; Azimahtol Hawariah, Lope Pihie

2012-01-01

Highlights: ► We isolated xanthorrhizol, a sesquiterpenoid compound from Curcuma xanthorrhiza. ► Xanthorrhizol induced apoptosis in HepG2 cells as observed using SEM. ► Apoptosis in xanthorrhizol-treated HepG2 cells involved Bcl-2 family proteins. ► DNA fragmentation was observed in xanthorrhizol-treated HepG2 cells. ► DNA fragmentation maybe due to cleavage of PARP and DFF45/ICAD proteins. -- Abstract: Xanthorrhizol is a plant-derived pharmacologically active sesquiterpenoid compound isolated from Curcuma xanthorrhiza. Previously, we have reported that xanthorrhizol inhibited the proliferation of HepG2 human hepatoma cells by inducing apoptotic cell death via caspase activation. Here, we attempt to further elucidate the mode of action of xanthorrhizol. Apoptosis in xanthorrhizol-treated HepG2 cells as observed by scanning electron microscopy was accompanied by truncation of BID; reduction of both anti-apoptotic Bcl-2 and Bcl-X L expression; cleavage of PARP and DFF45/ICAD proteins and DNA fragmentation. Taken together, these results suggest xanthorrhizol as a potent antiproliferative agent on HepG2 cells by inducing apoptosis via Bcl-2 family members. Hence we proposed that xanthorrhizol could be used as an anti-liver cancer drug for future studies.

Xanthorrhizol induced DNA fragmentation in HepG2 cells involving Bcl-2 family proteins

Energy Technology Data Exchange (ETDEWEB)

Tee, Thiam-Tsui, E-mail: thiamtsu@yahoo.com [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia); Cheah, Yew-Hoong [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia); Bioassay Unit, Herbal Medicine Research Center, Institute for Medical Research, Jalan Pahang, Kuala Lumpur (Malaysia); Meenakshii, Nallappan [Biology Department, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Mohd Sharom, Mohd Yusof; Azimahtol Hawariah, Lope Pihie [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia)

2012-04-20

Highlights: Black-Right-Pointing-Pointer We isolated xanthorrhizol, a sesquiterpenoid compound from Curcuma xanthorrhiza. Black-Right-Pointing-Pointer Xanthorrhizol induced apoptosis in HepG2 cells as observed using SEM. Black-Right-Pointing-Pointer Apoptosis in xanthorrhizol-treated HepG2 cells involved Bcl-2 family proteins. Black-Right-Pointing-Pointer DNA fragmentation was observed in xanthorrhizol-treated HepG2 cells. Black-Right-Pointing-Pointer DNA fragmentation maybe due to cleavage of PARP and DFF45/ICAD proteins. -- Abstract: Xanthorrhizol is a plant-derived pharmacologically active sesquiterpenoid compound isolated from Curcuma xanthorrhiza. Previously, we have reported that xanthorrhizol inhibited the proliferation of HepG2 human hepatoma cells by inducing apoptotic cell death via caspase activation. Here, we attempt to further elucidate the mode of action of xanthorrhizol. Apoptosis in xanthorrhizol-treated HepG2 cells as observed by scanning electron microscopy was accompanied by truncation of BID; reduction of both anti-apoptotic Bcl-2 and Bcl-X{sub L} expression; cleavage of PARP and DFF45/ICAD proteins and DNA fragmentation. Taken together, these results suggest xanthorrhizol as a potent antiproliferative agent on HepG2 cells by inducing apoptosis via Bcl-2 family members. Hence we proposed that xanthorrhizol could be used as an anti-liver cancer drug for future studies.

Involvement of Werner syndrome protein in MUTYH-mediated repair of oxidative DNA damage

Czech Academy of Sciences Publication Activity Database

Kanagaraj, R.; Parasuraman, P.; Mihaljevic, B.; van Loon, B.; Burdová, Kamila; König, C.; Furrer, A.; Bohr, V.A.; Hübscher, U.; Janscak, P.

2012-01-01

Roč. 40, č. 17 (2012), s. 8449-8459 ISSN 0305-1048 Grant - others:Swiss National Science Foundation(CH) 31003A-129747/1; Swiss National Science Foundation(CH) 3100-109312/2; Oncosuisse(CH) KLS-02344-02-2009; NIH(US) Z01-AG000726-17 Institutional research plan: CEZ:AV0Z50520514 Institutional support: RVO:68378050 Keywords : DNA repair * oxidative stress * MUTYH * WRN * Pol lambda Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 8.278, year: 2012

Repair of 8-methoxypsoralen + UVA-induced damage in specific sequences in chromosomal and episomal DNA in human cells

Energy Technology Data Exchange (ETDEWEB)

Dean, S.W.

1989-07-01

A study of the repair of DNA damage in the dihydrofolate reductase (dhfr) gene of SV40-transformed human fibroblasts after treatment with 8-methoxypsoralen (8MOP) and UVA is described. 8MOP+UVA-induced cross-links in the dhfr gene were completely repaired by 12 h in one normal and one Fanconi's anaemia (FA) group A cell line. In contrast, approximately 35% of cross-links in an episomally maintained Epstein--Barr virus derived plasmid remained unrepaired even after 48 h. Cross-linkable monoadducts in the dhfr gene were repaired more slowly than cross-links, and there was no detectable repair of cross-linkable monoadducts in the plasmid. Thus the ability of a cell to repair 8MOP+UVA-induced cross-links or cross-linkable monoadducts in an episome does not reflect its capacity to repair such lesions in genomic DNA.

Repair of 8-methoxypsoralen + UVA-induced damage in specific sequences in chromosomal and episomal DNA in human cells

International Nuclear Information System (INIS)

Dean, S.W.

1989-01-01

A study of the repair of DNA damage in the dihydrofolate reductase (dhfr) gene of SV40-transformed human fibroblasts after treatment with 8-methoxypsoralen (8MOP) and UVA is described. 8MOP+UVA-induced cross-links in the dhfr gene were completely repaired by 12 h in one normal and one Fanconi's anaemia (FA) group A cell line. In contrast, ∼35% of cross-links in an episomally maintained Epstein-Barr virus derived plasmid remained unrepaired even after 48 h. Cross-linkable monoadducts in the dhfr gene were repaired more slowly than cross-links, and there was no detectable repair of cross-linkable monoadducts in the plasmid. Thus the ability of a cell to repair 8MOP+UVA-induced cross-links or cross-linkable monoadducts in an episome does not reflect its capacity to repair such lesions in genomic DNA. (author)

Cellular responses of Saccharomyces cerevisiae to DNA damage

International Nuclear Information System (INIS)

Ciesla, Z.; Sledziewska-Gojska, E.; Nowicka, A.; Mieczkowski, P.; Fikus, M.U.; Koprowski, P.

1998-01-01

Full text. Several experimental strategies have been used to study responses of S. cerevisiae cells to DNA damage. One approach was based on the isolation of novel genes, the expression of which is induced by lesions in DNA. One of these genes, DIN7, was cloned and partially characterized previously. The product of DIN7 belongs to a large family of proteins involved in DNA repair and mutagenesis. This family includes Rad2, Rad27 and ExoI proteins of S. cerevisiae and their respective human homologues, all of which are endowed with DNA nuclease activity. To study cellular function of Din7 we constructed the pPK3 plasmid carrying DIN7 fused to the GAL1 promoter. Effects of DIN7 overproduction on the phenotypes of wild-type cells and of rad27 and exoI mutants were examined. Overproduction of Din7 does not seem to affect the proficiency of wild-type S. cerevisiae cells in recombination and mutagenesis. Also, overexpression of DIN7 does not suppress the deficiency of the EXOI gene product, the closest homologue of Din7, both in recombination and in controlling the fidelity of DNA replication. Unexpectedly, we found that elevated levels of Din7 result in a very high frequency of mitochondrial rho - mutants. A high frequency of production of rho - mutants wa s also observed in strains defective in the functioning of the Dun1 protein kinase involved in signal transmission in cells exposed to DNA damaging agents. Interestingly, deficiency of Dun1 results also in a significant derepression of the DIN7 gene. Experiments are under way to distinguish whether a high cellular level of Din7 specifically decreases stability of mitochondrial DNA or affects stability of chromosomal DNA as well. Analysis of previously constructed S. cerevisiae strains carrying random geno mic fusions with reporter lacZ gene, allowed us to identify the reading frame YBR173c, on chromosome II as a novel damage inducible gene - DIN8. We have shown that DIN8-lacZ fusion is induced in yeast cells treated

Comparative transcriptome analyses indicate molecular homology of zebrafish swimbladder and mammalian lung.

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Weiling Zheng

Full Text Available The fish swimbladder is a unique organ in vertebrate evolution and it functions for regulating buoyancy in most teleost species. It has long been postulated as a homolog of the tetrapod lung, but the molecular evidence is scarce. In order to understand the molecular function of swimbladder as well as its relationship with lungs in tetrapods, transcriptomic analyses of zebrafish swimbladder were carried out by RNA-seq. Gene ontology classification showed that genes in cytoskeleton and endoplasmic reticulum were enriched in the swimbladder. Further analyses depicted gene sets and pathways closely related to cytoskeleton constitution and regulation, cell adhesion, and extracellular matrix. Several prominent transcription factor genes in the swimbladder including hoxc4a, hoxc6a, hoxc8a and foxf1 were identified and their expressions in developing swimbladder during embryogenesis were confirmed. By comparison of enriched transcripts in the swimbladder with those in human and mouse lungs, we established the resemblance of transcriptome of the zebrafish swimbladder and mammalian lungs. Based on the transcriptomic data of zebrafish swimbladder, the predominant functions of swimbladder are in its epithelial and muscular tissues. Our comparative analyses also provide molecular evidence of the relatedness of the fish swimbladder and mammalian lung.

Direct Involvement of Retinoblastoma Family Proteins in DNA Repair by Non-homologous End-Joining

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Rebecca Cook

2015-03-01

Full Text Available Deficiencies in DNA double-strand break (DSB repair lead to genetic instability, a recognized cause of cancer initiation and evolution. We report that the retinoblastoma tumor suppressor protein (RB1 is required for DNA DSB repair by canonical non-homologous end-joining (cNHEJ. Support of cNHEJ involves a mechanism independent of RB1’s cell-cycle function and depends on its amino terminal domain with which it binds to NHEJ components XRCC5 and XRCC6. Cells with engineered loss of RB family function as well as cancer-derived cells with mutational RB1 loss show substantially reduced levels of cNHEJ. RB1 variants disabled for the interaction with XRCC5 and XRCC6, including a cancer-associated variant, are unable to support cNHEJ despite being able to confer cell-cycle control. Our data identify RB1 loss as a candidate driver of structural genomic instability and a causative factor for cancer somatic heterogeneity and evolution.

Altered DNA Methylation and Expression Profiles of 8-Oxoguanine DNA Glycosylase 1 in Lens Tissue from Age-related Cataract Patients.

Science.gov (United States)

Wang, Yong; Li, Fei; Zhang, Guowei; Kang, Lihua; Qin, Bai; Guan, Huaijin

2015-01-01

Oxidative stress and DNA damage contribute to the pathogenesis of age-related cataract (ARC). Most oxidative DNA lesions are repaired via the base excision repair (BER) proteins including 8-oxoguanine DNA glycosylase 1 (OGG1). This study examined DNA methylation of CpG islands upstream of OGG1 and their relation to the gene expression in lens cortex from ARC patients. The clinical case-control study consisted of 15 cortical type of ARC patients and 15 age-matched non-ARC controls who received transparent lens extraction due to vitreoretinal diseases. OGG1 expression in lens cortex was analyzed by qRT-PCR and Western blot. The localization and the proportion of cells positive for OGG1 were determined by immunofluorescence. Bisulfite-sequencing PCR (BSP) was performed to evaluate the methylation status of CpG islands near OGG1 in DNA extracted from lens cortex. To test relationship between the methylation and the expression of the gene of interest, 5-Aza-2'-deoxycytidine (5-Aza-dC) was used to induce demethylation of cultured human lens epithelium B-3 (HLE B-3). To test the role of OGG1 in the repair of cellular damage, HLE B-3 was transfected with OGG1 vector, followed by ultraviolet radiation b (UVB) exposure to induce apoptosis. The mRNA and protein levels of OGG1 were significantly reduced in the lens cortex of ARC. Immunofluorescence showed that the proportion of OGG1-positive cells decreased significantly in ARC cortex in comparison with the control. The CpG island in first exon of OGG1 displayed hypermethylation in the DNA extracted from the lens cortex of ARC. Treatment of HLEB-3 cells with 5-Aza-dC upregulated OGG1 expression. UVB-induced apoptosis was attenuated after transfection with OGG1. A reduced OGG1 expression was correlated with hypermethylation of a CpG island of OGG1 in lens cortex of ARC. The role of epigenetic change in OGG1 gene in the susceptibility to oxidative stress induced cortical ARC is warranted to further study.

Thymine DNA Glycosylase (TDG) is involved in the pathogenesis of intestinal tumors with reduced APC expression.

Science.gov (United States)

Xu, Jinfei; Cortellino, Salvatore; Tricarico, Rossella; Chang, Wen-Chi; Scher, Gabrielle; Devarajan, Karthik; Slifker, Michael; Moore, Robert; Bassi, Maria Rosaria; Caretti, Elena; Clapper, Margie; Cooper, Harry; Bellacosa, Alfonso

2017-10-27

Thymine DNA Glycosylase (TDG) is a base excision repair enzyme that acts as a thymine and uracil DNA N-glycosylase on G:T and G:U mismatches, thus protecting CpG sites in the genome from mutagenesis by deamination. In addition, TDG has an epigenomic function by removing the novel cytosine derivatives 5-formylcytosine and 5-carboxylcytosine (5caC) generated by Ten-Eleven Translocation (TET) enzymes during active DNA demethylation. We and others previously reported that TDG is essential for mammalian development. However, its involvement in tumor formation is unknown. To study the role of TDG in tumorigenesis, we analyzed the effects of its inactivation in a well-characterized model of tumor predisposition, the Apc Min mouse strain. Mice bearing a conditional Tdg flox allele were crossed with Fabpl ::Cre transgenic mice, in the context of the Apc Min mutation, in order to inactivate Tdg in the small intestinal and colonic epithelium. We observed an approximately 2-fold increase in the number of small intestinal adenomas in the test Tdg -mutant Apc Min mice in comparison to control genotypes (p=0.0001). This increase occurred in female mice, and is similar to the known increase in intestinal adenoma formation due to oophorectomy. In the human colorectal cancer (CRC) TCGA database, the subset of patients with TDG and APC expression in the lowest quartile exhibits an excess of female cases. We conclude that TDG inactivation plays a role in intestinal tumorigenesis initiated by mutation/underexpression of APC . Our results also indicate that TDG may be involved in sex-specific protection from CRC.

DUBbing Cancer: Deubiquitylating Enzymes Involved in Epigenetics, DNA Damage and the Cell Cycle As Therapeutic Targets.

Science.gov (United States)

Pinto-Fernandez, Adan; Kessler, Benedikt M

2016-01-01

Controlling cell proliferation is one of the hallmarks of cancer. A number of critical checkpoints ascertain progression through the different stages of the cell cycle, which can be aborted when perturbed, for instance by errors in DNA replication and repair. These molecular checkpoints are regulated by a number of proteins that need to be present at the right time and quantity. The ubiquitin system has emerged as a central player controlling the fate and function of such molecules such as cyclins, oncogenes and components of the DNA repair machinery. In particular, proteases that cleave ubiquitin chains, referred to as deubiquitylating enzymes (DUBs), have attracted recent attention due to their accessibility to modulation by small molecules. In this review, we describe recent evidence of the critical role of DUBs in aspects of cell cycle checkpoint control, associated DNA repair mechanisms and regulation of transcription, representing pathways altered in cancer. Therefore, DUBs involved in these processes emerge as potentially critical targets for the treatment of not only hematological, but potentially also solid tumors.

DUBbing cancer: Deubiquitylating enzymes involved in epigenetics, DNA damage and the cell cycle as therapeutic targets

Directory of Open Access Journals (Sweden)

Benedikt M Kessler

2016-07-01

Full Text Available Controlling cell proliferation is one of the hallmarks of cancer. A number of critical checkpoints ascertain progression through the different stages of the cell cycle, which can be aborted when perturbed, for instance by errors in DNA replication and repair. These molecular checkpoints are regulated by a number of proteins that need to be present at the right time and quantity. The ubiquitin system has emerged as a central player controlling the fate and function of such molecules such as cyclins, oncogenes and components of the DNA repair machinery. In particular, proteases that cleave ubiquitin chains, referred to as deubiquitylating enzymes (DUBs, have attracted recent attention due to their accessibility to modulation by small molecules. In this review, we describe recent evidence of the critical role of DUBs in aspects of cell cycle checkpoint control, associated DNA repair mechanisms and regulation of transcription, representing pathways altered in cancer. Therefore, DUBs involved in these processes emerge as potentially critical targets for the treatment of not only hematological, but potentially also solid tumors.

Histone peptide AKRHRK enhances H2O2-induced DNA damage and alters its site specificity

International Nuclear Information System (INIS)

Midorikawa, Kaoru; Murata, Mariko; Kawanishi, Shosuke

2005-01-01

Histone proteins are involved in compaction of DNA and the protection of cells from oxygen toxicity. However, several studies have demonstrated that the metal-binding histone reacts with H 2 O 2 , leading to oxidative damage to a nucleobase. We investigated whether histone can accelerate oxidative DNA damage, using a minimal model for the N-terminal tail of histone H4, CH 3 CO-AKRHRK-CONH 2 , which has a metal-binding site. This histone peptide enhanced DNA damage induced by H 2 O 2 and Cu(II), especially at cytosine residues, and induced additional DNA cleavage at the 5'-guanine of GGG sequences. The peptide also enhanced the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine and ESR spin-trapping signal from H 2 O 2 and Cu(II). Cyclic redox reactions involving histone-bound Cu(II) and H 2 O 2 , may give rise to multiple production of radicals leading to multiple hits in DNA. It is noteworthy that the histone H4 peptide with specific sequence AKRHRK can cause DNA damage rather than protection under metal-overloaded condition

An assay system for factors involved in mammalian DNA replication

International Nuclear Information System (INIS)

Reinhard, P.; Maillart, P.; Schluchter, M.; Gautschi, J.R.; Schindler, R.

1979-01-01

An assay for cellular factors stimulating DNA synthesis by partially lysed CHO cells is presented. The assay is based on the observation that in highly lysed cells, DNA synthesis, as determined by [ 3 H]dTTP incorporation, was only 2-5% of that in gently lysed cells, and that this low level of DNA synthesis could be increased by a factor of approx. 50 by the addition of CHO cell extract (i.e. supernatant of a cell homogenate subjected to high-speed centrifugation.) (Auth.)

Constitutional chromothripsis rearrangements involve clustered double-stranded DNA breaks and nonhomologous repair mechanisms.

Science.gov (United States)

Kloosterman, Wigard P; Tavakoli-Yaraki, Masoumeh; van Roosmalen, Markus J; van Binsbergen, Ellen; Renkens, Ivo; Duran, Karen; Ballarati, Lucia; Vergult, Sarah; Giardino, Daniela; Hansson, Kerstin; Ruivenkamp, Claudia A L; Jager, Myrthe; van Haeringen, Arie; Ippel, Elly F; Haaf, Thomas; Passarge, Eberhard; Hochstenbach, Ron; Menten, Björn; Larizza, Lidia; Guryev, Victor; Poot, Martin; Cuppen, Edwin

2012-06-28

Chromothripsis represents a novel phenomenon in the structural variation landscape of cancer genomes. Here, we analyze the genomes of ten patients with congenital disease who were preselected to carry complex chromosomal rearrangements with more than two breakpoints. The rearrangements displayed unanticipated complexity resembling chromothripsis. We find that eight of them contain hallmarks of multiple clustered double-stranded DNA breaks (DSBs) on one or more chromosomes. In addition, nucleotide resolution analysis of 98 breakpoint junctions indicates that break repair involves nonhomologous or microhomology-mediated end joining. We observed that these eight rearrangements are balanced or contain sporadic deletions ranging in size between a few hundred base pairs and several megabases. The two remaining complex rearrangements did not display signs of DSBs and contain duplications, indicative of rearrangement processes involving template switching. Our work provides detailed insight into the characteristics of chromothripsis and supports a role for clustered DSBs driving some constitutional chromothripsis rearrangements. Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.

PAH-DNA adducts in environmentally exposed population in relation to metabolic and DNA repair gene polymorphisms

International Nuclear Information System (INIS)

Binkova, Blanka; Chvatalova, Irena; Lnenickova, Zdena; Milcova, Alena; Tulupova, Elena; Farmer, Peter B.; Sram, Radim J.

2007-01-01

Epidemiologic studies indicate that prolonged exposure to particulate air pollution may be associated with increased risk of cardiovascular diseases and cancer in general population. These effects may be attributable to polycyclic aromatic hydrocarbons (PAHs) adsorbed to respirable air particles. It is expected that metabolic and DNA repair gene polymorphisms may modulate individual susceptibility to PAH exposure. This study investigates relationships between exposure to PAHs, polymorphisms of these genes and DNA adducts in group of occupationally exposed policemen (EXP, N = 53, males, aged 22-50 years) working outdoors in the downtown area of Prague and in matched 'unexposed' controls (CON, N = 52). Personal exposure to eight carcinogenic PAHs (c-PAHs) was evaluated by personal samplers during working shift prior to collection of biological samples. Bulky-aromatic DNA adducts were analyzed in lymphocytes by 32 P-postlabeling assay. Polymorphisms of metabolizing (GSTM1, GSTP1, GSTT1, EPHX1, CYP1A1-MspI) and DNA repair (XRCC1, XPD) genes were determined by PCR-based RFLP assays. As potential modifiers and/or cofounders, urinary cotinine levels were analyzed by radioimmunoassay, plasma levels of vitamins A, C, E and folates by HPLC, cholesterol and triglycerides using commercial kits. During the sampling period ambient particulate air pollution was as follows: PM10 32-55 μg/m 3 , PM2.5 27-38 μg/m 3 , c-PAHs 18-22 ng/m 3 ; personal exposure to c-PAHs: 9.7 ng/m 3 versus 5.8 ng/m 3 (P 8 nucleotides versus 0.82 ± 0.23 adducts/10 8 nucleotides, P = 0.065), whereas the level of the B[a]P-'like' adduct was significantly higher in exposed group (0.122 ± 0.036 adducts/10 8 nucleotides versus 0.099 ± 0.035 adducts/10 8 nucleotides, P = 0.003). A significant difference in both the total (P < 0.05) and the B[a]P-'like' DNA adducts (P < 0.01) between smokers and nonsmokers within both groups was observed. A significant positive association between DNA adduct and cotinine

In vitro gap-directed translesion DNA synthesis of an abasic site involving human DNA polymerases epsilon, lambda, and beta

Czech Academy of Sciences Publication Activity Database

Villani, G.; Hübscher, U.; Gironis, N.; Parkkinen, S.; Pospiech, H.; Shevelev, Igor; di Cicco, G.; Markennen, E.; Syvaaja, J.E.; Le Gac, N.T.

2011-01-01

Roč. 286, č. 37 (2011), s. 32094-32104 ISSN 0021-9258 Grant - others:Academy of Finland(FI) 106986; Academy of Finland(FI) 123082 Institutional research plan: CEZ:AV0Z50520514 Keywords : DNA damage * DNA polymerase * DNA repair * DNA replication * DNA -protein interaction Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.773, year: 2011

Targeting GLI by GANT61 involves mechanisms dependent on inhibition of both transcription and DNA licensing.

Science.gov (United States)

Zhang, Ruowen; Wu, Jiahui; Ferrandon, Sylvain; Glowacki, Katie J; Houghton, Janet A

2016-12-06

The GLI genes are transcription factors and in cancers are oncogenes, aberrantly and constitutively activated. GANT61, a specific GLI inhibitor, has induced extensive cytotoxicity in human models of colon cancer. The FOXM1 promoter was determined to be a transcriptional target of GLI1. In HT29 cells, inhibition of GLI1 binding at the GLI consensus sequence by GANT61 led to inhibited binding of Pol II, the pause-release factors DSIF, NELF and p-TEFb. The formation of R-loops (RNA:DNA hybrids, ssDNA), were reduced by GANT61 at the FOXM1 promoter. Pretreatment of HT29 cells with α-amanitin reduced GANT61-induced γH2AX foci. Co-localization of GLI1 and BrdU foci, inhibited by GANT61, indicated GLI1 and DNA replication to be linked. By co-immunoprecipitation and confocal microscopy, GLI1 co-localized with the DNA licensing factors ORC4, CDT1, and MCM2. Significant co-localization of GLI1 and ORC4 was inhibited by GANT61, and enrichment of ORC4 occurred at the GLI binding site in the FOXM1 promoter. CDT1 was found to be a transcription target of GLI1. Overexpression of CDT1 in HT29 and SW480 cells reduced GANT61-induced cell death, gH2AX foci, and cleavage of caspase-3. Data demonstrate involvement of transcription and of DNA replication licensing factors by non-transcriptional and transcriptional mechanisms in the GLI-dependent mechanism of action of GANT61.

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)

A newly identified DNA ligase of Saccharomyces cerevisiae involved in RAD52-independent repair of DNA double-strand breaks

Science.gov (United States)

Schär, Primo; Herrmann, Gernot; Daly, Graham; Lindahl, Tomas

1997-01-01

Eukaryotic DNA ligases are ATP-dependent DNA strand-joining enzymes that participate in DNA replication, repair, and recombination. Whereas mammalian cells contain several different DNA ligases, encoded by at least three distinct genes, only one DNA ligase has been detected previously in either budding yeast or fission yeast. Here, we describe a newly identified nonessential Saccharomyces cerevisiae gene that encodes a DNA ligase distinct from the CDC9 gene product. This DNA ligase shares significant amino acid sequence homology with human DNA ligase IV; accordingly, we designate the yeast gene LIG4. Recombinant LIG4 protein forms a covalent enzyme-AMP complex and can join a DNA single-strand break in a DNA/RNA hybrid duplex, the preferred substrate in vitro. Disruption of the LIG4 gene causes only marginally increased cellular sensitivity to several DNA damaging agents, and does not further sensitize cdc9 or rad52 mutant cells. In contrast, lig4 mutant cells have a 1000-fold reduced capacity for correct recircularization of linearized plasmids by illegitimate end-joining after transformation. Moreover, homozygous lig4 mutant diploids sporulate less efficiently than isogenic wild-type cells, and show retarded progression through meiotic prophase I. Spore viability is normal, but lig4 mutants appear to produce a higher proportion of tetrads with only three viable spores. The mutant phenotypes are consistent with functions of LIG4 in an illegitimate DNA end-joining pathway and ensuring efficient meiosis. PMID:9271115

Urinary 8-hydroxy-deoxyguanosine as a biomarker of oxidative DNA damage in employees of subway system.

Science.gov (United States)

Mehrdad, Ramin; Aghdaei, Sara; Pouryaghoub, Gholamreza

2015-01-01

Exposure to air pollutants, steel dust or other occupational and environmental hazards as oxidative stress have adverse effects on subway workers' health. Oxidative stress generates an excessive amount of reactive oxygen species (ROS) and Oxygen Free Radicals during their work time in the tunnels. Once DNA is repaired, Urinary 8-hydroxy-deoxyguanosine (8-OHdG) is excreted in the urine. Therefore, urinary level of 8-OHdG can reflect the extent of oxidative DNA damage. The aim of this study was to document the oxidative stress caused by exposure to these hazards by measuring 8-OHdG in workers urine. We collected urine samples of 81 male subway workers after their working shift. The concentration of urinary 8-OHdG was measured by ELISA method. We used linear regression analysis to compare the level of urinary 8-OHdG as a biomarker of oxidative stress between workers in tunnels and other staff. The mean concentration of urinary 8-OHdG for workers in the tunnel was 58.05 (SD=28.83) ng/mg creatinine and for another staff was 54.16 (SD =26.98) ng/mg creatinine.  After adjustment for age, smoking, driving and a second job in a linear regression model, the concentration of 8-OHdG for the exposed group was significantly higher than unexposed group (P=0.038). These findings confirm that the concentration of urinary 8-OHdG for workers who work in tunnels was significantly higher than the other staff. Additional investigations should be performed to understand that which ones of occupational exposures are more important to cause oxidative stress.

Error-Prone Translesion DNA Synthesis by Escherichia coli DNA Polymerase IV (DinB on Templates Containing 1,2-dihydro-2-oxoadenine

Directory of Open Access Journals (Sweden)

Masaki Hori

2010-01-01

Full Text Available Escherichia coli DNA polymerase IV (Pol IV is involved in bypass replication of damaged bases in DNA. Reactive oxygen species (ROS are generated continuously during normal metabolism and as a result of exogenous stress such as ionizing radiation. ROS induce various kinds of base damage in DNA. It is important to examine whether Pol IV is able to bypass oxidatively damaged bases. In this study, recombinant Pol IV was incubated with oligonucleotides containing thymine glycol (dTg, 5-formyluracil (5-fodU, 5-hydroxymethyluracil (5-hmdU, 7,8-dihydro-8-oxoguanine (8-oxodG and 1,2-dihydro-2-oxoadenine (2-oxodA. Primer extension assays revealed that Pol IV preferred to insert dATP opposite 5-fodU and 5-hmdU, while it inefficiently inserted nucleotides opposite dTg. Pol IV inserted dCTP and dATP opposite 8-oxodG, while the ability was low. It inserted dCTP more effectively than dTTP opposite 2-oxodA. Pol IV's ability to bypass these lesions decreased in the order: 2-oxodA > 5-fodU~5-hmdU > 8-oxodG > dTg. The fact that Pol IV preferred to insert dCTP opposite 2-oxodA suggests the mutagenic potential of 2-oxodA leading to A:T→G:C transitions. Hydrogen peroxide caused an ~2-fold increase in A:T→G:C mutations in E. coli, while the increase was significantly greater in E. coli overexpressing Pol IV. These results indicate that Pol IV may be involved in ROS-enhanced A:T→G:C mutations.

Effect of dietary factors on mutagenesis, metabolism, and binding to DNA of benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol

International Nuclear Information System (INIS)

Vance, R.E.

1988-01-01

Ellagic acid (EA), a naturally occurring plant phenol, at concentrations of 5 to 50 μg/plate, inhibited rate liver S9 protein dependent benzo[a]pyrene (B[a]P)-induced mutagenesis in Salmonella typhimurium TA 100 by 30-81% and B[a]P 7,8-dihydrodiol (DHD)-induced mutagenesis by 29 to 75%. EA did not significantly affect the metabolism of B[a]P or B[a]P 7,8-DHD as determined by high performance liquid chromatographic analysis of the organosoluble fraction and by the quantification of water-soluble conjugates. At these concentrations EA inhibited the covalent binding of [ 3 H] B[a]P and [ 3 H] B[a]P 7,8-DHD metabolites to calf thymus DNA by 5 to 42% and 27 to 64%, respectively. Formation of benzo[a]pyrene 7,8-dihydrodiol-9,10-epoxide:deoxyguanosine (BPDE:dG) adducts was inhibited by 13 to 56% for B[a]P for B[a]P and 11 to 38% for B[a]P 7,8-DHD. These results suggest that the antimutagenic effect of EA and its inhibition of B[a]P and B[a]P 7,8-DHD metabolite-binding to DNA is not due to the inhibition of S9-mediated metabolism of these compounds. The inhibitory effect may be by previously described scavenging mechanism or by a DNA-affinity binding mechanism that prevents BPDE:DNA adduct formation

JMJD1C demethylates MDC1 to regulate the RNF8 and BRCA1-mediated chromatin response to DNA breaks

DEFF Research Database (Denmark)

Watanabe, Sugiko; Watanabe, Kenji; Akimov, Vyacheslav

2013-01-01

Chromatin ubiquitylation flanking DNA double-strand breaks (DSBs), mediated by RNF8 and RNF168 ubiquitin ligases, orchestrates a two-branch pathway, recruiting repair factors 53BP1 or the RAP80-BRCA1 complex. We report that human demethylase JMJD1C regulates the RAP80-BRCA1 branch of this DNA...

Influence of 1.8-GHz (GSM) radiofrequency radiation (RFR) on DNA damage and repair induced by X-rays in human leukocytes in vitro.

Science.gov (United States)

Zhijian, Chen; Xiaoxue, Li; Yezhen, Lu; Deqiang, Lu; Shijie, Chen; Lifen, Jin; Jianlin, Lou; Jiliang, He

2009-01-01

In the present study, the in vitro comet assay was used to determine whether 1.8-GHz radiofrequency radiation (RFR) can influence DNA repair in human leukocytes exposed to X-rays. The specific energy absorption rate (SAR) of 2 W/kg (the current European safety limit) was applied. The leukocytes from four young healthy donors were intermittently exposed to RFR for 24 h (fields on for 5 min, fields off for 10 min), and then irradiated with X-rays at doses of 0.25, 0.5, 1.0 and 2.0 Gy. DNA damage to human leukocytes was detected using the comet assay at 0, 15, 45, 90, 150 and 240 min after exposure to X-rays. Using the comet assay, the percent of DNA in the tail (% tail DNA) served as the indicator of DNA damage; the DNA repair percentage (DRP) served as the indicator of the DNA repair speed. The results demonstrated that (1) the DNA repair speeds of human leukocytes after X-ray exposure exhibited individual differences among the four donors; (2) the intermittent exposures of 1.8-GHz RFR at the SAR of 2 W/kg for 24 h did not directly induce DNA damage or exhibit synergistic effects with X-rays on human leukocytes.

Cementum as a source of DNA in challenging forensic cases.

Science.gov (United States)

Mansour, Hussam; Krebs, Oliver; Sperhake, Jan Peter; Augustin, Christa; Koehne, Till; Amling, Michael; Püschel, Klaus

2018-02-01

Each forensic case is characterized by its own uniqueness. Deficient forensic cases require additional sources of human identifiers to assure the identity. We report on two different cases illustrating the role of teeth in answering challenging forensic questions. The first case involves identification of an adipocere male found in a car submersed in water for approximately 2 years. The second scenario, which involves paternity DNA testing of an exhumed body, was performed approximately 2.8 years post-mortem. The difficulty in anticipating the degradation of the DNA is one of the main obstacles. DNA profiling of dental tissues, DNA quantification by using real-time PCR (PowerQuant™ System/Promega) and a histological dental examination have been performed to address the encountered impediments of adverse post-mortem changes. Our results demonstrate that despite the adverse environmental conditions, a successful STR profile of DNA isolated from the root of teeth can be generated with respect to tooth type and apportion. We conclude that cementocytes are a fruitful source of DNA. Cementum resists DNA degradation in comparison to other tissues with respect to the intra- and inter-individual variation of histological and anatomical structures. Copyright © 2018 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

DNA2—An Important Player in DNA Damage Response or Just Another DNA Maintenance Protein?

Directory of Open Access Journals (Sweden)

Elzbieta Pawłowska

2017-07-01

Full Text Available The human DNA2 (DNA replication helicase/nuclease 2 protein is expressed in both the nucleus and mitochondria, where it displays ATPase-dependent nuclease and helicase activities. DNA2 plays an important role in the removing of long flaps in DNA replication and long-patch base excision repair (LP-BER, interacting with the replication protein A (RPA and the flap endonuclease 1 (FEN1. DNA2 can promote the restart of arrested replication fork along with Werner syndrome ATP-dependent helicase (WRN and Bloom syndrome protein (BLM. In mitochondria, DNA2 can facilitate primer removal during strand-displacement replication. DNA2 is involved in DNA double strand (DSB repair, in which it is complexed with BLM, RPA and MRN for DNA strand resection required for homologous recombination repair. DNA2 can be a major protein involved in the repair of complex DNA damage containing a DSB and a 5′ adduct resulting from a chemical group bound to DNA 5′ ends, created by ionizing radiation and several anticancer drugs, including etoposide, mitoxantrone and some anthracyclines. The role of DNA2 in telomere end maintenance and cell cycle regulation suggests its more general role in keeping genomic stability, which is impaired in cancer. Therefore DNA2 can be an attractive target in cancer therapy. This is supported by enhanced expression of DNA2 in many cancer cell lines with oncogene activation and premalignant cells. Therefore, DNA2 can be considered as a potential marker, useful in cancer therapy. DNA2, along with PARP1 inhibition, may be considered as a potential target for inducing synthetic lethality, a concept of killing tumor cells by targeting two essential genes.

Phylogeographic structure of cotton pest Adelphocoris suturalis (Hemiptera: Miridae): strong subdivision in China inferred from mtDNA and rDNA ITS markers

OpenAIRE

Zhang, Lijuan; Li, Hu; Li, Shujuan; Zhang, Aibing; Kou, Fei; Xun, Huaizhu; Wang, Pei; Wang, Ying; Song, Fan; Cui, Jianxin; Cui, Jinjie; Gouge, Dawn H.; Cai, Wanzhi

2015-01-01

Phylogeographic patterns of some extant plant and vertebrate species have been well studied; however, they are poorly understood in the majority of insects. The study documents analysis of mitochondrial (COI, CYTB and ND5) and nuclear (5.8S rDNA, ITS2 and 28S rDNA) data from 419 individuals of Adelphocoris suturalis, which is one of the main cotton pests found in the 31 locations in China and Japan involved in the study. Results show that the species is highly differentiated between populatio...

Thioredoxin suppresses microscopic hopping of T7 DNA polymerase on duplex DNA

NARCIS (Netherlands)

Etson, Candice M.; Hamdan, Samir M.; Richardson, Charles C.; Oijen, Antoine M. van; Richardson, Charles C.

2010-01-01

The DNA polymerases involved in DNA replication achieve high processivity of nucleotide incorporation by forming a complex with processivity factors. A model system for replicative DNA polymerases, the bacteriophage T7 DNA polymerase (gp5), encoded by gene 5, forms a tight, 1:1 complex with

Induction of SCE by DNA cross-links in human fibroblasts exposed to 8-MOP and UVA irradiation

International Nuclear Information System (INIS)

Bredberg, A.; Lambert, B.

1983-01-01

To study the SCE-inducing effect of psoralen cross-links in the DNA of normal, human fibroblasts, cell cultures were exposed to PUVA (0.2-1 μg of 8-MOP per ml, followed by UVA irradiation at 0.04 J/cm 2 ) and carefully washed to remove non-covalently bound psoralen. Some cell cultures were then given a second dose of UVA (1.1 J/cm 2 ), either immediately after PUVA or 1-3 days later. By this type of treatment, cells with different proportions of DNA cross-links are obtained. The initial PUVA treatment will mainly give rise to psoralen monoadducts and only few cross-links in the DNA, and the second UVA irradiation will convert a number of the psoralen monoadducts into cross-links. (orig./AJ)

HPLC analysis of 4',5'-monoadduct formation in calf thymus DNA and synthetic polynucleotides treated with UVA and 8-methoxypsoralen

International Nuclear Information System (INIS)

Gasparro, F.P.; Bagel, J.; Edelson, R.L.

1985-01-01

8-methoxypsoralen monoadduct formation in calf thymus DNA irradiated with subbands of ultraviolet A light has been quantitated by HPLC analysis of the enzymatic hydrolysates of the DNA. Normalization of the yield of monoadducts for the variation in source output and the absorptivity of 8-MOP at each of the irradiating wavelengths showed that the 4',5'-furan monoadduct was the principal photoproduct and the efficiency of its formation was independent of irradiating wavelength. Synthetic polynucleotides irradiated with ultraviolet A light demonstrated a base composition and sequence dependence for 8-MOP photoreactivity: (poly(dAdT.dAdT)>poly(dA.dT)>poly(dGdC.dGdC) in both the B and Z forms>poly(dT). (author)

Human herpes virus-8 DNA in bronchoalveolar lavage samples from patients with AIDS-associated pulmonary Kaposi's sarcoma

DEFF Research Database (Denmark)

Benfield, T L; Dodt, K K; Lundgren, Jens Dilling

1997-01-01

of KS. We hypothesized that these sequences are present in samples obtained by bronchoalveolar lavage (BAL) in patients with pulmonary KS. Utilizing a nested polymerase chain reaction (PCR), 7/12 BAL cell samples from HIV-infected patients with endobronchial KS were positive for HHV-8 DNA. In contrast......, and PCR amplification of HHV-8 in BAL cells provides a non-invasive method with a high positive predictive value....

Proteomic Identification of DNA-PK Involvement within the RET Signaling Pathway.

Directory of Open Access Journals (Sweden)

Lyle J Burdine

Full Text Available Constitutive activation of the Rearranged during Transfection (RET proto-oncogene leads to the development of MEN2A medullary thyroid cancer (MTC. The relatively clear genotype/phenotype relationship seen with RET mutations and the development of MEN2A is unusual in the fact that a single gene activity can drive the progression towards metastatic disease. Despite knowing the oncogene responsible for MEN2A, MTC, like most tumors of neural crest origin, remains largely resistant to chemotherapy. Constitutive activation of RET in a SK-N-MC cell line model reduces cell sensitivity to chemotherapy. In an attempt to identify components of the machinery responsible for the observed RET induced chemoresistance, we performed a proteomic screen of histones and associated proteins in cells with a constitutively active RET signaling pathway. The proteomic approach identified DNA-PKcs, a DNA damage response protein, as a target of the RET signaling pathway. Active DNA-PKcs, which is phosphorylated at site serine 2056 and localized to chromatin, was elevated within our model. Treatment with the RET inhibitor RPI-1 significantly reduced s2056 phosphorylation in RET cells as well as in a human medullary thyroid cancer cell line. Additionally, inhibition of DNA-PKcs activity diminished the chemoresistance observed in both cell lines. Importantly, we show that activated DNA-PKcs is elevated in medullary thyroid tumor samples and that expression correlates with expression of RET in thyroid tumors. These results highlight one mechanism by which RET signaling likely primes cells for rapid response to DNA damage and suggests DNA-PKcs as an additional target in MTC.

Mutation update for the CSB/ERCC6 and CSA/ERCC8 genes involved in Cockayne syndrome.

Science.gov (United States)

Laugel, V; Dalloz, C; Durand, M; Sauvanaud, F; Kristensen, U; Vincent, M C; Pasquier, L; Odent, S; Cormier-Daire, V; Gener, B; Tobias, E S; Tolmie, J L; Martin-Coignard, D; Drouin-Garraud, V; Heron, D; Journel, H; Raffo, E; Vigneron, J; Lyonnet, S; Murday, V; Gubser-Mercati, D; Funalot, B; Brueton, L; Sanchez Del Pozo, J; Muñoz, E; Gennery, A R; Salih, M; Noruzinia, M; Prescott, K; Ramos, L; Stark, Z; Fieggen, K; Chabrol, B; Sarda, P; Edery, P; Bloch-Zupan, A; Fawcett, H; Pham, D; Egly, J M; Lehmann, A R; Sarasin, A; Dollfus, H

2010-02-01

Cockayne syndrome is an autosomal recessive multisystem disorder characterized principally by neurological and sensory impairment, cachectic dwarfism, and photosensitivity. This rare disease is linked to mutations in the CSB/ERCC6 and CSA/ERCC8 genes encoding proteins involved in the transcription-coupled DNA repair pathway. The clinical spectrum of Cockayne syndrome encompasses a wide range of severity from severe prenatal forms to mild and late-onset presentations. We have reviewed the 45 published mutations in CSA and CSB to date and we report 43 new mutations in these genes together with the corresponding clinical data. Among the 84 reported kindreds, 52 (62%) have mutations in the CSB gene. Many types of mutations are scattered along the whole coding sequence of both genes, but clusters of missense mutations can be recognized and highlight the role of particular motifs in the proteins. Genotype-phenotype correlation hypotheses are considered with regard to these new molecular and clinical data. Additional cases of molecular prenatal diagnosis are reported and the strategy for prenatal testing is discussed. Two web-based locus-specific databases have been created to list all identified variants and to allow the inclusion of future reports (www.umd.be/CSA/ and www.umd.be/CSB/). (c) 2009 Wiley-Liss, Inc.

Altered epigenetic regulation of homeobox genes in human oral squamous cell carcinoma cells

Energy Technology Data Exchange (ETDEWEB)

Marcinkiewicz, Katarzyna M.; Gudas, Lorraine J., E-mail: ljgudas@med.cornell.edu

2014-01-01

To gain insight into oral squamous cell carcinogenesis, we performed deep sequencing (RNAseq) of non-tumorigenic human OKF6-TERT1R and tumorigenic SCC-9 cells. Numerous homeobox genes are differentially expressed between OKF6-TERT1R and SCC-9 cells. Data from Oncomine, a cancer microarray database, also show that homeobox (HOX) genes are dysregulated in oral SCC patients. The activity of Polycomb repressive complexes (PRC), which causes epigenetic modifications, and retinoic acid (RA) signaling can control HOX gene transcription. HOXB7, HOXC10, HOXC13, and HOXD8 transcripts are higher in SCC-9 than in OKF6-TERT1R cells; using ChIP (chromatin immunoprecipitation) we detected PRC2 protein SUZ12 and the epigenetic H3K27me3 mark on histone H3 at these genes in OKF6-TERT1R, but not in SCC-9 cells. In contrast, IRX1, IRX4, SIX2 and TSHZ3 transcripts are lower in SCC-9 than in OKF6-TERT1R cells. We detected SUZ12 and the H3K27me3 mark at these genes in SCC-9, but not in OKF6-TERT1R cells. SUZ12 depletion increased HOXB7, HOXC10, HOXC13, and HOXD8 transcript levels and decreased the proliferation of OKF6-TERT1R cells. Transcriptional responses to RA are attenuated in SCC-9 versus OKF6-TERT1R cells. SUZ12 and H3K27me3 levels were not altered by RA at these HOX genes in SCC-9 and OKF6-TERT1R cells. We conclude that altered activity of PRC2 is associated with dysregulation of homeobox gene expression in human SCC cells, and that this dysregulation potentially plays a role in the neoplastic transformation of oral keratinocytes. - Highlights: • RNAseq elucidates differences between non-tumorigenic and tumorigenic oral keratinocytes. • Changes in HOX mRNA in SCC-9 vs. OKF6-TERT1R cells are a result of altered epigenetic regulation. • RNAseq shows that retinoic acid (RA) influences gene expression in both OKF6-TERT1R and SCC-9 cells.

Altered epigenetic regulation of homeobox genes in human oral squamous cell carcinoma cells

International Nuclear Information System (INIS)

Marcinkiewicz, Katarzyna M.; Gudas, Lorraine J.

2014-01-01

To gain insight into oral squamous cell carcinogenesis, we performed deep sequencing (RNAseq) of non-tumorigenic human OKF6-TERT1R and tumorigenic SCC-9 cells. Numerous homeobox genes are differentially expressed between OKF6-TERT1R and SCC-9 cells. Data from Oncomine, a cancer microarray database, also show that homeobox (HOX) genes are dysregulated in oral SCC patients. The activity of Polycomb repressive complexes (PRC), which causes epigenetic modifications, and retinoic acid (RA) signaling can control HOX gene transcription. HOXB7, HOXC10, HOXC13, and HOXD8 transcripts are higher in SCC-9 than in OKF6-TERT1R cells; using ChIP (chromatin immunoprecipitation) we detected PRC2 protein SUZ12 and the epigenetic H3K27me3 mark on histone H3 at these genes in OKF6-TERT1R, but not in SCC-9 cells. In contrast, IRX1, IRX4, SIX2 and TSHZ3 transcripts are lower in SCC-9 than in OKF6-TERT1R cells. We detected SUZ12 and the H3K27me3 mark at these genes in SCC-9, but not in OKF6-TERT1R cells. SUZ12 depletion increased HOXB7, HOXC10, HOXC13, and HOXD8 transcript levels and decreased the proliferation of OKF6-TERT1R cells. Transcriptional responses to RA are attenuated in SCC-9 versus OKF6-TERT1R cells. SUZ12 and H3K27me3 levels were not altered by RA at these HOX genes in SCC-9 and OKF6-TERT1R cells. We conclude that altered activity of PRC2 is associated with dysregulation of homeobox gene expression in human SCC cells, and that this dysregulation potentially plays a role in the neoplastic transformation of oral keratinocytes. - Highlights: • RNAseq elucidates differences between non-tumorigenic and tumorigenic oral keratinocytes. • Changes in HOX mRNA in SCC-9 vs. OKF6-TERT1R cells are a result of altered epigenetic regulation. • RNAseq shows that retinoic acid (RA) influences gene expression in both OKF6-TERT1R and SCC-9 cells

Analysis of oxidative DNA damage, Part II: Synthesis of the internal standard 8-[18O]hydroxy-2'-deoxyguanosine

NARCIS (Netherlands)

Hermanns RCA; Zomer G; Stavenuiter JFC; Westra G; Visser T; van de Werken G

1993-01-01

In the project 'Oxidative DNA Damage' the first aim is to develop a mass spectrometric method for the quantification of 8-hydroxy-2'- deoxyguanosine (oh8dG). The required precision of the method requires the application of a labeled analogue as an internal standard. This report

PAH-DNA adducts in environmentally exposed population in relation to metabolic and DNA repair gene polymorphisms

Energy Technology Data Exchange (ETDEWEB)

Binkova, Blanka [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Chvatalova, Irena [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Lnenickova, Zdena [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Milcova, Alena [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Tulupova, Elena [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Cancer Biomarkers and Prevention Group, Biocentre, University of Leicester (United Kingdom); Farmer, Peter B. [Cancer Biomarkers and Prevention Group, Biocentre, University of Leicester (United Kingdom); Sram, Radim J. [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic)]. E-mail: sram@biomed.cas.cz

2007-07-01

Epidemiologic studies indicate that prolonged exposure to particulate air pollution may be associated with increased risk of cardiovascular diseases and cancer in general population. These effects may be attributable to polycyclic aromatic hydrocarbons (PAHs) adsorbed to respirable air particles. It is expected that metabolic and DNA repair gene polymorphisms may modulate individual susceptibility to PAH exposure. This study investigates relationships between exposure to PAHs, polymorphisms of these genes and DNA adducts in group of occupationally exposed policemen (EXP, N = 53, males, aged 22-50 years) working outdoors in the downtown area of Prague and in matched 'unexposed' controls (CON, N = 52). Personal exposure to eight carcinogenic PAHs (c-PAHs) was evaluated by personal samplers during working shift prior to collection of biological samples. Bulky-aromatic DNA adducts were analyzed in lymphocytes by {sup 32}P-postlabeling assay. Polymorphisms of metabolizing (GSTM1, GSTP1, GSTT1, EPHX1, CYP1A1-MspI) and DNA repair (XRCC1, XPD) genes were determined by PCR-based RFLP assays. As potential modifiers and/or cofounders, urinary cotinine levels were analyzed by radioimmunoassay, plasma levels of vitamins A, C, E and folates by HPLC, cholesterol and triglycerides using commercial kits. During the sampling period ambient particulate air pollution was as follows: PM10 32-55 {mu}g/m{sup 3}, PM2.5 27-38 {mu}g/m{sup 3}, c-PAHs 18-22 ng/m{sup 3}; personal exposure to c-PAHs: 9.7 ng/m{sup 3} versus 5.8 ng/m{sup 3} (P < 0.01) for EXP and CON groups, respectively. The total DNA adduct levels did not significantly differ between EXP and CON groups (0.92 {+-} 0.28 adducts/10{sup 8} nucleotides versus 0.82 {+-} 0.23 adducts/10{sup 8} nucleotides, P = 0.065), whereas the level of the B[a]P-'like' adduct was significantly higher in exposed group (0.122 {+-} 0.036 adducts/10{sup 8} nucleotides versus 0.099 {+-} 0.035 adducts/10{sup 8} nucleotides, P = 0

DNA topology and transcription

Science.gov (United States)

Kouzine, Fedor; Levens, David; Baranello, Laura

2014-01-01

Chromatin is a complex assembly that compacts DNA inside the nucleus while providing the necessary level of accessibility to regulatory factors conscripted by cellular signaling systems. In this superstructure, DNA is the subject of mechanical forces applied by variety of molecular motors. Rather than being a rigid stick, DNA possesses dynamic structural variability that could be harnessed during critical steps of genome functioning. The strong relationship between DNA structure and key genomic processes necessitates the study of physical constrains acting on the double helix. Here we provide insight into the source, dynamics, and biology of DNA topological domains in the eukaryotic cells and summarize their possible involvement in gene transcription. We emphasize recent studies that might inspire and impact future experiments on the involvement of DNA topology in cellular functions. PMID:24755522

Removal of oxygen free-radical-induced 5′,8-purine cyclodeoxynucleosides from DNA by the nucleotide excision-repair pathway in human cells

Science.gov (United States)

Kuraoka, Isao; Bender, Christina; Romieu, Anthony; Cadet, Jean; Wood, Richard D.; Lindahl, Tomas

2000-01-01

Exposure of cellular DNA to reactive oxygen species generates several classes of base lesions, many of which are removed by the base excision-repair pathway. However, the lesions include purine cyclodeoxynucleoside formation by intramolecular crosslinking between the C-8 position of adenine or guanine and the 5′ position of 2-deoxyribose. This distorting form of DNA damage, in which the purine is attached by two covalent bonds to the sugar-phosphate backbone, occurs as distinct diastereoisomers. It was observed here that both diastereoisomers block primer extension by mammalian and microbial replicative DNA polymerases, using DNA with a site-specific purine cyclodeoxynucleoside residue as template, and consequently appear to be cytotoxic lesions. Plasmid DNA containing either the 5′R or 5′S form of 5′,8-cyclo-2-deoxyadenosine was a substrate for the human nucleotide excision-repair enzyme complex. The R diastereoisomer was more efficiently repaired than the S isomer. No correction of the lesion by direct damage reversal or base excision repair was detected. Dual incision around the lesion depended on the core nucleotide excision-repair protein XPA. In contrast to several other types of oxidative DNA damage, purine cyclodeoxynucleosides are chemically stable and would be expected to accumulate at a slow rate over many years in the DNA of nonregenerating cells from xeroderma pigmentosum patients. High levels of this form of DNA damage might explain the progressive neurodegeneration seen in XPA individuals. PMID:10759556

Histone Modification Is Involved in Okadaic Acid (OA Induced DNA Damage Response and G2-M Transition Arrest in Maize.

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Hao Zhang

Full Text Available Histone modifications are involved in regulation of chromatin structure. To investigate the relationship between chromatin modification and cell cycle regulation during plant cell proliferation, Okadaic acid (OA, a specific inhibitor of serine/threonine protein phosphatase, was applied in this study. The results showed that OA caused the cell cycle arrest at preprophase, leading to seedling growth inhibition. Western blotting assay revealed that the spatial distribution of phosphorylation of Ser10 histone H3 tails (H3S10ph signals was altered under OA treatment. Reactive oxygen species (ROS was found to be at higher levels and TdT-mediated dUTP nick end labeling (TUNEL assay displayed DNA breaks happened at the chromatin after treatment with OA, companied with an increase in the acetylation of histone H4 at lysine 5 (H4K5ac level. From these observations, we speculated that the alteration of the spatial distribution of H3S10ph and the level of H4K5ac was involved in the procedure that OA induced DNA breaks and G2-M arrested by the accumulation of ROS, and that the histone H3S10ph and H4K5ac might facilitate DNA repair by their association with the chromatin decondensation.

8 CFR 1208.30 - Credible fear determinations involving stowaways and applicants for admission found inadmissible...

Science.gov (United States)

2010-01-01

... 8 Aliens and Nationality 1 2010-01-01 2010-01-01 false Credible fear determinations involving..., DEPARTMENT OF JUSTICE IMMIGRATION REGULATIONS PROCEDURES FOR ASYLUM AND WITHHOLDING OF REMOVAL Credible Fear of Persecution § 1208.30 Credible fear determinations involving stowaways and applicants for...

Human herpes virus-8 DNA in bronchoalveolar lavage samples from patients with AIDS-associated pulmonary Kaposi's sarcoma

DEFF Research Database (Denmark)

Benfield, T L; Dodt, K K; Lundgren, Jens Dilling

1997-01-01

Kaposi's sarcoma (KS) is the most frequent AIDS-associated neoplasm, and often disseminates to visceral organs, including the lungs. An ante-mortem diagnosis of pulmonary KS is difficult. Recently, DNA sequences resembling a new human herpes virus (HHV-8), have been identified in various forms...

N-acetylcysteine normalizes the urea cycle and DNA repair in cells from patients with Batten disease.

Science.gov (United States)

Kim, June-Bum; Lim, Nary; Kim, Sung-Jo; Heo, Tae-Hwe

2012-12-01

Batten disease is an inherited disorder characterized by early onset neurodegeneration due to the mutation of the CLN3 gene. The function of the CLN3 protein is not clear, but an association with oxidative stress has been proposed. Oxidative stress and DNA damage play critical roles in the pathogenesis of neurodegenerative diseases. Antioxidants are of interest because of their therapeutic potential for treating neurodegenerative diseases. We tested whether N-acetylcysteine (NAC), a well-known antioxidant, improves the pathology of cells from patients with Batten disease. At first, the expression levels of urea cycle components and DNA repair enzymes were compared between Batten disease cells and normal cells. We used both mRNA expression levels and Western blot analysis. We found that carbamoyl phosphate synthetase 1, an enzyme involved in the urea cycle, 8-oxoguanine DNA glycosylase 1 and DNA polymerase beta, enzymes involved in DNA repair, were expressed at higher levels in Batten disease cells than in normal cells. The treatment of Batten disease cells with NAC for 48 h attenuated activities of the urea cycle and of DNA repair, as indicated by the substantially decreased expression levels of carbamoyl phosphate synthetase 1, 8-oxoguanine DNA glycosylase 1 and DNA polymerase beta proteins compared with untreated Batten cells. NAC may serve in alleviating the burden of urea cycle and DNA repair processes in Batten disease cells. We propose that NAC may have beneficial effects in patients with Batten disease. Copyright © 2012 John Wiley & Sons, Ltd.

DNA methylation perturbations in genes involved in polyunsaturated Fatty Acid biosynthesis associated with depression and suicide risk.

Science.gov (United States)

Haghighi, Fatemeh; Galfalvy, Hanga; Chen, Sean; Huang, Yung-Yu; Cooper, Thomas B; Burke, Ainsley K; Oquendo, Maria A; Mann, J John; Sublette, M Elizabeth

2015-01-01

Polyunsaturated fatty acid (PUFA) status has been associated with neuropsychiatric disorders, including depression and risk of suicide. Long-chain PUFAs (LC-PUFAs) are obtained in the diet or produced by sequential desaturation and elongation of shorter-chain precursor fatty acids linoleic acid (LA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3). We compared DNA methylation patterns in genes involved in LC-PUFA biosynthesis in major depressive disorder (MDD) with (n = 22) and without (n = 39) history of suicide attempt, and age- and sex-matched healthy volunteers (n = 59). Plasma levels of selected PUFAs along the LC-PUFA biosynthesis pathway were determined by transesterification and gas chromatography. CpG methylation levels for the main human LC-PUFA biosynthetic genes, fatty acid desaturases 1 (Fads1) and 2 (Fads2), and elongation of very long-chain fatty acids protein 5 (Elovl5), were assayed by bisulfite pyrosequencing. Associations between PUFA levels and diagnosis or suicide attempt status did not survive correction for multiple testing. However, MDD diagnosis and suicide attempts were significantly associated with DNA methylation in Elovl5 gene regulatory regions. Also the relative roles of PUFA levels and DNA methylation with respect to diagnostic and suicide attempt status were determined by least absolute shrinkage and selection operator logistic regression analyses. We found that PUFA associations with suicide attempt status were explained by effects of Elovl5 DNA methylation within the regulatory regions. The observed link between plasma PUFA levels, DNA methylation, and suicide risk may have implications for modulation of disease-associated epigenetic marks by nutritional intervention.

DNA methylation perturbations in genes involved in polyunsaturated fatty acid biosynthesis associated with depression and suicide risk

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Fatemeh eHaghighi

2015-04-01

Full Text Available Polyunsaturated fatty acid (PUFA status has been associated with neuropsychiatric disorders, including depression and risk of suicide. Long-chain PUFAs (LC-PUFAs are obtained in the diet or produced by sequential desaturation and elongation of shorter-chain precursor fatty acids linoleic acid (LA, 18:2n-6 and α-linolenic acid (ALA, 18:3n-3. We compared DNA methylation patterns in genes involved in LC-PUFA biosynthesis in major depressive disorder (MDD with (n=22 and without (n=39 history of suicide attempt, and age- and sex-matched healthy volunteers (n=59. Plasma levels of selected PUFAs along the LC-PUFA biosynthesis pathway were determined by transesterification and gas chromatography. CpG methylation levels for the main human LC-PUFA biosynthetic genes, fatty acid desaturases 1 (Fads1 and 2 (Fads2, and elongation of very long chain fatty acids protein 5 (Elovl5, were assayed by bisulfite pyrosequencing. Associations between PUFA levels and diagnosis or suicide attempt status did not survive correction for multiple testing. However, MDD diagnosis and suicide attempts were significantly associated with DNA methylation in Elovl5 gene regulatory regions. Also the relative roles of PUFA levels and DNA methylation with respect to diagnostic and suicide attempt status were determined by least absolute shrinkage and selection operator (LASSO logistic regression analyses. We found that PUFA associations with suicide attempt status were explained by effects of Elovl5 DNA methylation within the regulatory regions. The observed link between plasma PUFA levels, DNA methylation, and suicide risk may have implications for modulation of disease-associated epigenetic marks by nutritional intervention.

SV40 host-substituted variants: a new look at the monkey DNA inserts and recombinant junctions.

Science.gov (United States)

Singer, Maxine; Winocour, Ernest

2011-04-10

The available monkey genomic data banks were examined in order to determine the chromosomal locations of the host DNA inserts in 8 host-substituted SV40 variant DNAs. Five of the 8 variants contained more than one linked monkey DNA insert per tandem repeat unit and in all cases but one, the 19 monkey DNA inserts in the 8 variants mapped to different locations in the monkey genome. The 50 parental DNAs (32 monkey and 18 SV40 DNA segments) which spanned the crossover and flanking regions that participated in monkey/monkey and monkey/SV40 recombinations were characterized by substantial levels of microhomology of up to 8 nucleotides in length; the parental DNAs also exhibited direct and inverted repeats at or adjacent to the crossover sequences. We discuss how the host-substituted SV40 variants arose and the nature of the recombination mechanisms involved. Copyright © 2011 Elsevier Inc. All rights reserved.

Decreased DNA Methylation in the Shati/Nat8l Promoter in Both Patients with Schizophrenia and a Methamphetamine-Induced Murine Model of Schizophrenia-Like Phenotype.

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Kyosuke Uno

Full Text Available The number of patients with schizophrenia has increased over the past decade. Previously, many studies have been performed to establish its diagnostic criteria, prophylactic methods, and effective therapies. In this study, we analyzed whether the ratios of DNA methylation in CpG islands of the Shati/Nat8l is decreased in model mice of schizophrenia-like phenotype using genomic DNA collected from brain regions and peripheral blood, since the mouse model of schizophrenia-like phenotype, mice treated repeatedly with methamphetamine showed increase of Shati/Nat8l mRNA expression in our previous experiment. The ratios of Shati/Nat8l CpG island methylation were significantly decreased in both the nucleus accumbens and the peripheral blood of model mice compared with those of control mice. We also investigated Shati/Nat8l methylation in the blood of patients with schizophrenia. We found that Shati/Nat8l CpG island methylation ratios were lower in the patients with schizophrenia than in the healthy controls, which is consistent with our findings in the mice model. To our knowledge, this is the first study to show similar alterations in methylation status of a particular genomic DNA site in both the brain and peripheral blood of mice. Furthermore, the same phenomenon was observed in corresponding human genomic sequences of the DNA extracted from the peripheral blood of patients with schizophrenia. Based on our findings, DNA methylation profiles of the CpG island of Shati/Nat8l might be a diagnostic biomarker of schizophrenia.

Radicals of DNA and DNA nucleotides generated by ionising radiation

International Nuclear Information System (INIS)

Przybytniak, G.

2004-01-01

A first stage of cell processes leading to DNA damage of initiated by radical reactions. In a model system such transformations were generated by ionising radiation which involves production of electron loss and electron gain centers of the substrate and radical formation. Using cryogenic ESR spectroscopy it was found that the DNA nucleotides, which convert to radical anions upon electron capture undergo the separation of unpaired spin and charge due to protonation. Circular and linear dichroism studies enabled to conclude that iron ions(III) induce strong changes in the DNA helical structure indicating their coordination with nitrogen bases. The repair of DNA radicals produced via radiolytic oxidation, i.e. the guanine radical cation and the allyl type radical of thymine, is possible at elevated temperatures due to the involvement of sulphydryl groups. The influence of the thiol charge is then limited

Involvement of DNA-PK and ATM in radiation- and heat-induced DNA damage recognition and apoptotic cell death

International Nuclear Information System (INIS)

Tomita, Masanori

2010-01-01

Exposure to ionizing radiation and hyperthermia results in important biological consequences, e.g. cell death, chromosomal aberrations, mutations, and DNA strand breaks. There is good evidence that the nucleus, specifically cellular DNA, is the principal target for radiation-induced cell lethality. DNA double-strand breaks (DSBs) are considered to be the most serious type of DNA damage induced by ionizing radiation. On the other hand, verifiable mechanisms which can lead to heat-induced cell death are damage to the plasma membrane and/or inactivation of heat-labile proteins caused by protein denaturation and subsequent aggregation. Recently, several reports have suggested that DSBs can be induced after hyperthermia because heat-induced phosphorylated histone H2AX (γ-H2AX) foci formation can be observed in several mammalian cell lines. In mammalian cells, DSBs are repaired primarily through two distinct and complementary mechanisms: non-homologous end joining (NHEJ), and homologous recombination (HR) or homology-directed repair (HDR). DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutated (ATM) are key players in the initiation of DSB repair and phosphorylate and/or activate many substrates, including themselves. These phosphorylated substrates have important roles in the functioning of cell cycle checkpoints and in cell death, as well as in DSB repair. Apoptotic cell death is a crucial cell suicide mechanism during development and in the defense of homeostasis. If DSBs are unrepaired or misrepaired, apoptosis is a very important system which can protect an organism against carcinogenesis. This paper reviews recently obtained results and current topics concerning the role of DNA-PK and ATM in heat- or radiation-induced apoptotic cell death. (author)

Site-directed mutational analysis of structural interactions of low molecule compounds binding to the N-terminal 8 kDa domain of DNA polymerase β

International Nuclear Information System (INIS)

Murakami, Shizuka; Kamisuki, Shinji; Takata, Kei-ichi; Kasai, Nobuyuki; Kimura, Seisuke; Mizushina, Yoshiyuki; Ohta, Keisuke; Sugawara, Fumio; Sakaguchi, Kengo

2006-01-01

We previously reported the mode of inhibition of DNA polymerase β (pol. β) by long chain fatty acids and a bile acid, involving binding analyses to the N-terminal 8-kDa DNA binding domain. Here we describe a site-directed mutational analysis in which the key amino acids (L11, K35, H51, K60, L77, and T79), which are direct interaction sites in the domain, were substituted with K, A, A, A, K, and A, respectively. And their pol. β interactions with a C24-long chain fatty acid, nervonic acid (NA), and a bile acid, lithocholic acid (LCA), were investigated by gel mobility shift assay and NMR spectroscopy. In the case of K35A, there was complete loss of DNA binding activity while K60A hardly has any activity. In contrast the other mutations had no appreciable effects. Thus, K35 and K60 are key amino acid sites for binding to template DNA. The DNA binding activities of L11K, H51A, and T79A as well as the wild type were inhibited by NA to the same extent. T79A demonstrated a disturbed interaction with LCA. 1 H- 15 N HSQC NMR analysis indicated that despite their many similarities, the wild-type and the mutant proteins displayed some significant chemical shift differences. Not only were the substituted amino acid residues three-dimensionally shifted, but some amino acids which are positioned far distant from the key amino acids showed a shift. These results suggest that the interaction surface was significantly distorted with the result that LCA could not bind to the domain. These findings confirm our previous biochemical and 3D structural proposals concerning inhibition by NA and LCA

Studies of DNA and chromosome damage in skin fibroblasts and blood lymphocytes from psoriasis patients treated with 8-methoxypsoralen and UVA irradiation

International Nuclear Information System (INIS)

Bredberg, A.; Lambert, B.; Lindblad, A.; Swanbeck, G.; Wennersten, G.

1983-01-01

Exposure of human lymphocytes and skin fibroblasts in vitro to a single, clinically used dose of PUVA, i.e., 0.1 micrograms/ml of 8-methoxypsoralen (8-MOP) plus 0.9-4 J/cm2 of longwave ultraviolet radiation (UVA), lead to the formation of DNA damage as determined by alkaline elution, and to chromosome aberrations and sister chromatid exchanges (SCE). When lymphocyte-enriched plasma was obtained from psoriasis patients 2 h after oral intake of 8-MOP and then UVA irradiated (1.8-3.6 J/cm2) in vitro, an increased frequency of chromosome aberrations and SCE was observed. Normal levels of chromosome aberrations and SCE were found in lymphocytes of psoriasis patients after 3-30 weeks of PUVA treatment in vivo. A small but statistically significant increase in the SCE frequency was observed in the lymphocytes of psoriasis patients treated for 1-6 years with PUVA (mean 18.0 SCE/cell) as compared with before PUVA (mean 15.8, p less than 0.05). Skin fibroblasts of psoriasis patients analyzed 5 years after the start of PUVA treatment showed a normal number of SCE but a high fraction of filter-retained DNA in the alkaline elution assay, suggesting the presence of cross-linked DNA

Evidence that OGG1 glycosylase protects neurons against oxidative DNA damage and cell death under ischemic conditions

DEFF Research Database (Denmark)

Liu, Dong; Croteau, Deborah L; Souza-Pinto, Nadja

2011-01-01

to ischemic and oxidative stress. After exposure of cultured neurons to oxidative and metabolic stress levels of OGG1 in the nucleus were elevated and mitochondria exhibited fragmentation and increased levels of the mitochondrial fission protein dynamin-related protein 1 (Drp1) and reduced membrane potential......7,8-Dihydro-8-oxoguanine DNA glycosylase (OGG1) is a major DNA glycosylase involved in base-excision repair (BER) of oxidative DNA damage to nuclear and mitochondrial DNA (mtDNA). We used OGG1-deficient (OGG1(-/-)) mice to examine the possible roles of OGG1 in the vulnerability of neurons....... Cortical neurons isolated from OGG1(-/-) mice were more vulnerable to oxidative insults than were OGG1(+/+) neurons, and OGG1(-/-) mice developed larger cortical infarcts and behavioral deficits after permanent middle cerebral artery occlusion compared with OGG1(+/+) mice. Accumulations of oxidative DNA...

The Q Motif Is Involved in DNA Binding but Not ATP Binding in ChlR1 Helicase.

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Hao Ding

Full Text Available Helicases are molecular motors that couple the energy of ATP hydrolysis to the unwinding of structured DNA or RNA and chromatin remodeling. The conversion of energy derived from ATP hydrolysis into unwinding and remodeling is coordinated by seven sequence motifs (I, Ia, II, III, IV, V, and VI. The Q motif, consisting of nine amino acids (GFXXPXPIQ with an invariant glutamine (Q residue, has been identified in some, but not all helicases. Compared to the seven well-recognized conserved helicase motifs, the role of the Q motif is less acknowledged. Mutations in the human ChlR1 (DDX11 gene are associated with a unique genetic disorder known as Warsaw Breakage Syndrome, which is characterized by cellular defects in genome maintenance. To examine the roles of the Q motif in ChlR1 helicase, we performed site directed mutagenesis of glutamine to alanine at residue 23 in the Q motif of ChlR1. ChlR1 recombinant protein was overexpressed and purified from HEK293T cells. ChlR1-Q23A mutant abolished the helicase activity of ChlR1 and displayed reduced DNA binding ability. The mutant showed impaired ATPase activity but normal ATP binding. A thermal shift assay revealed that ChlR1-Q23A has a melting point value similar to ChlR1-WT. Partial proteolysis mapping demonstrated that ChlR1-WT and Q23A have a similar globular structure, although some subtle conformational differences in these two proteins are evident. Finally, we found ChlR1 exists and functions as a monomer in solution, which is different from FANCJ, in which the Q motif is involved in protein dimerization. Taken together, our results suggest that the Q motif is involved in DNA binding but not ATP binding in ChlR1 helicase.

Perinatal transmission of human papilomavirus DNA

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Serafini Eduardo P

2009-06-01

Full Text Available Abstract The purpose was to study the perinatal transmission of human papillomavirus DNA (HPV-DNA in 63 mother-newborn pairs, besides looking at the epidemiological factors involved in the viral DNA transmission. The following sampling methods were used: (1 in the pregnant woman, when was recruited, in cervix and clinical lesions of the vagina, vulva and perineal region; (2 in the newborn, (a buccal, axillary and inguinal regions; (b nasopharyngeal aspirate, and (c cord blood; (3 in the children, buccal was repeated in the 4th week and 6th and 12th month of life. HPV-DNA was identified using two methodologies: multiplex PCR (PGMY09 and MY11 primers and nested-PCR (genotypes 6/11, 16, 18, 31, 33, 42, 52 and 58. Perinatal transmission was considered when concordance was found in type-specific HPV between mother/newborn or mother/child. HPV-DNA genital was detected in 49 pregnant women submitted to delivery. Eleven newborns (22.4%, n = 11/49 were HPV-DNA positive. In 8 cases (16.3%, n = 8/49 there was type specific HPV concordance between mother/newborn samples. At the end of the first month of life three children (6.1%, n = 3/49 became HPV-DNA positive, while two remained positive from birth. In 3 cases (100%, n = 3/3 there was type specific HPV concordance between mother/newborn samples. In the 6th month, a child (2%, n = 1/49 had become HPV-DNA positive between the 1st and 6th month of life, and there was type specific HPV concordance of mother/newborn samples. All the HPV-DNA positive children (22.4%, n = 11/49 at birth and at the end first month of life (6.1%, n = 3/49 became HPV-DNA negative at the age of 6 months. The HPV-DNA positive child (2%, n = 1/49 from 1st to the 6th month of life became HPV-DNA negative between the 6th and 12th month of life and one child had anogenital warts. In the twelfth month all (100%, n = 49/49 the children studied were HPV-DNA negative. A positive and significant correlation was observed between perinatal

Amplification of the Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 lytic origin of DNA replication is dependent upon a cis-acting AT-rich region and an ORF50 response element and the trans-acting factors ORF50 (K-Rta) and K8 (K-bZIP)

International Nuclear Information System (INIS)

AuCoin, David P.; Colletti, Kelly S.; Cei, Sylvia A.; Papouskova, Iva; Tarrant, Margaret; Pari, Gregory S.

2004-01-01

Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV8), has significant sequence homology to Epstein-Barr virus (EBV). In cell culture, HHV8 is primarily latent, and viral genes associated with lytic replication are not expressed. Two lytic origins of DNA replication (oriLyt) are present within the HHV8 genome and are composed of an AT-rich region adjacent to GC-rich DNA sequences. We have now identified essential cis- and trans-acting elements required for oriLyt-dependent DNA replication. The transient replication assay was used to show that two AT-rich elements, three consensus AP1 transcription factor-binding sites, an ORF50 response element (RE), and a consensus TATA box motif are essential for efficient origin-dependent DNA replication. Transient transfection of luciferase reporter constructs indicated that the downstream region of the HHV8 oriLyt responds to ORF50 and suggests that part of the oriLyt may be an enhancer/promoter. In addition, a transient cotransfection-replication assay elucidated the set of trans-acting factors required for lytic DNA replication. These factors consist of homologues to the core replication proteins: ORF6 (ssDNA binding protein), ORF9 (DNA polymerase), ORF40-41 (primase-associated factor), ORF44 (helicase), ORF56 (primase), and ORF59 (polymerase processivity factor) common to all herpesviruses along with ORF50 (K-Rta) and K8 (K-bZIP)

In situ analysis of Bacillus licheniformis biofilms: amyloid-like polymers and eDNA are involved in the adherence and aggregation of the extracellular matrix.

Science.gov (United States)

Randrianjatovo-Gbalou, I; Rouquette, P; Lefebvre, D; Girbal-Neuhauser, E; Marcato-Romain, C-E

2017-05-01

This study attempts to determine which of the exopolymeric substances are involved in the adherence and aggregation of a Bacillus licheniformis biofilm. The involvement of extracellular proteins and eDNA were particularly investigated using DNase and proteinase K treatment. The permeability of the biofilms increased fivefold after DNase I treatment. The quantification of the matrix components showed that, irrespective to the enzyme tested, eDNA and amyloid-like polymers were removed simultaneously. Size-exclusion chromatography analyses supported these observations and revealed the presence of associated nucleic acid and protein complexes in the biofilm lysates. These data suggest that some extracellular DNA and amyloid-like proteins were closely interlaced within the matrix. Finally, confocal laser scanning microscopy imaging gave supplementary clues about the 3D organization of the biofilms, confirming that eDNA and exoproteins were essentially layered under and around the bacterial cells, whereas the amyloid-like fractions were homogeneously distributed within the matrix. These results confirm that some DNA-amyloid complexes play a key role in the modulation of the mechanical resistance of B. licheniformis biofilms. The study highlights the need to consider the whole structure of biofilms and to target the interactions between matrix components. A better understanding of B. licheniformis biofilm physiology and the structural organization of the matrix will strengthen strategies of biofilm control. © 2017 The Society for Applied Microbiology.

DNA and protein binding, double-strand DNA cleavage and cytotoxicity of mixed ligand copper(II) complexes of the antibacterial drug nalidixic acid.

Science.gov (United States)

Loganathan, Rangasamy; Ganeshpandian, Mani; Bhuvanesh, Nattamai S P; Palaniandavar, Mallayan; Muruganantham, Amsaveni; Ghosh, Swapan K; Riyasdeen, Anvarbatcha; Akbarsha, Mohammad Abdulkader

2017-09-01

The water soluble mixed ligand complexes [Cu(nal)(diimine)(H 2 O)](ClO 4 ) 1-4, where H(nal) is nalidixic acid and diimine is 2,2'-bipyridine (1), 1,10-phenanthroline (2), 5,6-dimethyl-1,10-phenanthroline (3), and 3,4,7,8-tetramethyl-1,10-phenanthroline (4), have been isolated. The coordination geometry around Cu(II) in 1 and that in the Density Functional Theory optimized structures of 1-4 has been assessed as square pyramidal. The trend in DNA binding constants (K b ) determined using absorption spectral titration (K b : 1, 0.79±0.1base pair. In contrast, 3 and 4 are involved in intimate hydrophobic interaction with DNA through the methyl substituents on phen ring, which is supported by viscosity and protein binding studies. DNA docking studies imply that 4 is involved preferentially in DNA major groove binding while 1-3 in minor groove binding and that all the complexes, upon removing the axially coordinated water molecule, bind in the major groove. Interestingly, 3 and 4 display prominent double-strand DNA cleavage while 1 and 2 effect only single-strand DNA cleavage in the absence of an activator. The complexes 3 and 4 show cytotoxicity higher than 1 and 2 against human breast cancer cell lines (MCF-7). The complex 4 induces apoptotic mode of cell death in cancer cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Public involvement in pharmacogenomics research: a national survey on public attitudes towards pharmacogenomics research and the willingness to donate DNA samples to a DNA bank in Japan.

Science.gov (United States)

Kobayashi, Eriko; Satoh, Nobunori

2009-11-01

To assess the attitudes of the Japanese general public towards pharmacogenomics research and a DNA bank for identifying genomic markers associated with ADRs and their willingness to donate DNA samples, we conducted a national survey for 1,103 Japanese adults from the general public, not a patient population. The response rate was 36.8%. The majority of the respondents showed a positive attitude towards pharmacogenomics research (81.0%) and a DNA bank (70.4%). Considering fictitious clinical situations such as taking medications and experiencing ADRs, the willingness to donate DNA samples when experiencing ADRs (61.7%) was higher than when taking medications (45.3%). Older generations were significantly associated with a decreased willingness to donate (OR = 0.45, CI 0.28-0.72 in 50s. OR = 0.49, CI: 0.31-0.77 in 60s). Positive attitudes towards pharmacogenomics research, a DNA bank, blood/bone marrow/organ donation were significantly associated with an increased willingness. However, the respondents had the following concerns regarding a DNA bank: the confidentiality of their personal information, the manner by which research results were utilized and simply the use of their own DNA for research. In order to attain public understanding to overcome these concerns, a process of public awareness should be put into place to emphasize the beneficial aspects of identifying genomic markers associated with ADRs and to address these concerns raised in our study. Further study is needed to assess the willingness of actual patients taking medications in real situations, since the respondents in our study were from the general public, not a patient population, and their willingness was assessed on the condition of assuming that they were patients taking medications.

Effect of 8-MOP plus treatment on survival and repair of plasmid pBR322

International Nuclear Information System (INIS)

Bauluz, C.; Vidania, R.

1992-01-01

We have studied the lethality produced in pBR322 DNA after PUVA treatment (8-MOP+UVA). As recipients, we used a collection of E. coli strains differing in their repair capacities and analysed the involvement of several DNA repair pathways in the removal of plasmid lesions. We have also studied the effect of UVA radiation alone, in order to determine more precisely the effect attributable only to psoralen molecules. Results showed a strong lethal effect derived from PUVA treatment; however, some plasmid recovery was achieved in bacterial hosts proficient in Excision repair and SOS repair. another repair pathway, only detectable at high density of lesions, appeared to be relevant for the removal of 8-MOP:DNA adducts. (author)

Repair of 8-methoxypsoralen induced DNA interstrand cross-links in Tetrahymena thermophila. The effect of inhibitors of macromolecular synthesis

DEFF Research Database (Denmark)

Nielsen, P E; Køber, L

1985-01-01

-beta-D-arabinofuranoside (10 mM), puromycin (1 mM), hydroxyurea (5 mM) or 3-aminobenzamide (2.5 mM). None of the compounds showed any effect on the protein-DNA complexing step, and the ligation was partly inhibited only by nalidixic acid (150 micrograms/ml). The involvement of topoisomerases...

Effect of 8-MOP plus UVA treatment on survival and repair of plasmid pBR322; Efecto del tratamiento con 8-MOP mas UVA en la supervivencia y reparacion de pBR322

Energy Technology Data Exchange (ETDEWEB)

Bauluz, C; Vidania, R de

1991-07-01

We have studied the lethality produced in pBR322 DNA after PUVA treatment (8-MOP+UVA). As recipients, we used a collection of E. coli strains differing in their repair capacities and analysed the involvement of several DNA repair pathways in the removal of plasmid lesions. We have also studied the effect of UVA radiation alone, in order to determine more precisely the effect attributable only to psoralen molecules. Results showed a strong lethal effect derived from PUVA treatment; however, some plasmid recovery was achieved in bacterial hosts proficient in Excision repair and SOS repair. Another repair pathway, only detectable at high density of lesions, appeared to be relevant for the removal of 8-MOP:DNA adducts.(Author) 11 refs.

Cerebellar oxidative DNA damage and altered DNA methylation in the BTBR T+tf/J mouse model of autism and similarities with human post mortem cerebellum.

Directory of Open Access Journals (Sweden)

Svitlana Shpyleva

Full Text Available The molecular pathogenesis of autism is complex and involves numerous genomic, epigenomic, proteomic, metabolic, and physiological alterations. Elucidating and understanding the molecular processes underlying the pathogenesis of autism is critical for effective clinical management and prevention of this disorder. The goal of this study is to investigate key molecular alterations postulated to play a role in autism and their role in the pathophysiology of autism. In this study we demonstrate that DNA isolated from the cerebellum of BTBR T+tf/J mice, a relevant mouse model of autism, and from human post-mortem cerebellum of individuals with autism, are both characterized by an increased levels of 8-oxo-7-hydrodeoxyguanosine (8-oxodG, 5-methylcytosine (5mC, and 5-hydroxymethylcytosine (5hmC. The increase in 8-oxodG and 5mC content was associated with a markedly reduced expression of the 8-oxoguanine DNA-glycosylase 1 (Ogg1 and increased expression of de novo DNA methyltransferases 3a and 3b (Dnmt3a and Dnmt3b. Interestingly, a rise in the level of 5hmC occurred without changes in the expression of ten-eleven translocation expression 1 (Tet1 and Tet2 genes, but significantly correlated with the presence of 8-oxodG in DNA. This finding and similar elevation in 8-oxodG in cerebellum of individuals with autism and in the BTBR T+tf/J mouse model warrant future large-scale studies to specifically address the role of OGG1 alterations in pathogenesis of autism.

Enzymatic recognition of DNA damage induced by UVB-photosensitized titanium dioxide and biological consequences in Saccharomyces cerevisiae: Evidence for oxidatively DNA damage generation

International Nuclear Information System (INIS)

Pinto, A. Viviana; Deodato, Elder L.; Cardoso, Janine S.; Oliveira, Eliza F.; Machado, Sergio L.; Toma, Helena K.; Leitao, Alvaro C.; Padula, Marcelo de

2010-01-01

Although titanium dioxide (TiO 2 ) has been considered to be biologically inert, finding use in cosmetics, paints and food colorants, recent reports have demonstrated that when TiO 2 is attained by UVA radiation oxidative genotoxic and cytotoxic effects are observed in living cells. However, data concerning TiO 2 -UVB association is poor, even if UVB radiation represents a major environmental carcinogen. Herein, we investigated DNA damage, repair and mutagenesis induced by TiO 2 associated with UVB irradiation in vitro and in vivo using Saccharomyces cerevisiae model. It was found that TiO 2 plus UVB treatment in plasmid pUC18 generated, in addition to cyclobutane pyrimidine dimers (CPDs), specific damage to guanine residues, such as 8-oxo-7,8-dihydroguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), which are characteristic oxidatively generated lesions. In vivo experiments showed that, although the presence of TiO 2 protects yeast cells from UVB cytotoxicity, high mutation frequencies are observed in the wild-type (WT) and in an ogg1 strain (deficient in 8-oxoG and FapyG repair). Indeed, after TiO 2 plus UVB treatment, induced mutagenesis was drastically enhanced in ogg1 cells, indicating that mutagenic DNA lesions are repaired by the Ogg1 protein. This effect could be attenuated by the presence of metallic ion chelators: neocuproine or dipyridyl, which partially block oxidatively generated damage occurring via Fenton reactions. Altogether, the results indicate that TiO 2 plus UVB potentates UVB oxidatively generated damage to DNA, possibly via Fenton reactions involving the production of DNA base damage, such as 8-oxo-7,8-dihydroguanine.

Studying the synergistic damage effects induced by 1.8 GHz radiofrequency field radiation (RFR) with four chemical mutagens on human lymphocyte DNA using comet assay in vitro

International Nuclear Information System (INIS)

Wang Baohong; He Jiliang; Jin Lifen; Lu Deqiang; Zheng Wei; Lou Jianlin; Deng Hongping

2005-01-01

The aim of this investigation was to study the synergistic DNA damage effects in human lymphocytes induced by 1.8 GHz radiofrequency field radiation (RFR, SAR of 3 W/kg) with four chemical mutagens, i.e. mitomycin C (MMC, DNA crosslinker), bleomycin (BLM, radiomimetic agent), methyl methanesulfonate (MMS, alkylating agent), and 4-nitroquinoline-1-oxide (4NQO, UV-mimetic agent). The DNA damage of lymphocytes exposed to RFR and/or with chemical mutagens was detected at two incubation time (0 or 21 h) after treatment with comet assay in vitro. Three combinative exposure ways were used. Cells were exposed to RFR and chemical mutagens for 2 and 3 h, respectively. Tail length (TL) and tail moment (TM) were utilized as DNA damage indexes. The results showed no difference of DNA damage indexes between RFR group and control group at 0 and 21 h incubation after exposure (P > 0.05). There were significant difference of DNA damage indexes between MMC group and RFR + MMC co-exposure group at 0 and 21 h incubation after treatment (P 0.05). The experimental results indicated 1.8 GHz RFR (SAR, 3 W/kg) for 2 h did not induce the human lymphocyte DNA damage effects in vitro, but could enhance the human lymphocyte DNA damage effects induced by MMC and 4NQO. The synergistic DNA damage effects of 1.8 GHz RFR with BLM or MMS were not obvious

Dynamics of DNA conformations and DNA-protein interaction

DEFF Research Database (Denmark)

Metzler, R.; Ambjörnsson, T.; Lomholt, Michael Andersen

2005-01-01

Optical tweezers, atomic force microscopes, patch clamping, or fluorescence techniques make it possible to study both the equilibrium conformations and dynamics of single DNA molecules as well as their interaction with binding proteins. In this paper we address the dynamics of local DNA...... denaturation (bubble breathing), deriving its dynamic response to external physical parameters and the DNA sequence in terms of the bubble relaxation time spectrum and the autocorrelation function of bubble breathing. The interaction with binding proteins that selectively bind to the DNA single strand exposed...... in a denaturation bubble are shown to involve an interesting competition of time scales, varying between kinetic blocking of protein binding up to full binding protein-induced denaturation of the DNA. We will also address the potential to use DNA physics for the design of nanosensors. Finally, we report recent...

E2F1 induces p19INK4d, a protein involved in the DNA damage response, following UV irradiation.

Science.gov (United States)

Carcagno, Abel L; Giono, Luciana E; Marazita, Mariela C; Castillo, Daniela S; Pregi, Nicolás; Cánepa, Eduardo T

2012-07-01

Central to the maintenance of genomic integrity is the cellular DNA damage response. Depending on the type of genotoxic stress and through the activation of multiple signaling cascades, it can lead to cell cycle arrest, DNA repair, senescence, and apoptosis. p19INK4d, a member of the INK4 family of CDK inhibitors, plays a dual role in the DNA damage response, inhibiting cell proliferation and promoting DNA repair. Consistently, p19INK4d has been reported to become upregulated in response to UV irradiation and a great variety of genotoxic agents. Here, this induction is shown to result from a transcriptional stimulatory mechanism that can occur at every phase of the cell cycle except during mitosis. Moreover, evidence is presented that demonstrates that E2F1 is involved in the induction of p19INK4d following UV treatment, as it is prevented by E2F1 protein ablation and DNA-binding inhibition. Specific inhibition of this regulation using triplex-forming oligonucleotides that target the E2F response elements present in the p19INK4d promoter also block p19INK4d upregulation and sensitize cells to DNA damage. These results constitute the first description of a mechanism for the induction of p19INK4d in response to UV irradiation and demonstrate the physiological relevance of this regulation following DNA damage.

Damage to cellular and isolated DNA induced by a metabolite of aspirin

Energy Technology Data Exchange (ETDEWEB)

Oikawa, Shinji [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie 514-8507 (Japan)], E-mail: s-oikawa@doc.medic.mie-u.ac.jp; Kobayashi, Hatasu; Tada-Oikawa, Saeko [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie 514-8507 (Japan); JSPS Research Fellow (Japan); Isono, Yoshiaki [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie 514-8507 (Japan); Kawanishi, Shosuke [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie 514-8507 (Japan); Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Mie 513-8670 (Japan)

2009-02-10

Aspirin has been proposed as a possible chemopreventive agent. On the other hand, a recent cohort study showed that aspirin may increase the risk for pancreatic cancer. To clarify whether aspirin is potentially carcinogenic, we investigated the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), which is correlated with the incidence of cancer, in cultured cells treated with 2,3-dihydroxybenzoic acid (2,3-DHBA), a metabolite of aspirin. 2,3-DHBA induced 8-oxodG formation in the PANC-1 human pancreatic cancer cell line. 2,3-DHBA-induced DNA single-strand breaks were also revealed by comet assay using PANC-1 cells. Flow cytometric analyses showed that 2,3-DHBA increased the levels of intracellular reactive oxygen species (ROS) in PANC-1 cells. The 8-oxodG formation and ROS generation were also observed in the HL-60 leukemia cell line, but not in the hydrogen peroxide (H{sub 2}O{sub 2})-resistant clone HP100 cells, suggesting the involvement of H{sub 2}O{sub 2}. In addition, an hprt mutation assay supported the mutagenicity of 2,3-DHBA. We investigated the mechanism underlying the 2,3-DHBA-induced DNA damage using {sup 32}P-labeled DNA fragments of human tumor suppressor genes. 2,3-DHBA induced DNA damage in the presence of Cu(II) and NADH. DNA damage induced by 2,3-DHBA was enhanced by the addition of histone peptide-6 [AKRHRK]. Interestingly, 2,3-DHBA and histone peptide-6 caused base damage in the 5'-ACG-3' and 5'-CCG-3' sequences, hotspots of the p53 gene. Bathocuproine, a Cu(I) chelator, and catalase inhibited the DNA damage. Typical hydroxyl radical scavengers did not inhibit the DNA damage. These results suggest that ROS derived from the reaction of H{sub 2}O{sub 2} with Cu(I) participate in the DNA damage. In conclusion, 2,3-DHBA induces oxidative DNA damage and mutations, which may result in carcinogenesis.

Damage to cellular and isolated DNA induced by a metabolite of aspirin

International Nuclear Information System (INIS)

Oikawa, Shinji; Kobayashi, Hatasu; Tada-Oikawa, Saeko; Isono, Yoshiaki; Kawanishi, Shosuke

2009-01-01

Aspirin has been proposed as a possible chemopreventive agent. On the other hand, a recent cohort study showed that aspirin may increase the risk for pancreatic cancer. To clarify whether aspirin is potentially carcinogenic, we investigated the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), which is correlated with the incidence of cancer, in cultured cells treated with 2,3-dihydroxybenzoic acid (2,3-DHBA), a metabolite of aspirin. 2,3-DHBA induced 8-oxodG formation in the PANC-1 human pancreatic cancer cell line. 2,3-DHBA-induced DNA single-strand breaks were also revealed by comet assay using PANC-1 cells. Flow cytometric analyses showed that 2,3-DHBA increased the levels of intracellular reactive oxygen species (ROS) in PANC-1 cells. The 8-oxodG formation and ROS generation were also observed in the HL-60 leukemia cell line, but not in the hydrogen peroxide (H 2 O 2 )-resistant clone HP100 cells, suggesting the involvement of H 2 O 2 . In addition, an hprt mutation assay supported the mutagenicity of 2,3-DHBA. We investigated the mechanism underlying the 2,3-DHBA-induced DNA damage using 32 P-labeled DNA fragments of human tumor suppressor genes. 2,3-DHBA induced DNA damage in the presence of Cu(II) and NADH. DNA damage induced by 2,3-DHBA was enhanced by the addition of histone peptide-6 [AKRHRK]. Interestingly, 2,3-DHBA and histone peptide-6 caused base damage in the 5'-ACG-3' and 5'-CCG-3' sequences, hotspots of the p53 gene. Bathocuproine, a Cu(I) chelator, and catalase inhibited the DNA damage. Typical hydroxyl radical scavengers did not inhibit the DNA damage. These results suggest that ROS derived from the reaction of H 2 O 2 with Cu(I) participate in the DNA damage. In conclusion, 2,3-DHBA induces oxidative DNA damage and mutations, which may result in carcinogenesis

DNA Glycosylases Involved in Base Excision Repair May Be Associated with Cancer Risk in BRCA1 and BRCA2 Mutation Carriers

NARCIS (Netherlands)

A. Osorio (Ana); R.L. Milne (Roger); K.B. Kuchenbaecker (Karoline); T. Vaclová (Tereza); G. Pita (Guillermo); R. Alonso (Rosario); P. Peterlongo (Paolo); I. Blanco (Ignacio); M. de La Hoya (Miguel); M. Durán (Mercedes); O. Díez (Orland); T. Ramon Y Cajal; I. Konstantopoulou (I.); C. Martínez-Bouzas (Cristina); R. Andrés Conejero (Raquel); P. Soucy (Penny); L. McGuffog (Lesley); D. Barrowdale (Daniel); A. Lee (Andrew); B. Arver (Brita Wasteson); J. Rantala (Johanna); N. Loman (Niklas); H. Ehrencrona (Hans); O.I. Olopade (Olofunmilayo); M.S. Beattie (Mary); S.M. Domchek (Susan); K.L. Nathanson (Katherine); R. Rebbeck (Timothy); B.K. Arun (Banu); B.Y. Karlan (Beth); C.S. Walsh (Christine); K.J. Lester (Kathryn); E.M. John (Esther); A.S. Whittemore (Alice); M.B. Daly (Mary); M.C. Southey (Melissa); J.L. Hopper (John); M.-B. Terry (Mary-Beth); S.S. Buys (Saundra); R. Janavicius (Ramunas); C.M. Dorfling (Cecilia); E.J. van Rensburg (Elizabeth); L. Steele (Linda); S.L. Neuhausen (Susan); Y.C. Ding (Yuan); T.V.O. Hansen (Thomas); L. Jønson (Lars); B. Ejlertsen (Bent); A-M. Gerdes (Anne-Marie); J. Infante (Jon); B. Herráez (Belén); L.T. Moreno (Leticia Thais); J.N. Weitzel (Jeffrey); J. Herzog (Josef); K. Weeman (Kisa); S. Manoukian (Siranoush); B. Peissel (Bernard); D. Zaffaroni (D.); G. Scuvera (Giulietta); B. Bonnani (Bernardo); F. Mariette (F.); S. Volorio (Sara); A. Viel (Alessandra); L. Varesco (Liliana); L. Papi (Laura); L. Ottini (Laura); M.G. Tibiletti (Maria Grazia); P. Radice (Paolo); D. Yannoukakos (Drakoulis); J. Garber; S.D. Ellis (Steve); D. Frost (Debra); R. Platte (Radka); E. Fineberg (Elena); D.G. Evans (Gareth); F. Lalloo (Fiona); L. Izatt (Louise); R. Eeles (Rosalind); J.W. Adlard (Julian); R. Davidson (Rosemarie); T.J. Cole (Trevor); D. Eccles (Diana); J. Cook (Jackie); S.V. Hodgson (Shirley); C. Brewer (Carole); M. Tischkowitz (Marc); F. Douglas (Fiona); M.E. Porteous (Mary); L. Side (Lucy); L.J. Walker (Lisa); P.J. Morrison (Patrick); A. Donaldson (Alan); J. Kennedy (John); C. Foo (Claire); A.K. Godwin (Andrew); R.K. Schmutzler (Rita); B. Wapenschmidt (Barbara); K. Rhiem (Kerstin); C.W. Engel (Christoph); A. Meindl (Alfons); N. Ditsch (Nina); N. Arnold (Norbert); H. Plendl (Hansjoerg); D. Niederacher (Dieter); C. Sutter (Christian); S. Wang-Gohrke (Shan); D. Steinemann (Doris); S. Preisler-Adams (Sabine); K. Kast (Karin); R. Varon-Mateeva (Raymonda); P.A. Gehrig (Paola A.); D. Stoppa-Lyonnet (Dominique); O. Sinilnikova (Olga); S. Mazoyer (Sylvie); F. Damiola (Francesca); B. Poppe (Bruce); K. Claes (Kathleen); M. Piedmonte (Marion); K. Tucker (Kathryn); F.J. Backes (Floor); P.M. Rodríguez; W. Brewster (Wendy); K. Wakeley (Katie); T. Rutherford (Thomas); T. Caldes (Trinidad); H. Nevanlinna (Heli); K. Aittomäki (Kristiina); M.A. Rookus (Matti); T.A.M. van Os (Theo); L. van der Kolk (Lizet); J.L. de Lange (J.); E.J. Meijers-Heijboer (Hanne); A.H. van der Hout (Annemarie); C.J. van Asperen (Christi); E.B. Gómez García (Encarna); N. Hoogerbrugge (Nicoline); J.M. Collée (Margriet); C.H.M. van Deurzen (Carolien); R.B. van der Luijt (Rob); P. Devilee (Peter); E. Olah (Edith); C. Lazaro (Conxi); A. Teulé (A.); M. Menéndez (Mireia); A. Jakubowska (Anna); C. Cybulski (Cezary); J. Gronwald (Jacek); J. Lubinski (Jan); K. Durda (Katarzyna); K. Jaworska-Bieniek (Katarzyna); O.T. Johannson (Oskar); C. Maugard; M. Montagna (Marco); S. Tognazzo (Silvia); P.J. Teixeira; S. Healey (Sue); C. Olswold (Curtis); L. Guidugli (Lucia); N.M. Lindor (Noralane); S. Slager (Susan); C. Szabo (Csilla); J. Vijai (Joseph); M. Robson (Mark); N. Kauff (Noah); L. Zhang (Lingling); R. Rau-Murthy (Rohini); A. Fink-Retter (Anneliese); C.F. Singer (Christian); C. Rappaport (Christine); D. Geschwantler Kaulich (Daphne); G. Pfeiler (Georg); M.-K. Tea; A. Berger (Annemarie); C. Phelan (Catherine); M.H. Greene (Mark); P.L. Mai (Phuong); F. Lejbkowicz (Flavio); I.L. Andrulis (Irene); A.M. Mulligan (Anna Marie); G. Glendon (Gord); A.E. Toland (Amanda); S.E. Bojesen (Stig); I.S. Pedersen (Inge Sokilde); L. Sunde (Lone); M. Thomassen (Mads); T.A. Kruse (Torben); U.B. Jensen; E. Friedman (Eitan); Y. Laitman (Yael); S.P. Shimon (Shani Paluch); J. Simard (Jacques); D.F. Easton (Douglas); K. Offit (Kenneth); F.J. Couch (Fergus); G. Chenevix-Trench (Georgia); A.C. Antoniou (Antonis); J. Benítez (Javier)

2014-01-01

textabstractSingle Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between

Involvement of aberrant DNA methylation on reduced expression of lysophosphatidic acid receptor-1 gene in rat tumor cell lines

International Nuclear Information System (INIS)

Tsujiuchi, Toshifumi; Shimizu, Kyoko; Onishi, Mariko; Sugata, Eriko; Fujii, Hiromasa; Mori, Toshio; Honoki, Kanya; Fukushima, Nobuyuki

2006-01-01

Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors. Recently, it has been reported that alterations of LPA receptor expression might be important in the malignant transformation of tumor cells. Therefore, to assess an involvement of DNA methylation in reduced expression of the LPA receptor-1 (lpa1) gene, we investigated the expression of the lpa1 gene and its DNA methylation patterns in rat tumor cell lines. Both rat brain-derived neuroblastoma B103 and liver-derived hepatoma RH7777 cells used in this study indicated no expression of lpa1. For the analysis of methylation status, bisulfite sequencing was performed with B103 and RH7777 cells, comparing with other lpa1 expressed cells and normal tissues of brain and liver. The lpa1 expressed cells and tissues were all unmethylated in this region of lpa1. In contrast, both B103 and RH7777 cells were highly methylated, correlating with reduced expression of the lpa1. Treatment with 5-aza 2'-deoxycytidine induced expression of lpa1 gene in B103 and RH7777 cells after 24 h. In RH7777 cells treated with 5-aza 2'-deoxycytidine, stress fiber formation was also observed in response to LPA in RH7777 cells, but not in untreated RH7777 cells. These results suggest that aberrant DNA methylation of the lpa1 gene may be involved in its reduced expression in rat tumor cells

Spectroscopic studies on the interactions of 5-ethyl-6-phenyl-3,8-bis((3-aminoalkyl)propanamido)phenanthridin-5-ium derivatives with G-quadruplex DNA

Science.gov (United States)

Yalçın, Ergin; Duyar, Halil; Ihmels, Heiko; Seferoğlu, Zeynel

2018-05-01

An improved microwave-induced synthesis of five ethidium derivatives (Ethidium derivatives, 2a-d) is presented. As the derivatives 2a-d have been proposed previously to be telomerase inhibitors, the binding interactions of these ethidium derivatives with G-quadruplex DNA were evaluated by means of photometric and fluorimetric titration, thermal DNA denaturation, CD and 1H NMR spectroscopy. In particular, the compound bearing 3,8-bis(pyrrolidin-1-yl)propanamido substituent 2a exhibits high selectivity for G-quadruplex DNA relative to duplex DNA.

A small interfering RNA screen of genes involved in DNA repair identifies tumor-specific radiosensitization by POLQ knockdown

DEFF Research Database (Denmark)

Higgins, Geoff S; Prevo, Remko; Lee, Yin-Fai

2010-01-01

The effectiveness of radiotherapy treatment could be significantly improved if tumor cells could be rendered more sensitive to ionizing radiation (IR) without altering the sensitivity of normal tissues. However, many of the key therapeutically exploitable mechanisms that determine intrinsic tumor...... radiosensitivity are largely unknown. We have conducted a small interfering RNA (siRNA) screen of 200 genes involved in DNA damage repair aimed at identifying genes whose knockdown increased tumor radiosensitivity. Parallel siRNA screens were conducted in irradiated and unirradiated tumor cells (SQ20B......) and irradiated normal tissue cells (MRC5). Using gammaH2AX foci at 24 hours after IR, we identified several genes, such as BRCA2, Lig IV, and XRCC5, whose knockdown is known to cause increased cell radiosensitivity, thereby validating the primary screening end point. In addition, we identified POLQ (DNA...

DNA repair pathways involved in determining the level of cytotoxicity of environmentally relevant UV radiation

International Nuclear Information System (INIS)

Carpenter, L.

2000-01-01

The sensitivity of cell lines with defects in various DNA repair processes to different wavelengths of UV has been assessed in order to determine the importance of these repair pathways to the cytotoxicity of UV light. The cell lines used in this work were xrs-6 (a Chinese Hamster Ovary (CHO) cell line) mutant for XRCC5/Ku80, EM9 a CHO cell line mutant for XRCC1, UV61 a CHO cell line mutant for ERCC6/CSB, and E3p53-/-, a mouse embryonic fibroblast cell line null for p53. Xrs-6 (defective in Non Homologous End-Joining) was found to be sensitive to the cytotoxic effects of broadband UVA, but not narrowband UVA or narrowband UVB. EM9 (defective in Base Excision Repair/Single-Strand Break Repair) was not sensitive to the cytotoxic effects of both broadband and narrowband UVA, narrowband UVB or narrowband UVC. UV61 (defective in the Transcription Coupled Repair branch of Nucleotide Excision Repair) was sensitive to the cytotoxic effects of narrowband UVA, UVB and UVC. E3p53-/- was sensitive to the cytotoxic effects of narrowband UVA and UVB. Broadband UVA was found to induce high levels of chromosomal damage in xrs-6, as quantified by the micronucleus assay, most likely as a result of this cell lines inability to repair DNA double strand breaks. EM9 was found to be defective in the repair of broadband UVA-induced single strand breaks, as measured by the alkaline gel electrophoresis ('comet') assay. UV61 was unable to repair broadband UVB-induced DNA damage as measured by the alkaline gel electrophoresis ('comet') assay. These results provide evidence that: 1. DNA double-strand breaks contribute to the cytotoxicity of UVA to a greater extent than single-strand breaks. 2. Repair mechanisms that operate in response to UVA may be coupled to transcription. 3. UVB may directly induce SSBs. 4. P53 is involved in the response of the cell to both UVA and UVB radiation. (author)

DNA methylation patterns of candidate genes regulated by thymine DNA glycosylase in patients with TP53 germline mutations

Energy Technology Data Exchange (ETDEWEB)

Fortes, F.P. [CIPE, Laboratrio de Oncogentica Molecular, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Kuasne, H. [CIPE, Laboratrio NeoGene, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Departamento de Urologia, Faculdade de Medicina, Universidade Estadual Paulista, Botucatu, SP (Brazil); Marchi, F.A. [CIPE, Laboratrio NeoGene, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Programa Inter-Institucional em Bioinformtica, Instituto de Matemtica e Estatstica, Universidade So Paulo, So Paulo, SP (Brazil); Miranda, P.M. [CIPE, Laboratrio NeoGene, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Rogatto, S.R. [CIPE, Laboratrio NeoGene, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Departamento de Urologia, Faculdade de Medicina, Universidade Estadual Paulista, Botucatu, SP (Brazil); Achatz, M.I. [CIPE, Laboratrio de Oncogentica Molecular, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Departamento de Oncogentica, A.C. Camargo Cancer Center, So Paulo, SP (Brazil)

2015-04-28

Li-Fraumeni syndrome (LFS) is a rare, autosomal dominant, hereditary cancer predisposition disorder. In Brazil, the p.R337H TP53 founder mutation causes the variant form of LFS, Li-Fraumeni-like syndrome. The occurrence of cancer and age of disease onset are known to vary, even in patients carrying the same mutation, and several mechanisms such as genetic and epigenetic alterations may be involved in this variability. However, the extent of involvement of such events has not been clarified. It is well established that p53 regulates several pathways, including the thymine DNA glycosylase (TDG) pathway, which regulates the DNA methylation of several genes. This study aimed to identify the DNA methylation pattern of genes potentially related to the TDG pathway (CDKN2A, FOXA1, HOXD8, OCT4, SOX2, and SOX17) in 30 patients with germline TP53mutations, 10 patients with wild-type TP53, and 10 healthy individuals. We also evaluated TDG expression in patients with adrenocortical tumors (ADR) with and without the p.R337H TP53 mutation. Gene methylation patterns of peripheral blood DNA samples assessed by pyrosequencing revealed no significant differences between the three groups. However, increased TDG expression was observed by quantitative reverse transcription PCR in p.R337H carriers with ADR. Considering the rarity of this phenotype and the relevance of these findings, further studies using a larger sample set are necessary to confirm our results.

DNA glycosylases involved in base excision repair may be associated with cancer risk in BRCA1 and BRCA2 mutation carriers

NARCIS (Netherlands)

Osorio, A.; Milne, R.L.; Kuchenbaecker, K.; Vaclova, T.; Pita, G.; Alonso, R.; Peterlongo, P.; Blanco, I.; Hoya, M. de la; Duran, M.; Diez, O.; Ramon, Y.C.T.; Konstantopoulou, I.; Martinez-Bouzas, C.; Conejero, R. Andres; Soucy, P.; McGuffog, L.; Barrowdale, D.; Lee, A.; Swe, B.; Arver, B.; Rantala, J.; Loman, N.; Ehrencrona, H.; Olopade, O.I.; Beattie, M.S.; Domchek, S.M.; Nathanson, K.; Rebbeck, T.R.; Arun, B.K.; Karlan, B.Y.; Walsh, C.; Lester, J.; John, E.M.; Whittemore, A.S.; Daly, M.B.; Southey, M.; Hopper, J.; Terry, M.B.; Buys, S.S.; Janavicius, R.; Dorfling, C.M.; Rensburg, E.J. van; Steele, L.; Neuhausen, S.L.; Ding, Y.C.; Hansen, T.V.; Jonson, L.; Ejlertsen, B.; Gerdes, A.M.; Infante, M.; Herraez, B.; Moreno, L.T.; Weitzel, J.N.; Herzog, J.; Weeman, K.; Manoukian, S.; Peissel, B.; Zaffaroni, D.; Scuvera, G.; Bonanni, B.; Mariette, F.; Volorio, S.; Viel, A.; Varesco, L.; Papi, L.; Ottini, L.; Tibiletti, M.G.; Radice, P.; Yannoukakos, D.; Garber, J.; Ellis, S.; Frost, D.; Platte, R.; Fineberg, E.; Evans, G.; Lalloo, F.; Izatt, L.; Eeles, R.; Adlard, J.; Davidson, R.; Cole, T.; Eccles, D.; Cook, J; Hodgson, S.; Brewer, C.; Tischkowitz, M.; Douglas, F.; Porteous, M.; Side, L.; Walker, L.; Morrison, P.; Donaldson, A.; Kennedy, J.; Foo, C.; Godwin, A.K.; Schmutzler, R.K.; Wappenschmidt, B.; Rhiem, K.; Engel, C.; Hoogerbrugge-van der Linden, N.; et al.,

2014-01-01

Single Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of the

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

Detection of endonuclease III- and 8-oxoguanine glycosylase-sensitive base modifications in γ-irradiated DNA and cells by the aldehyde reactive probe (ARP) assay

International Nuclear Information System (INIS)

Mohsin Ali, M.; Kurisu, Satofumi; Yoshioka, Yoshihiro; Terato, Hiroaki; Ohyama, Yoshihiko; Ide Hiroshi; Kubo, Kihei

2004-01-01

Ionizing radiation generates diverse DNA lesions that differentially induce cell death and mutations. In the present study, calf thymus DNA (400 μg/ml) and HeLa cells were irradiated by 60 Co γ-rays, and abasic (AP) sites and endonuclease (Endo) III- and 8-oxoguanine glycosylase (hOGG1)-sensitive base modifications in DNA were quantitated by the aldehyde reactive probe (ARP) assay. The irradiation of calf thymus DNA in phosphate buffer generated 91 Endo III- and 100 hOGG1-sensitive base modifications and 110 AP sites per 10 6 base pairs (bp) per Gy. The yield of the lesions in Tris buffer was 41- to 91-fold lower than that in phosphate, demonstrating a radioprotective effect of Tris. The HeLa cell chromosomal DNA contained 12 Endo III- and 3.8 hOGG1-sensitive base modifications and less than 1 AP sites per 10 6 bp as endogenous damage, and their level was increased by irradiation. The yields of the damage at 1 Gy (roughly equivalent to the lethal dose of HeLa cells [1.6-1.8 Gy]) were 0.13 Endo III, 0.091 hOGG1, and 0.065 AP sites per 10 6 bp, showing that irradiation with a lethal dose brought about only a marginal increase in base damage relative to an endogenous one. A comparison of the present data with those reported for DNA strand breaks supports the primary importance of double-strand breaks and clustered lesions as lethal damages formed by ionizing radiation. (author)

TLR1/2 activation during heterologous prime-boost vaccination (DNA-MVA enhances CD8+ T Cell responses providing protection against Leishmania (Viannia.

Directory of Open Access Journals (Sweden)

Asha Jayakumar

2011-06-01

Full Text Available Leishmania (Viannia parasites present particular challenges, as human and murine immune responses to infection are distinct from other Leishmania species, indicating a unique interaction with the host. Further, vaccination studies utilizing small animal models indicate that modalities and antigens that prevent infection by other Leishmania species are generally not protective.Using a newly developed mouse model of chronic L. (Viannia panamensis infection and the heterologous DNA prime - modified vaccinia virus Ankara (MVA boost vaccination modality, we examined whether the conserved vaccine candidate antigen tryparedoxin peroxidase (TRYP could provide protection against infection/disease.Heterologous prime - boost (DNA/MVA vaccination utilizing TRYP antigen can provide protection against disease caused by L. (V. panamensis. However, protection is dependent on modulating the innate immune response using the TLR1/2 agonist Pam3CSK4 during DNA priming. Prime-boost vaccination using DNA alone fails to protect. Prior to infection protectively vaccinated mice exhibit augmented CD4 and CD8 IFNγ and memory responses as well as decreased IL-10 and IL-13 responses. IL-13 and IL-10 have been shown to be independently critical for disease in this model. CD8 T cells have an essential role in mediating host defense, as CD8 depletion reversed protection in the vaccinated mice; vaccinated mice depleted of CD4 T cells remained protected. Hence, vaccine-induced protection is dependent upon TLR1/2 activation instructing the generation of antigen specific CD8 cells and restricting IL-13 and IL-10 responses.Given the general effectiveness of prime-boost vaccination, the recalcitrance of Leishmania (Viannia to vaccine approaches effective against other species of Leishmania is again evident. However, prime-boost vaccination modality can with modulation induce protective responses, indicating that the delivery system is critical. Moreover, these results suggest that

Menadione-induced DNA fragmentation without 8-hydroxy-2'-deoxyguanosine formation in isolated rat hepatocytes

DEFF Research Database (Denmark)

Fischer-Nielsen, Anne; Corcoran, George B.; Poulsen, Henrik E.

1995-01-01

Farmakologi, frie iltradikaler, menadion, DNA fragmentering, rotteleverceller, oksidativ DNA skade......Farmakologi, frie iltradikaler, menadion, DNA fragmentering, rotteleverceller, oksidativ DNA skade...

Laboratory of Mutagenesis and DNA Repair

International Nuclear Information System (INIS)

2000-01-01

Full text: Two main lines of research were continued: the first one concerned the mechanisms controlling the fidelity of DNA replication in Escherichia coli; the second concerned cellular responses of Saccharomyces cerevisiae to DNA damaging agents. We have been investigating the question whether during chromosomal DNA replication in Escherichia coli the two DNA strands may be replicated with differential accuracy. To address this question we set up a new system that allows the examination of mutagenesis either of the leading strand or the lagging strand. Our results suggest that the lagging strand replication of the E. coli chromosome may be more accurate than leading strand replication. More recently, we studied mutagenesis of the two strands in recA730 strains which exhibit constitutive expression of the SOS system. Our results clearly indicate that in recA730 strains there is a significant difference in the fidelity of replication between the two replicating strands. Based on our data we propose a model describing a possible mechanism of SOS mutagenesis. To get more insight into cellular responses to DNA damage we have isolated several novel genes of S. cerevisiae, the transcription of which is induced by DNA lesions. Main effort was concentrated on the characterization of the DIN7 gene. We found that Din7p specifically affects the metabolism of mitochondrial DNA (mtDNA). The elevated level of Din7p results in an increased frequency of mitochondrial petite mutants, as well as in a higher frequency of mitochondrial point mutations. Din7p affects also the stability of microsatellite sequences present in the mitochondrial genome. As expected, Din7p was found to be located in mitochondria. In another project, we found that the DIN8 gene isolated in our laboratory is identical with the UMP1 gene encoding a chaperone-like protein involved in 20S proteasome maturation. Interestingly, induction of UMP1 expression in response to DNA damage is subject to regulation

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.

Enzymatic recognition of DNA damage induced by UVB-photosensitized titanium dioxide and biological consequences in Saccharomyces cerevisiae: evidence for oxidatively DNA damage generation.

Science.gov (United States)

Pinto, A Viviana; Deodato, Elder L; Cardoso, Janine S; Oliveira, Eliza F; Machado, Sérgio L; Toma, Helena K; Leitão, Alvaro C; de Pádula, Marcelo

2010-06-01

Although titanium dioxide (TiO(2)) has been considered to be biologically inert, finding use in cosmetics, paints and food colorants, recent reports have demonstrated that when TiO(2) is attained by UVA radiation oxidative genotoxic and cytotoxic effects are observed in living cells. However, data concerning TiO(2)-UVB association is poor, even if UVB radiation represents a major environmental carcinogen. Herein, we investigated DNA damage, repair and mutagenesis induced by TiO(2) associated with UVB irradiation in vitro and in vivo using Saccharomyces cerevisiae model. It was found that TiO(2) plus UVB treatment in plasmid pUC18 generated, in addition to cyclobutane pyrimidine dimers (CPDs), specific damage to guanine residues, such as 8-oxo-7,8-dihydroguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), which are characteristic oxidatively generated lesions. In vivo experiments showed that, although the presence of TiO(2) protects yeast cells from UVB cytotoxicity, high mutation frequencies are observed in the wild-type (WT) and in an ogg1 strain (deficient in 8-oxoG and FapyG repair). Indeed, after TiO(2) plus UVB treatment, induced mutagenesis was drastically enhanced in ogg1 cells, indicating that mutagenic DNA lesions are repaired by the Ogg1 protein. This effect could be attenuated by the presence of metallic ion chelators: neocuproine or dipyridyl, which partially block oxidatively generated damage occurring via Fenton reactions. Altogether, the results indicate that TiO(2) plus UVB potentates UVB oxidatively generated damage to DNA, possibly via Fenton reactions involving the production of DNA base damage, such as 8-oxo-7,8-dihydroguanine. Copyright 2010 Elsevier B.V. All rights reserved.

Enzymatic recognition of DNA damage induced by UVB-photosensitized titanium dioxide and biological consequences in Saccharomyces cerevisiae: Evidence for oxidatively DNA damage generation

Energy Technology Data Exchange (ETDEWEB)

Pinto, A. Viviana, E-mail: alicia.pinto@incqs.fiocruz.br [Laboratorio de Diagnostico Molecular e Hematologia, Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21941-540, Rio de Janeiro (Brazil); Laboratorio de Radiobiologia Molecular, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21949-900, Rio de Janeiro (Brazil); Deodato, Elder L. [Laboratorio de Diagnostico Molecular e Hematologia, Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21941-540, Rio de Janeiro (Brazil); Laboratorio de Radiobiologia Molecular, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21949-900, Rio de Janeiro (Brazil); Cardoso, Janine S. [Laboratorio de Radiobiologia Molecular, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21949-900, Rio de Janeiro (Brazil); Oliveira, Eliza F.; Machado, Sergio L.; Toma, Helena K. [Laboratorio de Diagnostico Molecular e Hematologia, Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21941-540, Rio de Janeiro (Brazil); Leitao, Alvaro C. [Laboratorio de Radiobiologia Molecular, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21949-900, Rio de Janeiro (Brazil); Padula, Marcelo de [Laboratorio de Diagnostico Molecular e Hematologia, Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21941-540, Rio de Janeiro (Brazil)

2010-06-01

Although titanium dioxide (TiO{sub 2}) has been considered to be biologically inert, finding use in cosmetics, paints and food colorants, recent reports have demonstrated that when TiO{sub 2} is attained by UVA radiation oxidative genotoxic and cytotoxic effects are observed in living cells. However, data concerning TiO{sub 2}-UVB association is poor, even if UVB radiation represents a major environmental carcinogen. Herein, we investigated DNA damage, repair and mutagenesis induced by TiO{sub 2} associated with UVB irradiation in vitro and in vivo using Saccharomyces cerevisiae model. It was found that TiO{sub 2} plus UVB treatment in plasmid pUC18 generated, in addition to cyclobutane pyrimidine dimers (CPDs), specific damage to guanine residues, such as 8-oxo-7,8-dihydroguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), which are characteristic oxidatively generated lesions. In vivo experiments showed that, although the presence of TiO{sub 2} protects yeast cells from UVB cytotoxicity, high mutation frequencies are observed in the wild-type (WT) and in an ogg1 strain (deficient in 8-oxoG and FapyG repair). Indeed, after TiO{sub 2} plus UVB treatment, induced mutagenesis was drastically enhanced in ogg1 cells, indicating that mutagenic DNA lesions are repaired by the Ogg1 protein. This effect could be attenuated by the presence of metallic ion chelators: neocuproine or dipyridyl, which partially block oxidatively generated damage occurring via Fenton reactions. Altogether, the results indicate that TiO{sub 2} plus UVB potentates UVB oxidatively generated damage to DNA, possibly via Fenton reactions involving the production of DNA base damage, such as 8-oxo-7,8-dihydroguanine.

Serum Level of Antibody against Benzo[a]pyrene-7,8-diol-9,10-epoxide-DNA Adducts in People Dermally Exposed to PAHs

Directory of Open Access Journals (Sweden)

Lenka Borska

2014-01-01

Full Text Available Some specific antibodies indicate the presence of antigenic structures on DNA (DNA adducts that can play an important role in the process of mutagenesis and/or carcinogenesis. They indicate the presence of increased genotoxic potential (hazard prior to the formation of disease (primary prevention. The present study was focused on the serum level of benzo[a]pyrene 7,8-diol-9,10-epoxide-DNA adducts antibodies (anti-BPDE-DNA in psoriatic patients (n=55 dermally exposed to different levels of polycyclic aromatic hydrocarbons (PAHs. The general goal of the study was to contribute to better understanding of the value of the assumed biomarker (anti-BPDE-DNA for evaluation of the organism's answer to genotoxic exposure to PAHs. Elevated level of exposure to PAHs resulted in the increased level of anti-BPDE-DNA. However, almost all levels of anti-BPDE-DNA ranged within the field of low values. Both variants of GT (CCT-3% and CCT-5% induced higher expression of anti-BPDE-DNA in the group of nonsmokers. Significant relations between the level of anti-BPDE-DNA and PASI score, total duration of the therapy, or time of UVR exposure were not found. Further studies are needed to reduce interpretation uncertainty of this promising bioindicator.

Nucleotide sequence determination of the region in adenovirus 5 DNA involved in cell transformation

International Nuclear Information System (INIS)

Maat, J.

1978-01-01

A description is given of investigations into the primary structure of the transforming region of adenovirus type 5 DNA. The phenomenon of cell transformation is discussed in general terms and the principles of a number of fairly recent techniques, which have been in use for DNA sequence determination since 1975 are dealt with. A few of the author's own techniques are described which deal both with nucleotide sequence analysis and with the determination of DNA cleavage sites of restriction endonucleases. The results are given of the mapping of cleavage sites in the HpaI-E fragment of adenovirus DNA of HpaII, HaeIII, AluI, HinfI and TaqI and of the determination of the nucleotide sequence in the transforming region of adenovirus type 5 DNA. The results of the sequence determination of the Ad5 HindIII-G fragment are discussed in relation with the investigation on the transforming proteins isolated from in vitro and in vivo synthesizing systems. Labelling procedures of DNA are described including the exonuclease III/DNA polymerase 1 method and TA polynucleotide kinase labelling of DNA fragments. (Auth.)

[DNA damage in human pleural mesothelial cells induced by exposure to carbon nanotubes].

Science.gov (United States)

Ogasawara, Yuki; Umezu, Noriaki; Ishii, Kazuyuki

2012-01-01

Nanomaterials are currently used in electronics, industrial materials, cosmetics, and medicine because they have useful physicochemical properties, such as strength, conductivity, durability, and chemical stability. As these materials have become widespread, many questions have arisen regarding their effects on health and the environment. In particular, recent studies have demonstrated that carbon nanotubes (CNTs) cause significant inflammation and mesothelioma in vivo. In this study, we investigated the potential risk posed by singlewalled carbon nanotube (SWCNT) and multiwalled carbon nanotube (MWCNT) exposure in human pleural mesothelial cells. CNT cytotoxicity was determined by a trypan blue exclusion assay, and DNA damage was detected by an alkaline comet assay. The concentration of 8-oxodeoxyguanosine (8-OHdG) in DNA was measured by high perhormance liquid chromatography with electrochemical detection. The expression of base excision repair enzymes in the cell was estimated by immunoblot analysis. We observed inhibitory effects on cell proliferation and the induction of DNA damage following exposure of cells to purified CNTs that were suspended in dispersion medium. However, accumulation of 8-OHdG in DNA was not found. In addition, the expression levels of base excision enzymes that are involved in hOGG1, hMTH1, and MYH in MeT-5A cells remained unchanged for 24 h after carbon nanotube exposure. CNTs significantly inhibit cell proliferation and decrease DNA damage in human pleural mesothelial cells. Our results indicate that the mechanism of CNT-induced genotoxicity is different from that following exposure to reactive oxygen species, which causes oxidative DNA modifications and 8-OHdG production. Further investigation is required to characterize the specific DNA mutations that occur following CNT exposure.

Efficient DNA ligation in DNA–RNA hybrid helices by Chlorella virus DNA ligase

Science.gov (United States)

Lohman, Gregory J. S.; Zhang, Yinhua; Zhelkovsky, Alexander M.; Cantor, Eric J.; Evans, Thomas C.

2014-01-01

Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with kcat ≈ 8 x 10−3 s−1 and KM DNA ligase produced only 5′-adenylylated DNA with a 20-fold lower kcat and a KM ≈ 300 nM. The rate of ligation increased with addition of Mn2+, but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5′-phosphorylated dC or dG residue on the 3′ side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA. PMID:24203707

Involvement of DNA methylation in the control of cell growth during heat stress in tobacco BY-2 cells.

Science.gov (United States)

Centomani, Isabella; Sgobba, Alessandra; D'Addabbo, Pietro; Dipierro, Nunzio; Paradiso, Annalisa; De Gara, Laura; Dipierro, Silvio; Viggiano, Luigi; de Pinto, Maria Concetta

2015-11-01

The alteration of growth patterns, through the adjustment of cell division and expansion, is a characteristic response of plants to environmental stress. In order to study this response in more depth, the effect of heat stress on growth was investigated in tobacco BY-2 cells. The results indicate that heat stress inhibited cell division, by slowing cell cycle progression. Cells were stopped in the pre-mitotic phases, as shown by the increased expression of CycD3-1 and by the decrease in the NtCycA13, NtCyc29 and CDKB1-1 transcripts. The decrease in cell length and the reduced expression of Nt-EXPA5 indicated that cell expansion was also inhibited. Since DNA methylation plays a key role in controlling gene expression, the possibility that the altered expression of genes involved in the control of cell growth, observed during heat stress, could be due to changes in the methylation state of their promoters was investigated. The results show that the altered expression of CycD3-1 and Nt-EXPA5 was consistent with changes in the methylation state of the upstream region of these genes. These results suggest that DNA methylation, controlling the expression of genes involved in plant development, contributes to growth alteration occurring in response to environmental changes.

DNA-binding proteins essential for protein-primed bacteriophage ø29 DNA replication

Directory of Open Access Journals (Sweden)

Margarita Salas

2016-08-01

Full Text Available Bacillus subtilis phage Φ29 has a linear, double-stranded DNA 19 kb long with an inverted terminal repeat of 6 nucleotides and a protein covalently linked to the 5’ ends of the DNA. This protein, called terminal protein (TP, is the primer for the initiation of replication, a reaction catalyzed by the viral DNA polymerase at the two DNA ends. The DNA polymerase further elongates the nascent DNA chain in a processive manner, coupling strand displacement with elongation. The viral protein p5 is a single-stranded DNA binding protein (SSB that binds to the single strands generated by strand displacement during the elongation process. Viral protein p6 is a double-stranded DNA binding protein (DBP that preferentially binds to the origins of replication at the Φ29 DNA ends and is required for the initiation of replication. Both SSB and DBP are essential for Φ29 DNA amplification. This review focuses on the role of these phage DNA-binding proteins in Φ29 DNA replication both in vitro and in vivo, as well as on the implication of several B. subtilis DNA-binding proteins in different processes of the viral cycle. We will revise the enzymatic activities of the Φ29 DNA polymerase: TP-deoxynucleotidylation, processive DNA polymerization coupled to strand displacement, 3’-5’ exonucleolysis and pyrophosphorolysis. The resolution of the Φ29 DNA polymerase structure has shed light on the translocation mechanism and the determinants responsible for processivity and strand displacement. These two properties have made Φ29 DNA polymerase one of the main enzymes used in the current DNA amplification technologies. The determination of the structure of Φ29 TP revealed the existence of three domains: the priming domain, where the primer residue Ser232, as well as Phe230, involved in the determination of the initiating nucleotide, are located, the intermediate domain, involved in DNA polymerase binding, and the N-terminal domain, responsible for DNA binding

Involvement of two microRNAs in the early immune response to DNA vaccination against a fish rhabdovirus

DEFF Research Database (Denmark)

Bela-ong, Dennis Berbulla; Schyth, Brian Dall; Zou, Jun

2015-01-01

Mechanisms that account for the high protective efficacy in teleost fish of a DNA vaccine expressing the glycoprotein (G) of Viral hemorrhagic septicemia virus (VHSV) are thought to involve early innate immune responses mediated by interferons (IFNs). Microribonucleic acids (miRNAs) are a diverse...... class of small (18–22 nucleotides) endogenous RNAs that potently mediate post-transcriptional silencing of a wide range of genes and are emerging as critical regulators of cellular processes, including immune responses. We have recently reported that miR-462 and miR-731 were strongly induced in rainbow......RNAs using anti-miRNA oligonucleotides was conducted in poly I:C-treated rainbow trout fingerlings. Following VHSV challenge, anti-miRNA-injected fish had faster development of disease and higher mortalities than control fish, indicating that miR-462/731 may be involved in IFN-mediated protection conferred...

Effect of 8-MOP plus UVA treatment on survival and repair of plasmid pBR322

International Nuclear Information System (INIS)

Bauluz, C.; Vidania, R. de

1991-01-01

We have studied the lethality produced in pBR322 DNA after PUVA treatment (8-MOP+UVA). As recipients, we used a collection of E. coli strains differing in their repair capacities and analysed the involvement of several DNA repair pathways in the removal of plasmid lesions. We have also studied the effect of UVA radiation alone, in order to determine more precisely the effect attributable only to psoralen molecules. Results showed a strong lethal effect derived from PUVA treatment; however, some plasmid recovery was achieved in bacterial hosts proficient in Excision repair and SOS repair. Another repair pathway, only detectable at high density of lesions, appeared to be relevant for the removal of 8-MOP:DNA adducts.(Author) 11 refs

Intercalation of a Zn(II) complex containing ciprofloxacin drug between DNA base pairs.

Science.gov (United States)

Shahabadi, Nahid; Asadian, Ali Ashraf; Mahdavi, Mryam

2017-11-02

In this study, an attempt has been made to study the interaction of a Zn(II) complex containing an antibiotic drug, ciprofloxacin, with calf thymus DNA using spectroscopic methods. It was found that Zn(II) complex could bind with DNA via intercalation mode as evidenced by: hyperchromism in UV-Vis spectrum; these spectral characteristics suggest that the Zn(II) complex interacts with DNA most likely through a mode that involves a stacking interaction between the aromatic chromophore and the base pairs of DNA. DNA binding constant (K b = 1.4 × 10 4 M -1 ) from spectrophotometric studies of the interaction of Zn(II) complex with DNA is comparable to those of some DNA intercalative polypyridyl Ru(II) complexes 1.0 -4.8 × 10 4 M -1 . CD study showed stabilization of the right-handed B form of DNA in the presence of Zn(II) complex as observed for the classical intercalator methylene blue. Thermodynamic parameters (ΔH DNA-MB, indicating that it binds to DNA in strong competition with MB for the intercalation.

Isolation and characterization of DNA probes from a flow-sorted human chromosome 8 library that detect restriction fragment length polymorphism (RFLP).

Science.gov (United States)

Wood, S; Starr, T V; Shukin, R J

1986-01-01

We have used a recombinant DNA library constructed from flow-sorted human chromosome 8 as a source of single-copy human probes. These probes have been screened for restriction fragment length polymorphism (RFLP) by hybridization to Southern transfers of genomic DNA from five unrelated individuals. We have detected six RFLPs distributed among four probes after screening 741 base pairs for restriction site variation. These RFLPs all behave as codominant Mendelian alleles. Two of the probes detect rare variants, while the other two detect RFLPs with PIC values of .36 and .16. Informative probes will be useful for the construction of a linkage map for chromosome 8 and for the localization of mutant alleles to this chromosome. Images Fig. 1 PMID:2879441

Role of DNA repair in repair of cytogenetic damages. Slowly repaired DNA injuries involved in cytogenetic damages repair

International Nuclear Information System (INIS)

Zaichkina, S.I.; Rozanova, O.M.; Aptikaev, G.F.; Ganassi, E.Eh.

1989-01-01

Caffeine was used to study the kinetics of cytogenetic damages repair in Chinese hamster fibroblasts. Its half-time (90 min) was shown to correlate with that of repair of slowly repaired DNA damages. The caffeine-induced increase in the number of irreparable DNA damages, attributed to inhibition of double-strand break repair, is in a quantitative correlation with the effect of the cytogenetic damage modification

One-electron oxidation reactions of purine and pyrimidine bases in cellular DNA.

Science.gov (United States)

Cadet, Jean; Wagner, J Richard; Shafirovich, Vladimir; Geacintov, Nicholas E

2014-06-01

The aim of this survey is to critically review the available information on one-electron oxidation reactions of nucleobases in cellular DNA with emphasis on damage induced through the transient generation of purine and pyrimidine radical cations. Since the indirect effect of ionizing radiation mediated by hydroxyl radical is predominant in cells, efforts have been made to selectively ionize bases using suitable one-electron oxidants that consist among others of high intensity UVC laser pulses. Thus, the main oxidation product in cellular DNA was found to be 8-oxo-7,8-dihydroguanine as a result of direct bi-photonic ionization of guanine bases and indirect formation of guanine radical cations through hole transfer reactions from other base radical cations. The formation of 8-oxo-7,8-dihydroguanine and other purine and pyrimidine degradation products was rationalized in terms of the initial generation of related radical cations followed by either hydration or deprotonation reactions in agreement with mechanistic pathways inferred from detailed mechanistic studies. The guanine radical cation has been shown to be implicated in three other nucleophilic additions that give rise to DNA-protein and DNA-DNA cross-links in model systems. Evidence was recently provided for the occurrence of these three reactions in cellular DNA. There is growing evidence that one-electron oxidation reactions of nucleobases whose mechanisms have been characterized in model studies involving aqueous solutions take place in a similar way in cells. It may also be pointed out that the above cross-linked lesions are only produced from the guanine radical cation and may be considered as diagnostic products of the direct effect of ionizing radiation.

Administration of HPV DNA vaccine via electroporation elicits the strongest CD8+ T cell immune responses compared to intramuscular injection and intradermal gene gun delivery

Science.gov (United States)

Best, Simon R.; Peng, Shiwen; Juang, Chi-Mou; Hung, Chien-Fu; Hannaman, Drew; Saunders, John R.; Wu, T.-C.; Pai, Sara I.

2009-01-01

DNA vaccines are an attractive approach to eliciting antigen-specific immunity. Intracellular targeting of tumor antigens through its linkage to immunostimulatory molecules such as calreticulin (CRT) can improve antigen processing and presentation through the MHC Class I pathway and increase cytotoxic CD8+ T cell production. However, even with these enhancements, the efficacy of such immunotherapeutic strategies is dependent on the identification of an effective route and method of DNA administration. Electroporation and gene gun-mediated particle delivery are leading methods of DNA vaccine delivery that can generate protective and therapeutic levels of immune responses in experimental models. In this study, we perform a head-to-head comparison of three methods of vaccination – conventional intramuscular injection, electroporation mediated intramuscular delivery, and epidermal gene gun-mediated particle delivery - in the ability to generate antigen specific cytotoxic CD8+ T cell responses as well as anti-tumor immune responses against an HPV-16 E7 expressing tumor cell line using the pNGVL4a-CRT/E7(detox) DNA vaccine. Vaccination via electroporation generated the highest number of E7-specific cytotoxic CD8+ T cells, which correlated to improved outcomes in the treatment of growing tumors. In addition, we demonstrate that electroporation results in significantly higher levels of circulating protein compared to gene gun or intramuscular vaccination, which likely enhances calreticulin’s role as a local tumor anti-angiogenesis agent. We conclude that electroporation is a promising method for delivery of HPV DNA vaccines and should be considered for DNA vaccine delivery in human clinical trials. PMID:19622402

Mitochondrial DNA Depletion in Respiratory Chain–Deficient Parkinson Disease Neurons

Science.gov (United States)

Rygiel, Karolina A.; Hepplewhite, Philippa D.; Morris, Christopher M.; Picard, Martin; Turnbull, Doug M.

2016-01-01

Objective To determine the extent of respiratory chain abnormalities and investigate the contribution of mtDNA to the loss of respiratory chain complexes (CI–IV) in the substantia nigra (SN) of idiopathic Parkinson disease (IPD) patients at the single‐neuron level. Methods Multiple‐label immunofluorescence was applied to postmortem sections of 10 IPD patients and 10 controls to quantify the abundance of CI–IV subunits (NDUFB8 or NDUFA13, SDHA, UQCRC2, and COXI) and mitochondrial transcription factors (TFAM and TFB2M) relative to mitochondrial mass (porin and GRP75) in dopaminergic neurons. To assess the involvement of mtDNA in respiratory chain deficiency in IPD, SN neurons, isolated with laser‐capture microdissection, were assayed for mtDNA deletions, copy number, and presence of transcription/replication‐associated 7S DNA employing a triplex real‐time polymerase chain reaction (PCR) assay. Results Whereas mitochondrial mass was unchanged in single SN neurons from IPD patients, we observed a significant reduction in the abundances of CI and II subunits. At the single‐cell level, CI and II deficiencies were correlated in patients. The CI deficiency concomitantly occurred with low abundances of the mtDNA transcription factors TFAM and TFB2M, which also initiate transcription‐primed mtDNA replication. Consistent with this, real‐time PCR analysis revealed fewer transcription/replication‐associated mtDNA molecules and an overall reduction in mtDNA copy number in patients. This effect was more pronounced in single IPD neurons with severe CI deficiency. Interpretation Respiratory chain dysfunction in IPD neurons not only involves CI, but also extends to CII. These deficiencies are possibly a consequence of the interplay between nDNA and mtDNA‐encoded factors mechanistically connected via TFAM. ANN NEUROL 2016;79:366–378 PMID:26605748

Identification of mitochondrial DNA sequence variation and development of single nucleotide polymorphic markers for CMS-D8 in cotton.

Science.gov (United States)

Suzuki, Hideaki; Yu, Jiwen; Wang, Fei; Zhang, Jinfa

2013-06-01

Cytoplasmic male sterility (CMS), which is a maternally inherited trait and controlled by novel chimeric genes in the mitochondrial genome, plays a pivotal role in the production of hybrid seed. In cotton, no PCR-based marker has been developed to discriminate CMS-D8 (from Gossypium trilobum) from its normal Upland cotton (AD1, Gossypium hirsutum) cytoplasm. The objective of the current study was to develop PCR-based single nucleotide polymorphic (SNP) markers from mitochondrial genes for the CMS-D8 cytoplasm. DNA sequence variation in mitochondrial genes involved in the oxidative phosphorylation chain including ATP synthase subunit 1, 4, 6, 8 and 9, and cytochrome c oxidase 1, 2 and 3 subunits were identified by comparing CMS-D8, its isogenic maintainer and restorer lines on the same nuclear genetic background. An allelic specific PCR (AS-PCR) was utilized for SNP typing by incorporating artificial mismatched nucleotides into the third or fourth base from the 3' terminus in both the specific and nonspecific primers. The result indicated that the method modifying allele-specific primers was successful in obtaining eight SNP markers out of eight SNPs using eight primer pairs to discriminate two alleles between AD1 and CMS-D8 cytoplasms. Two of the SNPs for atp1 and cox1 could also be used in combination to discriminate between CMS-D8 and CMS-D2 cytoplasms. Additionally, a PCR-based marker from a nine nucleotide insertion-deletion (InDel) sequence (AATTGTTTT) at the 59-67 bp positions from the start codon of atp6, which is present in the CMS and restorer lines with the D8 cytoplasm but absent in the maintainer line with the AD1 cytoplasm, was also developed. A SNP marker for two nucleotide substitutions (AA in AD1 cytoplasm to CT in CMS-D8 cytoplasm) in the intron (1,506 bp) of cox2 gene was also developed. These PCR-based SNP markers should be useful in discriminating CMS-D8 and AD1 cytoplasms, or those with CMS-D2 cytoplasm as a rapid, simple, inexpensive, and

Involvement of Opioid System, TRPM8, and ASIC Receptors in Antinociceptive Effect of Arrabidaea brachypoda (DC) Bureau.

Science.gov (United States)

Rodrigues, Vinícius Peixoto; Rocha, Cláudia Quintino da; Périco, Larissa Lucena; Santos, Raquel de Cássia Dos; Ohara, Rie; Nishijima, Catarine Massucato; Ferreira Queiroz, Emerson; Wolfender, Jean-Luc; Rocha, Lúcia Regina Machado da; Santos, Adair Roberto Soares; Vilegas, Wagner; Hiruma-Lima, Clélia Akiko

2017-11-02

Arrabidaea brachypoda (DC) Bureau is a medicinal plant found in Brazil. Known as "cipó-una", it is popularly used as a natural therapeutic agent against pain and inflammation. This study evaluated the chemical composition and antinociceptive activity of the dichloromethane fraction from the roots of A. brachypoda (DEAB) and its mechanism of action. The chemical composition was characterized by high-performance liquid chromatography, and this fraction is composed only of dimeric flavonoids. The antinociceptive effect was evaluated in formalin and hot plate tests after oral administration (10-100 mg/kg) in male Swiss mice. We also investigated the involvement of TRPV1 (transient receptor potential vanilloid 1), TRPA1 (transient receptor potential ankyrin 1), TRPM8 (transient receptor potential melastatin 8), and ASIC (acid-sensing ion channel), as well as the opioidergic, glutamatergic, and supraspinal pathways. Moreover, the nociceptive response was reduced (30 mg/kg) in the early and late phase of the formalin test. DEAB activity appears to involve the opioid system, TRPM8, and ASIC receptors, clearly showing that the DEAB alleviates acute pain in mice and suggesting the involvement of the TRPM8 and ASIC receptors and the opioid system in acute pain relief.

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

Effects of expression level of DNA repair-related genes involved in the NHEJ pathway on radiation-induced cognitive impairment

International Nuclear Information System (INIS)

Zhang Liyuan; Chen Liesong; Sun Rui; Ji Shengjun; Ding Yanyan; Wu Jia; Tian Ye

2013-01-01

Cranial radiation therapy can induce cognitive decline. Impairments of hippocampal neurogenesis are thought to be a paramountly important mechanism underlying radiation-induced cognitive dysfunction. In the mature nervous system, DNA double-strand breaks (DSBs) are mainly repaired by non-homologous end-joining (NHEJ) pathways. It has been demonstrated that NHEJ deficiencies are associated with impaired neurogenesis. In our study, rats were randomly divided into five groups to be irradiated by single doses of 0 (control), 0 (anesthesia control), 2, 10, and 20 Gy, respectively. The cognitive function of the irradiated rats was measured by open field, Morris water maze and passive avoidance tests. Real-time PCR was also used to detect the expression level of DNA DSB repair-related genes involved in the NHEJ pathway, such as XRCC4, XRCC5 and XRCC6, in the hippocampus. The influence of different radiation doses on cognitive function in rats was investigated. From the results of the behavior tests, we found that rats receiving 20 Gy irradiation revealed poorer learning and memory, while no significant loss of learning and memory existed in rats receiving irradiation from 0-10 Gy. The real-time PCR and Western blot results showed no significant difference in the expression level of DNA repair-related genes between the 10 and 20 Gy groups, which may help to explain the behavioral results, id est (i.e.) DNA damage caused by 0-10 Gy exposure was appropriately repaired, however, damage induced by 20 Gy exceeded the body's maximum DSB repair ability. Ionizing radiation-induced cognitive impairments depend on the radiation dose, and more directly on the body's own ability to repair DNA DSBs via the NHEJ pathway. (author)

DNA replication stress restricts ribosomal DNA copy number.

Science.gov (United States)

Salim, Devika; Bradford, William D; Freeland, Amy; Cady, Gillian; Wang, Jianmin; Pruitt, Steven C; Gerton, Jennifer L

2017-09-01

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

DNA replication stress restricts ribosomal DNA copy number

Science.gov (United States)

Salim, Devika; Bradford, William D.; Freeland, Amy; Cady, Gillian; Wang, Jianmin

2017-01-01

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number. PMID:28915237

DNA replication stress restricts ribosomal DNA copy number.

Directory of Open Access Journals (Sweden)

Devika Salim

2017-09-01

Full Text Available Ribosomal RNAs (rRNAs in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

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.

Genome-wide analysis of Pax8 binding provides new insights into thyroid functions

Directory of Open Access Journals (Sweden)

Ruiz-Llorente Sergio

2012-04-01

Full Text Available Abstract Background The transcription factor Pax8 is essential for the differentiation of thyroid cells. However, there are few data on genes transcriptionally regulated by Pax8 other than thyroid-related genes. To better understand the role of Pax8 in the biology of thyroid cells, we obtained transcriptional profiles of Pax8-silenced PCCl3 thyroid cells using whole genome expression arrays and integrated these signals with global cis-regulatory sequencing studies performed by ChIP-Seq analysis Results Exhaustive analysis of Pax8 immunoprecipitated peaks demonstrated preferential binding to intragenic regions and CpG-enriched islands, which suggests a role of Pax8 in transcriptional regulation of orphan CpG regions. In addition, ChIP-Seq allowed us to identify Pax8 partners, including proteins involved in tertiary DNA structure (CTCF and chromatin remodeling (Sp1, and these direct transcriptional interactions were confirmed in vivo. Moreover, both factors modulate Pax8-dependent transcriptional activation of the sodium iodide symporter (Nis gene promoter. We ultimately combined putative and novel Pax8 binding sites with actual target gene expression regulation to define Pax8-dependent genes. Functional classification suggests that Pax8-regulated genes may be directly involved in important processes of thyroid cell function such as cell proliferation and differentiation, apoptosis, cell polarity, motion and adhesion, and a plethora of DNA/protein-related processes. Conclusion Our study provides novel insights into the role of Pax8 in thyroid biology, exerted through transcriptional regulation of important genes involved in critical thyrocyte processes. In addition, we found new transcriptional partners of Pax8, which functionally cooperate with Pax8 in the regulation of thyroid gene transcription. Besides, our data demonstrate preferential location of Pax8 in non-promoter CpG regions. These data point to an orphan CpG island-mediated mechanism

Lead induces DNA damage and alteration of ALAD and antioxidant genes mRNA expression in construction site workers.

Science.gov (United States)

Akram, Zertashia; Riaz, Sadaf; Kayani, Mahmood Akhtar; Jahan, Sarwat; Ahmad, Malik Waqar; Ullah, Muhammad Abaid; Wazir, Hizbullah; Mahjabeen, Ishrat

2018-01-16

Oxidative stress and DNA damage are considered as possible mechanisms involved in lead toxicity. To test this hypothesis, DNA damage and expression variations of aminolevulinic acid dehydratase (ALAD), superoxide dismutase 2 (SOD2), and 8-oxoguanine DNA glycosylase 2a (OGG1-2a) genes was studied in a cohort of 100 exposed workers and 100 controls with comet assay and real-time polymerse chain reaction (PCR). Results indicated that increased number of comets was observed in exposed workers versus controls (p gene.

Photochemistry of psoralen-DNA adducts, biological effects of psoralen-DNA adducts, applications of psoralen-DNA photochemistry

Energy Technology Data Exchange (ETDEWEB)

Shi, Yun-bo

1988-03-01

This thesis consists of three main parts and totally eight chapters. In Part I, The author will present studies on the photochemistry of psoralen-DNA adducts, specifically, the wavelength dependencies for the photoreversals of thymidine-HMT (4'-hydroxymethyl-4, 5', 8-trimenthylpsoralen) monoadducts and diadduct and the same adducts incorporated in DNA helices and the wavelength dependecies for the photocrossslinking of thymidine-HMT monoadducts in double-stranded helices. In Part II, The author will report some biological effects of psoralen-DNA adducts, i.e., the effects on double-stranded DNA stability, DNA structure, and transcription by E. coli and T7 RNA polymerases. Finally, The author will focus on the applications of psoralen-DNA photochemistry to investigation of protein-DNA interaction during transcription, which includes the interaction of E. coli and T7 RNA polymerases with DNA in elongation complexes arrested at specific psoralen-DNA adduct sites as revealed by DNase I footprinting experiments. 123 refs., 52 figs., 12 tabs.

Photochemistry of psoralen-DNA adducts, biological effects of psoralen-DNA adducts, applications of psoralen-DNA photochemistry

International Nuclear Information System (INIS)

Shi, Yun-bo.

1988-03-01

This thesis consists of three main parts and totally eight chapters. In Part I, The author will present studies on the photochemistry of psoralen-DNA adducts, specifically, the wavelength dependencies for the photoreversals of thymidine-HMT (4'-hydroxymethyl-4, 5', 8-trimenthylpsoralen) monoadducts and diadduct and the same adducts incorporated in DNA helices and the wavelength dependecies for the photocrossslinking of thymidine-HMT monoadducts in double-stranded helices. In Part II, The author will report some biological effects of psoralen-DNA adducts, i.e., the effects on double-stranded DNA stability, DNA structure, and transcription by E. coli and T7 RNA polymerases. Finally, The author will focus on the applications of psoralen-DNA photochemistry to investigation of protein-DNA interaction during transcription, which includes the interaction of E. coli and T7 RNA polymerases with DNA in elongation complexes arrested at specific psoralen-DNA adduct sites as revealed by DNase I footprinting experiments. 123 refs., 52 figs., 12 tabs

HindIII RFLP on chromosome 8 detected with a calbindin 27 kDa cDNA probe, HBSC21

Energy Technology Data Exchange (ETDEWEB)

Parmentier, M; Vassart, G

1988-10-11

A 1.8 kb for EcoRI fragment of the human calbindin cDNA clone HBSC21 was subcloned into M13mp18 and used as a probe. HindIII identifies a 2 allele polymorphism with a band at 4.7 kb (A1) and a band at 4.3 kb (A2). A constant band is located at 5.3 kb. The calbindin 27 kDa gene was assigned to chromosome 8 using chinese hamster-human and mouse-human cell hybrids. Co-dominant segregation was demonstrated in 3 families (total of 20 individuals).

Distinct energetics and closing pathways for DNA polymerase β with 8-oxoG template and different incoming nucleotides

Directory of Open Access Journals (Sweden)

Wang Yanli

2007-02-01

Full Text Available Abstract Background 8-Oxoguanine (8-oxoG is a common oxidative lesion frequently encountered by DNA polymerases such as the repair enzyme DNA polymerase β (pol β. To interpret in atomic and energetic detail how pol β processes 8-oxoG, we apply transition path sampling to delineate closing pathways of pol β 8-oxoG complexes with dCTP and dATP incoming nucleotides and compare the results to those of the nonlesioned G:dCTP and G:dATPanalogues. Results Our analyses show that the closing pathways of the 8-oxoG complexes are different from one another and from the nonlesioned analogues in terms of the individual transition states along each pathway, associated energies, and the stability of each pathway's closed state relative to the corresponding open state. In particular, the closed-to-open state stability difference in each system establishes a hierarchy of stability (from high to low as G:C > 8-oxoG:C > 8-oxoG:A > G:A, corresponding to -3, -2, 2, 9 kBT, respectively. This hierarchy of closed state stability parallels the experimentally observed processing efficiencies for the four pairs. Network models based on the calculated rate constants in each pathway indicate that the closed species are more populated than the open species for 8-oxoG:dCTP, whereas the opposite is true for 8-oxoG:dATP. Conclusion These results suggest that the lower insertion efficiency (larger Km for dATP compared to dCTP opposite 8-oxoG is caused by a less stable closed-form of pol β, destabilized by unfavorable interactions between Tyr271 and the mispair. This stability of the closed vs. open form can also explain the higher insertion efficiency for 8-oxoG:dATP compared to the nonlesioned G:dATP pair, which also has a higher overall conformational barrier. Our study offers atomic details of the complexes at different states, in addition to helping interpret the different insertion efficiencies of dATP and dCTP opposite 8-oxoG and G.

Oxidatively damaged DNA in rats exposed by oral gavage to C60 fullerenes and single-walled carbon nanotubes

DEFF Research Database (Denmark)

Folkmann, Janne K; Risom, Lotte; Jacobsen, Nicklas R

2009-01-01

BACKGROUND: C60 fullerenes and single-walled carbon nanotubes (SWCNT) are projected to be used in medicine and consumer products with potential human exposure. The hazardous effects of these particles are expected to involve oxidative stress with generation of oxidatively damaged DNA that might...... be the initiating event in the development of cancer. OBJECTIVE: In this study we investigated the effect of a single oral administration of C60 fullerenes and SWCNT. METHODS: We measured the level of oxidative damage to DNA as the premutagenic 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in the colon mucosa...... of genotoxicity, whereas corn oil per se generated more genotoxicity than the particles. Although there was increased mRNA expression of 8-oxoguanine DNA glycosylase in the liver of C60 fullerene-treated rats, we found no significant increase in repair activity. CONCLUSIONS: Oral exposure to low doses of C60...

The role of mitochondrial DNA damage at skeletal muscle oxidative stress on the development of type 2 diabetes.

Science.gov (United States)

Dos Santos, Julia Matzenbacher; de Oliveira, Denise Silva; Moreli, Marcos Lazaro; Benite-Ribeiro, Sandra Aparecida

2018-04-20

Reduced cellular response to insulin in skeletal muscle is one of the major components of the development of type 2 diabetes (T2D). Mitochondrial dysfunction involves in the accumulation of toxic reactive oxygen species (ROS) that leads to insulin resistance. The aim of this study was to verify the involvement of mitochondrial DNA damage at ROS generation in skeletal muscle during development of T2D. Wistar rats were fed a diet containing 60% fat over 8 weeks and at day 14 a single injection of STZ (25 mg/kg) was administered (T2D-induced). Control rats received standard food and an injection of citrate buffer. Blood and soleus muscle were collected. Abdominal fat was quantified as well as glucose, triglyceride, LDL, HDL, and total cholesterol in plasma and mtDNA copy number, cytochrome b (cytb) mRNA, 8-hydroxyguanosine, and 8-isoprostane (a marker of ROS) in soleus muscle. T2D-induced animal presented similar characteristics to humans that develop T2D such as changes in blood glucose, abdominal fat, LDL, HDL and cholesterol total. In soleus muscle 8-isoprostane, mtDNA copy number and 8-hydroxyguanosine were increased, while cytb mRNA was decreased in T2D. Our results suggest that in the development of T2D, when risks factors of T2D are present, intracellular oxidative stress increases in skeletal muscle and is associated with a decrease in cytb transcription. To overcome this process mtDNA increased but due to the proximity of ROS generation, mtDNA remains damaged by oxidation leading to an increase in ROS in a vicious cycle accounting to the development of insulin resistance and further T2D.

An unstable donor-recipient DNA complex in transformation of Bacillus subtilis

International Nuclear Information System (INIS)

Popowski, J.; Venema, G.

1978-01-01

In re-extracted DNA obtained shortly after uptake of transforming DNA by Bacillus subtilis, increased amounts of donor DNA radioactivity banding at the position of donor-recipient DNA complex (DRC) are observed in CsCl gradients, if the cells are irradiated with high doses of UV prior to reextraction of the DNA. Qualitatively, the same phenomenon is observed if lysates of transforming cells are irradiated. UV-irradiation of lysates of competent cells to which single-stranded DNA is added after lysis, does not result in linkage of this DNA to the chromosomal DNA. Two observations argue in favour of the formation of a specific labile complex between donor and resident DNA during transformation. Firstly, heterologous donor DNA from Escherichia coli, although being processed to single-stranded DNA in competent B. subtilis, does not seem to be linked to the recipient chromosome upon UV-irradiation, and secondly, the labile complex of donor and recipient DNA can be stabilized by means of treatment of the lysates of transforming cells with 4, 5 1 , 8-trimethylpsoralen in conjuction with long-wave-ultra violet light irradiation. This indicates that basepairing is involved in the formation of the complex. On the basis of these results we assume that the unstable complex of donor and recipient DNA is an early intermediate in genetic recombination during transformation. (orig.) [de

Micrococcus luteus correndonucleases. III. Evidence for involvement in repair in vivo of two endonucleases specific for DNA containing pyrimidine dimers

International Nuclear Information System (INIS)

Riazuddin, S.; Grossman, L.; Mahler, I.

1977-01-01

Involvement of Py--Py correndonucleases I and II in repair of ultraviolet radiation damage in vivo by Micrococcus luteus has been demonstrated by their absence in the ultraviolet-sensitive mutant DB-7 derived by treatment of the wild type parent with N-methyl-N'-nitro-N-nitrosoguanidine. The necessity for their combined action in DNA repair in M. luteus is shown by: (a) reactivation of ultraviolet-damaged phiX174 RFI DNA in incision-defective hosts after in vivo treatment with both enzymes, (b) correlation between survival after ultraviolet irradiation and the level of the two enzymes, and (c) increased levels of repair synthesis after ultraviolet irradiation of toluenized cells DB-400 with wild type correndonuclease levels when compared with the transformant DB-200 and the mutant DB-7, which lack one or both enzymes

Effect of 8-Oxoguanine on DNA Structure and Deformability

Czech Academy of Sciences Publication Activity Database

Dršata, Tomáš; Kara, M.; Zacharias, M.; Lankaš, Filip

2013-01-01

Roč. 117, č. 39 (2013), s. 11617-11622 ISSN 1520-6106 Institutional support: RVO:61388963 Keywords : molecular-dynamics simulations * B-DNA * glycosylase MutM Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.377, year: 2013

Acute hypoxia and hypoxic exercise induce DNA strand breaks and oxidative DNA damage in humans

DEFF Research Database (Denmark)

Møller, P; Loft, S; Lundby, C

2001-01-01

; lymphocytes were isolated for analysis of DNA strand breaks and oxidatively altered nucleotides, detected by endonuclease III and formamidipyridine glycosylase (FPG) enzymes. Urine was collected for 24 h periods for analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a marker of oxidative DNA damage...... oxygen species, generated by leakage of the mitochondrial respiration or during a hypoxia-induced inflammation. Furthermore, the presence of DNA strand breaks may play an important role in maintaining hypoxia-induced inflammation processes. Hypoxia seems to deplete the antioxidant system of its capacity...

HPLC-MS/MS measurement of radiation and photo-induced damage in cellular DNA and human skin

International Nuclear Information System (INIS)

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

2010-01-01

Full text: The measurement of damage induced in cellular DNA by ionizing and solar radiations is of major importance to assess the molecular mode of action and the biological role (mutagenesis, DNA repair) of these genotoxic agents. For this purpose several analytical approaches including immunodetection, post-labeling and chromatographic assays have been designed. However most of them have been shown to suffer from a lack of specificity, sensitivity or quantitative response. It may be noted that the gas-chromatography method in its basal version has been found to lead to overestimated yields of oxidatively generated base lesions by two to three order of magnitude due to the occurrence of artifactual oxidation of the overwhelming purine and pyrimidine bases during the derivatization step of the assay. The advent of HPLC coupled to tandem mass spectrometry operating in the electrospray ionization mode has allowed overcoming most of these drawbacks. Thus, accurate determination of 11 oxidized bases and nucleosides has been achieved in cellular DNA upon exposure to radiation-induced hydroxyl radical and one-electron oxidation agents. This has involved quantitative enzymatic release of lesions from extracted DNA and their accurate detection at the output of the HPLC column using the highly quantitative isotopic dilution technique. Evidence was also provided for the generation of five clustered lesions that all involve a base modification and an altered 2-deoxyribose residue as the result of only one initial radical oxidation hit. These consist of (5'R)-5',8-cyclo-2'-deoxyadenosine and cytosinealdehyde adducts that arise from .OH-mediated hydrogen abstraction at C5 and C4 of the sugar moiety of cellular DNA respectively. The damaging effects of UVA radiation on cellular DNA and human skin were rationalized in terms of predominant 1 O 2 -mediated formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine. Other relevant types of DNA modifications consist in bipyrimidine

Involvement of Opioid System, TRPM8, and ASIC Receptors in Antinociceptive Effect of Arrabidaea brachypoda (DC Bureau

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Vinícius Peixoto Rodrigues

2017-11-01

Full Text Available Arrabidaea brachypoda (DC Bureau is a medicinal plant found in Brazil. Known as “cipó-una”, it is popularly used as a natural therapeutic agent against pain and inflammation. This study evaluated the chemical composition and antinociceptive activity of the dichloromethane fraction from the roots of A. brachypoda (DEAB and its mechanism of action. The chemical composition was characterized by high-performance liquid chromatography, and this fraction is composed only of dimeric flavonoids. The antinociceptive effect was evaluated in formalin and hot plate tests after oral administration (10–100 mg/kg in male Swiss mice. We also investigated the involvement of TRPV1 (transient receptor potential vanilloid 1, TRPA1 (transient receptor potential ankyrin 1, TRPM8 (transient receptor potential melastatin 8, and ASIC (acid-sensing ion channel, as well as the opioidergic, glutamatergic, and supraspinal pathways. Moreover, the nociceptive response was reduced (30 mg/kg in the early and late phase of the formalin test. DEAB activity appears to involve the opioid system, TRPM8, and ASIC receptors, clearly showing that the DEAB alleviates acute pain in mice and suggesting the involvement of the TRPM8 and ASIC receptors and the opioid system in acute pain relief.

1,8-Naphthyridine-2,7-diamine: a potential universal reader of Watson-Crick base pairs for DNA sequencing by electron tunneling.

Science.gov (United States)

Liang, Feng; Lindsay, Stuart; Zhang, Peiming

2012-11-21

With the aid of Density Functional Theory (DFT), we designed 1,8-naphthyridine-2,7-diamine as a recognition molecule to read DNA base pairs for genomic sequencing by electron tunneling. NMR studies show that it can form stable triplets with both A : T and G : C base pairs through hydrogen bonding. Our results suggest that the naphthyridine molecule should be able to function as a universal base pair reader in a tunneling gap, generating distinguishable signatures under electrical bias for each of DNA base pairs.

Prooxidant action of furanone compounds: implication of reactive oxygen species in the metal-dependent strand breaks and the formation of 8-hydroxy-2'-deoxyguanosine in DNA.

Science.gov (United States)

Murakami, K; Haneda, M; Makino, T; Yoshino, M

2007-07-01

Prooxidant properties of furanone compounds including 2,5-furanone (furaneol, 4-hydroxy-2,5-dimethyl-furan-3-one), 4,5-furanone (4,5-dimethyl-3-hydroxy-2(5H)-furanone) (sotolone) and cyclotene (2-hydroxy-3-methyl-2-cyclopenten-1-one) were analyzed in relation to the metal-reducing activity. Only 2.5-furanone known as a "strawberry or pineapple furanone" inactivated aconitase the most sensitive enzyme to active oxygen in the presence of ferrous sulfate, suggesting the furaneol/iron-mediated generation of reactive oxygen species. 2,5-Furanone caused strand scission of pBR322 DNA in the presence of copper. Treatment of calf thymus DNA with 2,5-furanone plus copper produced 8-hydroxy-2'-deoxyguanosine in DNA. 2,5-Furanone showed a potent copper-reducing activity, and thus, DNA strand breaks and the formation of 8-hydroxy-2'-deoxyguanosine by 2,5-furanone can be initiated by the production of superoxide radical through the reduction of cupric ion to cuprous ion, resulting in the conversion to hydrogen peroxide and hydroxyl radical. However, an isomer and analog of 2,5-furanone, 4,5-furanone and cyclotene, respectively, did not show an inactivation of aconitase, DNA injuries including strand breakage and the formation of 8-hydroxy-2'-deoxyguanosine, and copper-reducing activity. Cytotoxic effect of 2,5-furanone with hydroxyketone structure can be explained by its prooxidant properties: furaneol/transition metal complex generates reactive oxygen species causing the inactivation of aconitase and the formation of DNA base damage by hydroxyl radical.

Molecular interactions and residues involved in force generation in the T4 viral DNA packaging motor.

Science.gov (United States)

Migliori, Amy D; Smith, Douglas E; Arya, Gaurav

2014-12-12

Many viruses utilize molecular motors to package their genomes into preformed capsids. A striking feature of these motors is their ability to generate large forces to drive DNA translocation against entropic, electrostatic, and bending forces resisting DNA confinement. A model based on recently resolved structures of the bacteriophage T4 motor protein gp17 suggests that this motor generates large forces by undergoing a conformational change from an extended to a compact state. This transition is proposed to be driven by electrostatic interactions between complementarily charged residues across the interface between the N- and C-terminal domains of gp17. Here we use atomistic molecular dynamics simulations to investigate in detail the molecular interactions and residues involved in such a compaction transition of gp17. We find that although electrostatic interactions between charged residues contribute significantly to the overall free energy change of compaction, interactions mediated by the uncharged residues are equally if not more important. We identify five charged residues and six uncharged residues at the interface that play a dominant role in the compaction transition and also reveal salt bridging, van der Waals, and solvent hydrogen-bonding interactions mediated by these residues in stabilizing the compact form of gp17. The formation of a salt bridge between Glu309 and Arg494 is found to be particularly crucial, consistent with experiments showing complete abrogation in packaging upon Glu309Lys mutation. The computed contributions of several other residues are also found to correlate well with single-molecule measurements of impairments in DNA translocation activity caused by site-directed mutations. Copyright © 2014 Elsevier Ltd. All rights reserved.

Comparison on the interaction of Al3+/nano-Al13 with calf thymus DNA /salmon sperm DNA

Science.gov (United States)

Ma, Fei; Ma, Yue; Du, Changwen; Yang, Xiaodi; Shen, Renfang

2015-11-01

The conformation change, binding mode and binding site between Al3+/nano-Al13 and calf thymus DNA/salmon sperm DNA were investigated by UV-vis absorption, FTIR spectra, Raman spectroscopy and CD spectra, as well as melting curves measurement. The UV-vis spectra and circular dichroism spectra results suggested that the phosphate group structure was changed when Al3+ interacted with DNA, while the double-helix was distorted when nano-Al13 interacted with DNA. The FTIR and Raman spectroscopy revealed that the binding sites were Al3+ … PO2, Al3+ … N7/guanine PO2 … Al13 … N7-C8/guanine with calf thymus DNA, and Al3+ … N3-O2/cytosine, Al3+ … N7-C8/guanine, PO2 … Al13 … N7-C8/guanine, PO2 … Al13 … N1/adenine with salmon sperm DNA, respectively. The electrostatic binding was existed between Al3+ and DNA, and the electrostatic binding and complexing were found between nano-Al13 and DNA.

The mechanism of DNA ejection in the Bacillus anthracis spore-binding phage 8a revealed by cryo-electron tomography

Energy Technology Data Exchange (ETDEWEB)

Fu, Xiaofeng [Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, TX 77030 (United States); Walter, Michael H. [Department of Biology, University of Northern Iowa, Cedar Falls, IA 50614 (United States); Paredes, Angel [Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, TX 77030 (United States); Morais, Marc C., E-mail: mcmorais@utmb.edu [Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555 (United States); Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555 (United States); Liu, Jun, E-mail: Jun.Liu.1@uth.tmc.edu [Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, TX 77030 (United States)

2011-12-20

The structure of the Bacillus anthracis spore-binding phage 8a was determined by cryo-electron tomography. The phage capsid forms a T = 16 icosahedron attached to a contractile tail via a head-tail connector protein. The tail consists of a six-start helical sheath surrounding a central tail tube, and a structurally novel baseplate at the distal end of the tail that recognizes and attaches to host cells. The parameters of the icosahedral capsid lattice and the helical tail sheath suggest protein folds for the capsid and tail-sheath proteins, respectively, and indicate evolutionary relationships to other dsDNA viruses. Analysis of 2518 intact phage particles show four distinct conformations that likely correspond to four sequential states of the DNA ejection process during infection. Comparison of the four observed conformations suggests a mechanism for DNA ejection, including the molecular basis underlying coordination of tail sheath contraction and genome release from the capsid.

Autophagy in DNA Damage Response

Directory of Open Access Journals (Sweden)

Piotr Czarny

2015-01-01

Full Text Available DNA damage response (DDR involves DNA repair, cell cycle regulation and apoptosis, but autophagy is also suggested to play a role in DDR. Autophagy can be activated in response to DNA-damaging agents, but the exact mechanism underlying this activation is not fully understood, although it is suggested that it involves the inhibition of mammalian target of rapamycin complex 1 (mTORC1. mTORC1 represses autophagy via phosphorylation of the ULK1/2–Atg13–FIP200 complex thus preventing maturation of pre-autophagosomal structures. When DNA damage occurs, it is recognized by some proteins or their complexes, such as poly(ADPribose polymerase 1 (PARP-1, Mre11–Rad50–Nbs1 (MRN complex or FOXO3, which activate repressors of mTORC1. SQSTM1/p62 is one of the proteins whose levels are regulated via autophagic degradation. Inhibition of autophagy by knockout of FIP200 results in upregulation of SQSTM1/p62, enhanced DNA damage and less efficient damage repair. Mitophagy, one form of autophagy involved in the selective degradation of mitochondria, may also play role in DDR. It degrades abnormal mitochondria and can either repress or activate apoptosis, but the exact mechanism remains unknown. There is a need to clarify the role of autophagy in DDR, as this process may possess several important biomedical applications, involving also cancer therapy.

Overexpression of transcription factor AP-2 stimulates the PA promoter of the human uracil-DNA glycosylase (UNG) gene through a mechanism involving derepression

DEFF Research Database (Denmark)

Aas, Per Arne; Pena Diaz, Javier; Liabakk, Nina Beate

2009-01-01

within the region of DNA marked by PA. Footprinting analysis and electrophoretic mobility shift assays of PA and putative AP-2 binding regions with HeLa cell nuclear extract and recombinant AP-2alpha protein indicate that AP-2 transcription factors are central in the regulated expression of UNG2 m......The PA promoter in the human uracil-DNA glycosylase gene (UNG) directs expression of the nuclear form (UNG2) of UNG proteins. Using a combination of promoter deletion and mutation analyses, and transient transfection of HeLa cells, we show that repressor and derepressor activities are contained......alpha, lacking the activation domain but retaining the DNA binding and dimerization domains, stimulated PA to a level approaching that of full-length AP-2, suggesting that AP-2 overexpression stimulates PA activity by a mechanism involving derepression rather than activation, possibly by neutralizing...

Aging-dependent DNA hypermethylation and gene expression of GSTM1 involved in T cell differentiation.

Science.gov (United States)

Yeh, Shu-Hui; Liu, Cheng-Ling; Chang, Ren-Chieh; Wu, Chih-Chiang; Lin, Chia-Hsueh; Yang, Kuender D

2017-07-25

This study investigated whether aging was associated with epigenetic changes of DNA hypermethylation on immune gene expression and lymphocyte differentiation. We screened CG sites of methylation in blood leukocytes from different age populations, picked up genes with age-related increase of CG methylation content more than 15%, and validated immune related genes with CG hypermethylation involved in lymphocyte differentiation in the aged population. We found that 12 genes (EXHX1、 IL-10、 TSP50、 GSTM1、SLC5A5、SPI1、F2R、LMO2、PTPN6、FGFR2、MMP9、MET) were associated with promoter or exon one DNA hypermethylation in the aged group. Two immune related genes, GSTM1 and LMO2, were chosen to validate its aging-related CG hypermethylation in different leukocytes. We are the first to validate that GSTM1_P266 and LMO2_E128 CG methylation contents in T lymphocytes but not polymorphonuclear cells (PMNs) or mononuclear cells (MNCs) were significantly increased in the aged population. The GSTM1 mRNA expression in T lymphocytes but not PMNs or MNCs was inversely associated with the GSTM1 CG hypermethylation levels in the aged population studied. Further studies showed that lower GSTM1 CG methylation content led to the higher GSTM1 mRNA expression in T cells and knockdown of GSTM1 mRNA expression decreased type 1 T helper cell (Th1) differentiation in Jurkat T cells and normal adult CD4 T cells. The GSTM1_P266 hypermethylation in the aged population associated with lower GSTM1 mRNA expression was involved in Th1 differentiation, highlighting that modulation of aging-associated GSTM1 methylation may be able to enhance T helper cell immunity in the elders.

A new non-catalytic role for ubiquitin ligase RNF8 in unfolding higher-order chromatin structure

DEFF Research Database (Denmark)

Luijsterburg, Martijn S; Acs, Klara; Ackermann, Leena

2012-01-01

The ubiquitin ligases RNF8 and RNF168 orchestrate DNA damage signalling through the ubiquitylation of histone H2A and the recruitment of downstream repair factors. Here, we demonstrate that RNF8, but not RNF168 or the canonical H2A ubiquitin ligase RNF2, mediates extensive chromatin decondensation....... Our data show that CHD4, the catalytic subunit of the NuRD complex, interacts with RNF8 and is essential for RNF8-mediated chromatin unfolding. The chromatin remodelling activity of CHD4 promotes efficient ubiquitin conjugation and assembly of RNF168 and BRCA1 at DNA double-strand breaks....... Interestingly, RNF8-mediated recruitment of CHD4 and subsequent chromatin remodelling were independent of the ubiquitin-ligase activity of RNF8, but involved a non-canonical interaction with the forkhead-associated (FHA) domain. Our study reveals a new mechanism of chromatin remodelling-assisted ubiquitylation...

Flexible DNA Path in the MCM Double Hexamer Loaded on DNA.

Science.gov (United States)

Hizume, Kohji; Kominami, Hiroaki; Kobayashi, Kei; Yamada, Hirofumi; Araki, Hiroyuki

2017-05-16

The formation of the pre-replicative complex (pre-RC) during the G1 phase, which is also called the licensing of DNA replication, is the initial and essential step of faithful DNA replication during the subsequent S phase. It is widely accepted that in the pre-RC, double-stranded DNA passes through the holes of two ring-shaped minichromosome maintenance (MCM) 2-7 hexamers; however, the spatial organization of the DNA and proteins involved in pre-RC formation is unclear. Here we reconstituted the pre-RC from purified DNA and proteins and visualized the complex using atomic force microscopy (AFM). AFM revealed that the MCM double hexamers formed elliptical particles on DNA. Analysis of the angle of binding of DNA to the MCM double hexamer suggests that the DNA does not completely pass through both holes of the MCM hexamers, possibly because the DNA exited from the gap between Mcm2 and Mcm5. A DNA loop fastened by the MCM double hexamer was detected in pre-RC samples reconstituted from purified proteins as well as those purified from yeast cells, suggesting a higher-order architecture of the loaded MCM hexamers and DNA strands.

DNA methylation

DEFF Research Database (Denmark)

Williams, Kristine; Christensen, Jesper; Helin, Kristian

2012-01-01

DNA methylation is involved in key cellular processes, including X-chromosome inactivation, imprinting and transcriptional silencing of specific genes and repetitive elements. DNA methylation patterns are frequently perturbed in human diseases such as imprinting disorders and cancer. The recent...... discovery that the three members of the TET protein family can convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) has provided a potential mechanism leading to DNA demethylation. Moreover, the demonstration that TET2 is frequently mutated in haematopoietic tumours suggests that the TET...... proteins are important regulators of cellular identity. Here, we review the current knowledge regarding the function of the TET proteins, and discuss various mechanisms by which they contribute to transcriptional control. We propose that the TET proteins have an important role in regulating DNA methylation...

55K isoform of CDK9 associates with Ku70 and is involved in DNA repair

International Nuclear Information System (INIS)

Liu, Hongbing; Herrmann, Christine H.; Chiang, Karen; Sung, Tzu-Ling; Moon, Sung-Hwan; Donehower, Lawrence A.; Rice, Andrew P.

2010-01-01

Positive elongation factor b (P-TEFb) is a cellular protein kinase that is required for RNA polymerase II (RNAP II) transcriptional elongation of protein coding genes. P-TEFb is a set of different molecular complexes, each containing CDK9 as the catalytic subunit. There are two isoforms of the CDK9 protein - the major 42 KDa CDK9 isoform and the minor 55KDa isoform that is translated from an in-frame mRNA that arises from an upstream transcriptional start site. We found that shRNA depletion of the 55K CDK9 protein in HeLa cells induces apoptosis and double-strand DNA breaks (DSBs). The levels of apoptosis and DSBs induced by the depletion were reduced by expression of a 55K CDK9 protein variant resistant to the shRNA, indicating that these phenotypes are the consequence of depletion of the 55K protein and not off-target effects. We also found that the 55K CDK9 protein, but not the 42K CDK9 protein, specifically associates with Ku70, a protein involved in DSB repair. Our findings suggest that the 55K CDK9 protein may function in repair of DNA through an association with Ku70.

cDNA Library Screening Identifies Protein Interactors Potentially Involved in Non-telomeric Roles of Arabidopsis Telomerase

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Ladislav eDokládal

2015-11-01

Full Text Available Telomerase-reverse transcriptase (TERT plays an essential catalytic role in maintaining telomeres. However, in animal systems telomerase plays additional non-telomeric functional roles. We previously screened an Arabidopsis cDNA library for proteins that interact with the C-terminal extension (CTE TERT domain and identified a nuclear-localized protein that contains a RNA recognition motif (RRM. This RRM-protein forms homodimers in both plants and yeast. Mutation of the gene encoding the RRM-protein had no detectable effect on plant growth and development, nor did it affect telomerase activity or telomere length in vivo, suggesting a non-telomeric role for TERT/RRM-protein complexes. The gene encoding the RRM-protein is highly expressed in leaf and reproductive tissues. We further screened an Arabidopsis cDNA library for proteins that interact with the RRM-protein and identified five interactors. These proteins are involved in numerous non-telomere-associated cellular activities. In plants, the RRM-protein, both alone and in a complex with its interactors, localizes to nuclear speckles. Transcriptional analyses in wild-type and rrm mutant plants, as well as transcriptional co-analyses, suggest that TERT, the RRM-protein, and the RRM-protein interactors may play important roles in non-telomeric cellular functions.

Prognostic value of CtIP/RBBP8 expression in breast cancer

International Nuclear Information System (INIS)

Soria-Bretones, Isabel; Sáez, Carmen; Ruíz-Borrego, Manuel; Japón, Miguel A; Huertas, Pablo

2013-01-01

CtIP/RBBP8 is a multifunctional protein involved in transcription, DNA replication, DNA repair by homologous recombination and the G1 and G2 checkpoints. Its multiple roles are controlled by its interaction with several specific factors, including the tumor suppressor proteins BRCA1 and retinoblastoma. Both its functions and interactors point to a putative oncogenic potential of CtIP/RBBP8 loss. However, CtIP/RBBP8 relevance in breast tumor appearance, development, and prognosis has yet to be established. We performed a retrospective analysis of CtIP/RBBP8 and RB1 levels by immunohistochemistry using 384 paraffin-embedded breast cancer biopsies obtained during tumor removal surgery. We have observed that low or no expression of CtIP/RBBP8 correlates with high-grade breast cancer and with nodal metastasis. Reduction on CtIP/RBBP8 is most common in hormone receptor (HR)-negative, HER2-positive, and basal-like tumors. We observed lower levels of RB1 on those tumors with reduced CtIP/RBBP8 levels. On luminal tumors, decreased but not absence of CtIP/RBBP8 levels correlate with increased disease-free survival when treated with a combination of hormone, radio, and chemo therapies

Hox code in embryos of Chinese soft-shelled turtle Pelodiscus sinensis correlates with the evolutionary innovation in the turtle.

Science.gov (United States)

Ohya, Yoshie Kawashima; Kuraku, Shigehiro; Kuratani, Shigeru

2005-03-15

Turtles have the most unusual body plan of the amniotes, with a dorsal shell consisting of modified ribs. Because this morphological change in the ribs can be described as an axial-level specific alteration, the evolution of the turtle carapace should depend on changes in the Hox code. To identify turtle-specific changes in developmental patterns, we cloned several Hox genes from the Chinese soft-shelled turtle, Pelodiscus sinensis, examined their expression patterns during embryogenesis, and compared them with those of chicken and mouse embryos. We detected possibly turtle-specific derived traits in Hoxc-6 expression, which is restricted to the paraxial part of the embryo; in the expression of Hoxa-5 and Hoxb-5, the transcripts of which were detected only at the cervical level; and in Hoxc-8 and Hoxa-7 expression, which is shifted anteriorly relative to that of the other two amniote groups. From the known functions of the Hox orthologs in model animals, these P. sinensis-specific changes apparently correlate with specializations in the turtle-specific body plan. Copyright 2005 Wiley-Liss, Inc.

The involvement of nuclear nucleases in rat thymocyte DNA degradation after γ-irradiation

International Nuclear Information System (INIS)

Nikonova, L.V.; Nelipovich, P.A.; Umansky, S.R.

1982-01-01

Possible mechanisms of internucleosomal DNA fragmentation in thymocytes of irradiated rats were studied. It was shown that thymocyte nuclei contain at least two nucleases that cleave DNA between nucleosomes - a Ca 2+ /Mg 2+ -dependent nuclease and an acidic one which does not depend on bivalent ions. 2 and 3 h after irradiation at a dose of 10 Gy the initial rate of DNA cleavage by Ca 2+ /Mg 2+ -dependent nuclease in isolated nuclei increased three and seven times, respectively, but the kinetics of DNA digestion by acidic nuclease did not change. The experiments with cycloheximide indicated that Ca 2+ /Mg 2+ -dependent endonuclease turns over at a high rate. The activity of the cytoplasmic acidic and Mg 2+ -dependent nucleases was shown to increase (by 40 and 50%, respectively) 3h after irradiation. The effect is caused by the de novo synthesis of the nucleases. At the same time the activity of nuclear nucleases did not essentially change. The chromatin isolated from rat thymocytes 3 h after irradiation did not differ in its sensitivity to some exogenic nucleases (DNAase I, micrococcal nuclease and nuclease from Serratia marcescens) from the control. Thus, Ca 2+ /Mg 2+ -dependent endonuclease seems to be responsible for the postirradiation internucleosomal DNA fragmentation in dying thymocytes. (Auth.)

Purification, crystallization and preliminary X-ray analysis of OsAREB8 from rice, a member of the AREB/ABF family of bZIP transcription factors, in complex with its cognate DNA

International Nuclear Information System (INIS)

Miyazono, Ken-ichi; Koura, Tsubasa; Kubota, Keiko; Yoshida, Takuya; Fujita, Yasunari; Yamaguchi-Shinozaki, Kazuko; Tanokura, Masaru

2012-01-01

OsAREB8 from rice (O. sativa), a member of the AREB/ABF family of bZIP transcription factors, was expressed, purified and crystallized using the sitting-drop vapour-diffusion method. A crystal of OsAREB8 in complex with its cognate DNA diffracted X-rays to 3.65 Å resolution. The AREB/ABF family of bZIP transcription factors play a key role in drought stress response and tolerance during the vegetative stage in plants. To reveal the DNA-recognition mechanism of the AREB/ABF family of proteins, the bZIP domain of OsAREB8, an AREB/ABF-family protein from Oryza sativa, was expressed in Escherichia coli, purified and crystallized with its cognate DNA. Crystals of the OsAREB8–DNA complex were obtained by the sitting-drop vapour-diffusion method at 277 K with a reservoir solution consisting of 50 mM MES pH 6.4, 29% MPD, 2 mM spermidine, 20 mM magnesium acetate and 100 mM sodium chloride. A crystal diffracted X-rays to 3.65 Å resolution and belonged to space group C222, with unit-cell parameters a = 155.1, b = 206.7, c = 38.5 Å. The crystal contained one OsAREB8–DNA complex in the asymmetric unit

Radiation-induced DNA damage as a function of DNA hydration

International Nuclear Information System (INIS)

Swarts, S.G.; Miao, L.; Wheeler, K.T.; Sevilla, M.D.; Becker, D.

1995-01-01

Radiation-induced DNA damage is produced from the sum of the radicals generated by the direct ionization of the DNA (direct effect) and by the reactions of the DNA with free radicals formed in the surrounding environment (indirect effect). The indirect effect has been believed to be the predominant contributor to radiation-induced intracellular DNA damage, mainly as the result of reactions of bulk water radicals (e.g., OH·) with DNA. However, recent evidence suggests that DNA damage, derived from the irradiation of water molecules that are tightly bound in the hydration layer, may occur as the result of the transfer of electron-loss centers (e.g. holes) and electrons from these water molecules to the DNA. Since this mechanism for damaging DNA more closely parallels that of the direct effect, the irradiation of these tightly bound water molecules may contribute to a quasi-direct effect. These water molecules comprise a large fraction of the water surrounding intracellular DNA and could account for a significant proportion of intracellular radiation-induced DNA damage. Consequently, the authors have attempted to characterize this quasi-direct effect to determine: (1) the extent of the DNA hydration layer that is involved with this effect, and (2) what influence this effect has on the types and quantities of radiation-induced DNA damage

SIRT3 restricts HBV transcription and replication via epigenetic regulation of cccDNA involving SUV39H1 and SETD1A histone methyltransferases.

Science.gov (United States)

Ren, Ji-Hua; Hu, Jie-Li; Cheng, Sheng-Tao; Yu, Hai-Bo; Wong, Vincent Kam Wai; Law, Betty Yuen Kwan; Yang, Yong-Feng; Huang, Ying; Liu, Yi; Chen, Wei-Xian; Cai, Xue-Fei; Tang, Hua; Hu, Yuan; Zhang, Wen-Lu; Liu, Xiang; Long, Quan-Xin; Zhou, Li; Tao, Na-Na; Zhou, Hong-Zhong; Yang, Qiu-Xia; Ren, Fang; He, Lin; Gong, Rui; Huang, Ai-Long; Chen, Juan

2018-04-06

Hepatitis B virus (HBV) infection remains a major health problem worldwide. Maintenance of the covalently closed circular DNA (cccDNA) which serves as a template for HBV RNA transcription is responsible for the failure of eradicating chronic HBV during current antiviral therapy. cccDNA is assembled with cellular histone proteins into chromatin, but little is known about the regulation of HBV chromatin by histone posttranslational modifications. In this study, we identified SIRT3 as a host factor restricting HBV transcription and replication by screening seven members of Sirtuin family which is the class III histone deacetylase. Ectopic SIRT3 expression significantly reduced total HBV RNAs, 3.5-kb RNA as well as replicative intermediate DNA in HBV-infected HepG2-NTCP cells and PHH. In contrast, gene silencing of SIRT3 promoted HBV transcription and replication. Mechanistic study found nuclear SIRT3 was recruited to the HBV cccDNA, where it deacetylated histone 3 lysine 9 (H3K9). Importantly, occupancy of SIRT3 onto cccDNA could increase the recruitment of histone methyltransferase SUV39H1 to cccDNA and decrease recruitment of SETD1A, leading to a marked increase of H3K9me3 and a decrease of H3K4me3 on cccDNA. Moreover, SIRT3-mediated HBV cccDNA transcriptional repression involved decreased binding of host RNA polymerase II and transcription factor YY1 to cccDNA. Finally, viral protein HBx could relieve SIRT3-mediated cccDNA transcriptional repression by inhibiting both SIRT3 expression and its recruitment to cccDNA. SIRT3 is a novel host factor epigenetically restricting HBV cccDNA transcription by acting cooperatively with histone methyltransferase. These data provided a rational for the use of SIRT3 activators in the prevention or treatment of HBV infection. This article is protected by copyright. All rights reserved. © 2018 by the American Association for the Study of Liver Diseases.

8-Hydroxydeoxyguanosine in vitro

DEFF Research Database (Denmark)

Fischer-Nielsen, A; Poulsen, H E; Loft, S

1992-01-01

Oxidative DNA damage, as expressed by 8-hydroxydeoxyguanosine (8-OHdG), was investigated in calf thymus DNA exposed to either ultraviolet radiation or to FeCl2/H2O2 in a Fenton-like reaction. The influence of iron (absent in the UV system and present in the FeCl2/H2O2 system) and pH (7.4 and 4.0)...

DNA-membrane complex restoration in Micrococcus radiodurans after X-irradiation: relation to repair, DNA synthesis and DNA degradation

Energy Technology Data Exchange (ETDEWEB)

Dardalhon-Samsonoff, M; Averbeck, D [Institut du Radium, 75 - Paris (France). Lab. Curie

1980-07-01

The DNA-membrane complex in Micrococcus radiodurans was shown to be essentially constituted of proteins, lipids and DNA. The complex was dissociated immediately after X-irradiation of cells and restored during post-incubation in complete medium. In X-irradiated protoplasts some DNA remained associated with the complex. Restoration of the complex during post-incubation was only seen in a medium favouring DNA polymerase and ligase activities. Under this condition no DNA synthesis occurred, suggesting that complex restoration may involve ligase activity. The complex restoration in the wild type and the X-ray sensitive mutant UV17 of M. radiodurans was strictly dependent on the X-ray dose. It was correlated with survival and DNA degradation but always preceded the onset of DNA synthesis after X-irradiation. At the same dose the complex restoration was about 2 fold lower in mutant than in wild type cells indicating that the restoration of the complex is related to repair capacity. The results are consistent with the idea that the complex protects X-irradiated DNA of M. radiodurans from further breakdown and, subsequently, permits DNA synthesis and repair to occur.

Oxidative DNA damage and mammary cell proliferation by alcohol-derived salsolinol.

Science.gov (United States)

Murata, Mariko; Midorikawa, Kaoru; Kawanishi, Shosuke

2013-10-21

Drinking alcohol is a risk factor for breast cancer. Salsolinol (SAL) is endogenously formed by a condensation reaction of dopamine with acetaldehyde, a major ethanol metabolite, and SAL is detected in blood and urine after alcohol intake. We investigated the possibility that SAL can participate in tumor initiation and promotion by causing DNA damage and cell proliferation, leading to alcohol-associated mammary carcinogenesis. SAL caused oxidative DNA damage including 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), in the presence of transition metal ions, such as Cu(II) and Fe(III)EDTA. Inhibitory effects of scavengers on SAL-induced DNA damage and the electron spin resonance study indicated the involvement of H₂O₂, which is generated via the SAL radical. Experiments on scavengers and site specificity of DNA damage suggested ·OH generation via a Fenton reaction and copper-peroxide complexes in the presence of Fe(III)EDTA and Cu(II), respectively. SAL significantly increased 8-oxodG formation in normal mammary epithelial MCF-10A cells. In addition, SAL induced cell proliferation in estrogen receptor (ER)-negative MCF-10A cells, and the proliferation was inhibited by an antioxidant N-acetylcysteine and an epidermal growth factor receptor (EGFR) inhibitor AG1478, suggesting that reactive oxygen species may participate in the proliferation of MCF-10A cells via EGFR activation. Furthermore, SAL induced proliferation in estrogen-sensitive breast cancer MCF-7 cells, and a surface plasmon resonance sensor revealed that SAL significantly increased the binding activity of ERα to the estrogen response element but not ERβ. In conclusion, SAL-induced DNA damage and cell proliferation may play a role in tumor initiation and promotion of multistage mammary carcinogenesis in relation to drinking alcohol.

Effect of γ-irradiated DNA on the activity of DNA polymerase

International Nuclear Information System (INIS)

Leadon, S.A.; Ward, J.F.

1981-01-01

A cell-free assay was developed to measure the effect of γ-irradiated DNA template on the ability of DNA polymerase to copy unirradiated template. Doses as low as 1 krad were able to decrease (approx. 15%) the activity of both bacterial and mammalian DNA polymerases in the assay. The percentage of polymerase activity decreased as the dose received by the template increased. The reduction in DNA polymerase activity was shown to be due to an inhibition of the enzyme by the irradiated DNA. Irradiated poly(dA-dT) was more effective in reducing polymerase activity than calf thymus DNA. Thus the polymerase-inhibition site(s) appears to be associated with base damage, specifically adenine or thymine. Using a free-radical scavenger, OH radicals were found to be involved in producing the damage sites. The interaction between irradiated DNA and DNA polymerase was found to be specific for the enzyme and not for other proteins present in the assay. The inhibition of DNA polymerase occurred prior to or during the initiation of DNA synthesis rather than after initiation of synthesis, i.e., during elongation

Harmonising measurements of 8-oxo-7,8-dihydro-2'-deoxyguanosine in cellular DNA and urine

DEFF Research Database (Denmark)

Møller, Peter; Cooke, Marcus S; Collins, Andrew

2012-01-01

Levels of oxidatively damaged cellular DNA and urinary excretion of damaged 2'-deoxyribonuclosides are widely measured in biomonitoring studies examining the role of oxidative stress induced by environmental exposures, lifestyle factors and development of disease. This has promoted efforts to har...

RAD51 interconnects between DNA replication, DNA repair and immunity.

Science.gov (United States)

Bhattacharya, Souparno; Srinivasan, Kalayarasan; Abdisalaam, Salim; Su, Fengtao; Raj, Prithvi; Dozmorov, Igor; Mishra, Ritu; Wakeland, Edward K; Ghose, Subroto; Mukherjee, Shibani; Asaithamby, Aroumougame

2017-05-05

RAD51, a multifunctional protein, plays a central role in DNA replication and homologous recombination repair, and is known to be involved in cancer development. We identified a novel role for RAD51 in innate immune response signaling. Defects in RAD51 lead to the accumulation of self-DNA in the cytoplasm, triggering a STING-mediated innate immune response after replication stress and DNA damage. In the absence of RAD51, the unprotected newly replicated genome is degraded by the exonuclease activity of MRE11, and the fragmented nascent DNA accumulates in the cytosol, initiating an innate immune response. Our data suggest that in addition to playing roles in homologous recombination-mediated DNA double-strand break repair and replication fork processing, RAD51 is also implicated in the suppression of innate immunity. Thus, our study reveals a previously uncharacterized role of RAD51 in initiating immune signaling, placing it at the hub of new interconnections between DNA replication, DNA repair, and immunity. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

A chromatin modifying enzyme, SDG8, is involved in morphological, gene expression, and epigenetic responses to mechanical stimulation.

Science.gov (United States)

Cazzonelli, Christopher I; Nisar, Nazia; Roberts, Andrea C; Murray, Kevin D; Borevitz, Justin O; Pogson, Barry J

2014-01-01

Thigmomorphogenesis is viewed as being a response process of acclimation to short repetitive bursts of mechanical stimulation or touch. The underlying molecular mechanisms that coordinate changes in how touch signals lead to long-term morphological changes are enigmatic. Touch responsive gene expression is rapid and transient, and no transcription factor or DNA regulatory motif has been reported that could confer a genome wide mechanical stimulus. We report here on a chromatin modifying enzyme, SDG8/ASHH2, which can regulate the expression of many touch responsive genes identified in Arabidopsis. SDG8 is required for the permissive expression of touch induced genes; and the loss of function of sdg8 perturbs the maximum levels of induction on selected touch gene targets. SDG8 is required to maintain permissive H3K4 trimethylation marks surrounding the Arabidopsis touch-inducible gene TOUCH 3 (TCH3), which encodes a calmodulin-like protein (CML12). The gene neighboring was also slightly down regulated, revealing a new target for SDG8 mediated chromatin modification. Finally, sdg8 mutants show perturbed morphological response to wind-agitated mechanical stimuli, implicating an epigenetic memory-forming process in the acclimation response of thigmomorphogenesis.

Significance of 8-oxoG in the spectrum of DNA damages caused by ionising radiation of different quality

Czech Academy of Sciences Publication Activity Database

Štěpán, Václav; Davídková, Marie

2007-01-01

Roč. 122, 1-4 (2007), s. 113-115 ISSN 0144-8420. [Symposium on Microdosimetry /14./. Venezia, 13.11.2005-18.11.2005] R&D Projects: GA AV ČR KJB4048401; GA ČR GA202/05/2728 Institutional research plan: CEZ:AV0Z10480505 Keywords : DNA damage * 8-oxoguanine * ionizing radiation * theoretical modeling Subject RIV: BO - Biophysics Impact factor: 0.528, year: 2007

Evaluation of the 8th TNM classification on p16-positive oropharyngeal squamous cell carcinomas in the Netherlands, and the importance of additional HPV DNA-testing.

Science.gov (United States)

Nauta, I H; Rietbergen, M M; van Bokhoven, A A J D; Bloemena, E; Witte, B I; Heideman, D A M; Baatenburg de Jong, R J; Brakenhoff, R H; Leemans, C R

2018-02-09

Oropharyngeal squamous cell carcinomas (OPSCCs) are traditionally caused by smoking and excessive alcohol consumption. However, in the last decades high-risk human papillomavirus (HR-HPV) infections play an increasingly important role in tumorigenesis. HPV-driven OPSCCs are known to have a more favorable prognosis, which has led to important and marked changes in the recently released TNM-8. In this edition, OPSCCs are divided based on p16-immunostaining, with p16-overexpression as surrogate marker for the presence of HPV. The aims of this study are to evaluate TNM-8 on a Dutch consecutive cohort of patients with p16-positive OPSCC and to determine the relevance of additional HPV DNA-testing. All OPSCC patients without distant metastases at diagnosis and treated with curative intent at VU University Medical Center (2000-2015) and Erasmus Medical Center (2000-2006) were included (N = 1,204). HPV-status was established by p16-immunostaining followed by HPV DNA-PCR on the p16-immunopositive cases. We compared TNM-7 and TNM-8 using the Harrell's C index. In total, 388 of 1,204 (32.2%) patients were p16-immunopositive. In these patients, TNM-8 had a markedly better predictive prognostic power than TNM-7 (Harrell's C index 0.63 versus 0.53). Of the 388 p16-positive OPSCCs, 48 tumors (12.4%) were HPV DNA-negative. This subgroup had distinct demographic, clinical and morphologic characteristics and showed a significantly worse five-year overall survival compared to the HPV DNA-positive tumors (P HPV DNA-negative subgroup with distinct features and a worse overall survival, indicating the importance to perform additional HPV DNA-testing when predicting prognosis and particularly for selecting patients for de-intensified treatment regimens. © The Author 2018. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Salt-Dependent DNA-DNA Spacings in Intact Bacteriophage lambda Reflect Relative Importance of DNA Self-Repulsion and Bending Energies

Energy Technology Data Exchange (ETDEWEB)

X Qiu; D Rau; V Parsegian; L Fang; C Knobler; W Gelbart

2011-12-31

Using solution synchrotron x-ray scattering, we measure the variation of DNA-DNA d spacings in bacteriophage {lambda} with mono-, di-, and polyvalent salt concentrations, for wild-type [48.5 x 10{sup 3} base pairs (bp)] and short-genome-mutant (37.8 kbp) strains. From the decrease in d spacings with increasing salt, we deduce the relative contributions of DNA self-repulsion and bending to the energetics of packaged phage genomes. We quantify the DNA-DNA interaction energies within the intact phage by combining the measured d spacings in the capsid with measurements of osmotic pressure in DNA assemblies under the same salt conditions in bulk solution. In the commonly used Tris-Mg buffer, the DNA-DNA interaction energies inside the phage capsids are shown to be about 1 kT/bp, an order of magnitude larger than the bending energies.

Quantification of cell-free DNA in blood plasma and DNA damage degree in lymphocytes to evaluate dysregulation of apoptosis in schizophrenia patients.

Science.gov (United States)

Ershova, E S; Jestkova, E M; Chestkov, I V; Porokhovnik, L N; Izevskaya, V L; Kutsev, S I; Veiko, N N; Shmarina, G; Dolgikh, O; Kostyuk, S V

2017-04-01

Oxidative DNA damage has been proposed as one of the causes of schizophrenia (SZ), and post mortem data indicate a dysregulation of apoptosis in SZ patients. To evaluate apoptosis in vivo we quantified the concentration of plasma cell-free DNA (cfDNA index, determined using fluorescence), the levels of 8-oxodG in cfDNA (immunoassay) and lymphocytes (FL1-8-oxodG index, flow cytometry) of male patients with acute psychotic disorders: paranoid SZ (total N = 58), schizophreniform (N = 11) and alcohol-induced (N = 14) psychotic disorder, and 30 healthy males. CfDNA in SZ (N = 58) does not change compared with controls. In SZ patients. Elevated levels of 8-oxodG were found in cfDNA (N = 58) and lymphocytes (n = 45). The main sources of cfDNA are dying cells with oxidized DNA. Thus, the cfDNA/FL1-8-oxodG ratio shows the level of apoptosis in damaged cells. Two subgroups were identified among the SZ patients (n = 45). For SZ-1 (31%) and SZ-2 (69%) median values of cfDNA/FL1-8-oxodG index are related as 1:6 (p DNA in the patient's body tissues and may be a contributing cause of acute psychotic disorder. Copyright © 2016 Elsevier Ltd. All rights reserved.

Involvement of the 5'-leader sequence in coupling the stability of a human H3 histone mRNA with DNA replication

International Nuclear Information System (INIS)

Morris, T.; Marashi, F.; Weber, L.; Hickey, E.; Greenspan, D.; Bonner, J.; Stein, J.; Stein, G.

1986-01-01

Two lines of evidence derived from fusion gene constructs indicate that sequences residing in the 5'-nontranslated region of a cell cycle-dependent human H3 histone mRNA are involved in the selective destabilization that occurs when DNA synthesis is terminated. The experimental approach was to construct chimeric genes in which fragments of the mRNA coding regions of the H3 histone gene were fused with fragments of genes not expressed in a cell cycle-dependent manner. After transfection in HeLa S3 cells with the recombinant plasmids, levels of fusion mRNAs were determined by S1 nuclease analysis prior to and following DNA synthesis inhibition. When the first 20 nucleotides of an H3 histone mRNA leader were replaced with 89 nucleotides of the leader from a Drosophila heat-shock (hsp70) mRNA, the fusion transcript remained stable during inhibition of DNA synthesis, in contrast to the rapid destabilization of the endogenous histone mRNA in these cells. In a reciprocal experiment, a histone-globin fusion gene was constructed that produced a transcript with the initial 20 nucleotides of the H3 histone mRNA substituted for the human β-globin mRNA leader. In HeLa cells treated with inhibitors of DNA synthesis and/or protein synthesis, cellular levels of this histone-globin fusion mRNA appeared to be regulated in a manner similar to endogenous histone mRNA levels. These results suggest that the first 20 nucleotides of the leader are sufficient to couple histone mRNA stability with DNA replication

Involvement of interleukin-8 in dialysis-related arthritis.

Science.gov (United States)

Takayama, F; Miyazaki, T; Aoyama, I; Tsukushi, S; Sato, M; Yamazaki, C; Shimokata, K; Niwa, T

1998-04-01

To elucidate the role of interleukin (IL)-8, a chemotactic factor for neutrophils, in dialysis-related arthritis (DRA) of patients on long-term hemodialysis, the concentration of IL-8 was measured in the synovial fluids of DRA patients with acute arthralgia and joint swelling, and was compared with those in patients with rheumatoid arthritis (RA) and patients with osteoarthritis (OA). We noted a marked elevation of IL-8 in the joint fluids of patients with DRA and RA as compared with OA. Furthermore, to determine the role of IL-8 in synovitis, we examined the in vivo effect of intra-articular injection of human recombinant IL-8 on leukocyte infiltration into the joint space of rabbits. A single injection of IL-8 to the joints of rabbits induced rapid infiltration of neutrophils into the joint space and synovial tissues, which reached a maximum in four hours. The oral administration of indometacin farnesil (a prodrug that is converted to indomethacin after intestinal absorption) before the injection of IL-8 alleviated the infiltration of neutrophils. When human synovial cells were incubated with tumor necrosis factor (TNF)-alpha, the expression of IL-8 mRNA and IL-8 production in the cultured synovial cells were increased. The TNF-alpha-stimulated expression of IL-8 mRNA and IL-8 production in the cultured synovial cells were markedly inhibited by dexamethasone. In conclusion, IL-8 levels were markedly elevated in the joint fluids of patients with DRA. Interleukin-8 released from synovial cells may be an important factor to induce acute inflammation in DRA. Dexamethasone and indomethacin may be effective for DRA by inhibiting the production and chemotactic actions of IL-8, respectively.

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.

Secreted single‐stranded DNA is involved in the initial phase of biofilm formation by Neisseria gonorrhoeae

DEFF Research Database (Denmark)

Zweig, Maria; Schork, Sabine; Koerdt, Andrea

2014-01-01

plays an important role in biofilm formation. Many clinical isolates contain a gonococcal genetic island that encodes a type IV secretion system (T4SS). The T4SS of N. gonorrhoeae strain MS11 secretes ssDNA directly into the medium. Biofilm formation, studied in continuous flow‐chamber systems...... was developed in which thermostable fluorescently labelled ssDNA‐ and ss/dsDNA‐binding proteins were used to visualize ssDNA and total DNA in biofilms and planktonic cultures. Remarkably, mainly dsDNA was detected in biofilms of the ssDNA secreting strain. We conclude that the secreted ssDNA facilitates initial...

Nickel exposure induces oxidative damage to mitochondrial DNA in Neuro2a cells: the neuroprotective roles of melatonin.

Science.gov (United States)

Xu, Shang-Cheng; He, Min-Di; Lu, Yong-Hui; Li, Li; Zhong, Min; Zhang, Yan-Wen; Wang, Yuan; Yu, Zheng-Ping; Zhou, Zhou

2011-11-01

Recent studies suggest that oxidative stress and mitochondrial dysfunction play important roles in the neurotoxicity of nickel. Because mitochondrial DNA (mtDNA) is highly vulnerable to oxidative stress and melatonin can efficiently protect mtDNA against oxidative damage in various pathological conditions, the aims of this study were to determine whether mtDNA oxidative damage was involved in the neurotoxicity of nickel and to assay the neuroprotective effects of melatonin in mtDNA. In this study, we exposed mouse neuroblastoma cell lines (Neuro2a) to different concentrations of nickel chloride (NiCl(2), 0.125, 0.25, and 0.5 mm) for 24 hr. We found that nickel significantly increased reactive oxygen species (ROS) production and mitochondrial superoxide levels. In addition, nickel exposure increased mitochondrial 8-hydroxyguanine (8-OHdG) content and reduced mtDNA content and mtDNA transcript levels. Consistent with this finding, nickel was found to destroy mtDNA nucleoid structure and decrease protein levels of Tfam, a key protein component for nucleoid organization. However, all the oxidative damage to mtDNA induced by nickel was efficiently attenuated by melatonin pretreatment. Our results suggest that oxidative damage to mtDNA may account for the neurotoxicity of nickel. Melatonin has great pharmacological potential in protecting mtDNA against the adverse effects of nickel in the nervous system. © 2011 John Wiley & Sons A/S.

Identification of transcription coactivator OCA-B-dependent genes involved in antigen-dependent B cell differentiation by cDNA array analyses.

Science.gov (United States)

Kim, Unkyu; Siegel, Rachael; Ren, Xiaodi; Gunther, Cary S; Gaasterland, Terry; Roeder, Robert G

2003-07-22

The tissue-specific transcriptional coactivator OCA-B is required for antigen-dependent B cell differentiation events, including germinal center formation. However, the identity of OCA-B target genes involved in this process is unknown. This study has used large-scale cDNA arrays to monitor changes in gene expression patterns that accompany mature B cell differentiation. B cell receptor ligation alone induces many genes involved in B cell expansion, whereas B cell receptor and helper T cell costimulation induce genes associated with B cell effector function. OCA-B expression is induced by both B cell receptor ligation alone and helper T cell costimulation, suggesting that OCA-B is involved in B cell expansion as well as B cell function. Accordingly, several genes involved in cell proliferation and signaling, such as Lck, Kcnn4, Cdc37, cyclin D3, B4galt1, and Ms4a11, have been identified as OCA-B-dependent genes. Further studies on the roles played by these genes in B cells will contribute to an understanding of B cell differentiation.

APOBEC3 cytidine deaminases in double-strand DNA break repair and cancer promotion.

Science.gov (United States)

Nowarski, Roni; Kotler, Moshe

2013-06-15

High frequency of cytidine to thymidine conversions was identified in the genome of several types of cancer cells. In breast cancer cells, these mutations are clustered in long DNA regions associated with single-strand DNA (ssDNA), double-strand DNA breaks (DSB), and genomic rearrangements. The observed mutational pattern resembles the deamination signature of cytidine to uridine carried out by members of the APOBEC3 family of cellular deaminases. Consistently, APOBEC3B (A3B) was recently identified as the mutational source in breast cancer cells. A3G is another member of the cytidine deaminases family predominantly expressed in lymphoma cells, where it is involved in mutational DSB repair following ionizing radiation treatments. This activity provides us with a new paradigm for cancer cell survival and tumor promotion and a mechanistic link between ssDNA, DSBs, and clustered mutations. Cancer Res; 73(12); 3494-8. ©2013 AACR. ©2013 AACR.

Exploring translocation of proteins on DNA by NMR

International Nuclear Information System (INIS)

Marius Clore, G.

2011-01-01

While an extensive body of knowledge has accumulated on the structures of transcription factors, DNA and their complexes from both NMR and crystallography, much less is known at a molecular level regarding the mechanisms whereby transcription factors locate their specific DNA target site within an overwhelming sea of non-specific DNA sites. Indirect kinetic data suggested that three processes are involved in the search procedure: jumping by dissociation of the protein from the DNA followed by re-association at another site, direct transfer from one DNA molecule or segment to another, and one-dimensional sliding. In this brief perspective I summarize recent NMR developments from our laboratory that have permitted direct characterization of the species and molecular mechanisms involved in the target search process, including the detection of highly transient sparsely-populated states. The main tool in these studies involves the application of paramagnetic relaxation enhancement, supplemented by z-exchange spectroscopy, lineshape analysis and residual dipolar couplings. These studies led to the first direct demonstration of rotation-coupled sliding of a protein along the DNA and the direct transfer of a protein from one DNA molecule to another without dissociating into free solution.

Effect of dactyloscopic powders on DNA profiling from enhanced fingerprints: results from an experimental study.

Science.gov (United States)

Tozzo, Pamela; Giuliodori, Alice; Rodriguez, Daniele; Caenazzo, Luciana

2014-03-01

We conducted a study on the effect of fingerprint enhancement methods on subsequent short tandem repeat profiling. First, we performed a study typing blood traces deposited on 5 different surfaces, treated with 8 types of dactyloscopic powders. Three different DNA extraction methods were used. Subsequently, we analyzed latent fingerprints on the same 5 surfaces enhanced with the 8 different powders used in the first part of the study. This study has demonstrated that DNA profiling can be performed on fingerprints left on different substrates, and the substrate will affect the amount of DNA that can be recovered for DNA typing. In the first phase of the study, a profile was obtained in 92% of the 120 samples analyzed; in the second part, in 55% of the 80 samples analyzed, we obtained a profile complete in 32.5% of the cases. From the results obtained, it seems that the powders used in latent fingerprints enhancement, rather than having a direct inhibitory effect on extraction and amplification of DNA, may cause partial degradation of DNA, reducing the efficiency of amplification reaction. It should not be forgotten that these results were obtained under laboratory conditions, and in real caseworks, there may still be different problems involved.

Origin of DNA in human serum and usefulness of serum as a material for DNA typing.

Science.gov (United States)

Takayama, T; Yamada, S; Watanabe, Y; Hirata, K; Nagai, A; Nakamura, I; Bunai, Y; Ohya, I

2001-06-01

The aims of this study were to clarify the origin of DNA in human serum and to investigate whether serum is a material available for DNA typing in routine forensic practice. Blood was donated from 10 healthy adult volunteers and stored for up to 8 days, at 4 degrees C and at room temperature. The serum DNA concentration at zero time was in the range of 5.6 to 21.8 ng/ml with a mean of 12.2+/-1.6 ng/ml. The concentrations increased with storage time. On agarose gel electrophoresis, all serum samples showed ladder patterns and the size of each band was an integer multiple of approximately 180 bp considered to be characteristic of apoptosis. DNA typing from DNA released by apoptosis was possible. Exact DNA typing of D1S80, HLA DQA1, PM, CSF1PO, TPOX, TH01 and vWA was possible for each sample. These results indicate that serum contains fragmented DNA derived from apoptosis of leukocytes, especially neutrophils, and that fragmented DNA is an appropriate material for DNA typing.

Biological activity of SV40 DNA

International Nuclear Information System (INIS)

Abrahams, P.J.

1978-01-01

This thesis deals with a study on the biological activity of SV40 DNA. The transforming activity of SV40 DNA and DNA fragments is investigated in order to define as precisely as possible the area of the viral genome that is involved in the transformation. The infectivity of SV40 DNA is used to study the defective repair mechanisms of radiation damages of human xeroderma pigmentosum cells. (C.F.)

Differential effects of silver nanoparticles on DNA damage and DNA repair gene expression in Ogg1-deficient and wild type mice.

Science.gov (United States)

Nallanthighal, Sameera; Chan, Cadia; Murray, Thomas M; Mosier, Aaron P; Cady, Nathaniel C; Reliene, Ramune

2017-10-01

Due to extensive use in consumer goods, it is important to understand the genotoxicity of silver nanoparticles (AgNPs) and identify susceptible populations. 8-Oxoguanine DNA glycosylase 1 (OGG1) excises 8-oxo-7,8-dihydro-2-deoxyguanine (8-oxoG), a pro-mutagenic lesion induced by oxidative stress. To understand whether defects in OGG1 is a possible genetic factor increasing an individual's susceptibly to AgNPs, we determined DNA damage, genome rearrangements, and expression of DNA repair genes in Ogg1-deficient and wild type mice exposed orally to 4 mg/kg of citrate-coated AgNPs over a period of 7 d. DNA damage was examined at 3 and 7 d of exposure and 7 and 14 d post-exposure. AgNPs induced 8-oxoG, double strand breaks (DSBs), chromosomal damage, and DNA deletions in both genotypes. However, 8-oxoG was induced earlier in Ogg1-deficient mice and 8-oxoG levels were higher after 7-d treatment and persisted longer after exposure termination. AgNPs downregulated DNA glycosylases Ogg1, Neil1, and Neil2 in wild type mice, but upregulated Myh, Neil1, and Neil2 glycosylases in Ogg1-deficient mice. Neil1 and Neil2 can repair 8-oxoG. Thus, AgNP-mediated downregulation of DNA glycosylases in wild type mice may contribute to genotoxicity, while upregulation thereof in Ogg1-deficient mice could serve as an adaptive response to AgNP-induced DNA damage. However, our data show that Ogg1 is indispensable for the efficient repair of AgNP-induced damage. In summary, citrate-coated AgNPs are genotoxic in both genotypes and Ogg1 deficiency exacerbates the effect. These data suggest that humans with genetic polymorphisms and mutations in OGG1 may have increased susceptibility to AgNP-mediated DNA damage.

DNA methylation in inflammatory genes among children with obstructive sleep apnea.

Science.gov (United States)

Kim, Jinkwan; Bhattacharjee, Rakesh; Khalyfa, Abdelnaby; Kheirandish-Gozal, Leila; Capdevila, Oscar Sans; Wang, Yang; Gozal, David

2012-02-01

Pediatric obstructive sleep apnea (OSA) leads to multiple end-organ morbidities that are mediated by the cumulative burden of oxidative stress and inflammation. Because not all children with OSA exhibit increased systemic inflammation, genetic and environmental factors may be affecting patterns of DNA methylation in genes subserving inflammatory functions. DNA from matched children with OSA with and without high levels of high-sensitivity C-reactive protein (hsCRP) were assessed for DNA methylation levels of 24 inflammatory-related genes. Primer-based polymerase chain reaction assays in a case-control setting involving 47 OSA cases and 31 control subjects were conducted to confirm the findings; hsCRP and myeloid-related protein (MRP) 8/14 levels were also assayed. Forkhead box P3 (FOXP3) and interferon regulatory factor 1 (IRF1) showed higher methylation in six children with OSA and high hsCRP levels compared with matched children with OSA and low hsCRP levels (P DNA methylation levels compared with children with OSA and low CRP levels and control subjects. IRF1 did not exhibit significant differences. FOXP3 DNA methylation levels correlated with hsCRP and MRP 8/14 levels and with apnea-hypopnea index (AHI), BMI z score, and apolipoprotein B levels. A stepwise multiple regression model showed that AHI was independently associated with FOXP3 DNA methylation levels (P gene, which regulates expression of T regulatory lymphocytes, is more likely to display increased methylation among children with OSA who exhibit increased systemic inflammatory responses. Thus, epigenetic modifications may constitute an important determinant of inflammatory phenotype in OSA, and FOXP3 DNA methylation levels may provide a potential biomarker for end-organ vulnerability.

Anthraquinones quinizarin and danthron unwind negatively supercoiled DNA and lengthen linear DNA

International Nuclear Information System (INIS)

Verebová, Valéria; Adamcik, Jozef; Danko, Patrik; Podhradský, Dušan; Miškovský, Pavol; Staničová, Jana

2014-01-01

Highlights: • Anthraquinones quinizarin and danthron unwind negatively supercoiled DNA. • Anthraquinones quinizarin and danthron lengthen linear DNA. • Anthraquinones quinizarin and danthron possess middle binding affinity to DNA. • Anthraquinones quinizarin and danthron interact with DNA by intercalating mode. - Abstract: The intercalating drugs possess a planar aromatic chromophore unit by which they insert between DNA bases causing the distortion of classical B-DNA form. The planar tricyclic structure of anthraquinones belongs to the group of chromophore units and enables anthraquinones to bind to DNA by intercalating mode. The interactions of simple derivatives of anthraquinone, quinizarin (1,4-dihydroxyanthraquinone) and danthron (1,8-dihydroxyanthraquinone), with negatively supercoiled and linear DNA were investigated using a combination of the electrophoretic methods, fluorescence spectrophotometry and single molecule technique an atomic force microscopy. The detection of the topological change of negatively supercoiled plasmid DNA, unwinding of negatively supercoiled DNA, corresponding to appearance of DNA topoisomers with the low superhelicity and an increase of the contour length of linear DNA in the presence of quinizarin and danthron indicate the binding of both anthraquinones to DNA by intercalating mode

Anthraquinones quinizarin and danthron unwind negatively supercoiled DNA and lengthen linear DNA

Energy Technology Data Exchange (ETDEWEB)

Verebová, Valéria [Institute of Biophysics, University of Veterinary Medicine and Pharmacy, Komenského 73, 041 81 Košice (Slovakia); Adamcik, Jozef [Food and Soft Materials Science, Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 9, CH-8092 Zürich (Switzerland); Danko, Patrik; Podhradský, Dušan [Department of Biochemistry, Institute of Chemistry, Faculty of Sciences, P.J. Šafárik University, Moyzesova 11, 041 54 Košice (Slovakia); Miškovský, Pavol [Department of Biophysics, Faculty of Sciences, P.J. Šafárik University, Jesenná 5, 041 54 Košice (Slovakia); Center for Interdisciplinary Biosciences, Faculty of Sciences, P.J. Šafárik University, Jesenná 5, 041 54 Košice (Slovakia); Staničová, Jana, E-mail: jana.stanicova@uvlf.sk [Institute of Biophysics, University of Veterinary Medicine and Pharmacy, Komenského 73, 041 81 Košice (Slovakia)

2014-01-31

Highlights: • Anthraquinones quinizarin and danthron unwind negatively supercoiled DNA. • Anthraquinones quinizarin and danthron lengthen linear DNA. • Anthraquinones quinizarin and danthron possess middle binding affinity to DNA. • Anthraquinones quinizarin and danthron interact with DNA by intercalating mode. - Abstract: The intercalating drugs possess a planar aromatic chromophore unit by which they insert between DNA bases causing the distortion of classical B-DNA form. The planar tricyclic structure of anthraquinones belongs to the group of chromophore units and enables anthraquinones to bind to DNA by intercalating mode. The interactions of simple derivatives of anthraquinone, quinizarin (1,4-dihydroxyanthraquinone) and danthron (1,8-dihydroxyanthraquinone), with negatively supercoiled and linear DNA were investigated using a combination of the electrophoretic methods, fluorescence spectrophotometry and single molecule technique an atomic force microscopy. The detection of the topological change of negatively supercoiled plasmid DNA, unwinding of negatively supercoiled DNA, corresponding to appearance of DNA topoisomers with the low superhelicity and an increase of the contour length of linear DNA in the presence of quinizarin and danthron indicate the binding of both anthraquinones to DNA by intercalating mode.

Developmental exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin alters DNA methyltransferase (dnmt) expression in zebrafish (Danio rerio)

International Nuclear Information System (INIS)

Aluru, Neelakanteswar; Kuo, Elaine; Helfrich, Lily W.; Karchner, Sibel I.; Linney, Elwood A.; Pais, June E.; Franks, Diana G.

2015-01-01

DNA methylation is one of the most important epigenetic modifications involved in the regulation of gene expression. The DNA methylation reaction is catalyzed by DNA methyltransferases (DNMTs). Recent studies have demonstrated that toxicants can affect normal development by altering DNA methylation patterns, but the mechanisms of action are poorly understood. Hence, we tested the hypothesis that developmental exposure to TCDD affects dnmt gene expression patterns. Zebrafish embryos were exposed to 5 nM TCDD for 1 h from 4 to 5 h post-fertilization (hpf) and sampled at 12, 24, 48, 72, and 96 hpf to determine dnmt gene expression and DNA methylation patterns. We performed a detailed analysis of zebrafish dnmt gene expression during development and in adult tissues. Our results demonstrate that dnmt3b genes are highly expressed in early stages of development, and dnmt3a genes are more abundant in later stages. TCDD exposure upregulated dnmt1 and dnmt3b2 expression, whereas dnmt3a1, 3b1, and 3b4 are downregulated following exposure. We did not observe any TCDD-induced differences in global methylation or hydroxymethylation levels, but the promoter methylation of aryl hydrocarbon receptor (AHR) target genes was altered. In TCDD-exposed embryos, AHR repressor a (ahrra) and c-fos promoters were differentially methylated. To characterize the TCDD effects on DNMTs, we cloned the dnmt promoters with xenobiotic response elements and conducted AHR transactivation assays using a luciferase reporter system. Our results suggest that ahr2 can regulate dnmt3a1, dnmt3a2, and dnmt3b2 expression. Overall, we demonstrate that developmental exposure to TCDD alters dnmt expression and DNA methylation patterns. - Highlights: • TCDD altered the dnmt expression in a gene and developmental time-specific manner. • TCDD hypermethylated ahrra and hypomethylated c-fos proximal promoter regions. • Functional analysis suggests that ahr2 can regulate dnmt3a1, 3a2, and 3b2 expression. • Dnmt

Developmental exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin alters DNA methyltransferase (dnmt) expression in zebrafish (Danio rerio)

Energy Technology Data Exchange (ETDEWEB)

Aluru, Neelakanteswar, E-mail: naluru@whoi.edu [Biology Department and Woods Hole Center for Oceans and Human Health, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Kuo, Elaine [Biology Department and Woods Hole Center for Oceans and Human Health, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Stanford University, 450 Serra Mall, Stanford, CA 94305 (United States); Helfrich, Lily W. [Biology Department and Woods Hole Center for Oceans and Human Health, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Northwestern University, 633 Clark St, Evanston, IL 60208 (United States); Karchner, Sibel I. [Biology Department and Woods Hole Center for Oceans and Human Health, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Linney, Elwood A. [Department of Molecular Genetics and Microbiology, Duke University Medical Center, Box 3020, Durham, NC 27710 (United States); Pais, June E. [New England Biolabs, 240 County Road, Ipswich, MA 01938 (United States); Franks, Diana G. [Biology Department and Woods Hole Center for Oceans and Human Health, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States)

2015-04-15

DNA methylation is one of the most important epigenetic modifications involved in the regulation of gene expression. The DNA methylation reaction is catalyzed by DNA methyltransferases (DNMTs). Recent studies have demonstrated that toxicants can affect normal development by altering DNA methylation patterns, but the mechanisms of action are poorly understood. Hence, we tested the hypothesis that developmental exposure to TCDD affects dnmt gene expression patterns. Zebrafish embryos were exposed to 5 nM TCDD for 1 h from 4 to 5 h post-fertilization (hpf) and sampled at 12, 24, 48, 72, and 96 hpf to determine dnmt gene expression and DNA methylation patterns. We performed a detailed analysis of zebrafish dnmt gene expression during development and in adult tissues. Our results demonstrate that dnmt3b genes are highly expressed in early stages of development, and dnmt3a genes are more abundant in later stages. TCDD exposure upregulated dnmt1 and dnmt3b2 expression, whereas dnmt3a1, 3b1, and 3b4 are downregulated following exposure. We did not observe any TCDD-induced differences in global methylation or hydroxymethylation levels, but the promoter methylation of aryl hydrocarbon receptor (AHR) target genes was altered. In TCDD-exposed embryos, AHR repressor a (ahrra) and c-fos promoters were differentially methylated. To characterize the TCDD effects on DNMTs, we cloned the dnmt promoters with xenobiotic response elements and conducted AHR transactivation assays using a luciferase reporter system. Our results suggest that ahr2 can regulate dnmt3a1, dnmt3a2, and dnmt3b2 expression. Overall, we demonstrate that developmental exposure to TCDD alters dnmt expression and DNA methylation patterns. - Highlights: • TCDD altered the dnmt expression in a gene and developmental time-specific manner. • TCDD hypermethylated ahrra and hypomethylated c-fos proximal promoter regions. • Functional analysis suggests that ahr2 can regulate dnmt3a1, 3a2, and 3b2 expression. • Dnmt

Retroviral DNA Integration

Science.gov (United States)

2016-01-01

The integration of a DNA copy of the viral RNA genome into host chromatin is the defining step of retroviral replication. This enzymatic process is catalyzed by the virus-encoded integrase protein, which is conserved among retroviruses and LTR-retrotransposons. Retroviral integration proceeds via two integrase activities: 3′-processing of the viral DNA ends, followed by the strand transfer of the processed ends into host cell chromosomal DNA. Herein we review the molecular mechanism of retroviral DNA integration, with an emphasis on reaction chemistries and architectures of the nucleoprotein complexes involved. We additionally discuss the latest advances on anti-integrase drug development for the treatment of AIDS and the utility of integrating retroviral vectors in gene therapy applications. PMID:27198982

DNA polymerases κ and ζ cooperatively perform mutagenic translesion synthesis of the C8–2′-deoxyguanosine adduct of the dietary mutagen IQ in human cells

Science.gov (United States)

Bose, Arindam; Pande, Paritosh; Jasti, Vijay P.; Millsap, Amy D.; Hawkins, Edward K.; Rizzo, Carmelo J.; Basu, Ashis K.

2015-01-01

The roles of translesion synthesis (TLS) DNA polymerases in bypassing the C8–2′-deoxyguanosine adduct (dG-C8-IQ) formed by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a highly mutagenic and carcinogenic heterocyclic amine found in cooked meats, were investigated. Three plasmid vectors containing the dG-C8-IQ adduct at the G1-, G2- or G3-positions of the NarI site (5′-G1G2CG3CC-3′) were replicated in HEK293T cells. Fifty percent of the progeny from the G3 construct were mutants, largely G→T, compared to 18% and 24% from the G1 and G2 constructs, respectively. Mutation frequency (MF) of dG-C8-IQ was reduced by 38–67% upon siRNA knockdown of pol κ, whereas it was increased by 10–24% in pol η knockdown cells. When pol κ and pol ζ were simultaneously knocked down, MF of the G1 and G3 constructs was reduced from 18% and 50%, respectively, to IQ. We conclude that pol κ and pol ζ cooperatively carry out the majority of the error-prone TLS of dG-C8-IQ, whereas pol η is involved primarily in its error-free bypass. PMID:26220181

Macromolecule oxidation and DNA repair in mussel (Mytilus edulis L.) gill following exposure to Cd and Cr(VI)

International Nuclear Information System (INIS)

Emmanouil, C.; Sheehan, T.M.T.; Chipman, J.K.

2007-01-01

The oxidation of DNA and lipid was analysed in the marine mussel (Mytilus edulis) in response to exposure (10 μg/l and 200 μg/l) to cadmium (Cd) and chromium [Cr(VI)]. Concentration dependent uptake of both metals into mussel tissues was established and levels of gill ATP were not depleted at these exposure levels. DNA strand breakage in gill cells (analysed by the comet assay) was elevated by both metals, however, DNA oxidation [measured by DNA strand breakage induced by the DNA repair enzyme formamidopyrimidine glycosylase (FPG)] was not elevated. This was despite a statistically significant increase in both malondialdehyde and 4-hydroxynonenal - indicative of lipid peroxidation - following treatment with Cd. In contrast, both frank DNA stand breaks and FPG-induced DNA strand breaks (indicative of DNA oxidation) were increased following injection of mussels with sodium dichromate (10.4 μg Cr(VI)/mussel). The metals also showed differential inhibitory potential towards DNA repair enzyme activity with Cd exhibiting inhibition of DNA cutting activity towards an oligonucleotide containing 8-oxo-7,8-dihydro-2'-deoxyguanosine and Cr(VI) showing inhibition of such activity towards an oligonucleotide containing ethenoadenosine, both at 200 μg/l. The metals thus show DNA damage activity in mussel gill with distinct mechanisms involving both direct and indirect (oxidative) DNA damage, as well as impairing different DNA repair capacities. A combination of these activities can contribute to adverse effects in these organisms

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.

DNA repair mechanism in radioresistant bacteria

International Nuclear Information System (INIS)

Kitayama, Shigeru

1992-01-01

Many radiation resistant bacteria have been isolated from various sources which are not in high background field. Since Deinococcus radiodurans had been isolated first in 1956, studies on the mechanism for radioresistance were carried out mostly using this bacterium. DNA in this bacterium isn't protected against injury induced by not only ionizing radiation but also ultraviolet light. Therefore, DNA damages induced by various treatments are efficiently and accurately repaired in this cells. Damages in base and/or sugar in DNA are removed by endonucleases which, if not all, are synthesized during postirradiation incubation. Following the endonucleolytic cleavage the strand scissions in DNA are seemed to be rejoined by a process common for the repair of strand scissions induced by such as ionizing radiations. Induce protein(s) is also involved in this rejoining process of strand scissions. DNA repair genes were classified into three phenotypic groups. (1)Genes which are responsible for the endonucleolytic activities. (2) Genes involved in the rejoining of DNA strand scissions. (3) Genes which participate in genetic recombination and repair. Three genes belong to (1) and (2) were cloned onto approximately 1 kbp DNA fragments which base sequences have been determined. (author)

DNA repair mechanism in radioresistant bacteria

International Nuclear Information System (INIS)

Kitayama, Shigeru

1992-01-01

Many radiation resistant bacteria have been isolated from various sources which are not in high background field. Since Deinococcus radiodurans had been isolated first in 1956, the studies on the mechanism of radioresistance were mostly carried out using this bacterium. DNA in this bacterium isn't protected against injury induced by not only ionizing radiation but also ultraviolet light. Therefore, DNA damages induced by various treatments are efficiently and accurately repaired in this cells. Damages in base and/or sugar in DNA are removed by endonucleases which, if not all, are synthesized during postirradiation incubation. Following the endonucleolytic cleavage the strand scissions in DNA are seemed to be rejoined by a process common for the repair of strand scissions induced by such as ionizing radiations. Induce protein(s) is also involved in this rejoining process of strand scissions. DNA repair genes were classified into three phenotypic groups. (1) Genes which are responsible for the endonucleolytic activities. (2) Genes involved in the rejoining of DNA strand scissions. (3) Genes which participate in genetic recombination and repair. Three genes belong to (1) and (2) were cloned onto approximately 1 kbp DNA fragments which base sequences have been determined. (author)

Differential sensitivity to aphidicolin of replicative DNA synthesis and ultraviolet-induced unscheduled DNA synthesis in vivo in mammalian cells

International Nuclear Information System (INIS)

Seki, Shuji; Hosogi, Nobuo; Oda, Takuzo

1984-01-01

In vivo in mammalian cells, ultraviolet-induced unscheduled DNA synthesis was less sensitive to aphidicolin than was replicative DNA synthesis. Replicative DNA synthesis in HeLa, HEp-2, WI-38 VA-13 and CV-1 cells was inhibited more than 97 % by aphidicolin at 10 μg/ml, whereas aphidicolin inhibition of DNA synthesis in ultraviolet-irradiated cells varied between 30 % and 90 % depending on cell types and assay conditions. Aphidicolin inhibition of unscheduled DNA synthesis (UDS) in HeLa cells increased gradually with increasing aphidicolin concentration and reached approximately 90 % at 100 μg/ml aphidicolin. A significant fraction of UDS in ultraviolet-irradiated HEp-2 cells was resistant to aphidicolin even at 300 μg/ml. Considered along with related information reported previously, the present results suggest that both aphidicolin-sensitive and insensitive DNA polymerases, DNA polymerase α and a non-α DNA polymerase (possibly DNA polymerase β), are involved in in situ UDS in these ultraviolet-irradiated cells. Comparison of staphylococcal nuclease sensitivity between DNAs repaired in the presence and in the absence of aphidicolin in HEp-2 cells suggested that the involvement of DNA polymerase α in UDS favored DNA synthesis in the intranucleosomal region. (author)

Identification and characterization of a DNA primase activity present in herpes simplex virus type 1-infected HeLa cells

International Nuclear Information System (INIS)

Holmes, A.M.; Wietstock, S.M.; Ruyechan, W.T.

1988-01-01

A novel DNA primase activity has been identified in HeLa cells infected with herpes simplex virus type 1 (HSV-1). Such an activity has not been detected in mock-infected cells. The primase activity coeluted with a portion of HSV-1 DNA polymerase from single-stranded DNA agarose columns loaded with high-salt extracts derived from infected cells. This DNA primase activity could be distinguished from host HeLa cell DNA primase by several criteria. First, the pH optimum of the HSV primase was relatively broad and peaked at 8.2 to 8.7 pH units. Second, freshly isolated HSV DNA primase was less salt sensitive than the HeLa primase. Third, antibodies raised against individual peptides of the calf thymus DNA polymerase:primase complex cross-reacted with the HeLa primase but did not react with the HSV DNA primase. Fourth, freshly prepared HSV DNA primase appeared to be associated with the HSV polymerase, but after storage at 4 degree C for several weeks, the DNA primase separated from the viral DNA polymerase. This free DNA primase had an apparent molecular size of approximately 40 kilodaltons, whereas free HeLa DNA primase had an apparent molecular size of approximately 110 kilodaltons. On the basis of these data, the authors believe that the novel DNA primase activity in HSV-infected cells may be virus coded and that this enzyme represents a new and important function involved in the replication of HSV DNA

Lead-induced DNA damage in Vicia faba root cells: Potential involvement of oxidative stress

OpenAIRE

Pourrut, Bertrand; Jean, Séverine; Silvestre, Jérôme; Pinelli, Eric

2011-01-01

Genotoxic effects of lead (0–20 µM) were investigated in whole-plant roots of Vicia faba L., grown hydroponically under controlled conditions. Lead-induced DNA damage in V. faba roots was evaluated by use of the comet assay, which allowed the detection of DNA strand-breakage and with the V. faba micronucleus test, which revealed chromosome aberrations. The results clearly indicate that lead induced DNA fragmentation in a dose-dependant manner with a maximum effect at 10 µM. In addition, at th...

DNA-Binding Properties of African Swine Fever Virus pA104R, a Histone-Like Protein Involved in Viral Replication and Transcription.

Science.gov (United States)

Frouco, Gonçalo; Freitas, Ferdinando B; Coelho, João; Leitão, Alexandre; Martins, Carlos; Ferreira, Fernando

2017-06-15

African swine fever virus (ASFV) codes for a putative histone-like protein (pA104R) with extensive sequence homology to bacterial proteins that are implicated in genome replication and packaging. Functional characterization of purified recombinant pA104R revealed that it binds to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) over a wide range of temperatures, pH values, and salt concentrations and in an ATP-independent manner, with an estimated binding site size of about 14 to 16 nucleotides. Using site-directed mutagenesis, the arginine located in pA104R's DNA-binding domain, at position 69, was found to be relevant for efficient DNA-binding activity. Together, pA104R and ASFV topoisomerase II (pP1192R) display DNA-supercoiling activity, although none of the proteins by themselves do, indicating that the two cooperate in this process. In ASFV-infected cells, A104R transcripts were detected from 2 h postinfection (hpi) onward, reaching a maximum concentration around 16 hpi. pA104R was detected from 12 hpi onward, localizing with viral DNA replication sites and being found exclusively in the Triton-insoluble fraction. Small interfering RNA (siRNA) knockdown experiments revealed that pA104R plays a critical role in viral DNA replication and gene expression, with transfected cells showing lower viral progeny numbers (up to a reduction of 82.0%), lower copy numbers of viral genomes (-78.3%), and reduced transcription of a late viral gene (-47.6%). Taken together, our results strongly suggest that pA104R participates in the modulation of viral DNA topology, probably being involved in viral DNA replication, transcription, and packaging, emphasizing that ASFV mutants lacking the A104R gene could be used as a strategy to develop a vaccine against ASFV. IMPORTANCE Recently reintroduced in Europe, African swine fever virus (ASFV) causes a fatal disease in domestic pigs, causing high economic losses in affected countries, as no vaccine or treatment is currently

NMR structural studies of the ionizing radiation adduct 7-hydro-8-oxodeoxyguanosine (8-oxo-7H-dG) opposite deoxyadenosine in a DNA duplex. 8-oxo-7H-dG(syn)·dA(anti) alignment at lesion site

International Nuclear Information System (INIS)

Kouchakdjian, M.; Patel, D.J.; Bodepudi, V.; Shibutani, S.; Eisenberg, M.; Johnson, F.; Grollman, A.P.

1991-01-01

Proton NMR studies are reported on the complementary d(C1-C2-A3-C4-T5-A6-oxo-G7-T8-C9-A10-C11-C12)·d(G13-G14-T15-G16-A17-A18-T19-A20-G21-T22-G23-G24) dodecanucleotide duplex (designated 8-oxo-7H-dG·dA 12-mer), which contains a centrally located 7-hydro-8-oxodeoxyguanosine (8-oxo-7H-dG) residue, a group commonly found in DNA that has been exposed to ionizing radiation or oxidizing free radicals. From the NMR spectra it can be deduced that this moiety exists as two tautomers, or gives rise to two DNA conformations, that are in equilibrium and that exchange slowly. The present study focuses on the major component of the equilibrium that originates in the 6,8-dioxo tautomer of 8-oxo-7H-dG. The authors have assigned the exchangeable NH1, NH7, and NH 2 -2 base protons located on the Watson-Crick and Hoogsteen edges of 8-oxo-7H-dG7 in the 8-oxo-7H-dG·dA 12-mer duplex, using an analysis of one- and two-dimensional nuclear Overhauser enhancement (NOE) data in H 2 O solution. They were able to detect a set of intra- and interstrand NOEs between protons (exchangeable and nonexchangeable) on adjacent residues in the d(A6-oxo-G7-T8)·d(A17-A18-T19) trinucleotide segment centered about the lesion site that establishes stacking of the oxo-dG7(syn)·dA(anti) pair between stable Watson-Crick dA6·dT19 and dT8·A17 base pairs with minimal perturbation of the helix. The structural studies demonstrate that 8-oxo-7H-dG(syn)·dA(anti) forms a stable pair in the interior of the helix, providing a basis for the observed incorporation of dA opposite 8-oxo-7H-dG when readthrough occurs past this oxidized nucleoside base

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

Exercise-Induced Neuroprotection of Hippocampus in APP/PS1 Transgenic Mice via Upregulation of Mitochondrial 8-Oxoguanine DNA Glycosylase

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Hai Bo

2014-01-01

Full Text Available Improving mitochondrial function has been proposed as a reasonable therapeutic strategy to reduce amyloid-β (Aβ load and to modify the progression of Alzheimer’s disease (AD. However, the relationship between mitochondrial adaptation and brain neuroprotection caused by physical exercise in AD is poorly understood. This study was undertaken to investigate the effects of long-term treadmill exercise on mitochondrial 8-oxoguanine DNA glycosylase-1 (OGG1 level, mtDNA oxidative damage, and mitochondrial function in the hippocampus of APP/PS1 transgenic mouse model of AD. In the present study, twenty weeks of treadmill training significantly improved the cognitive function and reduced the expression of Aβ-42 in APP/PS1 transgenic (Tg mice. Training also ameliorated mitochondrial respiratory function by increasing the complexes I, and IV and ATP synthase activities, whereas it attenuated ROS generation and mtDNA oxidative damage in Tg mice. Furthermore, the impaired mitochondrial antioxidant enzymes and mitochondrial OGG1 activities seen in Tg mice were restored with training. Acetylation level of mitochondrial OGG1 and MnSOD was markedly suppressed in Tg mice after exercise training, in parallel with increased level of SIRT3. These findings suggest that exercise training could increase mtDNA repair capacity in the mouse hippocampus, which in turn would result in protection against AD-related mitochondrial dysfunction and phenotypic deterioration.

Serum Level of Antibodies (IgG, IgM Against Benzo[a]pyrene-7,8-diol-9,10-epoxide-DNA Adducts in Children Dermatologically Exposed to Coal Tar

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Pavel Borský

2017-06-01

Full Text Available Crude coal tar (CCT contains polycyclic aromatic hydrocarbons (PAHs. Benzo[a]pyrene (BaP is metabolized into a highly reactive metabolite benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE that is able to bind to DNA and creates BPDE-DNA adducts. Adducted DNA becomes immunogenic and induces immune response by production of antibodies against BPDE-DNA adducts (Ab-BPDE-DNA. Circulating Ab-BPDE-DNA was proposed as potential biomarker of genotoxic exposure to BaP (PAHs. Goeckerman therapy (GT of psoriasis uses dermal application of CCT ointment (PAHs. In presented study (children with psoriasis treated by GT; n = 19 the therapy significantly increased the level of Ab-BPDE-DNA (EI = 0.29/0.19–0.34 vs. 0.31/0.25–0.40; median/lower–upper quartile; p < 0.01. The results support the idea of Ab-BPDE-DNA level as a possible tentative indicator of exposure, effects and susceptibility of the organism to the exposure of BaP (PAHs.

PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases.

Science.gov (United States)

Cline, J; Braman, J C; Hogrefe, H H

1996-09-15

The replication fidelities of Pfu, Taq, Vent, Deep Vent and UlTma DNA polymerases were compared using a PCR-based forward mutation assay. Average error rates (mutation frequency/bp/duplication) increased as follows: Pfu (1.3 x 10(-6)) Pfu and UlTma (approximately 5 x 10(-5)). Buffer optimization experiments indicated that Pfu fidelity was highest in the presence of 2-3 mM MgSO4 and 100-300 microM each dNTP and at pH 8.5-9.1. Under these conditions, the error rate of exo- Pfu was approximately 40-fold higher (5 x 10(-5)) than the error rate of Pfu. As the reaction pH was raised from pH 8 to 9, the error rate of Pfu decreased approximately 2-fold, while the error rate of exo- Pfu increased approximately 9-fold. An increase in error rate with pH has also been noted for the exonuclease-deficient DNA polymerases Taq and exo- Klenow, suggesting that the parameters which influence replication error rates may be similar in pol l- and alpha-like polymerases. Finally, the fidelity of 'long PCR' DNA polymerase mixtures was examined. The error rates of a Taq/Pfu DNA polymerase mixture and a Klentaq/Pfu DNA polymerase mixture were found to be less than the error rate of Taq DNA polymerase, but approximately 3-4-fold higher than the error rate of Pfu DNA polymerase.

Transfer and persistence of non-self DNA on hands over time: Using empirical data to evaluate DNA evidence given activity level propositions.

Science.gov (United States)

Szkuta, Bianca; Ballantyne, Kaye N; Kokshoorn, Bas; van Oorschot, Roland A H

2018-03-01

Questions relating to how DNA from an individual got to where it was recovered from and the activities associated with its pickup, retention and deposition are increasingly relevant to criminal investigations and judicial considerations. To address activity level propositions, investigators are typically required to assess the likelihood that DNA was transferred indirectly and not deposited through direct contact with an item or surface. By constructing a series of Bayesian networks, we demonstrate their use in assessing activity level propositions derived from a recent legal case involving the alleged secondary transfer of DNA to a surface following a handshaking event. In the absence of data required to perform the assessment, a set of handshaking simulations were performed to obtain probabilities on the persistence of non-self DNA on the hands following a 40min, 5h or 8h delay between the handshake and contact with the final surface (an axe handle). Variables such as time elapsed, and the activities performed and objects contacted between the handshake and contact with the axe handle, were also considered when assessing the DNA results. DNA from a known contributor was transferred to the right hand of an opposing hand-shaker (as a depositor), and could be subsequently transferred to, and detected on, a surface contacted by the depositor 40min to 5h post-handshake. No non-self DNA from the known contributor was detected in deposits made 8h post-handshake. DNA from the depositor was generally detected as the major or only contributor in the profiles generated. Contributions from the known contributor were minor, decreasing in presence and in the strength of support for inclusion as the time between the handshake and transfer event increased. The construction of a series of Bayesian networks based on the case circumstances provided empirical estimations of the likelihood of direct or indirect deposition. The analyses and conclusions presented demonstrate both the

Gene activation of heavy ion treated bacillus subtilis 168 endospores during germination involved DNA-repair

International Nuclear Information System (INIS)

Moeller, R.; Berger, T.; Reitz, G.; Okayasu, Ryuichi

2006-01-01

This research project is aimed at correlating radiation effects induced DNA damage in Bacillus subtilis endospores with the linear energy transfer (LET) of the used radiation by investigating survival and gene activation after irradiation with high-LET particles. During the stationary growth phase Bacillus subtilis change their metabolic active state from the vegetative cells to the metabolic inactive but even more resistant endospores. If spores find optimal conditions, they could germinate and switch to the vegetative growth. With these outgrowth spores can and/or must repair the induced formed DNA damage. During germination spores lose their most resistance. In more detail, DNA repair and mutation induction events investigated will include the survivability, behaviour against specific antibiotics and their germination. DNA repair pattern will be detected during germination by using DNA microarrays, which contain the whole genome of Bacillus subtilis 168. (author)

Identification of mammalian proteins cross-linked to DNA by ionizing radiation.

Science.gov (United States)

Barker, Sharon; Weinfeld, Michael; Zheng, Jing; Li, Liang; Murray, David

2005-10-07

Ionizing radiation (IR) is an important environmental risk factor for various cancers and also a major therapeutic agent for cancer treatment. Exposure of mammalian cells to IR induces several types of damage to DNA, including double- and single-strand breaks, base and sugar damage, as well as DNA-DNA and DNA-protein cross-links (DPCs). Little is known regarding the biological consequences of DPCs. Identifying the proteins that become cross-linked to DNA by IR would be an important first step in this regard. We have therefore undertaken a proteomics study to isolate and identify proteins involved in IR-induced DPCs. DPCs were induced in AA8 Chinese hamster ovary or GM00637 human fibroblast cells using 0-4 gray of gamma-rays under either aerated or hypoxic conditions. DPCs were isolated using a recently developed method, and proteins were identified by mass spectrometry. We identified 29 proteins as being cross-linked to DNA by IR under aerated and/or hypoxic conditions. The identified proteins include structural proteins, actin-associated proteins, transcription regulators, RNA-splicing components, stress-response proteins, cell cycle regulatory proteins, and GDP/GTP-binding proteins. The involvement of several proteins (actin, histone H2B, and others) in DPCs was confirmed by using Western blot analysis. The dose responsiveness of DPC induction was examined by staining one-dimensional SDS-polyacrylamide gels with SYPRO Tangerine followed by analysis using fluorescence imaging. Quantitation of the fluorescence signal indicated no significant difference in total yields of IR-induced DPCs generated under aerated or hypoxic conditions, although differences were observed for several individual protein bands.

Critical involvement of the ATM-dependent DNA damage response in the apoptotic demise of HIV-1-elicited syncytia.

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Jean-Luc Perfettini

Full Text Available DNA damage can activate the oncosuppressor protein ataxia telangiectasia mutated (ATM, which phosphorylates the histone H2AX within characteristic DNA damage foci. Here, we show that ATM undergoes an activating phosphorylation in syncytia elicited by the envelope glycoprotein complex (Env of human immunodeficiency virus-1 (HIV-1 in vitro. This was accompanied by aggregation of ATM in discrete nuclear foci that also contained phospho-histone H2AX. DNA damage foci containing phosphorylated ATM and H2AX were detectable in syncytia present in the brain or lymph nodes from patients with HIV-1 infection, as well as in a fraction of blood leukocytes, correlating with viral status. Knockdown of ATM or of its obligate activating factor NBS1 (Nijmegen breakage syndrome 1 protein, as well as pharmacological inhibition of ATM with KU-55933, inhibited H2AX phosphorylation and prevented Env-elicited syncytia from undergoing apoptosis. ATM was found indispensable for the activation of MAP kinase p38, which catalyzes the activating phosphorylation of p53 on serine 46, thereby causing p53 dependent apoptosis. Both wild type HIV-1 and an HIV-1 mutant lacking integrase activity induced syncytial apoptosis, which could be suppressed by inhibiting ATM. HIV-1-infected T lymphoblasts from patients with inactivating ATM or NBS1 mutations also exhibited reduced syncytial apoptosis. Altogether these results indicate that apoptosis induced by a fusogenic HIV-1 Env follows a pro-apoptotic pathway involving the sequential activation of ATM, p38MAPK and p53.

[DNA-dependent DNA polymerase induced by herpes virus papio (HVP) in producing cells].

Science.gov (United States)

D'iachenko, A G; Beriia, L Ia; Matsenko, L D; Kakubava, V V; Kokosh, L V

1980-11-01

A new DNA polymerase was found in the cells of suspension lymphoblastoid cultures, which produce lymphotropic baboon herpes virus (HVP). The enzyme was isolated in a partially purified form. In some properties the enzyme differs from other cellular DNA polymerases. The HVP-induced DNA polymerase has the molecular weight of 1,6 x 10(5) and sedimentation coefficient of about 8S. The enzyme is resistant to high salt concentrations and N-ethylmaleimide, but shows a pronounced sensitivity to phosphonoacetate. The enzyme effectively copies "activated" DNA and synthetic deoxyribohomopolymers. The attempts to detect the DNA polymerase activity in HVP virions were unsuccessful.

4β-Hydroxywithanolide E selectively induces oxidative DNA damage for selective killing of oral cancer cells.

Science.gov (United States)

Tang, Jen-Yang; Huang, Hurng-Wern; Wang, Hui-Ru; Chan, Ya-Ching; Haung, Jo-Wen; Shu, Chih-Wen; Wu, Yang-Chang; Chang, Hsueh-Wei

2018-03-01

Reactive oxygen species (ROS) induction had been previously reported in 4β-hydroxywithanolide (4βHWE)-induced selective killing of oral cancer cells, but the mechanism involving ROS and the DNA damage effect remain unclear. This study explores the role of ROS and oxidative DNA damage of 4βHWE in the selective killing of oral cancer cells. Changes in cell viability, morphology, ROS, DNA double strand break (DSB) signaling (γH2AX foci in immunofluorescence and DSB signaling in western blotting), and oxidative DNA damage (8-oxo-2'deoxyguanosine [8-oxodG]) were detected in 4βHWE-treated oral cancer (Ca9-22) and/or normal (HGF-1) cells. 4βHWE decreased cell viability, changed cell morphology and induced ROS generation in oral cancer cells rather than oral normal cells, which were recovered by a free radical scavenger N-acetylcysteine (NAC). For immunofluorescence, 4βHWE also accumulated more of the DSB marker, γH2AX foci, in oral cancer cells than in oral normal cells. For western blotting, DSB signaling proteins such as γH2AX and MRN complex (MRE11, RAD50, and NBS1) were overexpressed in 4βHWE-treated oral cancer cells in different concentrations and treatment time. In the formamidopyrimidine-DNA glycolyase (Fpg)-based comet assay and 8-oxodG-based flow cytometry, the 8-oxodG expressions were higher in 4βHWE-treated oral cancer cells than in oral normal cells. All the 4βHWE-induced DSB and oxidative DNA damage to oral cancer cells were recovered by NAC pretreatment. Taken together, the 4βHWE selectively induced DSB and oxidative DNA damage for the ROS-mediated selective killing of oral cancer cells. © 2017 Wiley Periodicals, Inc.

Detection and Quantification of 8-Hydroxy-2′-Deoxyguanosine in Alzheimer’s Transgenic Mouse Urine using Capillary Electrophoresis

Science.gov (United States)

Zhang, Cheng; Nestorova, Gergana; Rissman, Robert A.; Feng, June

2013-01-01

8-Hydroxy-2′-deoxyguanosine (8-OHdG) is one of the major forms of oxidative deoxyribonucleic acid (DNA) damage, and is commonly analyzed as an excellent marker of DNA lesions. The purpose of this study was to develop a sensitive method to accurately and rapidly quantify the 8-OHdG by using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). The method involved the use of specific antibody to detect DNA lesions (8-OHdG) and consecutive fluorescence labeling. Next, the urine sample with 8-OHdG fluorescently labeled along with other constituents was resolved by capillary electrophoretic system and the lesion of interest was detected using fluorescence detector. The limit of detection was 0.18 fmol, which is sufficient sensitivity for detection and quantification of 8-OHdG in untreated urine samples. The relative standard deviation (RSD) was found to be 11.32 % for migration time, and 5.52 % for peak area. To demonstrate the utility of this method, the urinary concentration of 8-OHdG in an Alzheimer’s transgenic mouse model was determined. Collectively, our results indicate that this methodology offers great advantages such as high separation efficiency, good selectivity, low limit of detection (LOD), simplicity and low cost of analysis. PMID:23712533

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.

Investigation of the Mitochondrial ATPase 6/8 and tRNA(Lys) Genes Mutations in Autism.

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Piryaei, Fahimeh; Houshmand, Massoud; Aryani, Omid; Dadgar, Sepideh; Soheili, Zahra-Soheila

2012-01-01

Autism results from developmental factors that affect many or all functional brain systems. Brain is one of tissues which are crucially in need of adenosine triphosphate (ATP). Autism is noticeably affected by mitochondrial dysfunction which impairs energy metabolism. Considering mutations within ATPase 6, ATPase 8 and tRNA(Lys) genes, associated with different neural diseases, and the main role of ATPase 6/8 in energy generation, we decided to investigate mutations on these mtDNA-encoded genes to reveal their roles in autism pathogenesis. In this experimental study, mutation analysis for the mentioned genes were performed in a cohort of 24 unrelated patients with idiopathic autism by employing amplicon sequencing of mtDNA fragments. In this study, 12 patients (50%) showed point mutations that represent a significant correlation between autism and mtDNA variations. Most of the identified substitutions (55.55%) were observed on MT-ATP6, altering some conserved amino acids to other ones which could potentially affect ATPase 6 function. Mutations causing amino acid replacement denote involvement of mtDNA genes, especially ATPase 6 in autism pathogenesis. MtDNA mutations in relation with autism could be remarkable to realize an understandable mechanism of pathogenesis in order to achieve therapeutic solutions.

DNA Glycosylases Involved in Base Excision Repair May Be Associated with Cancer Risk in BRCA1 and BRCA2 Mutation Carriers

DEFF Research Database (Denmark)

Osorio, Ana; Milne, Roger L; Kuchenbaecker, Karoline

2014-01-01

Single Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of th...

Small molecules, inhibitors of DNA-PK, targeting DNA repair and beyond

Directory of Open Access Journals (Sweden)

David eDavidson

2013-01-01

Full Text Available Many current chemotherapies function by damaging genomic DNA in rapidly dividing cells ultimately leading to cell death. This therapeutic approach differentially targets cancer cells that generally display rapid cell division compared to normal tissue cells. However, although these treatments are initially effective in arresting tumor growth and reducing tumor burden, resistance and disease progression eventually occur. A major mechanism underlying this resistance is increased levels of cellular DNA repair. Most cells have complex mechanisms in place to repair DNA damage that occurs due to environmental exposures or normal metabolic processes. These systems, initially overwhelmed when faced with chemotherapy induced DNA damage, become more efficient under constant selective pressure and as a result chemotherapies become less effective. Thus, inhibiting DNA repair pathways using target specific small molecule inhibitors may overcome cellular resistance to DNA damaging chemotherapies. Non-homologous end joining (NHEJ a major mechanism for the repair of double strand breaks (DSB in DNA is regulated in part by the serine/threonine kinase, DNA dependent protein kinase (DNA-PK. The DNA-PK holoenzyme acts as a scaffold protein tethering broken DNA ends and recruiting other repair molecules. It also has enzymatic activity that may be involved in DNA damage signaling. Because of its’ central role in repair of DSBs, DNA-PK has been the focus of a number of small molecule studies. In these studies specific DNA-PK inhibitors have shown efficacy in synergizing chemotherapies in vitro. However, compounds currently known to specifically inhibit DNA-PK are limited by poor pharmacokinetics: these compounds have poor solubility and have high metabolic lability in vivo leading to short serum half-lives. Future improvement in DNA-PK inhibition will likely be achieved by designing new molecules based on the recently reported crystallographic structure of DNA

DNA Topology and the Initiation of Virus DNA Packaging.

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Choon Seok Oh

Full Text Available During progeny assembly, viruses selectively package virion genomes from a nucleic acid pool that includes host nucleic acids. For large dsDNA viruses, including tailed bacteriophages and herpesviruses, immature viral DNA is recognized and translocated into a preformed icosahedral shell, the prohead. Recognition involves specific interactions between the viral packaging enzyme, terminase, and viral DNA recognition sites. Generally, viral DNA is recognized by terminase's small subunit (TerS. The large terminase subunit (TerL contains translocation ATPase and endonuclease domains. In phage lambda, TerS binds a sequence repeated three times in cosB, the recognition site. TerS binding to cosB positions TerL to cut the concatemeric DNA at the adjacent nicking site, cosN. TerL introduces staggered nicks in cosN, generating twelve bp cohesive ends. Terminase separates the cohesive ends and remains bound to the cosB-containing end, in a nucleoprotein structure called Complex I. Complex I docks on the prohead's portal vertex and translocation ensues. DNA topology plays a role in the TerSλ-cosBλ interaction. Here we show that a site, I2, located between cosN and cosB, is critically important for an early DNA packaging step. I2 contains a complex static bend. I2 mutations block DNA packaging. I2 mutant DNA is cut by terminase at cosN in vitro, but in vivo, no cos cleavage is detected, nor is there evidence for Complex I. Models for what packaging step might be blocked by I2 mutations are presented.

Mitochondrial DNA maintenance is regulated in human hepatoma cells by glycogen synthase kinase 3β and p53 in response to tumor necrosis factor α.

Science.gov (United States)

Vadrot, Nathalie; Ghanem, Sarita; Braut, Françoise; Gavrilescu, Laura; Pilard, Nathalie; Mansouri, Abdellah; Moreau, Richard; Reyl-Desmars, Florence

2012-01-01

During chronic liver inflammation, up-regulated Tumor Necrosis Factor alpha (TNF-α) targets hepatocytes and induces abnormal reactive oxygen species (ROS) production responsible for mitochondrial DNA (mtDNA) alterations. The serine/threonine Glycogen Synthase Kinase 3 beta (GSK3β) plays a pivotal role during inflammation but its involvement in the maintenance of mtDNA remains unknown. The aim of this study was to investigate its involvement in TNF-α induced mtDNA depletion and its interrelationship with p53 a protein known to maintain mtDNA copy numbers. Using quantitative polymerase chain reaction (qPCR) we found that at 30 min in human hepatoma HepG2 cells TNF-α induced 0.55±0.10 mtDNA lesions per 10 Kb and a 52.4±2.8% decrease in mtDNA content dependent on TNF-R1 receptor and ROS production. Both lesions and depletion returned to baseline from 1 to 6 h after TNF-α exposure. Luminol-amplified chemiluminescence (LAC) was used to measure the rapid (10 min) and transient TNF-α induced increase in ROS production (168±15%). A transient 8-oxo-dG level of 1.4±0.3 ng/mg DNA and repair of abasic sites were also measured by ELISA assays. Translocation of p53 to mitochondria was observed by Western Blot and co-immunoprecipitations showed that TNF-α induced p53 binding to GSK3β and mitochondrial transcription factor A (TFAM). In addition, mitochondrial D-loop immunoprecipitation (mtDIP) revealed that TNF-α induced p53 binding to the regulatory D-loop region of mtDNA. The knockdown of p53 by siRNAs, inhibition by the phosphoSer(15)p53 antibody or transfection of human mutant active GSK3βS9A pcDNA3 plasmid inhibited recovery of mtDNA content while blockade of GSK3β activity by SB216763 inhibitor or knockdown by siRNAs suppressed mtDNA depletion. This study is the first to report the involvement of GSK3β in TNF-α induced mtDNA depletion. We suggest that p53 binding to GSK3β, TFAM and D-loop could induce recovery of mtDNA content through mtDNA repair.

Mitochondrial DNA maintenance is regulated in human hepatoma cells by glycogen synthase kinase 3β and p53 in response to tumor necrosis factor α.

Directory of Open Access Journals (Sweden)

Nathalie Vadrot

Full Text Available During chronic liver inflammation, up-regulated Tumor Necrosis Factor alpha (TNF-α targets hepatocytes and induces abnormal reactive oxygen species (ROS production responsible for mitochondrial DNA (mtDNA alterations. The serine/threonine Glycogen Synthase Kinase 3 beta (GSK3β plays a pivotal role during inflammation but its involvement in the maintenance of mtDNA remains unknown. The aim of this study was to investigate its involvement in TNF-α induced mtDNA depletion and its interrelationship with p53 a protein known to maintain mtDNA copy numbers. Using quantitative polymerase chain reaction (qPCR we found that at 30 min in human hepatoma HepG2 cells TNF-α induced 0.55±0.10 mtDNA lesions per 10 Kb and a 52.4±2.8% decrease in mtDNA content dependent on TNF-R1 receptor and ROS production. Both lesions and depletion returned to baseline from 1 to 6 h after TNF-α exposure. Luminol-amplified chemiluminescence (LAC was used to measure the rapid (10 min and transient TNF-α induced increase in ROS production (168±15%. A transient 8-oxo-dG level of 1.4±0.3 ng/mg DNA and repair of abasic sites were also measured by ELISA assays. Translocation of p53 to mitochondria was observed by Western Blot and co-immunoprecipitations showed that TNF-α induced p53 binding to GSK3β and mitochondrial transcription factor A (TFAM. In addition, mitochondrial D-loop immunoprecipitation (mtDIP revealed that TNF-α induced p53 binding to the regulatory D-loop region of mtDNA. The knockdown of p53 by siRNAs, inhibition by the phosphoSer(15p53 antibody or transfection of human mutant active GSK3βS9A pcDNA3 plasmid inhibited recovery of mtDNA content while blockade of GSK3β activity by SB216763 inhibitor or knockdown by siRNAs suppressed mtDNA depletion. This study is the first to report the involvement of GSK3β in TNF-α induced mtDNA depletion. We suggest that p53 binding to GSK3β, TFAM and D-loop could induce recovery of mtDNA content through mtDNA repair.

Translocation and gross deletion breakpoints in human inherited disease and cancer II: Potential involvement of repetitive sequence elements in secondary structure formation between DNA ends.

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Chuzhanova, Nadia; Abeysinghe, Shaun S; Krawczak, Michael; Cooper, David N

2003-09-01

Translocations and gross deletions are responsible for a significant proportion of both cancer and inherited disease. Although such gene rearrangements are nonuniformly distributed in the human genome, the underlying mutational mechanisms remain unclear. We have studied the potential involvement of various types of repetitive sequence elements in the formation of secondary structure intermediates between the single-stranded DNA ends that recombine during rearrangements. Complexity analysis was used to assess the potential of these ends to form secondary structures, the maximum decrease in complexity consequent to a gross rearrangement being used as an indicator of the type of repeat and the specific DNA ends involved. A total of 175 pairs of deletion/translocation breakpoint junction sequences available from the Gross Rearrangement Breakpoint Database [GRaBD; www.uwcm.ac.uk/uwcm/mg/grabd/grabd.html] were analyzed. Potential secondary structure was noted between the 5' flanking sequence of the first breakpoint and the 3' flanking sequence of the second breakpoint in 49% of rearrangements and between the 5' flanking sequence of the second breakpoint and the 3' flanking sequence of the first breakpoint in 36% of rearrangements. Inverted repeats, inversions of inverted repeats, and symmetric elements were found in association with gross rearrangements at approximately the same frequency. However, inverted repeats and inversions of inverted repeats accounted for the vast majority (83%) of deletions plus small insertions, symmetric elements for one-half of all antigen receptor-mediated translocations, while direct repeats appear only to be involved in mediating simple deletions. These findings extend our understanding of illegitimate recombination by highlighting the importance of secondary structure formation between single-stranded DNA ends at breakpoint junctions. Copyright 2003 Wiley-Liss, Inc.

DNA repair in Mycobacterium tuberculosis revisited.

Science.gov (United States)

Dos Vultos, Tiago; Mestre, Olga; Tonjum, Tone; Gicquel, Brigitte

2009-05-01

Our understanding of Mycobacterium tuberculosis DNA repair mechanisms is still poor compared with that of other bacterial organisms. However, the publication of the first complete M. tuberculosis genome sequence 10 years ago boosted the study of DNA repair systems in this organism. A first step in the elucidation of M. tuberculosis DNA repair mechanisms was taken by Mizrahi and Andersen, who identified homologs of genes involved in the reversal or repair of DNA damage in Escherichia coli and related organisms. Genes required for nucleotide excision repair, base excision repair, recombination, and SOS repair and mutagenesis were identified. Notably, no homologs of genes involved in mismatch repair were identified. Novel characteristics of the M. tuberculosis DNA repair machinery have been found over the last decade, such as nonhomologous end joining, the presence of Mpg, ERCC3 and Hlr - proteins previously presumed to be produced exclusively in mammalian cells - and the recently discovered bifunctional dCTP deaminase:dUTPase. The study of these systems is important to develop therapeutic agents that can counteract M. tuberculosis evolutionary changes and to prevent adaptive events resulting in antibiotic resistance. This review summarizes our current understanding of the M. tuberculosis DNA repair system.

Mechanism for iron control of the Vibrio fischeri luminescence system: involvement of cyclic AMP and cyclic AMP receptor protein and modulation of DNA level.

Science.gov (United States)

Dunlap, P V

1992-07-01

Iron controls luminescence in Vibrio fischeri by an indirect but undefined mechanism. To gain insight into that mechanism, the involvement of cyclic AMP (cAMP) and cAMP receptor protein (CRP) and of modulation of DNA levels in iron control of luminescence were examined in V. fischeri and in Escherichia coli containing the cloned V. fischeri lux genes on plasmids. For V. fischeri and E. coli adenylate cyclase (cya) and CRP (crp) mutants containing intact lux genes (luxR luxICDABEG), presence of the iron chelator ethylenediamine-di(o-hydroxyphenyl acetic acid) (EDDHA) increased expression of the luminescence system like in the parent strains only in the cya mutants in the presence of added cAMP. In the E. coli strains containing a plasmid with a Mu dl(lacZ) fusion in luxR, levels of beta-galactosidase activity (expression from the luxR promoter) and luciferase activity (expression from the lux operon promoter) were both 2-3-fold higher in the presence of EDDHA in the parent strain, and for the mutants this response to EDDHA was observed only in the cya mutant in the presence of added cAMP. Therefore, cAMP and CRP are required for the iron restriction effect on luminescence, and their involvement in iron control apparently is distinct from the known differential control of transcription from the luxR and luxICDABEG promoters by cAMP-CRP. Furthermore, plasmid and chromosomal DNA levels were higher in E. coli and V. fischeri in the presence of EDDHA. The higher DNA levels correlated with an increase in expression of chromosomally encoded beta-galactosidase in E. coli and with a higher level of autoinducer in cultures of V. fischeri. These results implicate cAMP-CRP and modulation of DNA levels in the mechanism of iron control of the V. fischeri luminescence system.

Mutagenesis of the lac promoter region in M13 mp10 phage DNA by 4'-hydroxymethyl-4,5',8-trimethylpsoralen