DNA alkylation lesions and their repair in human cells: modification of the comet assay with 3-methyladenine DNA glycosylase (AlkD).

Science.gov (United States)

Hašplová, Katarína; Hudecová, Alexandra; Magdolénová, Zuzana; Bjøras, Magnar; Gálová, Eliška; Miadoková, Eva; Dušinská, Mária

2012-01-05

3-methyladenine DNA glycosylase (AlkD) belongs to a new family of DNA glycosylases; it initiates repair of cytotoxic and promutagenic alkylated bases (its main substrates being 3-methyladenine and 7-methylguanine). The modification of the comet assay (single cell gel electrophoresis) using AlkD enzyme thus allows assessment of specific DNA alkylation lesions. The resulting baseless sugars are alkali-labile, and under the conditions of the alkaline comet assay they appear as DNA strand breaks. The alkylating agent methyl methanesulfonate (MMS) was used to induce alkylation lesions and to optimize conditions for the modified comet assay method with AlkD on human lymphoblastoid (TK6) cells. We also studied cellular and in vitro DNA repair of alkylated bases in DNA in TK6 cells after treatment with MMS. Results from cellular repair indicate that 50% of DNA alkylation is repaired in the first 60 min. The in vitro repair assay shows that while AlkD recognises most alkylation lesions after 60 min, a cell extract from TK6 cells recognises most of the MMS-induced DNA adducts already in the first 15 min of incubation, with maximum detection of lesions after 60 min' incubation. Additionally, we tested the in vitro repair capacity of human lymphocyte extracts from 5 individuals and found them to be able to incise DNA alkylations in the same range as AlkD. The modification of the comet assay with AlkD can be useful for in vitro and in vivo genotoxicity studies to detect alkylation damage and repair and also for human biomonitoring and molecular epidemiology studies. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Structure of uracil-DNA glycosylase from Mycobacterium tuberculosis: insights into interactions with ligands

International Nuclear Information System (INIS)

Kaushal, Prem Singh; Talawar, Ramappa K.; Varshney, Umesh; Vijayan, M.

2010-01-01

The molecule of uracil-DNA glycosylase from M. tuberculosis exhibits domain motion on binding to DNA or a proteinaceous inhibitor. The highly conserved DNA-binding region interacts with a citrate ion in the structure. Uracil N-glycosylase (Ung) is the most thoroughly studied of the group of uracil DNA-glycosylase (UDG) enzymes that catalyse the first step in the uracil excision-repair pathway. The overall structure of the enzyme from Mycobacterium tuberculosis is essentially the same as that of the enzyme from other sources. However, differences exist in the N- and C-terminal stretches and some catalytic loops. Comparison with appropriate structures indicate that the two-domain enzyme closes slightly when binding to DNA, while it opens slightly when binding to the proteinaceous inhibitor Ugi. The structural changes in the catalytic loops on complexation reflect the special features of their structure in the mycobacterial protein. A comparative analysis of available sequences of the enzyme from different sources indicates high conservation of amino-acid residues in the catalytic loops. The uracil-binding pocket in the structure is occupied by a citrate ion. The interactions of the citrate ion with the protein mimic those of uracil, in addition to providing insights into other possible interactions that inhibitors could be involved in

Synthesis and characterization of DNA minor groove binding alkylating agents.

Science.gov (United States)

Iyer, Prema; Srinivasan, Ajay; Singh, Sreelekha K; Mascara, Gerard P; Zayitova, Sevara; Sidone, Brian; Fouquerel, Elise; Svilar, David; Sobol, Robert W; Bobola, Michael S; Silber, John R; Gold, Barry

2013-01-18

Derivatives of methyl 3-(1-methyl-5-(1-methyl-5-(propylcarbamoyl)-1H-pyrrol-3-ylcarbamoyl)-1H-pyrrol-3-ylamino)-3-oxopropane-1-sulfonate (1), a peptide-based DNA minor groove binding methylating agent, were synthesized and characterized. In all cases, the N-terminus was appended with an O-methyl sulfonate ester, while the C-terminus group was varied with nonpolar and polar side chains. In addition, the number of pyrrole rings was varied from 2 (dipeptide) to 3 (tripeptide). The ability of the different analogues to efficiently generate N3-methyladenine was demonstrated as was their selectivity for minor groove (N3-methyladenine) versus major groove (N7-methylguanine) methylation. Induced circular dichroism studies were used to measure the DNA equilibrium binding properties of the stable sulfone analogues; the tripeptide binds with affinity that is >10-fold higher than that of the dipeptide. The toxicities of the compounds were evaluated in alkA/tag glycosylase mutant E. coli and in human WT glioma cells and in cells overexpressing and under-expressing N-methylpurine-DNA glycosylase, which excises N3-methyladenine from DNA. The results show that equilibrium binding correlates with the levels of N3-methyladenine produced and cellular toxicity. The toxicity of 1 was inversely related to the expression of MPG in both the bacterial and mammalian cell lines. The enhanced toxicity parallels the reduced activation of PARP and the diminished rate of formation of aldehyde reactive sites observed in the MPG knockdown cells. It is proposed that unrepaired N3-methyladenine is toxic due to its ability to directly block DNA polymerization.

Two DNA glycosylases in Esherichia coli which release primarily 3-methyladenine

International Nuclear Information System (INIS)

Thomas, L.; Yang, C.; Goldthwait, D.A.

1982-01-01

Two enzymes have been partially purified from Escherichia coli and designated 3-methyladenine DNA glycosylases I and II. The apparent molecular weight of glycosylase I is 20,000, and that of II is 27,000. Glycosylase I releases 3-methyladenine (3-MeA) while II releases 3-MeA, 3-methylguanine (3-MeG), 7-methylguanine (7-MeG), and 7-methyladenine (7-MeA). The rate of release of 3-MeA by glycosylase II is 30 times that of 7-MeG. Glycosylase I is missing in mutants tag 1 and tag 2. In crude extracts, the 3-MeA activity of II is approximately 10% of the total 3-MeA activity. A 50% inactivation at 48 0 C required 5 min for I and 65 min for II. The 3-MeA and 7-MeG activities of the glycosylase II preparation could not be separated by isoelectric focusing, by chromatography of DEAE, Sephadex G-100, phosphocellulose, DNA-cellulose, or carboxymethylcellulose, or by heating at 50 0 C

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

Tungsten disulfide nanosheet and exonuclease III co-assisted amplification strategy for highly sensitive fluorescence polarization detection of DNA glycosylase activity

International Nuclear Information System (INIS)

Zhao, Jingjin; Ma, Yefei; Kong, Rongmei; Zhang, Liangliang; Yang, Wen; Zhao, Shulin

2015-01-01

Herein, we introduced a tungsten disulfide (WS 2 ) nanosheet and exonuclease III (Exo III) co-assisted signal amplification strategy for highly sensitive fluorescent polarization (FP) assay of DNA glycosylase activity. Two DNA glycosylases, uracil-DNA glycosylase (UDG) and human 8-oxoG DNA glycosylase 1 (hOGG1), were tested. A hairpin-structured probe (HP) which contained damaged bases in the stem was used as the substrate. The removal of damaged bases from substrate by DNA glycosylase would lower the melting temperature of HP. The HP was then opened and hybridized with a FAM dye-labeled single strand DNA (DP), generating a duplex with a recessed 3′-terminal of DP. This design facilitated the Exo III-assisted amplification by repeating the hybridization and digestion of DP, liberating numerous FAM fluorophores which could not be adsorbed on WS 2 nanosheet. Thus, the final system exhibited a small FP signal. However, in the absence of DNA glycosylases, no hybridization between DP and HP was occurred, hampering the hydrolysis of DP by Exo III. The intact DP was then adsorbed on the surface of WS 2 nanosheet that greatly amplified the mass of the labeled-FAM fluorophore, resulting in a large FP value. With the co-assisted amplification strategy, the sensitivity was substantially improved. In addition, this method was applied to detect UDG activity in cell extracts. The study of the inhibition of UDG was also performed. Furthermore, this method is simple in design, easy in implementation, and selective, which holds potential applications in the DNA glycosylase related mechanism research and molecular diagnostics. - Highlights: • A fluorescence polarization strategy for DNA glycosylase activity detection was developed. • The present method was based on WS 2 nanosheet and exonuclease III co-assisted signal amplification. • A high sensitivity and desirable selectivity were achieved. • This method provides a promising universal platform for DNA glycosylase

Tungsten disulfide nanosheet and exonuclease III co-assisted amplification strategy for highly sensitive fluorescence polarization detection of DNA glycosylase activity

Energy Technology Data Exchange (ETDEWEB)

Zhao, Jingjin; Ma, Yefei [Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources of Education Ministry, Guangxi Normal University, Guilin, 541004 (China); Kong, Rongmei [The Key Laboratory of Life-Organic Analysis, College of Chemistry and Chemical Engineering, Qufu Normal University, Qufu, Shandong 273165 (China); Zhang, Liangliang, E-mail: liangzhang319@163.com [Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources of Education Ministry, Guangxi Normal University, Guilin, 541004 (China); Yang, Wen; Zhao, Shulin [Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources of Education Ministry, Guangxi Normal University, Guilin, 541004 (China)

2015-08-05

Herein, we introduced a tungsten disulfide (WS{sub 2}) nanosheet and exonuclease III (Exo III) co-assisted signal amplification strategy for highly sensitive fluorescent polarization (FP) assay of DNA glycosylase activity. Two DNA glycosylases, uracil-DNA glycosylase (UDG) and human 8-oxoG DNA glycosylase 1 (hOGG1), were tested. A hairpin-structured probe (HP) which contained damaged bases in the stem was used as the substrate. The removal of damaged bases from substrate by DNA glycosylase would lower the melting temperature of HP. The HP was then opened and hybridized with a FAM dye-labeled single strand DNA (DP), generating a duplex with a recessed 3′-terminal of DP. This design facilitated the Exo III-assisted amplification by repeating the hybridization and digestion of DP, liberating numerous FAM fluorophores which could not be adsorbed on WS{sub 2} nanosheet. Thus, the final system exhibited a small FP signal. However, in the absence of DNA glycosylases, no hybridization between DP and HP was occurred, hampering the hydrolysis of DP by Exo III. The intact DP was then adsorbed on the surface of WS{sub 2} nanosheet that greatly amplified the mass of the labeled-FAM fluorophore, resulting in a large FP value. With the co-assisted amplification strategy, the sensitivity was substantially improved. In addition, this method was applied to detect UDG activity in cell extracts. The study of the inhibition of UDG was also performed. Furthermore, this method is simple in design, easy in implementation, and selective, which holds potential applications in the DNA glycosylase related mechanism research and molecular diagnostics. - Highlights: • A fluorescence polarization strategy for DNA glycosylase activity detection was developed. • The present method was based on WS{sub 2} nanosheet and exonuclease III co-assisted signal amplification. • A high sensitivity and desirable selectivity were achieved. • This method provides a promising universal platform for DNA

DNA translocation by human uracil DNA glycosylase: the case of single-stranded DNA and clustered uracils.

Science.gov (United States)

Schonhoft, Joseph D; Stivers, James T

2013-04-16

Human uracil DNA glycosylase (hUNG) plays a central role in DNA repair and programmed mutagenesis of Ig genes, requiring it to act on sparsely or densely spaced uracil bases located in a variety of contexts, including U/A and U/G base pairs, and potentially uracils within single-stranded DNA (ssDNA). An interesting question is whether the facilitated search mode of hUNG, which includes both DNA sliding and hopping, changes in these different contexts. Here we find that hUNG uses an enhanced local search mode when it acts on uracils in ssDNA, and also, in a context where uracils are densely clustered in duplex DNA. In the context of ssDNA, hUNG performs an enhanced local search by sliding with a mean sliding length larger than that of double-stranded DNA (dsDNA). In the context of duplex DNA, insertion of high-affinity abasic product sites between two uracil lesions serves to significantly extend the apparent sliding length on dsDNA from 4 to 20 bp and, in some cases, leads to directionally biased 3' → 5' sliding. The presence of intervening abasic product sites mimics the situation where hUNG acts iteratively on densely spaced uracils. The findings suggest that intervening product sites serve to increase the amount of time the enzyme remains associated with DNA as compared to nonspecific DNA, which in turn increases the likelihood of sliding as opposed to falling off the DNA. These findings illustrate how the search mechanism of hUNG is not predetermined but, instead, depends on the context in which the uracils are located.

In vivo repair of methylation damage in Aag 3-methyladenine DNA glycosylase null mouse cells

OpenAIRE

Smith, Stephen A.; Engelward, Bevin P.

2000-01-01

3-Methyladenine (3MeA) DNA glycosylases initiate base excision repair by removing 3MeA. These glycosylases also remove a broad spectrum of spontaneous and environmentally induced base lesions in vitro. Mouse cells lacking the Aag 3MeA DNA glycosylase (also known as the Mpg, APNG or ANPG DNA glycosylase) are susceptible to 3MeA-induced S phase arrest, chromosome aberrations and apoptosis, but it is not known if Aag is solely responsible for repair of 3MeA in vivo. Here we show that in Aag–/– c...

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

Energy Technology Data Exchange (ETDEWEB)

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

2017-10-20

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

Structure of a DNA glycosylase that unhooks interstrand cross-links

Energy Technology Data Exchange (ETDEWEB)

Mullins, Elwood A.; Warren, Garrett M.; Bradley, Noah P.; Eichman, Brandt F. (Vanderbilt)

2017-04-10

DNA glycosylases are important editing enzymes that protect genomic stability by excising chemically modified nucleobases that alter normal DNA metabolism. These enzymes have been known only to initiate base excision repair of small adducts by extrusion from the DNA helix. However, recent reports have described both vertebrate and microbial DNA glycosylases capable of unhooking highly toxic interstrand cross-links (ICLs) and bulky minor groove adducts normally recognized by Fanconi anemia and nucleotide excision repair machinery, although the mechanisms of these activities are unknown. Here we report the crystal structure of Streptomyces sahachiroi AlkZ (previously Orf1), a bacterial DNA glycosylase that protects its host by excising ICLs derived from azinomycin B (AZB), a potent antimicrobial and antitumor genotoxin. AlkZ adopts a unique fold in which three tandem winged helix-turn-helix motifs scaffold a positively charged concave surface perfectly shaped for duplex DNA. Through mutational analysis, we identified two glutamine residues and a β-hairpin within this putative DNA-binding cleft that are essential for catalytic activity. Additionally, we present a molecular docking model for how this active site can unhook either or both sides of an AZB ICL, providing a basis for understanding the mechanisms of base excision repair of ICLs. Given the prevalence of this protein fold in pathogenic bacteria, this work also lays the foundation for an emerging role of DNA repair in bacteria-host pathogenesis.

Dietary elevated sucrose modulation of diesel-induced genotoxicity in the colon and liver of Big Blue rats

DEFF Research Database (Denmark)

Risom, L.; Moller, P.; Hansen, Max

2003-01-01

Earlier studies have indicated that sucrose possesses either co-carcinogenic or tumor-promoter effects in colon carcinogenesis induced by genotoxic carcinogens. In this study we investigated the role of sucrose on diesel exhaust particle (DEP)-induced genotoxicity in the colonic mucosa and liver......-breaks and DNA adducts in liver. DEP and sucrose treatment did not have any effect on mutation frequency in colon and liver. Oxidative DNA damage detected as 8-oxodG (8-oxo-7,8-dihydro-2'-deoxyguanosine) and endonuclease III or formamidopyrimidine DNA glycosylase sensitive sites was unaltered in colon and liver....... The mRNA expression levels of the DNA repair enzymes N-methylpurine DNA glycosylase (MPG), 8-oxoguanine DNA glycosylase (OGG1) and ERCC1 (part of the nucleotide excision repair complex) measured by reverse transcription-polymerase chain reaction were increased in liver by DEP feeding. In colon...

Accelerated repair and reduced mutagenicity of DNA damage induced by cigarette smoke in human bronchial cells transfected with E.coli formamidopyrimidine DNA glycosylase.

Directory of Open Access Journals (Sweden)

Mara Foresta

Full Text Available Cigarette smoke (CS is associated to a number of pathologies including lung cancer. Its mutagenic and carcinogenic effects are partially linked to the presence of reactive oxygen species and polycyclic aromatic hydrocarbons (PAH inducing DNA damage. The bacterial DNA repair enzyme formamidopyrimidine DNA glycosylase (FPG repairs both oxidized bases and different types of bulky DNA adducts. We investigated in vitro whether FPG expression may enhance DNA repair of CS-damaged DNA and counteract the mutagenic effects of CS in human lung cells. NCI-H727 non small cell lung carcinoma cells were transfected with a plasmid vector expressing FPG fused to the Enhanced Green Fluorescent Protein (EGFP. Cells expressing the fusion protein EGFP-FPG displayed accelerated repair of adducts and DNA breaks induced by CS condensate. The mutant frequencies induced by low concentrations of CS condensate to the Na(+K(+-ATPase locus (oua(r were significantly reduced in cells expressing EGFP-FPG. Hence, expression of the bacterial DNA repair protein FPG stably protects human lung cells from the mutagenic effects of CS by improving cells' capacity to repair damaged DNA.

Sensitization of human carcinoma cells to alkylating agents by small interfering RNA suppression of 3-alkyladenine-DNA glycosylase.

Science.gov (United States)

Paik, Johanna; Duncan, Tod; Lindahl, Tomas; Sedgwick, Barbara

2005-11-15

One of the major cytotoxic lesions generated by alkylating agents is DNA 3-alkyladenine, which can be excised by 3-alkyladenine DNA glycosylase (AAG). Inhibition of AAG may therefore result in increased cellular sensitivity to chemotherapeutic alkylating agents. To investigate this possibility, we have examined the role of AAG in protecting human tumor cells against such agents. Plasmids that express small interfering RNAs targeted to two different regions of AAG mRNA were transfected into HeLa cervical carcinoma cells and A2780-SCA ovarian carcinoma cells. Stable derivatives of both cell types with low AAG protein levels were sensitized to alkylating agents. Two HeLa cell lines with AAG protein levels reduced by at least 80% to 90% displayed a 5- to 10-fold increase in sensitivity to methyl methanesulfonate, N-methyl-N-nitrosourea, and the chemotherapeutic drugs temozolomide and 1,3-bis(2-chloroethyl)-1-nitrosourea. These cells showed no increase in sensitivity to UV light or ionizing radiation. After treatment with methyl methanesulfonate, AAG knockdown HeLa cells were delayed in S phase but accumulated in G2-M. Our data support the hypothesis that ablation of AAG activity in human tumor cells may provide a useful strategy to enhance the efficacy of current chemotherapeutic regimens that include alkylating agents.

A combinatorial role for MutY and Fpg DNA glycosylases in mutation avoidance in Mycobacterium smegmatis

International Nuclear Information System (INIS)

Hassim, Farzanah; Papadopoulos, Andrea O.; Kana, Bavesh D.; Gordhan, Bhavna G.

2015-01-01

did not result in any growth/survival defects or changes in mutation rates. Taken together these data indicate that the mycobacterial mutY, in combination with the Fpg DNA N-glycosylases, plays an important role in controlling mutagenesis under oxidative stress

A combinatorial role for MutY and Fpg DNA glycosylases in mutation avoidance in Mycobacterium smegmatis

Energy Technology Data Exchange (ETDEWEB)

Hassim, Farzanah; Papadopoulos, Andrea O.; Kana, Bavesh D.; Gordhan, Bhavna G., E-mail: bhavna.gordhan@nhls.ac.za

2015-09-15

homologues did not result in any growth/survival defects or changes in mutation rates. Taken together these data indicate that the mycobacterial mutY, in combination with the Fpg DNA N-glycosylases, plays an important role in controlling mutagenesis under oxidative stress.

Expression of human oxoguanine glycosylase 1 or formamidopyrimidine glycosylase in human embryonic kidney 293 cells exacerbates methylmercury toxicity in vitro

Energy Technology Data Exchange (ETDEWEB)

Ondovcik, Stephanie L.; Preston, Thomas J.; McCallum, Gordon P. [Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario M5S 3M2 (Canada); Wells, Peter G., E-mail: pg.wells@utoronto.ca [Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario M5S 3M2 (Canada); Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario M5S 1A8 (Canada)

2013-08-15

Exposure to methylmercury (MeHg) acutely at high levels, or via chronic low-level dietary exposure from daily fish consumption, can lead to adverse neurological effects in both the adult and developing conceptus. To determine the impact of variable DNA repair capacity, and the role of reactive oxygen species (ROS) and oxidatively damaged DNA in the mechanism of toxicity, transgenic human embryonic kidney (HEK) 293 cells that stably express either human oxoguanine glycosylase 1 (hOgg1) or its bacterial homolog, formamidopyrimidine glycosylase (Fpg), which primarily repair the oxidative lesion 8-oxo-2′-deoxyguanosine (8-oxodG), were used to assess the in vitro effects of MeHg. Western blotting confirmed the expression of hOgg1 or Fpg in both the nuclear and mitochondrial compartments of their respective cell lines. Following acute (1–2 h) incubations with 0–10 μM MeHg, concentration-dependent decreases in clonogenic survival and cell growth accompanied concentration-dependent increases in lactate dehydrogenase (LDH) release, ROS formation, 8-oxodG levels and apurinic/apyrimidinic (AP) sites, consistent with the onset of cytotoxicity. Paradoxically, hOgg1- and Fpg-expressing HEK 293 cells were more sensitive than wild-type cells stably transfected with the empty vector control to MeHg across all cellular and biochemical parameters, exhibiting reduced clonogenic survival and cell growth, and increased LDH release and DNA damage. Accordingly, upregulation of specific components of the base excision repair (BER) pathway may prove deleterious potentially due to the absence of compensatory enhancement of downstream processes to repair toxic intermediary abasic sites. Thus, interindividual variability in DNA repair activity may constitute an important risk factor for environmentally-initiated, oxidatively damaged DNA and its pathological consequences. - Highlights: • hOgg1 and Fpg repair oxidatively damaged DNA. • hOgg1- and Fpg-expressing cells are more

A novel monofunctional DNA glycosylase activity against thymine glycol in mouse cell nuclei

International Nuclear Information System (INIS)

Yamamoto, Ryohei; Akiyama, Miyuki; Matsuyama, Satoshi; Kubo, Kihei; Ide, Hiroshi; Yamamoto, Kazuo

2008-01-01

Reactive oxygen species continuously oxidize DNA bases and threaten the genetic integrity. Thymine glycol (TG), one of the representative oxidized products, is repaired mainly by base excision repair (BER). In Escherichia coli, endonuclease III (Nth) and endonuclease VIII (Nei) are known to actively remove TG from DNA, and the homologs are well conserved in various organisms. These are bifunctional glycosylases, also associated with apurinic/apyrimidinic (AP) lyase activity. In the present study, a monofunctional TG-DNA glycosylase activity is shown to be one of the predominant nuclear activities present in some mouse tissues. By combining hypertonic extraction and column chromatography, we successfully separated the novel activity from majority of the bifunctional activities. Since it has been reported that mNTH1 may not be a dominant nuclear activity, the monofunctional glycosylase activity, together with mNEIL1, may be the major TG-DNA glycosylases in the mouse nucleus. The optimal reaction conditions for the monofunctional activity were found to be pH 7-8 and 100-150 mM KC1, and the activity was resistant to 20 mM EDTA. High monofunctional activity was detected in the spleen and stomach, while the level was significantly lower in the liver, suggesting that the contribution of the monofunctional activity is variable among organs. (author)

A 5-methylcytosine DNA glycosylase/lyase demethylates the retrotransposon Tos17 and promotes its transposition in rice

KAUST Repository

La, Honggui; Ding, Bo; Mishra, Gyan Prakash; Zhou, Bo; Yang, Hongmei; Bellizzi, Maria Del Rosario; Chen, Songbiao; Meyers, Blake C.; Peng, Zhaohua; Zhu, Jian-Kang; Wang, Guoliang

2011-01-01

DNA 5-methylcytosine (5-meC) is an important epigenetic mark for transcriptional gene silencing in many eukaryotes. In Arabidopsis, 5-meC DNA glycosylase/lyases actively remove 5-meC to counter-act transcriptional gene silencing in a locus-specific manner, and have been suggested to maintain the expression of transposons. However, it is unclear whether plant DNA demethylases can promote the transposition of transposons. Here we report the functional characterization of the DNA glycosylase/lyase DNG701 in rice. DNG701 encodes a large (1,812 amino acid residues) DNA glycosylase domain protein. Recombinant DNG701 protein showed 5-meC DNA glycosylase and lyase activities in vitro. Knockout or knockdown of DNG701 in rice plants led to DNA hypermethylation and reduced expression of the retrotransposon Tos17. Tos17 showed less transposition in calli derived from dng701 knockout mutant seeds compared with that in wild-type calli. Overexpression of DNG701 in both rice calli and transgenic plants substantially reduced DNA methylation levels of Tos17 and enhanced its expression. The overexpression also led to more frequent transposition of Tos17 in calli. Our results demonstrate that rice DNG701 is a 5-meC DNA glycosylase/lyase responsible for the demethylation of Tos17 and this DNA demethylase plays a critical role in promoting Tos17 transposition in rice calli.

A 5-methylcytosine DNA glycosylase/lyase demethylates the retrotransposon Tos17 and promotes its transposition in rice

KAUST Repository

La, Honggui

2011-09-06

DNA 5-methylcytosine (5-meC) is an important epigenetic mark for transcriptional gene silencing in many eukaryotes. In Arabidopsis, 5-meC DNA glycosylase/lyases actively remove 5-meC to counter-act transcriptional gene silencing in a locus-specific manner, and have been suggested to maintain the expression of transposons. However, it is unclear whether plant DNA demethylases can promote the transposition of transposons. Here we report the functional characterization of the DNA glycosylase/lyase DNG701 in rice. DNG701 encodes a large (1,812 amino acid residues) DNA glycosylase domain protein. Recombinant DNG701 protein showed 5-meC DNA glycosylase and lyase activities in vitro. Knockout or knockdown of DNG701 in rice plants led to DNA hypermethylation and reduced expression of the retrotransposon Tos17. Tos17 showed less transposition in calli derived from dng701 knockout mutant seeds compared with that in wild-type calli. Overexpression of DNG701 in both rice calli and transgenic plants substantially reduced DNA methylation levels of Tos17 and enhanced its expression. The overexpression also led to more frequent transposition of Tos17 in calli. Our results demonstrate that rice DNG701 is a 5-meC DNA glycosylase/lyase responsible for the demethylation of Tos17 and this DNA demethylase plays a critical role in promoting Tos17 transposition in rice calli.

RPA physically interacts with the human DNA glycosylase NEIL1 to regulate excision of oxidative DNA base damage in primer-template structures.

Science.gov (United States)

Theriot, Corey A; Hegde, Muralidhar L; Hazra, Tapas K; Mitra, Sankar

2010-06-04

The human DNA glycosylase NEIL1, activated during the S-phase, has been shown to excise oxidized base lesions in single-strand DNA substrates. Furthermore, our previous work demonstrating functional interaction of NEIL1 with PCNA and flap endonuclease 1 (FEN1) suggested its involvement in replication-associated repair. Here we show interaction of NEIL1 with replication protein A (RPA), the heterotrimeric single-strand DNA binding protein that is essential for replication and other DNA transactions. The NEIL1 immunocomplex isolated from human cells contains RPA, and its abundance in the complex increases after exposure to oxidative stress. NEIL1 directly interacts with the large subunit of RPA (K(d) approximately 20 nM) via the common interacting interface (residues 312-349) in NEIL1's disordered C-terminal region. RPA inhibits the base excision activity of both wild-type NEIL1 (389 residues) and its C-terminal deletion CDelta78 mutant (lacking the interaction domain) for repairing 5-hydroxyuracil (5-OHU) in a primer-template structure mimicking the DNA replication fork. This inhibition is reduced when the damage is located near the primer-template junction. Contrarily, RPA moderately stimulates wild-type NEIL1 but not the CDelta78 mutant when 5-OHU is located within the duplex region. While NEIL1 is inhibited by both RPA and Escherichia coli single-strand DNA binding protein, only inhibition by RPA is relieved by PCNA. These results showing modulation of NEIL1's activity on single-stranded DNA substrate by RPA and PCNA support NEIL1's involvement in repairing the replicating genome. Copyright 2010 Elsevier B.V. All rights reserved.

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

DENV gene of bacteriophage T4 codes for both pyrimidine dimer-DNA glycosylase and apyrimidinic endonuclease activities

International Nuclear Information System (INIS)

McMillan, S.; Edenberg, H.J.; Radany, E.H.; Friedberg, R.C.; Friedberg, E.C.

1981-01-01

Recent studies have shown that purified preparations of phage T4 UV DNA-incising activity (T4 UV endonuclease or endonuclease V of phase T4) contain a pyrimidine dimer-DNA glycosylase activity that catalyzes hydrolysis of the 5' glycosyl bond of dimerized pyrimidines in UV-irradiated DNA. Such enzyme preparations have also been shown to catalyze the hydrolysis of phosphodiester bonds in UV-irradiated DNA at a neutral pH, presumably reflecting the action of an apurinic/apyrimidinic endonuclease at the apyrimidinic sites created by the pyrimidine dimer-DNA glycosylase. In this study we found that preparations of T4 UV DNA-incising activity contained apurinic/apyrimidinic endonuclease activity that nicked depurinated form I simian virus 40 DNA. Apurinic/apyrimidinic endonuclease activity was also found in extracts of Escherichia coli infected with T4 denV + phage. Extracts of cells infected with T4 denV mutants contained significantly lower levels of apurinic/apyrimidinic endonuclease activity; these levels were no greater than the levels present in extracts of uninfected cells. Furthermore, the addition of DNA containing UV-irradiated DNA and T4 enzyme resulted in competition for pyrimidine dimer-DNA glycosylase activity against the UV-irradiated DNA. On the basis of these results, we concluded that apurinic/apyrimidinic endonuclease activity is encoded by the denV gene of phage T4, the same gene that codes for pyrimidine dimer-DNA glycosylase activity

Excision of deaminated cytosine from the vertebrate genome: role of the SMUG1 uracil–DNA glycosylase

Science.gov (United States)

Nilsen, Hilde; Haushalter, Karl A.; Robins, Peter; Barnes, Deborah E.; Verdine, Gregory L.; Lindahl, Tomas

2001-01-01

Gene-targeted mice deficient in the evolutionarily conserved uracil–DNA glycosylase encoded by the UNG gene surprisingly lack the mutator phenotype characteristic of bacterial and yeast ung– mutants. A complementary uracil–DNA glycosylase activity detected in ung–/– murine cells and tissues may be responsible for the repair of deaminated cytosine residues in vivo. Here, specific neutralizing antibodies were used to identify the SMUG1 enzyme as the major uracil–DNA glycosylase in UNG-deficient mice. SMUG1 is present at similar levels in cell nuclei of non-proliferating and proliferating tissues, indicating a replication- independent role in DNA repair. The SMUG1 enzyme is found in vertebrates and insects, whereas it is absent in nematodes, plants and fungi. We propose a model in which SMUG1 has evolved in higher eukaryotes as an anti-mutator distinct from the UNG enzyme, the latter being largely localized to replication foci in mammalian cells to counteract de novo dUMP incorporation into DNA. PMID:11483530

Impact of psychostimulants and atomoxetine on the expression of 8-hydroxyguanine glycosylase 1 in human cells.

Science.gov (United States)

Schmidt, Andreas Johannes; Clement, Hans-Willi; Gebhardt, Stefan; Hemmeter, Ulrich Michael; Schulz, Eberhard; Krieg, Jürgen-Christian; Kircher, Tilo; Heiser, Philip

2010-06-01

Oxidative DNA damage as one sign of reactive oxygen species induced oxidative stress is an important factor in the pathogenesis of various psychiatric disorders. Altered levels of DNA base damage products as well as the expression of the main repair enzyme 8-hydroxyguanine glycosylase 1 have been described. The aim of the present study was to examine the effects of drugs (amphetamine, methylphenidate and atomoxetine) used in the treatment of attention deficit-hyperactivity disorder on the expression of this enzyme via reverse transcriptase-polymerase chain reaction in human neuroblastoma SH-SY5Y and human monocytic U-937 cells at concentrations of 50, 500 and 5,000 ng/ml. We observed decreased expression of this enzyme for all applied substances. In U-937 cells, the significance level was reached after treatment with 5,000 ng/ml amphetamine as well as after treatment with 50, 500 and 5,000 ng/ml atomoxetine. Incubation of SH-SY5Y cells with 50 and 5,000 ng/ml amphetamine and 5,000 ng/ml methylphenidate led to significant decreases of 8-hydroxyguanine glycosylase 1. As a positive correlation between the expression of 8-hydroxyguanine glycosylase 1 and the level of oxidative DNA damage products has been described, we accordingly consider these substances (amphetamine, methylphenidate and atomoxetine) to possibly play a protective role in this process.

A combinatorial role for MutY and Fpg DNA glycosylases in mutation avoidance in Mycobacterium smegmatis.

Science.gov (United States)

Hassim, Farzanah; Papadopoulos, Andrea O; Kana, Bavesh D; Gordhan, Bhavna G

2015-09-01

Hydroxyl radical (OH) among reactive oxygen species cause damage to nucleobases with thymine being the most susceptible, whilst in contrast, the singlet oxygen ((1)02) targets only guanine bases. The high GC content of mycobacterial genomes predisposes these organisms to oxidative damage of guanine. The exposure of cellular DNA to OH and one-electron oxidants results in the formation of two main degradation products, the pro-mutagenic 8-oxo-7,8-dihydroguanine (8-oxoGua) and the cytotoxic 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). These lesions are repaired through the base excision repair (BER) pathway and we previously, demonstrated a combinatorial role for the mycobacterial Endonuclease III (Nth) and the Nei family of DNA glycosylases in mutagenesis. In addition, the formamidopyrimidine (Fpg/MutM) and MutY DNA glycosylases have also been implicated in mutation avoidance and BER in mycobacteria. In this study, we further investigate the combined role of MutY and the Fpg/Nei DNA glycosylases in Mycobacterium smegmatis and demonstrate that deletion of mutY resulted in enhanced sensitivity to oxidative stress, an effect which was not exacerbated in Δfpg1 Δfpg2 or Δnei1 Δnei2 double mutant backgrounds. However, combinatorial loss of the mutY, fpg1 and fpg2 genes resulted in a significant increase in mutation rates suggesting interplay between these enzymes. Consistent with this, there was a significant increase in C → A mutations with a corresponding change in cell morphology of rifampicin resistant mutants in the Δfpg1 Δfpg2 ΔmutY deletion mutant. In contrast, deletion of mutY together with the nei homologues did not result in any growth/survival defects or changes in mutation rates. Taken together these data indicate that the mycobacterial mutY, in combination with the Fpg DNA N-glycosylases, plays an important role in controlling mutagenesis under oxidative stress. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

The DNA glycosylases OGG1 and NEIL3 influence differentiation potential, proliferation, and senescence-associated signs in neural stem cells

International Nuclear Information System (INIS)

Reis, Amilcar; Hermanson, Ola

2012-01-01

Highlights: ► DNA glycosylases OGG1 and NEIL3 are required for neural stem cell state. ► No effect on cell viability by OGG1 or NEIL3 knockdown in neural stem cells. ► OGG1 or NEIL3 RNA knockdown result in decreased proliferation and differentiation. ► Increased HP1γ immunoreactivity after NEIL3 knockdown suggests premature senescence. -- Abstract: Embryonic neural stem cells (NSCs) exhibit self-renewal and multipotency as intrinsic characteristics that are key parameters for proper brain development. When cells are challenged by oxidative stress agents the resulting DNA lesions are repaired by DNA glycosylases through the base excision repair (BER) pathway as a means to maintain the fidelity of the genome, and thus, proper cellular characteristics. The functional roles for DNA glycosylases in NSCs have however remained largely unexplored. Here we demonstrate that RNA knockdown of the DNA glycosylases OGG1 and NEIL3 decreased NSC differentiation ability and resulted in decreased expression of both neuronal and astrocytic genes after mitogen withdrawal, as well as the stem cell marker Musashi-1. Furthermore, while cell survival remained unaffected, NEIL3 deficient cells displayed decreased cell proliferation rates along with an increase in HP1γ immunoreactivity, a sign of premature senescence. Our results suggest that DNA glycosylases play multiple roles in governing essential neural stem cell characteristics.

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.

Alkyladenine DNA glycosylase (AAG) localizes to mitochondria and interacts with mitochondrial single-stranded binding protein (mtSSB).

Science.gov (United States)

van Loon, Barbara; Samson, Leona D

2013-03-01

Due to a harsh environment mitochondrial genomes accumulate high levels of DNA damage, in particular oxidation, hydrolytic deamination, and alkylation adducts. While repair of alkylated bases in nuclear DNA has been explored in detail, much less is known about the repair of DNA alkylation damage in mitochondria. Alkyladenine DNA glycosylase (AAG) recognizes and removes numerous alkylated bases, but to date AAG has only been detected in the nucleus, even though mammalian mitochondria are known to repair DNA lesions that are specific substrates of AAG. Here we use immunofluorescence to show that AAG localizes to mitochondria, and we find that native AAG is present in purified human mitochondrial extracts, as well as that exposure to alkylating agent promotes AAG accumulation in the mitochondria. We identify mitochondrial single-stranded binding protein (mtSSB) as a novel interacting partner of AAG; interaction between mtSSB and AAG is direct and increases upon methyl methanesulfonate (MMS) treatment. The consequence of this interaction is specific inhibition of AAG glycosylase activity in the context of a single-stranded DNA (ssDNA), but not a double-stranded DNA (dsDNA) substrate. By inhibiting AAG-initiated processing of damaged bases, mtSSB potentially prevents formation of DNA breaks in ssDNA, ensuring that base removal primarily occurs in dsDNA. In summary, our findings suggest the existence of AAG-initiated BER in mitochondria and further support a role for mtSSB in DNA repair. Copyright © 2012. Published by Elsevier B.V.

Human cytomegalovirus uracil DNA glycosylase associates with ppUL44 and accelerates the accumulation of viral DNA

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Dixon Melissa

2005-07-01

Full Text Available Abstract Background Human cytomegalovirus UL114 encodes a uracil-DNA glycosylase homolog that is highly conserved in all characterized herpesviruses that infect mammals. Previous studies demonstrated that the deletion of this nonessential gene delays significantly the onset of viral DNA synthesis and results in a prolonged replication cycle. The gene product, pUL114, also appears to be important in late phase DNA synthesis presumably by introducing single stranded breaks. Results A series of experiments was performed to formally assign the observed phenotype to pUL114 and to characterize the function of the protein in viral replication. A cell line expressing pUL114 complemented the observed phenotype of a UL114 deletion virus in trans, confirming that the observed defects were the result of a deficiency in this gene product. Stocks of recombinant viruses without elevated levels of uracil were produced in the complementing cells; however they retained the phenotype of poor growth in normal fibroblasts suggesting that poor replication was unrelated to uracil content of input genomes. Recombinant viruses expressing epitope tagged versions of this gene demonstrated that pUL114 was expressed at early times and that it localized to viral replication compartments. This protein also coprecipitated with the DNA polymerase processivity factor, ppUL44 suggesting that these proteins associate in infected cells. This apparent interaction did not appear to require other viral proteins since ppUL44 could recruit pUL114 to the nucleus in uninfected cells. An analysis of DNA replication kinetics revealed that the initial rate of DNA synthesis and the accumulation of progeny viral genomes were significantly reduced compared to the parent virus. Conclusion These data suggest that pUL114 associates with ppUL44 and that it functions as part of the viral DNA replication complex to increase the efficiency of both early and late phase viral DNA synthesis.

The DNA glycosylases OGG1 and NEIL3 influence differentiation potential, proliferation, and senescence-associated signs in neural stem cells

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Reis, Amilcar [Linnaeus Center in Developmental Biology for Regenerative Medicine (DBRM), Department of Neuroscience, Karolinska Institutet, SE 17177 Stockholm (Sweden); Hermanson, Ola, E-mail: ola.hermanson@ki.se [Linnaeus Center in Developmental Biology for Regenerative Medicine (DBRM), Department of Neuroscience, Karolinska Institutet, SE 17177 Stockholm (Sweden)

2012-07-13

Highlights: Black-Right-Pointing-Pointer DNA glycosylases OGG1 and NEIL3 are required for neural stem cell state. Black-Right-Pointing-Pointer No effect on cell viability by OGG1 or NEIL3 knockdown in neural stem cells. Black-Right-Pointing-Pointer OGG1 or NEIL3 RNA knockdown result in decreased proliferation and differentiation. Black-Right-Pointing-Pointer Increased HP1{gamma} immunoreactivity after NEIL3 knockdown suggests premature senescence. -- Abstract: Embryonic neural stem cells (NSCs) exhibit self-renewal and multipotency as intrinsic characteristics that are key parameters for proper brain development. When cells are challenged by oxidative stress agents the resulting DNA lesions are repaired by DNA glycosylases through the base excision repair (BER) pathway as a means to maintain the fidelity of the genome, and thus, proper cellular characteristics. The functional roles for DNA glycosylases in NSCs have however remained largely unexplored. Here we demonstrate that RNA knockdown of the DNA glycosylases OGG1 and NEIL3 decreased NSC differentiation ability and resulted in decreased expression of both neuronal and astrocytic genes after mitogen withdrawal, as well as the stem cell marker Musashi-1. Furthermore, while cell survival remained unaffected, NEIL3 deficient cells displayed decreased cell proliferation rates along with an increase in HP1{gamma} immunoreactivity, a sign of premature senescence. Our results suggest that DNA glycosylases play multiple roles in governing essential neural stem cell characteristics.

Overexpression, purification, crystallization and preliminary X-ray analysis of uracil N-glycosylase from Mycobacterium tuberculosis in complex with a proteinaceous inhibitor

Energy Technology Data Exchange (ETDEWEB)

Singh, Prem [Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 (India); Talawar, Ramappa K.; Krishna, P. D. V.; Varshney, Umesh [Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012 (India); Vijayan, M., E-mail: mv@mbu.iisc.ernet.in [Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 (India)

2006-12-01

Uracil N-glycosylase from M. tuberculosis has been crystallized in complex with a proteinaceous inhibitor (Ugi) and X-ray diffraction data have been collected. Uracil N-glycosylase is an enzyme which initiates the pathway of uracil-excision repair of DNA. The enzyme from Mycobacterium tuberculosis was co-expressed with a proteinaceous inhibitor from Bacillus subtilis phage and was crystallized in monoclinic space group C2, with unit-cell parameters a = 201.14, b = 64.27, c = 203.68 Å, β = 109.7°. X-ray data from the crystal have been collected for structure analysis.

Overexpression, purification, crystallization and preliminary X-ray analysis of uracil N-glycosylase from Mycobacterium tuberculosis in complex with a proteinaceous inhibitor

International Nuclear Information System (INIS)

Singh, Prem; Talawar, Ramappa K.; Krishna, P. D. V.; Varshney, Umesh; Vijayan, M.

2006-01-01

Uracil N-glycosylase from M. tuberculosis has been crystallized in complex with a proteinaceous inhibitor (Ugi) and X-ray diffraction data have been collected. Uracil N-glycosylase is an enzyme which initiates the pathway of uracil-excision repair of DNA. The enzyme from Mycobacterium tuberculosis was co-expressed with a proteinaceous inhibitor from Bacillus subtilis phage and was crystallized in monoclinic space group C2, with unit-cell parameters a = 201.14, b = 64.27, c = 203.68 Å, β = 109.7°. X-ray data from the crystal have been collected for structure analysis

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...

Repair of Alkylation Damage in Eukaryotic Chromatin Depends on Searching Ability of Alkyladenine DNA Glycosylase.

Science.gov (United States)

Zhang, Yaru; O'Brien, Patrick J

2015-11-20

Human alkyladenine DNA glycosylase (AAG) initiates the base excision repair pathway by excising alkylated and deaminated purine lesions. In vitro biochemical experiments demonstrate that AAG uses facilitated diffusion to efficiently search DNA to find rare sites of damage and suggest that electrostatic interactions are critical to the searching process. However, it remains an open question whether DNA searching limits the rate of DNA repair in vivo. We constructed AAG mutants with altered searching ability and measured their ability to protect yeast from alkylation damage in order to address this question. Each of the conserved arginine and lysine residues that are near the DNA binding interface were mutated, and the functional impacts were evaluated using kinetic and thermodynamic analysis. These mutations do not perturb catalysis of N-glycosidic bond cleavage, but they decrease the ability to capture rare lesion sites. Nonspecific and specific DNA binding properties are closely correlated, suggesting that the electrostatic interactions observed in the specific recognition complex are similarly important for DNA searching complexes. The ability of the mutant proteins to complement repair-deficient yeast cells is positively correlated with the ability of the proteins to search DNA in vitro, suggesting that cellular resistance to DNA alkylation is governed by the ability to find and efficiently capture cytotoxic lesions. It appears that chromosomal access is not restricted and toxic sites of alkylation damage are readily accessible to a searching protein.

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...

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.

Alkyladenine DNA glycosylase (Aag) in somatic hypermutation and class switch recombination.

Science.gov (United States)

Longerich, Simonne; Meira, Lisiane; Shah, Dharini; Samson, Leona D; Storb, Ursula

2007-12-01

Somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes require the cytosine deaminase AID, which deaminates cytosine to uracil in Ig gene DNA. Paradoxically, proteins involved normally in error-free base excision repair and mismatch repair, seem to be co-opted to facilitate SHM and CSR, by recruiting error-prone translesion polymerases to DNA sequences containing deoxy-uracils created by AID. Major evidence supports at least one mechanism whereby the uracil glycosylase Ung removes AID-generated uracils creating abasic sites which may be used either as uninformative templates for DNA synthesis, or processed to nicks and gaps that prime error-prone DNA synthesis. We investigated the possibility that deamination at adenines also initiates SHM. Adenosine deamination would generate hypoxanthine (Hx), a substrate for the alkyladenine DNA glycosylase (Aag). Aag would generate abasic sites which then are subject to error-prone repair as above for AID-deaminated cytosine processed by Ung. If the action of an adenosine deaminase followed by Aag were responsible for significant numbers of mutations at A, we would find a preponderance of A:T>G:C transition mutations during SHM in an Aag deleted background. However, this was not observed and we found that the frequencies of SHM and CSR were not significantly altered in Aag-/- mice. Paradoxically, we found that Aag is expressed in B lymphocytes undergoing SHM and CSR and that its activity is upregulated in activated B cells. Moreover, we did find a statistically significant, albeit low increase of T:A>C:G transition mutations in Aag-/- animals, suggesting that Aag may be involved in creating the SHM A>T bias seen in wild type mice.

Analysis of substrate specificity of Schizosaccharomyces pombe Mag1 alkylpurine DNA glycosylase

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Adhikary, Suraj; Eichman, Brandt F. (Vanderbilt)

2014-10-02

DNA glycosylases specialized for the repair of alkylation damage must identify, with fine specificity, a diverse array of subtle modifications within DNA. The current mechanism involves damage sensing through interrogation of the DNA duplex, followed by more specific recognition of the target base inside the active site pocket. To better understand the physical basis for alkylpurine detection, we determined the crystal structure of Schizosaccharomyces pombe Mag1 (spMag1) in complex with DNA and performed a mutational analysis of spMag1 and the close homologue from Saccharomyces cerevisiae (scMag). Despite strong homology, spMag1 and scMag differ in substrate specificity and cellular alkylation sensitivity, although the enzymological basis for their functional differences is unknown. We show that Mag preference for 1,N{sup 6}-ethenoadenine ({var_epsilon}A) is influenced by a minor groove-interrogating residue more than the composition of the nucleobase-binding pocket. Exchanging this residue between Mag proteins swapped their {var_epsilon}A activities, providing evidence that residues outside the extrahelical base-binding pocket have a role in identification of a particular modification in addition to sensing damage.

Direct detection of chicken genomic DNA for gender determination by thymine-DNA glycosylase.

Science.gov (United States)

Porat, N; Bogdanov, K; Danielli, A; Arie, A; Samina, I; Hadani, A

2011-02-01

1. Birds, especially nestlings, are generally difficult to sex by morphology and early detection of chick gender in ovo in the hatchery would facilitate removal of unwanted chicks and diminish welfare objections regarding culling after hatch. 2. We describe a method to determine chicken gender without the need for PCR via use of Thymine-DNA Glycosylase (TDG). TDG restores thymine (T)/guanine (G) mismatches to cytosine (C)/G. We show here, that like DNA Polymerase, TDG can recognise, bind and function on a primer hybridised to chicken genomic DNA. 3. The primer contained a T to mismatch a G in a chicken genomic template and the T/G was cleaved with high fidelity by TDG. Thus, the chicken genomic DNA can be identified without PCR amplification via direct and linear detection. Sensitivity was increased using gender specific sequences from the chicken genome. 4. Currently, these are laboratory results, but we anticipate that further development will allow this method to be used in non-laboratory settings, where PCR cannot be employed.

A unique uracil-DNA binding protein of the uracil DNA glycosylase superfamily.

Science.gov (United States)

Sang, Pau Biak; Srinath, Thiruneelakantan; Patil, Aravind Goud; Woo, Eui-Jeon; Varshney, Umesh

2015-09-30

Uracil DNA glycosylases (UDGs) are an important group of DNA repair enzymes, which pioneer the base excision repair pathway by recognizing and excising uracil from DNA. Based on two short conserved sequences (motifs A and B), UDGs have been classified into six families. Here we report a novel UDG, UdgX, from Mycobacterium smegmatis and other organisms. UdgX specifically recognizes uracil in DNA, forms a tight complex stable to sodium dodecyl sulphate, 2-mercaptoethanol, urea and heat treatment, and shows no detectable uracil excision. UdgX shares highest homology to family 4 UDGs possessing Fe-S cluster. UdgX possesses a conserved sequence, KRRIH, which forms a flexible loop playing an important role in its activity. Mutations of H in the KRRIH sequence to S, G, A or Q lead to gain of uracil excision activity in MsmUdgX, establishing it as a novel member of the UDG superfamily. Our observations suggest that UdgX marks the uracil-DNA for its repair by a RecA dependent process. Finally, we observed that the tight binding activity of UdgX is useful in detecting uracils in the genomes. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Processing of free radical damaged DNA bases

International Nuclear Information System (INIS)

Wallace, S.

2003-01-01

while cells overexpressing one of the oxidative DNA glycosylases are radiosensitive. Human TK6 lymphoblastoid cells overexpressing the oxidative DNA glycosylases are also radiosensitive. Furthermore, both radioresistance and radiosensitivity correlated with the enzymatic production of double strand breaks produced after irradiation during attempted repair. Because these data indicated the potential harmful effects of the ordinarily beneficial presence of the oxidative DNA glycosylases after treatment of cells with ionizing radiation, it was of interest to see if the base excision repair enzymes were induced by ionizing radiation. We found that ionizing radiation does not induce the oxidative DNA glycosylases in either bacterial or in human TK6 lymphoblastoid cells. Interestingly, during this past year, three novel DNA glycosylases that recognize free radical-damaged bases have been identified. The characterization of these activities will also be discussed

Alkyladenine DNA glycosylase (AAG) localizes to mitochondria and interacts with mitochondrial single-stranded binding protein (mtSSB)

OpenAIRE

van Loon, Barbara; Samson, Leona D.

2013-01-01

Due to a harsh environment mitochondrial genomes accumulate high levels of DNA damage, in particular oxidation, hydrolytic deamination, and alkylation adducts. While repair of alkylated bases in nuclear DNA has been explored in detail, much less is known about the repair of DNA alkylation damage in mitochondria. Alkyladenine DNA glycosylase (AAG) recognizes and removes numerous alkylated bases, but to date AAG has only been detected in the nucleus, even though mammalian mitochondria are known...

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

Directory of Open Access Journals (Sweden)

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.

Destabilization of the PCNA trimer mediated by its interaction with the NEIL1 DNA glycosylase

Energy Technology Data Exchange (ETDEWEB)

Prakash, Aishwarya; Moharana, Kedar; Wallace, Susan S.; Doublié, Sylvie

2016-12-19

The base excision repair (BER) pathway repairs oxidized lesions in the DNA that result from reactive oxygen species generated in cells. If left unrepaired, these damaged DNA bases can disrupt cellular processes such as replication. NEIL1 is one of the 11 human DNA glycosylases that catalyze the first step of the BER pathway, i.e. recognition and excision of DNA lesions. NEIL1 interacts with essential replication proteins such as the ring-shaped homotrimeric proliferating cellular nuclear antigen (PCNA). We isolated a complex formed between NEIL1 and PCNA (±DNA) using size exclusion chromatography (SEC). This interaction was confirmed using native gel electrophoresis and mass spectrometry. Stokes radii measured by SEC hinted that PCNA in complex with NEIL1 (±DNA) was no longer a trimer. Height measurements and images obtained by atomic force microscopy also demonstrated the dissociation of the PCNA homotrimer in the presence of NEIL1 and DNA, while small-angle X-ray scattering analysis confirmed the NEIL1 mediated PCNA trimer dissociation and formation of a 1:1:1 NEIL1-DNA-PCNA(monomer) complex. Furthermore, ab initio shape reconstruction provides insights into the solution structure of this previously unreported complex. Together, these data point to a potential mechanistic switch between replication and BER.

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

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D'ham, Cedric

1998-01-01

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

The oxidative DNA glycosylases of Mycobacterium tuberculosis exhibit different substrate preferences from their Escherichia coli counterparts.

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Guo, Yin; Bandaru, Viswanath; Jaruga, Pawel; Zhao, Xiaobei; Burrows, Cynthia J; Iwai, Shigenori; Dizdaroglu, Miral; Bond, Jeffrey P; Wallace, Susan S

2010-02-04

The DNA glycosylases that remove oxidized DNA bases fall into two general families: the Fpg/Nei family and the Nth superfamily. Based on protein sequence alignments, we identified four putative Fpg/Nei family members, as well as a putative Nth protein in Mycobacterium tuberculosis H37Rv. All four Fpg/Nei proteins were successfully overexpressed using a bicistronic vector created in our laboratory. The MtuNth protein was also overexpressed in soluble form. The substrate specificities of the purified enzymes were characterized in vitro with oligodeoxynucleotide substrates containing single lesions. Some were further characterized by gas chromatography/mass spectrometry (GC/MS) analysis of products released from gamma-irradiated DNA. MtuFpg1 has substrate specificity similar to that of EcoFpg. Both EcoFpg and MtuFpg1 are more efficient at removing spiroiminodihydantoin (Sp) than 7,8-dihydro-8-oxoguanine (8-oxoG). However, MtuFpg1 shows a substantially increased opposite base discrimination compared to EcoFpg. MtuFpg2 contains only the C-terminal domain of an Fpg protein and has no detectable DNA binding activity or DNA glycosylase/lyase activity and thus appears to be a pseudogene. MtuNei1 recognizes oxidized pyrimidines on both double-stranded and single-stranded DNA and exhibits uracil DNA glycosylase activity. MtuNth recognizes a variety of oxidized bases, including urea, 5,6-dihydrouracil (DHU), 5-hydroxyuracil (5-OHU), 5-hydroxycytosine (5-OHC) and methylhydantoin (MeHyd). Both MtuNei1 and MtuNth excise thymine glycol (Tg); however, MtuNei1 strongly prefers the (5R) isomers, whereas MtuNth recognizes only the (5S) isomers. MtuNei2 did not demonstrate activity in vitro as a recombinant protein, but like MtuNei1 when expressed in Escherichia coli, it decreased the spontaneous mutation frequency of both the fpg mutY nei triple and nei nth double mutants, suggesting that MtuNei2 is functionally active in vivo recognizing both guanine and cytosine oxidation products

The ada operon of Mycobacterium tuberculosis encodes two DNA methyltransferases for inducible repair of DNA alkylation damage.

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Yang, Mingyi; Aamodt, Randi M; Dalhus, Bjørn; Balasingham, Seetha; Helle, Ina; Andersen, Pernille; Tønjum, Tone; Alseth, Ingrun; Rognes, Torbjørn; Bjørås, Magnar

2011-06-10

The ada operon of Mycobacterium tuberculosis, which encodes a composite protein of AdaA and AlkA and a separate AdaB/Ogt protein, was characterized. M. tuberculosis treated with N-methyl-N'-nitro-N-nitrosoguanidine induced transcription of the adaA-alkA and adaB genes, suggesting that M. tuberculosis mount an inducible response to methylating agents. Survival assays of the methyltransferase defective Escherichia coli mutant KT233 (ada ogt), showed that expression of the adaB gene rescued the alkylation sensitivity. Further, adaB but not adaA-alkA complemented the hypermutator phenotype of KT233. Purified AdaA-AlkA and AdaB possessed methyltransferase activity. These data suggested that AdaB counteract the cytotoxic and mutagenic effect of O(6)-methylguanine, while AdaA-AlkA most likely transfers methyl groups from innocuous methylphosphotriesters. AdaA-AlkA did not possess alkylbase DNA glycosylase activity nor rescue the alkylation sensitivity of the E. coli mutant BK2118 (tag alkA). We propose that AdaA-AlkA is a positive regulator of the adaptive response in M. tuberculosis. It thus appears that the ada operon of M. tuberculosis suppresses the mutagenic effect of alkylation but not the cytotoxic effect of lesions such as 3-methylpurines. Collectively, these data indicate that M. tuberculosis hypermutator strains with defective adaptive response genes might sustain robustness to cytotoxic alkylation DNA damage and confer a selective advantage contributing to host adaptation. Copyright © 2011 Elsevier B.V. All rights reserved.

A TetR family transcriptional factor directly regulates the expression of a 3-methyladenine DNA glycosylase and physically interacts with the enzyme to stimulate its base excision activity in Mycobacterium bovis BCG.

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Liu, Lei; Huang, Cheng; He, Zheng-Guo

2014-03-28

3-Methyladenine DNA glycosylase recognizes and excises a wide range of damaged bases and thus plays a critical role in base excision repair. However, knowledge on the regulation of DNA glycosylase in prokaryotes and eukaryotes is limited. In this study, we successfully characterized a TetR family transcriptional factor from Mycobacterium bovis bacillus Calmette-Guerin (BCG), namely BCG0878c, which directly regulates the expression of 3-methyladenine DNA glycosylase (designated as MbAAG) and influences the base excision activity of this glycosylase at the post-translational level. Using electrophoretic mobility shift assay and DNase I footprinting experiments, we identified two conserved motifs within the upstream region of mbaag specifically recognized by BCG0878c. Significant down-regulation of mbaag was observed in BCG0878c-overexpressed M. bovis BCG strains. By contrast, about 12-fold up-regulation of mbaag expression was found in bcg0878c-deleted mutant M. bovis BCG strains. β-Galactosidase activity assays also confirmed these results. Thus, BCG0878c can function as a negative regulator of mbaag expression. In addition, the regulator was shown to physically interact with MbAAG to enhance the ability of the glycosylase to bind damaged DNA. Interaction between the two proteins was further found to facilitate AAG-catalyzed removal of hypoxanthine from DNA. These results indicate that a TetR family protein can dually regulate the function of 3-methyladenine DNA glycosylase in M. bovis BCG both at the transcriptional and post-translational levels. These findings enhance our understanding of the expression and regulation of AAG in mycobacteria.

Improved Synthesis of β-D-6-Methylpurine Riboside and Antitumor Effects of the β-D- and α-D-Anomers

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J. Sufrin

2005-08-01

Full Text Available 6-Methylpurine-β-D-riboside (β-D-MPR has been synthesized by coupling6-methylpurine and 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribose using conditions that producethe β-D-anomer exclusively. The in vitro antitumor effects of β-D-MPR and 6-methyl-purine-α-D-riboside (α-D-MPR in five human tumor cell lines showed that β-D-MPR washighly active (IC50 values ranging from 6 to 34 nM. α-D-MPR, although less active than β-D-MPR, also exhibited significant antitumor effects (IC50 values ranging from 1.47 to 4.83"µ"M.

Detrimental effects of hypoxia-specific expression of uracil DNA glycosylase (Ung) in Mycobacterium smegmatis.

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Kurthkoti, Krishna; Varshney, Umesh

2010-12-01

Mycobacterium tuberculosis is known to reside latently in a significant fraction of the human population. Although the bacterium possesses an aerobic mode of metabolism, it adapts to persistence under hypoxic conditions such as those encountered in granulomas. While in mammalian systems hypoxia is a recognized DNA-damaging stress, aspects of DNA repair in mycobacteria under such conditions have not been studied. We subjected Mycobacterium smegmatis, a model organism, to the Wayne's protocol of hypoxia. Analysis of the mRNA of a key DNA repair enzyme, uracil DNA glycosylase (Ung), by real-time reverse transcriptase PCR (RT-PCR) revealed its downregulation during hypoxia. However, within an hour of recovery of the culture under normal oxygen levels, the Ung mRNA was restored. Analysis of Ung by immunoblotting and enzyme assays supported the RNA analysis results. To understand its physiological significance, we misexpressed Ung in M. smegmatis by using a hypoxia-responsive promoter of narK2 from M. tuberculosis. Although the misexpression of Ung during hypoxia decreased C-to-T mutations, it compromised bacterial survival upon recovery at normal oxygen levels. RT-PCR analysis of other base excision repair gene transcripts (UdgB and Fpg) suggested that these DNA repair functions also share with Ung the phenomenon of downregulation during hypoxia and recovery with return to normal oxygen conditions. We discuss the potential utility of this phenomenon in developing attenuated strains of mycobacteria.

The disordered C-terminal domain of human DNA glycosylase NEIL1 contributes to its stability via intramolecular interactions.

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Hegde, Muralidhar L; Tsutakawa, Susan E; Hegde, Pavana M; Holthauzen, Luis Marcelo F; Li, Jing; Oezguen, Numan; Hilser, Vincent J; Tainer, John A; Mitra, Sankar

2013-07-10

NEIL1 [Nei (endonuclease VIII)-like protein 1], one of the five mammalian DNA glycosylases that excise oxidized DNA base lesions in the human genome to initiate base excision repair, contains an intrinsically disordered C-terminal domain (CTD; ~100 residues), not conserved in its Escherichia coli prototype Nei. Although dispensable for NEIL1's lesion excision and AP lyase activities, this segment is required for efficient in vivo enzymatic activity and may provide an interaction interface for many of NEIL1's interactions with other base excision repair proteins. Here, we show that the CTD interacts with the folded domain in native NEIL1 containing 389 residues. The CTD is poised for local folding in an ordered structure that is induced in the purified fragment by osmolytes. Furthermore, deletion of the disordered tail lacking both Tyr and Trp residues causes a red shift in NEIL1's intrinsic Trp-specific fluorescence, indicating a more solvent-exposed environment for the Trp residues in the truncated protein, which also exhibits reduced stability compared to the native enzyme. These observations are consistent with stabilization of the native NEIL1 structure via intramolecular, mostly electrostatic, interactions that were disrupted by mutating a positively charged (Lys-rich) cluster of residues (amino acids 355-360) near the C-terminus. Small-angle X-ray scattering (SAXS) analysis confirms the flexibility and dynamic nature of NEIL1's CTD, a feature that may be critical to providing specificity for NEIL1's multiple, functional interactions. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

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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.

Release of 3-methyladenine from linker and core DNA of chromatin by a purified DNA glycosylase

International Nuclear Information System (INIS)

Heller, E.P.; Goldthwait, D.A.

1983-01-01

Oligonucleosomes were isolated from [ 14 C]thymidine-labeled HeLa cells by digestion of the nuclei with micrococcal nuclease and were then alkylated with [ 3 H]methylnitrosourea. Nucleosome core particles were also prepared by further digestion of the oligonucleosomes. The distribution of 3 H-labeled methyl groups in the linker versus the core DNA was established by a determination of 3 H: 14 C ratios in oligonucleosome and core DNA. The ratios in the core DNA of 145 and 165 base pair DNA fragments were 5.2 and 5.4, respectively, while the ratio in the oligonucleosomal DNA was 8.2. Assuming an equal mixture (as determined) of 145 and 165 base pair fragments of DNA in the 185 base pair repeat, the relative concentration of 3 H methyl groups in the linker versus the core DNA was 4.2. Thus, 45% of the 3 H methyl groups were in the linker DNA, and 55% were in the core DNA. Some shielding of the DNA was evident during alkylation. The concentrations of alkyl groups on the linker and core DNA were 67 and 12% of that found on free DNA alkylated under comparable conditions. No evidence for preferential shielding of the major or minor groove was observed. The purified 3-methyladenine DNA glycosylase I of Escherichia coli released approximately 37% of the 3-methyladenine from the linker DNA and 13% from the core DNA. The limited enzymatic removal of 3-methyladenine in vitro compared to the efficient removal in vivo suggests that conformational changes of the oligonucleosome and core structure must occur for total repair

Pms2 and uracil-DNA glycosylases act jointly in the mismatch repair pathway to generate Ig gene mutations at A-T base pairs.

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Girelli Zubani, Giulia; Zivojnovic, Marija; De Smet, Annie; Albagli-Curiel, Olivier; Huetz, François; Weill, Jean-Claude; Reynaud, Claude-Agnès; Storck, Sébastien

2017-04-03

During somatic hypermutation (SHM) of immunoglobulin genes, uracils introduced by activation-induced cytidine deaminase are processed by uracil-DNA glycosylase (UNG) and mismatch repair (MMR) pathways to generate mutations at G-C and A-T base pairs, respectively. Paradoxically, the MMR-nicking complex Pms2/Mlh1 is apparently dispensable for A-T mutagenesis. Thus, how detection of U:G mismatches is translated into the single-strand nick required for error-prone synthesis is an open question. One model proposed that UNG could cooperate with MMR by excising a second uracil in the vicinity of the U:G mismatch, but it failed to explain the low impact of UNG inactivation on A-T mutagenesis. In this study, we show that uracils generated in the G1 phase in B cells can generate equal proportions of A-T and G-C mutations, which suggests that UNG and MMR can operate within the same time frame during SHM. Furthermore, we show that Ung -/- Pms2 -/- mice display a 50% reduction in mutations at A-T base pairs and that most remaining mutations at A-T bases depend on two additional uracil glycosylases, thymine-DNA glycosylase and SMUG1. These results demonstrate that Pms2/Mlh1 and multiple uracil glycosylases act jointly, each one with a distinct strand bias, to enlarge the immunoglobulin gene mutation spectrum from G-C to A-T bases. © 2017 Girelli Zubani et al.

The contribution of Nth and Nei DNA glycosylases to mutagenesis in Mycobacterium smegmatis.

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Moolla, Nabiela; Goosens, Vivianne J; Kana, Bavesh D; Gordhan, Bhavna G

2014-01-01

The increased prevalence of drug resistant strains of Mycobacterium tuberculosis (Mtb) indicates that significant mutagenesis occurs during tuberculosis disease in humans. DNA damage by host-derived reactive oxygen/nitrogen species is hypothesized to be critical for the mutagenic process in Mtb thus, highlighting an important role for DNA repair enzymes in maintenance of genome fidelity. Formamidopyrimidine (Fpg/MutM/Fapy) and EndonucleaseVIII (Nei) constitute the Fpg/Nei family of DNA glycosylases and together with EndonucleaseIII (Nth) are central to the base excision repair pathway in bacteria. In this study we assess the contribution of Nei and Nth DNA repair enzymes in Mycobacterium smegmatis (Msm), which retains a single nth homologue and duplications of the Fpg (fpg1 and fpg2) and Nei (nei1 and nei2) homologues. Using an Escherichia coli nth deletion mutant, we confirm the functionality of the mycobacterial nth gene in the base excision repair pathway. Msm mutants lacking nei1, nei2 and nth individually or in combination did not display aberrant growth in broth culture. Deletion of nth individually results in increased UV-induced mutagenesis and combinatorial deletion with the nei homologues results in reduced survival under oxidative stress conditions and an increase in spontaneous mutagenesis to rifampicin. Deletion of nth together with the fpg homolgues did not result in any growth/survival defects or changes in mutation rate. Furthermore, no differential emergence of the common rifampicin resistance conferring genotypes were noted. Collectively, these data confirm a role for Nth in base excision repair in mycobacteria and further highlight a novel interplay between the Nth and Nei homologues in spontaneous mutagenesis. Copyright © 2013 Elsevier B.V. All rights reserved.

Is Human Oxoguanine Glycosylase 1 Genetic Variant Successful Even on Oral Squamous Cell Carcinoma?

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Aydemir, Levent; Bireller, Elif Sinem; Avci, Hakan; Boy Metin, Zeynep; Deger, Kemal; Unur, Meral; Cakmakoglu, Bedia

2017-01-01

Oral squamous cell carcinoma (OSCC) is one of the most widespread cancer types that arise from different sites of oral cavity and has a 5-year survival rate. This study is aimed at investigating the human oxoguanine glycosylase 1 (hOGG1)-Ser326Cys and APE-Asp148Glu polymorphisms of DNA repair genes in OSCC. We investigated the hOGG1-Ser326Cys and APE-Asp148Glu polymorphisms of DNA repair genes in the oral cavity. Genotyping was conducted using polymerase chain reaction-restriction fragment length polymorphism analysis based on 132 patients who were diagnosed as having OSCC and 160 healthy subjects. Individuals with the genotype hOGG1-Ser326Cys, Cys allele carriers, were found significantly more frequently in the patient group compared to the control group as increase in risk (p oral squamous cancer. In view of our results, further studies including expression levels are required in which hOGG1-Ser326Cys should be investigated as molecular biomarkers for the early prediction of squamous cell carcinoma. © 2017 S. Karger AG, Basel.

Structural plasticity in Mycobacterium tuberculosis uracil-DNA glycosylase (MtUng) and its functional implications.

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Arif, S M; Geethanandan, K; Mishra, P; Surolia, A; Varshney, U; Vijayan, M

2015-07-01

17 independent crystal structures of family I uracil-DNA glycosylase from Mycobacterium tuberculosis (MtUng) and its complexes with uracil and its derivatives, distributed among five distinct crystal forms, have been determined. Thermodynamic parameters of binding in the complexes have been measured using isothermal titration calorimetry. The two-domain protein exhibits open and closed conformations, suggesting that the closure of the domain on DNA binding involves conformational selection. Segmental mobility in the enzyme molecule is confined to a 32-residue stretch which plays a major role in DNA binding. Uracil and its derivatives can bind to the protein in two possible orientations. Only one of them is possible when there is a bulky substituent at the 5' position. The crystal structures of the complexes provide a reasonable rationale for the observed thermodynamic parameters. In addition to providing fresh insights into the structure, plasticity and interactions of the protein molecule, the results of the present investigation provide a platform for structure-based inhibitor design.

Uracil DNA glycosylase counteracts APOBEC3G-induced hypermutation of hepatitis B viral genomes: excision repair of covalently closed circular DNA.

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Kouichi Kitamura

Full Text Available The covalently closed circular DNA (cccDNA of the hepatitis B virus (HBV plays an essential role in chronic hepatitis. The cellular repair system is proposed to convert cytoplasmic nucleocapsid (NC DNA (partially double-stranded DNA into cccDNA in the nucleus. Recently, antiviral cytidine deaminases, AID/APOBEC proteins, were shown to generate uracil residues in the NC-DNA through deamination, resulting in cytidine-to-uracil (C-to-U hypermutation of the viral genome. We investigated whether uracil residues in hepadnavirus DNA were excised by uracil-DNA glycosylase (UNG, a host factor for base excision repair (BER. When UNG activity was inhibited by the expression of the UNG inhibitory protein (UGI, hypermutation of NC-DNA induced by either APOBEC3G or interferon treatment was enhanced in a human hepatocyte cell line. To assess the effect of UNG on the cccDNA viral intermediate, we used the duck HBV (DHBV replication model. Sequence analyses of DHBV DNAs showed that cccDNA accumulated G-to-A or C-to-T mutations in APOBEC3G-expressing cells, and this was extensively enhanced by UNG inhibition. The cccDNA hypermutation generated many premature stop codons in the P gene. UNG inhibition also enhanced the APOBEC3G-mediated suppression of viral replication, including reduction of NC-DNA, pre-C mRNA, and secreted viral particle-associated DNA in prolonged culture. Enhancement of APOBEC3G-mediated suppression by UNG inhibition was not observed when the catalytic site of APOBEC3G was mutated. Transfection experiments of recloned cccDNAs revealed that the combination of UNG inhibition and APOBEC3G expression reduced the replication ability of cccDNA. Taken together, these data indicate that UNG excises uracil residues from the viral genome during or after cccDNA formation in the nucleus and imply that BER pathway activities decrease the antiviral effect of APOBEC3-mediated hypermutation.

Toehold-mediated strand displacement reaction-dependent fluorescent strategy for sensitive detection of uracil-DNA glycosylase activity.

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Wu, Yushu; Wang, Lei; Jiang, Wei

2017-03-15

Sensitive detection of uracil-DNA glycosylase (UDG) activity is beneficial for evaluating the repairing process of DNA lesions. Here, toehold-mediated strand displacement reaction (TSDR)-dependent fluorescent strategy was constructed for sensitive detection of UDG activity. A single-stranded DNA (ssDNA) probe with two uracil bases and a trigger sequence were designed. A hairpin probe with toehold domain was designed, and a reporter probe was also designed. Under the action of UDG, two uracil bases were removed from ssDNA probe, generating apurinic/apyrimidinic (AP) sites. Then, the AP sites could inhibit the TSDR between ssDNA probe and hairpin probe, leaving the trigger sequence in ssDNA probe still free. Subsequently, the trigger sequence was annealed with the reporter probe, initiating the polymerization and nicking amplification reaction. As a result, numerous G-quadruplex (G4) structures were formed, which could bind with N-methyl-mesoporphyrin IX (NMM) to generate enhanced fluorescent signal. In the absence of UDG, the ssDNA probe could hybridize with the toehold domain of the hairpin probe to initiate TSDR, blocking the trigger sequence, and then the subsequent amplification reaction would not occur. The proposed strategy was successfully implemented for detecting UDG activity with a detection limit of 2.7×10 -5 U/mL. Moreover, the strategy could distinguish UDG well from other interference enzymes. Furthermore, the strategy was also applied for detecting UDG activity in HeLa cells lysate with low effect of cellular components. These results indicated that the proposed strategy offered a promising tool for sensitive quantification of UDG activity in UDG-related function study and disease prognosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Oxidative DNA damage and repair in skeletal muscle of humans exposed to high-altitude hypoxia

International Nuclear Information System (INIS)

Lundby, Carsten; Pilegaard, Henriette; Hall, Gerrit van; Sander, Mikael; Calbet, Jose; Loft, Steffen; Moeller, Peter

2003-01-01

Recent research suggests that high-altitude hypoxia may serve as a model for prolonged oxidative stress in healthy humans. In this study, we investigated the consequences of prolonged high-altitude hypoxia on the basal level of oxidative damage to nuclear DNA in muscle cells, a major oxygen-consuming tissue. Muscle biopsies from seven healthy humans were obtained at sea level and after 2 and 8 weeks of hypoxia at 4100 m.a.s.l. We found increased levels of strand breaks and endonuclease III-sensitive sites after 2 weeks of hypoxia, whereas oxidative DNA damage detected by formamidopyrimidine DNA glycosylase (FPG) protein was unaltered. The expression of 8-oxoguanine DNA glycosylase 1 (OGG1), determined by quantitative RT-PCR of mRNA levels did not significantly change during high-altitude hypoxia, although the data could not exclude a minor upregulation. The expression of heme oxygenase-1 (HO-1) was unaltered by prolonged hypoxia, in accordance with the notion that HO-1 is an acute stress response protein. In conclusion, our data indicate high-altitude hypoxia may serve as a good model for oxidative stress and that antioxidant genes are not upregulated in muscle tissue by prolonged hypoxia despite increased generation of oxidative DNA damage

Aag DNA glycosylase promotes alkylation-induced tissue damage mediated by Parp1.

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Calvo, Jennifer A; Moroski-Erkul, Catherine A; Lake, Annabelle; Eichinger, Lindsey W; Shah, Dharini; Jhun, Iny; Limsirichai, Prajit; Bronson, Roderick T; Christiani, David C; Meira, Lisiane B; Samson, Leona D

2013-04-01

Alkylating agents comprise a major class of front-line cancer chemotherapeutic compounds, and while these agents effectively kill tumor cells, they also damage healthy tissues. Although base excision repair (BER) is essential in repairing DNA alkylation damage, under certain conditions, initiation of BER can be detrimental. Here we illustrate that the alkyladenine DNA glycosylase (AAG) mediates alkylation-induced tissue damage and whole-animal lethality following exposure to alkylating agents. Aag-dependent tissue damage, as observed in cerebellar granule cells, splenocytes, thymocytes, bone marrow cells, pancreatic β-cells, and retinal photoreceptor cells, was detected in wild-type mice, exacerbated in Aag transgenic mice, and completely suppressed in Aag⁻/⁻ mice. Additional genetic experiments dissected the effects of modulating both BER and Parp1 on alkylation sensitivity in mice and determined that Aag acts upstream of Parp1 in alkylation-induced tissue damage; in fact, cytotoxicity in WT and Aag transgenic mice was abrogated in the absence of Parp1. These results provide in vivo evidence that Aag-initiated BER may play a critical role in determining the side-effects of alkylating agent chemotherapies and that Parp1 plays a crucial role in Aag-mediated tissue damage.

Aag DNA glycosylase promotes alkylation-induced tissue damage mediated by Parp1.

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Jennifer A Calvo

2013-04-01

Full Text Available Alkylating agents comprise a major class of front-line cancer chemotherapeutic compounds, and while these agents effectively kill tumor cells, they also damage healthy tissues. Although base excision repair (BER is essential in repairing DNA alkylation damage, under certain conditions, initiation of BER can be detrimental. Here we illustrate that the alkyladenine DNA glycosylase (AAG mediates alkylation-induced tissue damage and whole-animal lethality following exposure to alkylating agents. Aag-dependent tissue damage, as observed in cerebellar granule cells, splenocytes, thymocytes, bone marrow cells, pancreatic β-cells, and retinal photoreceptor cells, was detected in wild-type mice, exacerbated in Aag transgenic mice, and completely suppressed in Aag⁻/⁻ mice. Additional genetic experiments dissected the effects of modulating both BER and Parp1 on alkylation sensitivity in mice and determined that Aag acts upstream of Parp1 in alkylation-induced tissue damage; in fact, cytotoxicity in WT and Aag transgenic mice was abrogated in the absence of Parp1. These results provide in vivo evidence that Aag-initiated BER may play a critical role in determining the side-effects of alkylating agent chemotherapies and that Parp1 plays a crucial role in Aag-mediated tissue damage.

Probing the DNA Structural Requirements for Facilitated Diffusion

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2015-01-01

DNA glycosylases perform a genome-wide search to locate damaged nucleotides among a great excess of undamaged nucleotides. Many glycosylases are capable of facilitated diffusion, whereby multiple sites along the DNA are sampled during a single binding encounter. Electrostatic interactions between positively charged amino acids and the negatively charged phosphate backbone are crucial for facilitated diffusion, but the extent to which diffusing proteins rely on the double-helical structure DNA is not known. Kinetic assays were used to probe the DNA searching mechanism of human alkyladenine DNA glycosylase (AAG) and to test the extent to which diffusion requires B-form duplex DNA. Although AAG excises εA lesions from single-stranded DNA, it is not processive on single-stranded DNA because dissociation is faster than N-glycosidic bond cleavage. However, the AAG complex with single-stranded DNA is sufficiently stable to allow for DNA annealing when a complementary strand is added. This observation provides evidence of nonspecific association of AAG with single-stranded DNA. Single-strand gaps, bubbles, and bent structures do not impede the search by AAG. Instead, these flexible or bent structures lead to the capture of a nearby site of damage that is more efficient than that of a continuous B-form duplex. The ability of AAG to negotiate these helix discontinuities is inconsistent with a sliding mode of diffusion but can be readily explained by a hopping mode that involves microscopic dissociation and reassociation. These experiments provide evidence of relatively long-range hops that allow a searching protein to navigate around DNA binding proteins that would serve as obstacles to a sliding protein. PMID:25495964

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

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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.

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

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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.

Substitution of Active Site Tyrosines with Tryptophan Alters the Free Energy for Nucleotide Flipping by Human Alkyladenine DNA Glycosylase†

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Hendershot, Jenna M.; Wolfe, Abigail E.; O'Brien, Patrick J.

2011-01-01

Human alkyladenine DNA glycosylase (AAG) locates and excises a wide variety of structurally diverse alkylated and oxidized purine lesions from DNA to initiate the base excision repair pathway. Recognition of a base lesion requires flipping of the damaged nucleotide into a relatively open active site pocket between two conserved tyrosine residues, Y127 and Y159. We have mutated each of these amino acids to tryptophan and measured the kinetic effects on the nucleotide flipping and base excision steps. The Y127W and Y159W mutant proteins have robust glycosylase activity toward DNA containing 1,N6-ethenoadenine (εA), within 4-fold of that of the wildtype enzyme, raising the possibility that tryptophan fluorescence could be used to probe the DNA binding and nucleotide flipping steps. Stopped-flow fluorescence was used to compare the time-dependent changes in tryptophan fluorescence and εA fluorescence. For both mutants, the tryptophan fluorescence exhibited two-step binding with essentially identical rate constants as were observed for the εA fluorescence changes. These results provide evidence that AAG forms an initial recognition complex in which the active site pocket is perturbed and the stacking of the damaged base is disrupted. Upon complete nucleotide flipping, there is further quenching of the tryptophan fluorescence with coincident quenching of the εA fluorescence. Although these mutations do not have large effects on the rate constant for excision of εA, there are dramatic effects on the rate constants for nucleotide flipping that result in 40 to 100-fold decreases in the flipping equilibrium relative to wildtype. Most of this effect is due to an increased rate of unflipping, but surprisingly the Y159W mutation causes a 5-fold increase in the rate constant for flipping. The large effect on the equilibrium for nucleotide flipping explains the greater deleterious effects that these mutations have on the glycosylase activity toward base lesions that are in

Selective inhibition by methoxyamine of the apurinic/apyrimidinic endonuclease activity associated with pyrimidine dimer-DNA glycosylases from Micrococcus luteus and bacteriophage T4

International Nuclear Information System (INIS)

Liuzzi, M.; Weinfeld, M.; Paterson, M.C.

1987-01-01

The UV endonucleases from Micrococcus luteus and bacteriophage T4 possess two catalytic activities specific for the site of cyclobutane pyrimidine dimers in UV-irradiated DNA: a DNA glycosylase that cleaves the 5'-glycosyl bond of the dimerized pyrimidines and an apurinic/apyrimidinic (AP) endonuclease that thereupon incises the phosphodiester bond 3' to the resulting apyrimidinic site. The authors have explored the potential use of methoxyamine, a chemical that reacts at neutral pH with AP sites in DNA, as a selective inhibitor of the AP endonuclease activities residing in the M. luteus and T4 enzymes. The presence of 50 mM methoxyamine during incubation of UV-treated, [ 3 H]thymine-labeled poly(dA) x poly(dT) with either enzyme preparation was found to protect completely the irradiated copolymer from endonucleolytic attack at dimer sites, as assayed by yield of acid-soluble radioactivity. In contrast, the dimer-DNA glycosylase activity of each enzyme remained fully functional, as monitored retrospectively by release of free thymine after either photochemical-(5 kJ/m 2 , 254 nm) or photoenzymic- (Escherichia coli photolyase plus visible light) induced reversal of pyrimidine dimers in the UV-damaged substrate. The data demonstrate that the inhibition of the strand-incision reaction arises because of chemical modification of the AP sites and is not due to inactivation of the enzyme by methoxyamine. The results, combined with earlier findings for 5'-acting AP endonucleases, strongly suggest that methoxyamine is a highly specific inhibitor of virtually all AP endonucleases, irrespective of their modes of action, and may therefore prove useful in a wide variety of DNA repair studies

Unique features of the structure and interactions of mycobacterial uracil-DNA glycosylase: structure of a complex of the Mycobacterium tuberculosis enzyme in comparison with those from other sources.

Science.gov (United States)

Kaushal, Prem Singh; Talawar, Ramappa K; Krishna, P D V; Varshney, Umesh; Vijayan, M

2008-05-01

Uracil-DNA glycosylase (UNG), a repair enzyme involved in the excision of uracil from DNA, from mycobacteria differs from UNGs from other sources, particularly in the sequence in the catalytically important loops. The structure of the enzyme from Mycobacterium tuberculosis (MtUng) in complex with a proteinaceous inhibitor (Ugi) has been determined by X-ray analysis of a crystal containing seven crystallographically independent copies of the complex. This structure provides the first geometric characterization of a mycobacterial UNG. A comparison of the structure with those of other UNG proteins of known structure shows that a central core region of the molecule is relatively invariant in structure and sequence, while the N- and C-terminal tails exhibit high variability. The tails are probably important in folding and stability. The mycobacterial enzyme exhibits differences in UNG-Ugi interactions compared with those involving UNG from other sources. The MtUng-DNA complex modelled on the basis of the known structure of the complex involving the human enzyme indicates a domain closure in the enzyme when binding to DNA. The binding involves a larger burial of surface area than is observed in binding by human UNG. The DNA-binding site of MtUng is characterized by the presence of a higher proportion of arginyl residues than is found in the binding site of any other UNG of known structure. In addition to the electrostatic effects produced by the arginyl residues, the hydrogen bonds in which they are involved compensate for the loss of some interactions arising from changes in amino-acid residues, particularly in the catalytic loops. The results arising from the present investigation represent unique features of the structure and interaction of mycobacterial Ungs.

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

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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.

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.

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.

Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins.

Science.gov (United States)

Zdżalik, Daria; Domańska, Anna; Prorok, Paulina; Kosicki, Konrad; van den Born, Erwin; Falnes, Pål Ø; Rizzo, Carmelo J; Guengerich, F Peter; Tudek, Barbara

2015-06-01

AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno(ɛ)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ɛ-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ɛ-adducts, 1,N(6)-ethenoadenine (ɛA), 3,N(4)-ethenocytosine (ɛC) and 1,N(2)-ethenoguanine (1,N(2)-ɛG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate. Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ɛ-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed ɛA and ɛC from ds and ssDNA but were inactive toward 1,N(2)-ɛG. SC-1A repaired only ɛA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ɛ-adducts in dsDNA, while only ɛA and ɛC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only ɛC in ssDNA. Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N(2)-ɛG and that ALKBH3 removes only ɛC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins. Copyright © 2015 Elsevier B

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...

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.

Elucidation of the mechanism of X-ray induced DNA duplication observed in human Gorlin cells

International Nuclear Information System (INIS)

Nomura, J.; Suzuki, N.; Kita, K.; Sugaya, S.

2004-01-01

A phenomenon in which DNA synthesis level increases rapidly after x-ray irradiation has found out in the cells which originate in Gorlin patients. A gene, by which an expression level changes after x-ray irradiation, is searched in the human Gorlin cells by the mRNA differential display method. The DNA synthesis level decreases in normal human cell after x-ray irradiation of 2 Gy dose, but increases twice in the Gorlin cell. Expression levels of gene SMT3A, however decrease clearly in the Gorlin cells after the irradiation. The relations between expression levels of gene SMT3M, a protein like ubichitin, and DNA synthesis levels are searched. DNA synthesis activity in normal human cells, which are treated by antisese oligonucleotide and suppressed expression of the genes SMT3A, increases after x-ray irradiation. An increase of the DNA synthesis level after the irradiation is not a phenomenon in particular cells, but indicates the possibility of general phenomena in normal human cells. It is reported that the gene SMT3A combines with a glycosylase which operates in DNA repairing process. The protein modification of gene SMT3A indicates a possibility for controlling of stress protection mechanism in the cells. (M. Suetake)

Reaction products from N-methyl-N-nitrosourea and deoxyribonucleic acid containing thymidine residues. Synthesis and identification of a new methylation product, O4-methyl-thymidine

Science.gov (United States)

Lawley, P. D.; Orr, D. J.; Shah, S. A.; Farmer, P. B.; Jarman, M.

1973-01-01

1. DNA was treated with N-methyl-N-nitrosourea at pH7–8, 37°C, degraded to yield 3- and 7-methylpurines and deoxyribonucleosides and the reaction products were separated by chromatography on ion-exchange resins. The following methods for identification and determination of products were used: with unlabelled N-methyl-N-nitrosourea, u.v. absorption; use of methyl-14C-labelled N-methyl-N-nitrosourea and use of [14C]thymine-labelled DNA. 2. The synthesis of O4-methylthymidine and its identification by u.v. and mass spectroscopy are reported. 3. 3-Methylthymidine and O4-methylthymidine were found as methylation products from N-methyl-N-nitrosourea with thymidine and with DNA, in relatively small yields. Unidentified products containing thymine were found in enzymic digests of N-methyl-N-nitrosourea-treated DNA, which may be phosphotriesters. 4. The possible role of formation of methylthymines in mutagenesis by N-methyl-N-nitrosourea is discussed. PMID:4798180

Benzene-derived N2-(4-hydroxyphenyl)-deoxyguanosine adduct: UvrABC incision and its conformation in DNA

Energy Technology Data Exchange (ETDEWEB)

Hang, Bo; Rodriguez, Ben; Yang, Yanu; Guliaev, Anton B.; Chenna, Ahmed

2010-06-14

Benzene, a ubiquitous human carcinogen, forms DNA adducts through its metabolites such as p-benzoquinone (p-BQ) and hydroquinone (HQ). N(2)-(4-Hydroxyphenyl)-2'-deoxyguanosine (N(2)-4-HOPh-dG) is the principal adduct identified in vivo by (32)P-postlabeling in cells or animals treated with p-BQ or HQ. To study its effect on repair specificity and replication fidelity, we recently synthesized defined oligonucleotides containing a site-specific adduct using phosphoramidite chemistry. We here report the repair of this adduct by Escherichia coli UvrABC complex, which performs the initial damage recognition and incision steps in the nucleotide excision repair (NER) pathway. We first showed that the p-BQ-treated plasmid was efficiently cleaved by the complex, indicating the formation of DNA lesions that are substrates for NER. Using a 40-mer substrate, we found that UvrABC incises the DNA strand containing N(2)-4-HOPh-dG in a dose- and time-dependent manner. The specificity of such repair was also compared with that of DNA glycosylases and damage-specific endonucleases of E. coli, both of which were found to have no detectable activity toward N(2)-4-HOPh-dG. To understand why this adduct is specifically recognized and processed by UvrABC, molecular modeling studies were performed. Analysis of molecular dynamics trajectories showed that stable G:C-like hydrogen bonding patterns of all three Watson-Crick hydrogen bonds are present within the N(2)-4-HOPh-G:C base pair, with the hydroxyphenyl ring at an almost planar position. In addition, N(2)-4-HOPh-dG has a tendency to form more stable stacking interactions than a normal G in B-type DNA. These conformational properties may be critical in differential recognition of this adduct by specific repair enzymes.

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.

Age and metabolic risk factors associated with oxidatively damaged DNA in human peripheral blood mononuclear cells

DEFF Research Database (Denmark)

Løhr, Mille; Jensen, Annie; Eriksen, Louise

2015-01-01

Aging is associated with oxidative stress-generated damage to DNA and this could be related to metabolic disturbances. This study investigated the association between levels of oxidatively damaged DNA in peripheral blood mononuclear cells (PBMCs) and metabolic risk factors in 1,019 subjects, aged...... 18-93 years. DNA damage was analyzed as strand breaks by the comet assay and levels of formamidopyrimidine (FPG-) and human 8-oxoguanine DNA glycosylase 1 (hOGG1)-sensitive sites There was an association between age and levels of FPG-sensitive sites for women, but not for men. The same tendency......, cholesterol and glycosylated hemoglobin (HbA1c). In the group of men, there were significant positive associations between alcohol intake, HbA1c and FPG-sensitive sites in multivariate analysis. The levels of metabolic risk factors were positively associated with age, yet only few subjects fulfilled all...

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

Important role of the nucleotide excision repair pathway in Mycobacterium smegmatis in conferring protection against commonly encountered DNA-damaging agents.

Science.gov (United States)

Kurthkoti, Krishna; Kumar, Pradeep; Jain, Ruchi; Varshney, Umesh

2008-09-01

Mycobacteria are an important group of human pathogens. Although the DNA repair mechanisms in mycobacteria are not well understood, these are vital for the pathogen's persistence in the host macrophages. In this study, we generated a null mutation in the uvrB gene of Mycobacterium smegmatis to allow us to compare the significance of the nucleotide excision repair (NER) pathway with two important base excision repair pathways, initiated by uracil DNA glycosylase (Ung) and formamidopyrimidine DNA glycosylase (Fpg or MutM), in an isogenic strain background. The strain deficient in NER was the most sensitive to commonly encountered DNA-damaging agents such as UV, low pH, reactive oxygen species, hypoxia, and was also sensitive to acidified nitrite. Taken together with previous observations on NER-deficient M. tuberculosis, these results suggest that NER is an important DNA repair pathway in mycobacteria.

Ionizing radiation-induced DNA damage and its repair in human cells. Final performance report, July 1992 - June 1995

International Nuclear Information System (INIS)

Dizdaroglu, M.

1995-01-01

The studies of DNA damage in living cells in vitro and in vivo were continued. A variety of systems including cultured mammalian cells, animals, and human tissues were used to conduct these studies. In addition, enzymatic repair of DNA base damage was studied using several DNA glycosylases. To this end, substrate specificities of these enzymes were examined in terms of a large number of base lesions in DNA. In the first phase of the studies, the author sought to introduce improvements to his methodologies for measurement of DNA damage using the technique of gas chromatography/mass spectrometry (GC/MS). In particular, the quantitative measurement of DNA base damage and DNA-protein crosslinks was improved by incorporation of isotope-dilution mass spectrometry into the methodologies. This is one of the most accurate techniques for quantification of organic compounds. Having improved the measurement technique, studies of DNA damage in living cells and DNA repair by repair enzymes were pursued. This report provides a summary of these studies with references to the original work

Base excision DNA repair in the embryonic development of the sea urchin, Strongylocentrotus intermedius.

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Torgasheva, Natalya A; Menzorova, Natalya I; Sibirtsev, Yurii T; Rasskazov, Valery A; Zharkov, Dmitry O; Nevinsky, Georgy A

2016-06-21

In actively proliferating cells, such as the cells of the developing embryo, DNA repair is crucial for preventing the accumulation of mutations and synchronizing cell division. Sea urchin embryo growth was analyzed and extracts were prepared. The relative activity of DNA polymerase, apurinic/apyrimidinic (AP) endonuclease, uracil-DNA glycosylase, 8-oxoguanine-DNA glycosylase, and other glycosylases was analyzed using specific oligonucleotide substrates of these enzymes; the reaction products were resolved by denaturing 20% polyacrylamide gel electrophoresis. We have characterized the profile of several key base excision repair activities in the developing embryos (2 blastomers to mid-pluteus) of the grey sea urchin, Strongylocentrotus intermedius. The uracil-DNA glycosylase specific activity sharply increased after blastula hatching, whereas the specific activity of 8-oxoguanine-DNA glycosylase steadily decreased over the course of the development. The AP-endonuclease activity gradually increased but dropped at the last sampled stage (mid-pluteus 2). The DNA polymerase activity was high at the first cleavage division and then quickly decreased, showing a transient peak at blastula hatching. It seems that the developing sea urchin embryo encounters different DNA-damaging factors early in development within the protective envelope and later as a free-floating larva, with hatching necessitating adaptation to the shift in genotoxic stress conditions. No correlation was observed between the dynamics of the enzyme activities and published gene expression data from developing congeneric species, S. purpuratus. The results suggest that base excision repair enzymes may be regulated in the sea urchin embryos at the level of covalent modification or protein stability.

MutY DNA Glycosylase Protects Cells From Tumor Necrosis Factor Alpha-Induced Necroptosis.

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Tran, An Hue Vy; Han, Se Hee; Kim, Joon; Grasso, Francesca; Kim, In San; Han, Ye Sun

2017-07-01

Numerous studies have implied that mutY DNA glycosylase (MYH) is involved in the repair of post-replicative mispairs and plays a critical role in the base excision repair pathway. Recent in vitro studies have shown that MYH interacts with tumor necrosis factor receptor type 1-associated death domain (TRADD), a key effector protein of tumor necrosis factor receptor-1 (TNFR1) signaling. The association between MYH and TRADD is reversed during tumor necrosis factor alpha (TNF-α)- and camptothecin (CPT)-induced apoptosis, and enhanced during TNF-α-induced survival. After investigating the role of MYH interacts with various proteins following TNF-α stimulation, here, we focus on MYH and TRADD interaction functions in necroptosis and its effects to related proteins. We report that the level of the MYH and TRADD complex was also reduced during necroptosis induced by TNF-α and zVAD-fmk. In particular, we also found that MYH is a biologically important necrosis suppressor. Under combined TNF-α and zVAD-fmk treatment, MYH-deficient cells were induced to enter the necroptosis pathway but primary mouse embryonic fibroblasts (MEFs) were not. Necroptosis in the absence of MYH proceeds via the inactivation of caspase-8, followed by an increase in the formation of the kinase receptor- interacting protein 1 (RIP1)-RIP3 complex. Our results suggested that MYH, which interacts with TRADD, inhibits TNF-α necroptotic signaling. Therefore, MYH inactivation is essential for necroptosis via the downregulation of caspase-8. J. Cell. Biochem. 118: 1827-1838, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Transmission of the PabI family of restriction DNA glycosylase genes: mobility and long-term inheritance.

Science.gov (United States)

Kojima, Kenji K; Kobayashi, Ichizo

2015-10-19

R.PabI is an exceptional restriction enzyme that functions as a DNA glycosylase. The enzyme excises an unmethylated base from its recognition sequence to generate apurinic/apyrimidinic (AP) sites, and also displays AP lyase activity, cleaving the DNA backbone at the AP site to generate the 3'-phospho alpha, beta-unsaturated aldehyde end in addition to the 5'-phosphate end. The resulting ends are difficult to religate with DNA ligase. The enzyme was originally isolated in Pyrococcus, a hyperthermophilic archaeon, and additional homologs subsequently identified in the epsilon class of the Gram-negative bacterial phylum Proteobacteria, such as Helicobacter pylori. Systematic analysis of R.PabI homologs and their neighboring genes in sequenced genomes revealed co-occurrence of R.PabI with M.PabI homolog methyltransferase genes. R.PabI and M.PabI homolog genes are occasionally found at corresponding (orthologous) loci in different species, such as Helicobacter pylori, Helicobacter acinonychis and Helicobacter cetorum, indicating long-term maintenance of the gene pair. One R.PabI and M.PabI homolog gene pair is observed immediately after the GMP synthase gene in both Campylobacter and Helicobacter, representing orthologs beyond genera. The mobility of the PabI family of restriction-modification (RM) system between genomes is evident upon comparison of genomes of sibling strains/species. Analysis of R.PabI and M.PabI homologs in H. pylori revealed an insertion of integrative and conjugative elements (ICE), and replacement with a gene of unknown function that may specify a membrane-associated toxin (hrgC). In view of the similarity of HrgC with toxins in type I toxin-antitoxin systems, we addressed the biological significance of this substitution. Our data indicate that replacement with hrgC occurred in the common ancestor of hspAmerind and hspEAsia. Subsequently, H. pylori with and without hrgC were intermixed at this locus, leading to complex distribution of hrgC in East

Systematic analysis of DEMETER-like DNA glycosylase genes shows lineage-specific Smi-miR7972 involved in SmDML1 regulation in Salvia miltiorrhiza.

Science.gov (United States)

Li, Jiang; Li, Caili; Lu, Shanfa

2018-05-08

DEMETER-like DNA glycosylases (DMLs) initiate the base excision repair-dependent DNA demethylation to regulate a wide range of biological processes in plants. Six putative SmDML genes, termed SmDML1-SmDML6, were identified from the genome of S. miltiorrhiza, an emerging model plant for Traditional Chinese Medicine (TCM) studies. Integrated analysis of gene structures, sequence features, conserved domains and motifs, phylogenetic analysis and differential expression showed the conservation and divergence of SmDMLs. SmDML1, SmDML2 and SmDML4 were significantly down-regulated by the treatment of 5Aza-dC, a general DNA methylation inhibitor, suggesting involvement of SmDMLs in genome DNA methylation change. SmDML1 was predicted and experimentally validated to be target of Smi-miR7972. Computational analysis of forty whole genome sequences and almost all of RNA-seq data from Lamiids revealed that MIR7972s were only distributed in some plants of the three orders, including Lamiales, Solanales and Boraginales, and the number of MIR7972 genes varied among species. It suggests that MIR7972 genes underwent expansion and loss during the evolution of some Lamiids species. Phylogenetic analysis of MIR7972s showed closer evolutionary relationships between MIR7972s in Boraginales and Solanales in comparison with Lamiales. These results provide a valuable resource for elucidating DNA demethylation mechanism in S. miltiorrhiza.

Human NTH1 physically interacts with p53 and proliferating cell nuclear antigen

International Nuclear Information System (INIS)

Oyama, Masaki; Wakasugi, Mitsuo; Hama, Takashi; Hashidume, Hatsuho; Iwakami, Yasutaka; Imai, Rika; Hoshino, Sanae; Morioka, Hiroshi; Ishigaki, Yasuhito; Nikaido, Osamu; Matsunaga, Tsukasa

2004-01-01

Thymine glycol (Tg) is one of predominant oxidative DNA lesions caused by ionizing radiation and other oxidative stresses. Human NTH1 is a bifunctional enzyme with DNA glycosylase and AP lyase activities and removes Tg as the first step of base excision repair (BER). We have searched for the factors interacting with NTH1 by using a pull-down assay and found that GST-NTH1 fusion protein precipitates proliferating cell nuclear antigen (PCNA) and p53 as well as XPG from human cell-free extracts. GST-NTH1 also bound to recombinant FLAG-tagged XPG, PCNA, and (His) 6 -tagged p53 proteins, indicating direct protein-protein interaction between those proteins. Furthermore, His-p53 and FLAG-XPG, but not PCNA, stimulated the Tg DNA glycosylase/AP lyase activity of GST-NTH1 or NTH1. These results provide an insight into the positive regulation of BER reaction and also suggest a possible linkage between BER of Tg and other cellular mechanisms

Characterization of the major formamidopyrimidine-DNA glycosylase homolog in Mycobacterium tuberculosis and its linkage to variable tandem repeats.

Science.gov (United States)

Olsen, Ingrid; Balasingham, Seetha V; Davidsen, Tonje; Debebe, Ephrem; Rødland, Einar A; van Soolingen, Dick; Kremer, Kristin; Alseth, Ingrun; Tønjum, Tone

2009-07-01

The ability to repair DNA damage is likely to play an important role in the survival of facultative intracellular parasites because they are exposed to high levels of reactive oxygen species and nitrogen intermediates inside phagocytes. Correcting oxidative damage in purines and pyrimidines is the primary function of the enzymes formamidopyrimidine (faPy)-DNA glycosylase (Fpg) and endonuclease VIII (Nei) of the base excision repair pathway, respectively. Four gene homologs, belonging to the fpg/nei family, have been identified in Mycobacterium tuberculosis H37Rv. The recombinant protein encoded by M. tuberculosis Rv2924c, termed Mtb-Fpg1, was overexpressed, purified and biochemically characterized. The enzyme removed faPy and 5-hydroxycytosine lesions, as well as 8-oxo-7,8-dihydroguanine (8oxoG) opposite to C, T and G. Mtb-Fpg1 thus exhibited substrate specificities typical for Fpg enzymes. Although Mtb-fpg1 showed nearly complete nucleotide sequence conservation in 32 M. tuberculosis isolates, the region upstream of Mtb-fpg1 in these strains contained tandem repeat motifs of variable length. A relationship between repeat length and Mtb-fpg1 expression level was demonstrated in M. tuberculosis strains, indicating that an increased length of the tandem repeats positively influenced the expression levels of Mtb-fpg1. This is the first example of such a tandem repeat region of variable length being linked to the expression level of a bacterial gene.

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

Science.gov (United States)

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

2017-11-20

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

The N-terminal domain of human DNA helicase Rtel1 contains a redox active iron-sulfur cluster.

Science.gov (United States)

Landry, Aaron P; Ding, Huangen

2014-01-01

Human telomere length regulator Rtel1 is a superfamily II DNA helicase and is essential for maintaining proper length of telomeres in chromosomes. Here we report that the N-terminal domain of human Rtel1 (RtelN) expressed in Escherichia coli cells produces a protein that contains a redox active iron-sulfur cluster with the redox midpoint potential of -248 ± 10 mV (pH 8.0). The iron-sulfur cluster in RtelN is sensitive to hydrogen peroxide and nitric oxide, indicating that reactive oxygen/nitrogen species may modulate the DNA helicase activity of Rtel1 via modification of its iron-sulfur cluster. Purified RtelN retains a weak binding affinity for the single-stranded (ss) and double-stranded (ds) DNA in vitro. However, modification of the iron-sulfur cluster by hydrogen peroxide or nitric oxide does not significantly affect the DNA binding activity of RtelN, suggesting that the iron-sulfur cluster is not directly involved in the DNA interaction in the N-terminal domain of Rtel1.

The N-Terminal Domain of Human DNA Helicase Rtel1 Contains a Redox Active Iron-Sulfur Cluster

Directory of Open Access Journals (Sweden)

Aaron P. Landry

2014-01-01

Full Text Available Human telomere length regulator Rtel1 is a superfamily II DNA helicase and is essential for maintaining proper length of telomeres in chromosomes. Here we report that the N-terminal domain of human Rtel1 (RtelN expressed in Escherichia coli cells produces a protein that contains a redox active iron-sulfur cluster with the redox midpoint potential of −248 ± 10 mV (pH 8.0. The iron-sulfur cluster in RtelN is sensitive to hydrogen peroxide and nitric oxide, indicating that reactive oxygen/nitrogen species may modulate the DNA helicase activity of Rtel1 via modification of its iron-sulfur cluster. Purified RtelN retains a weak binding affinity for the single-stranded (ss and double-stranded (ds DNA in vitro. However, modification of the iron-sulfur cluster by hydrogen peroxide or nitric oxide does not significantly affect the DNA binding activity of RtelN, suggesting that the iron-sulfur cluster is not directly involved in the DNA interaction in the N-terminal domain of Rtel1.

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)

DNA repair synthesis in human fibroblasts requires DNA polymerase delta

International Nuclear Information System (INIS)

Nishida, C.; Reinhard, P.; Linn, S.

1988-01-01

When UV-irradiated cultured diploid human fibroblasts were permeabilized with Brij-58 then separated from soluble material by centrifugation, conservative DNA repair synthesis could be restored by a soluble factor obtained from the supernatant of similarly treated HeLa cells. Extensive purification of this factor yielded a 10.2 S, 220,000-dalton polypeptide with the DNA polymerase and 3'- to 5'-exonuclease activities reported for DNA polymerase delta II. Monoclonal antibody to KB cell DNA polymerase alpha, while binding to HeLa DNA polymerase alpha, did not bind to the HeLa DNA polymerase delta. Moreover, at micromolar concentrations N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP) and 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) were potent inhibitors of DNA polymerase alpha, but did not inhibit the DNA polymerase delta. Neither purified DNA polymerase alpha nor beta could promote repair DNA synthesis in the permeabilized cells. Furthermore, under conditions which inhibited purified DNA polymerase alpha by greater than 90%, neither monoclonal antibodies to DNA polymerase alpha, BuPdGTP, nor BuAdATP was able to inhibit significantly the DNA repair synthesis mediated by the DNA polymerase delta. Thus, it appears that a major portion of DNA repair synthesis induced by UV irradiation might be catalyzed by DNA polymerase delta. When xeroderma pigmentosum human diploid fibroblasts were utilized, DNA repair synthesis dependent upon ultraviolet light could be restored by addition of both T4 endonuclease V and DNA polymerase delta, but not by addition of either one alone

Regulation and function of DNA methylation in plants and animals

KAUST Repository

He, Xinjian; Chen, Taiping; Zhu, Jian-Kang

2011-01-01

) pathway. In animals, multiple mechanisms of active DNA demethylation have been proposed, including a deaminase- and DNA glycosylase-initiated BER pathway. New information concerning the effects of various histone modifications on the establishment

NEIL2 protects against oxidative DNA damage induced by sidestream smoke in human cells.

Directory of Open Access Journals (Sweden)

Altaf H Sarker

Full Text Available Secondhand smoke (SHS is a confirmed lung carcinogen that introduces thousands of toxic chemicals into the lungs. SHS contains chemicals that have been implicated in causing oxidative DNA damage in the airway epithelium. Although DNA repair is considered a key defensive mechanism against various environmental attacks, such as cigarette smoking, the associations of individual repair enzymes with susceptibility to lung cancer are largely unknown. This study investigated the role of NEIL2, a DNA glycosylase excising oxidative base lesions, in human lung cells treated with sidestream smoke (SSS, the main component of SHS. To do so, we generated NEIL2 knockdown cells using siRNA-technology and exposed them to SSS-laden medium. Representative SSS chemical compounds in the medium were analyzed by mass spectrometry. An increased production of reactive oxygen species (ROS in SSS-exposed cells was detected through the fluorescent detection and the induction of HIF-1α. The long amplicon-quantitative PCR (LA-QPCR assay detected significant dose-dependent increases of oxidative DNA damage in the HPRT gene of cultured human pulmonary fibroblasts (hPF and BEAS-2B epithelial cells exposed to SSS for 24 h. These data suggest that SSS exposure increased oxidative stress, which could contribute to SSS-mediated toxicity. siRNA knockdown of NEIL2 in hPF and HEK 293 cells exposed to SSS for 24 h resulted in significantly more oxidative DNA damage in HPRT and POLB than in cells with control siRNA. Taken together, our data strongly suggest that decreased repair of oxidative DNA base lesions due to an impaired NEIL2 expression in non-smokers exposed to SSS would lead to accumulation of mutations in genomic DNA of lung cells over time, thus contributing to the onset of SSS-induced lung cancer.

Radiolysis of DNA-protein complexes

Energy Technology Data Exchange (ETDEWEB)

Begusova, Marie [Department of Radiation Dosimetry, Nuclear Physics Institute, Na Truhlarce 39/64, CZ-18086, Prague 8 (Czech Republic)]. E-mail: begusova@ujf.cas.cz; Gillard, Nathalie [Centre de Biophysique Moleculaire, CNRS, rue Charles-Sadron, F-45071 Orleans Cedex 2 (France); Sy, Denise [Centre de Biophysique Moleculaire, CNRS, rue Charles-Sadron, F-45071 Orleans Cedex 2 (France); Castaing, Bertrand [Centre de Biophysique Moleculaire, CNRS, rue Charles-Sadron, F-45071 Orleans Cedex 2 (France); Charlier, Michel [Centre de Biophysique Moleculaire, CNRS, rue Charles-Sadron, F-45071 Orleans Cedex 2 (France); Spotheim-Maurizot, Melanie [Centre de Biophysique Moleculaire, CNRS, rue Charles-Sadron, F-45071 Orleans Cedex 2 (France)

2005-02-01

We discuss here modifications of DNA and protein radiolysis due to the interaction of these two partners in specific complexes. Experimental patterns of frank strand breaks (FSB) and alkali revealed breaks (ARB) obtained for DNA lac operator bound to the lac repressor and for a DNA containing an abasic site analog bound to the formamidopyrimidine-DNA glycosylase are reported. Experimental data are compared to predicted damage distribution obtained using the theoretical model RADACK.

Radiolysis of DNA-protein complexes

International Nuclear Information System (INIS)

Begusova, Marie; Gillard, Nathalie; Sy, Denise; Castaing, Bertrand; Charlier, Michel; Spotheim-Maurizot, Melanie

2005-01-01

We discuss here modifications of DNA and protein radiolysis due to the interaction of these two partners in specific complexes. Experimental patterns of frank strand breaks (FSB) and alkali revealed breaks (ARB) obtained for DNA lac operator bound to the lac repressor and for a DNA containing an abasic site analog bound to the formamidopyrimidine-DNA glycosylase are reported. Experimental data are compared to predicted damage distribution obtained using the theoretical model RADACK

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

International Nuclear Information System (INIS)

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

2013-01-01

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

Oxidative Stress, DNA Damage, and Inflammation Induced by Ambient Air and Wood Smoke Particulate Matter in Human A549 and THP-1 Cell Lines

DEFF Research Database (Denmark)

Danielsen, Pernille Høgh; Møller, Peter; Jensen, Keld Alstrup

2011-01-01

PM (WSPM) is poorly assessed. We assessed a wide spectrum of toxicity end points in human A549 lung epithelial and THP-1 monocytic cell lines comparingWSPM from high or low oxygen combustion and ambient PM collected in a village with many operating wood stoves and from a rural background area...... from the wood stove area. Expression of oxoguanine glycosylase 1, lymphocyte function-associated antigen-1, and interleukin-6 did not change. We conclude that WSPM has small particle size, high level of PAH, low level of water-soluble metals, and produces high levels of free radicals, DNA damage...

Potent protection of gallic acid against DNA oxidation: Results of human and animal experiments

International Nuclear Information System (INIS)

Ferk, Franziska; Chakraborty, Asima; Jaeger, Walter; Kundi, Michael; Bichler, Julia; Misik, Miroslav; Wagner, Karl-Heinz; Grasl-Kraupp, Bettina; Sagmeister, Sandra; Haidinger, Gerald; Hoelzl, Christine; Nersesyan, Armen; Dusinska, Maria; Simic, Tatjana; Knasmueller, Siegfried

2011-01-01

Gallic acid (3,4,5-trihydroxybenzoic acid, GA) is a constituent of plant derived foods, beverages and herbal remedies. We investigated its DNA protective properties in a placebo controlled human intervention trial in single cell gel electrophoresis experiments. Supplementation of drinking water with GA (12.8 mg/person/d) for three days led to a significant reduction of DNA migration attributable to oxidised pyrimidines (endonuclease III sensitive sites) and oxidised purines (formamidopyrimidine glycosylase sensitive sites) in lymphocytes of healthy individuals by 75% and 64% respectively. Also DNA damage caused by treatment of the cells with reactive oxygen species (ROS) was reduced after GA consumption (by 41%). These effects were paralleled by an increase of the activities of antioxidant enzymes (superoxide dismutase, glutathione peroxidase and glutathion-S-transferase-π) and a decrease of intracellular ROS concentrations in lymphocytes, while no alterations of the total antioxidant capacity (TAC), of malondialdehyde levels in serum and of the urinary excretion of isoprostanes were found. Experiments with rats showed that GA reduces oxidatively damaged DNA in lymphocytes, liver, colon and lungs and protects these organs against γ-irradiation-induced strand breaks and formation of oxidatively damaged DNA-bases. Furthermore, the number of radiation-induced preneoplastic hepatic foci was decreased by 43% after oral administration of the phenolic. Since we did not find alterations of the TAC in plasma and lipid peroxidation of cell membranes but intracellular effects it is likely that the antioxidant properties of GA seen in vivo are not due to direct scavenging of radicals but rather to indirect mechanisms (e.g. protection against ROS via activation of transcription factors). As the amount of GA used in the intervention trial is similar to the daily intake in Middle Europe (18 mg/person/day), our findings indicate that it may contribute to prevention of formation

Potent protection of gallic acid against DNA oxidation: Results of human and animal experiments

Energy Technology Data Exchange (ETDEWEB)

Ferk, Franziska; Chakraborty, Asima [Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, A-1090 Vienna (Austria); Jaeger, Walter [Department of Clinical Pharmacy and Diagnostic, University of Vienna, Vienna (Austria); Kundi, Michael [Institute of Environmental Health, Center for Public Health, Medical University of Vienna, A-1090 Vienna (Austria); Bichler, Julia; Misik, Miroslav [Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, A-1090 Vienna (Austria); Wagner, Karl-Heinz [Department of Nutritional Sciences, University of Vienna, 1090 Vienna (Austria); Grasl-Kraupp, Bettina; Sagmeister, Sandra [Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, A-1090 Vienna (Austria); Haidinger, Gerald [Department of Epidemiology, Center for Public Health, Medical University of Vienna, A-1090 Vienna (Austria); Hoelzl, Christine; Nersesyan, Armen [Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, A-1090 Vienna (Austria); Dusinska, Maria [Health Effect Laboratory, Center for Ecological Economics, Norwegian Institute for Air Research, NO-2027 Kjeller (Norway); Simic, Tatjana [Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, A-1090 Vienna (Austria); Knasmueller, Siegfried, E-mail: siegfried.knasmueller@meduniwien.ac.at [Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, A-1090 Vienna (Austria)

2011-10-01

Gallic acid (3,4,5-trihydroxybenzoic acid, GA) is a constituent of plant derived foods, beverages and herbal remedies. We investigated its DNA protective properties in a placebo controlled human intervention trial in single cell gel electrophoresis experiments. Supplementation of drinking water with GA (12.8 mg/person/d) for three days led to a significant reduction of DNA migration attributable to oxidised pyrimidines (endonuclease III sensitive sites) and oxidised purines (formamidopyrimidine glycosylase sensitive sites) in lymphocytes of healthy individuals by 75% and 64% respectively. Also DNA damage caused by treatment of the cells with reactive oxygen species (ROS) was reduced after GA consumption (by 41%). These effects were paralleled by an increase of the activities of antioxidant enzymes (superoxide dismutase, glutathione peroxidase and glutathion-S-transferase-{pi}) and a decrease of intracellular ROS concentrations in lymphocytes, while no alterations of the total antioxidant capacity (TAC), of malondialdehyde levels in serum and of the urinary excretion of isoprostanes were found. Experiments with rats showed that GA reduces oxidatively damaged DNA in lymphocytes, liver, colon and lungs and protects these organs against {gamma}-irradiation-induced strand breaks and formation of oxidatively damaged DNA-bases. Furthermore, the number of radiation-induced preneoplastic hepatic foci was decreased by 43% after oral administration of the phenolic. Since we did not find alterations of the TAC in plasma and lipid peroxidation of cell membranes but intracellular effects it is likely that the antioxidant properties of GA seen in vivo are not due to direct scavenging of radicals but rather to indirect mechanisms (e.g. protection against ROS via activation of transcription factors). As the amount of GA used in the intervention trial is similar to the daily intake in Middle Europe (18 mg/person/day), our findings indicate that it may contribute to prevention of

A unique dual recognition hairpin probe mediated fluorescence amplification method for sensitive detection of uracil-DNA glycosylase and endonuclease IV activities.

Science.gov (United States)

Wu, Yushu; Yan, Ping; Xu, Xiaowen; Jiang, Wei

2016-03-07

Uracil-DNA glycosylase (UDG) and endonuclease IV (Endo IV) play cooperative roles in uracil base-excision repair (UBER) and inactivity of either will interrupt the UBER to cause disease. Detection of UDG and Endo IV activities is crucial to evaluate the UBER process in fundamental research and diagnostic application. Here, a unique dual recognition hairpin probe mediated fluorescence amplification method was developed for sensitively and selectively detecting UDG and Endo IV activities. For detecting UDG activity, the uracil base in the probe was excised by the target enzyme to generate an apurinic/apyrimidinic (AP) site, achieving the UDG recognition. Then, the AP site was cleaved by a tool enzyme Endo IV, releasing a primer to trigger rolling circle amplification (RCA) reaction. Finally, the RCA reaction produced numerous repeated G-quadruplex sequences, which interacted with N-methyl-mesoporphyrin IX to generate an enhanced fluorescence signal. Alternatively, for detecting Endo IV activity, the uracil base in the probe was first converted into an AP site by a tool enzyme UDG. Next, the AP site was cleaved by the target enzyme, achieving the Endo IV recognition. The signal was then generated and amplified in the same way as those in the UDG activity assay. The detection limits were as low as 0.00017 U mL(-1) for UDG and 0.11 U mL(-1) for Endo IV, respectively. Moreover, UDG and Endo IV can be well distinguished from their analogs. This method is beneficial for properly evaluating the UBER process in function studies and disease prognoses.

My journey to DNA repair.

Science.gov (United States)

Lindahl, Tomas

2013-02-01

I completed my medical studies at the Karolinska Institute in Stockholm but have always been devoted to basic research. My longstanding interest is to understand fundamental DNA repair mechanisms in the fields of cancer therapy, inherited human genetic disorders and ancient DNA. I initially measured DNA decay, including rates of base loss and cytosine deamination. I have discovered several important DNA repair proteins and determined their mechanisms of action. The discovery of uracil-DNA glycosylase defined a new category of repair enzymes with each specialized for different types of DNA damage. The base excision repair pathway was first reconstituted with human proteins in my group. Cell-free analysis for mammalian nucleotide excision repair of DNA was also developed in my laboratory. I found multiple distinct DNA ligases in mammalian cells, and led the first genetic and biochemical work on DNA ligases I, III and IV. I discovered the mammalian exonucleases DNase III (TREX1) and IV (FEN1). Interestingly, expression of TREX1 was altered in some human autoimmune diseases. I also showed that the mutagenic DNA adduct O(6)-methylguanine (O(6)mG) is repaired without removing the guanine from DNA, identifying a surprising mechanism by which the methyl group is transferred to a residue in the repair protein itself. A further novel process of DNA repair discovered by my research group is the action of AlkB as an iron-dependent enzyme carrying out oxidative demethylation. Copyright © 2013. Production and hosting by Elsevier Ltd.

Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA

DEFF Research Database (Denmark)

Cowell, G M; Kønigshøfer, E; Danielsen, E M

1988-01-01

The complete primary structure (967 amino acids) of an intestinal human aminopeptidase N (EC 3.4.11.2) was deduced from the sequence of a cDNA clone. Aminopeptidase N is anchored to the microvillar membrane via an uncleaved signal for membrane insertion. A domain constituting amino acid 250...

DNA Base Excision Repair (BER) and Cancer Gene Therapy: Use of the Human N-mythlpurien DNA Glycosylase (MPG) to Sensitize Breast Cancer Cells to Low Dose Chemotherapy

National Research Council Canada - National Science Library

Harvey, Tia

2003-01-01

The DNA Base Excision Repair (PER) pathway is responsible for the repair of alkylation and oxidative DNA damage resulting in protection against the deleterious effects of endogenous and exogenous agents encountered on a daily basis...

2- and 4-Aminobiphenyls induce oxidative DNA damage in human hepatoma (Hep G2) cells via different mechanisms

International Nuclear Information System (INIS)

Wang Shuchi; Chung, Jing-Gung; Chen, C.-H.; Chen, S.-C.

2006-01-01

4-Aminobiphenyl (4-ABP) and its analogue, 2-aminobiphenyl (2-ABP), were examined for their ability to induce oxidative DNA damage in Hep G2 cells. Using the alkaline comet assay, we showed that 2-ABP and 4-ABP (25-200 μM) were able to induce the DNA damage in Hep G2 cells. With both compounds, formation of intracellular reactive oxygen species (ROS) was detected using flow cytometry analysis. Post-treatment of 2-ABP and 4-ABP-treated cells by endonuclease III (Endo III) or formamidopyrimidine-DNA glycosylase (Fpg) to determine the formation of oxidized pyrimidines or oxidized purines showed a significant increase of the extent of DNA migration. This indicated that oxidative DNA damage occurs in Hep G2 cells after exposure to 2-ABP and 4-ABP. This assumption was further substantiated by the fact that the spin traps, 5,5-dimethyl-pyrroline-N-oxide (DMPO) and N-tert-butyl-α-phenylnitrone (PBN), decreased DNA damage significantly. Furthermore, addition of the catalase (100 U/ml) caused a decrease in the DNA damage induced by 2-ABP or 4-ABP, indicating that H 2 O 2 is involved in ABP-induced DNA damage. Pre-incubation of the cells with the iron chelator desferrioxamine (DFO) (1 mM) and with the copper chelator neocupronine (NC) (100 μM) also decreased DNA damage in cells treated with 200 μM 2-ABP or 200 μM 4-ABP, while the calcium chelator {1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester}(BAPTA/AM) (10 μM) decreased only DNA strand breaks in cells exposed to 4-ABP. This suggested that ions are involved in the formation of DNA strand breaks. Using RT-PCR and Western blotting, lower inhibition of the expression of the OGG1 gene and of the OGG1 protein was observed in cells treated with 4-ABP, and 2-ABP-treated cells showed a marked reduction in the expression of OGG1 gene and OGG1 protein. Taken together, our finding indicated the mechanisms of induced oxidative DNA damage in Hep G2 cell by 2-ABP and 4-ABP are different, although both

Cloning and characterization of a functional human homolog of Escherichia coli endonuclease III

Science.gov (United States)

Aspinwall, Richard; Rothwell, Dominic G.; Roldan-Arjona, Teresa; Anselmino, Catherine; Ward, Christopher J.; Cheadle, Jeremy P.; Sampson, Julian R.; Lindahl, Tomas; Harris, Peter C.; Hickson, Ian D.

1997-01-01

Repair of oxidative damage to DNA bases is essential to prevent mutations and cell death. Endonuclease III is the major DNA glycosylase activity in Escherichia coli that catalyzes the excision of pyrimidines damaged by ring opening or ring saturation, and it also possesses an associated lyase activity that incises the DNA backbone adjacent to apurinic/apyrimidinic sites. During analysis of the area adjacent to the human tuberous sclerosis gene (TSC2) in chromosome region 16p13.3, we identified a gene, OCTS3, that encodes a 1-kb transcript. Analysis of OCTS3 cDNA clones revealed an open reading frame encoding a predicted protein of 34.3 kDa that shares extensive sequence similarity with E. coli endonuclease III and a related enzyme from Schizosaccharomyces pombe, including a conserved active site region and an iron/sulfur domain. The product of the OCTS3 gene was therefore designated hNTH1 (human endonuclease III homolog 1). The hNTH1 protein was overexpressed in E. coli and purified to apparent homogeneity. The recombinant protein had spectral properties indicative of the presence of an iron/sulfur cluster, and exhibited DNA glycosylase activity on double-stranded polydeoxyribonucleotides containing urea and thymine glycol residues, as well as an apurinic/apyrimidinic lyase activity. Our data indicate that hNTH1 is a structural and functional homolog of E. coli endonuclease III, and that this class of enzymes, for repair of oxidatively damaged pyrimidines in DNA, is highly conserved in evolution from microorganisms to human cells. PMID:8990169

Selective inhibition by harmane of the apurinic apyrimidinic endonuclease activity of phage T4-induced UV endonuclease.

Science.gov (United States)

Warner, H R; Persson, M L; Bensen, R J; Mosbaugh, D W; Linn, S

1981-11-25

1-Methyl-9H-pyrido-[3,4-b]indole (harmane) inhibits the apurinic/apyrimidinic (AP) endonuclease activity of the UV endonuclease induced by phage T4, whereas it stimulates the pyrimidine dimer-DNA glycosylase activity of that enzyme. E. coli endonuclease IV, E. coli endonuclease VI (the AP endonuclease activity associated with E. coli exonuclease III), and E. coli uracil-DNA glycosylase were not inhibited by harmane. Human fibroblast AP endonucleases I and II also were only slightly inhibited. Therefore, harmane is neither a general inhibitor of AP endonucleases, nor a general inhibitor of Class I AP endonucleases which incise DNA on the 3'-side of AP sites. However, E. coli endonuclease III and its associated dihydroxythymine-DNA glycosylase activity were both inhibited by harmane. This observation suggests that harmane may inhibit only AP endonucleases which have associated glycosylase activities.

DNA repair in human bronchial epithelial cells

International Nuclear Information System (INIS)

Fornace, A.J. Jr.; Lechner, J.F.; Grafstrom, R.C.; Harris, C.C.

1982-01-01

The purpose of this investigation was to compare the response of human cell types (bronchial epithelial cells and fibroblasts and skin fibroblasts) to various DNA damaging agents. Repair of DNA single strand breaks (SSB) induced by 5 krads of X-ray was similar for all cell types; approximately 90% of the DNA SSB were rejoined within one hour. During excision repair of DNA damage from u.v.-radiation, the frequencies of DNA SSB as estimated by the alkaline elution technique, were similar in all cell types. Repair replication as measured by BND cellulose chromatography was also similar in epithelial and fibroblastic cells after u.v.-irradiation. Similar levels of SSB were also observed in epithelial and fibroblastic cells after exposure to chemical carcinogens: 7,12-dimethylbenz[a]anthracene; benzo[a]pyrene diol epoxide (BPDE); or N-methyl-N-nitro-N-nitrosoguanidine. Significant repair replication of BPDE-induced DNA damage was detected in both bronchial epithelial and fibroblastic cells, although the level in fibroblasts was approximately 40% of that in epithelial cells. The pulmonary carcinogen asbestos did not damage DNA. DNA-protein crosslinks induced by formaldehyde were rapidly removed in bronchial cells. Further, epithelial and fibroblastic cells, which were incubated with formaldehyde and the polymerase inhibitor combination of cytosine arabinoside and hydroxyurea, accumulated DNA SSB at approximately equal frequencies. These results should provide a useful background for further investigations of the response of human bronchial cells to various DNA damaging agents

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

International Nuclear Information System (INIS)

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

2014-01-01

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

Ginsenoside Rg3 induces DNA damage in human osteosarcoma cells and reduces MNNG-induced DNA damage and apoptosis in normal human cells.

Science.gov (United States)

Zhang, Yue-Hui; Li, Hai-Dong; Li, Bo; Jiang, Sheng-Dan; Jiang, Lei-Sheng

2014-02-01

Panax ginseng is a Chinese medicinal herb. Ginsenosides are the main bioactive components of P. ginseng, and ginsenoside Rg3 is the primary ginsenoside. Ginsenosides can potently kill various types of cancer cells. The present study was designed to evaluate the potential genotoxicity of ginsenoside Rg3 in human osteosarcoma cells and the protective effect of ginsenoside Rg3 with respect to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced DNA damage and apoptosis in a normal human cell line (human fibroblasts). Four human osteosarcoma cell lines (MG-63, OS732, U-2OS and HOS cells) and a normal human cell line (human fibroblasts) were employed to investigate the cytotoxicity of ginsenosides Rg3 by MTT assay. Alkaline comet assay and γH2AX focus staining were used to detect the DNA damage in MG-63 and U-2OS cells. The extent of cell apoptosis was determined by flow cytometry and a DNA ladder assay. Our results demonstrated that the cytotoxicity of ginsenoside Rg3 was dose-dependent in the human osteosarcoma cell lines, and MG-63 and U-2OS cells were the most sensitive to ginsenoside Rg3. As expected, compared to the negative control, ginsenoside Rg3 significantly increased DNA damage in a concentration-dependent manner. In agreement with the comet assay data, the percentage of γH2AX-positive MG-63 and U-2OS cells indicated that ginsenoside Rg3 induced DNA double-strand breaks in a concentration-dependent manner. The results also suggest that ginsenoside Rg3 reduces the extent of MNNG-induced DNA damage and apoptosis in human fibroblasts.

Regulation and function of DNA methylation in plants and animals

KAUST Repository

He, Xinjian

2011-02-15

DNA methylation is an important epigenetic mark involved in diverse biological processes. In plants, DNA methylation can be established through the RNA-directed DNA methylation pathway, an RNA interference pathway for transcriptional gene silencing (TGS), which requires 24-nt small interfering RNAs. In mammals, de novo DNA methylation occurs primarily at two developmental stages: during early embryogenesis and during gametogenesis. While it is not clear whether establishment of DNA methylation patterns in mammals involves RNA interference in general, de novo DNA methylation and suppression of transposons in germ cells require 24-32-nt piwi-interacting small RNAs. DNA methylation status is dynamically regulated by DNA methylation and demethylation reactions. In plants, active DNA demethylation relies on the repressor of silencing 1 family of bifunctional DNA glycosylases, which remove the 5-methylcytosine base and then cleave the DNA backbone at the abasic site, initiating a base excision repair (BER) pathway. In animals, multiple mechanisms of active DNA demethylation have been proposed, including a deaminase- and DNA glycosylase-initiated BER pathway. New information concerning the effects of various histone modifications on the establishment and maintenance of DNA methylation has broadened our understanding of the regulation of DNA methylation. The function of DNA methylation in plants and animals is also discussed in this review. © 2011 IBCB, SIBS, CAS All rights reserved.

Gadd45a promotes DNA demethylation through TDG

OpenAIRE

Li, Zheng; Gu, Tian-Peng; Weber, Alain R.; Shen, Jia-Zhen; Li, Bin-Zhong; Xie, Zhi-Guo; Yin, Ruichuan; Guo, Fan; Liu, Xiaomeng; Tang, Fuchou; Wang, Hailin; Sch?r, Primo; Xu, Guo-Liang

2015-01-01

Growth arrest and DNA-damage-inducible protein 45 (Gadd45) family members have been implicated in DNA demethylation in vertebrates. However, it remained unclear how they contribute to the demethylation process. Here, we demonstrate that Gadd45a promotes active DNA demethylation through thymine DNA glycosylase (TDG) which has recently been shown to excise 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) generated in Ten-eleven-translocation (Tet)?initiated oxidative demethylation. The conn...

Activation of apurinic/apyrimidinic endonuclease in human cells by reactive oxygen species and its correlation with their adaptive response to genotoxicity of free radicals

OpenAIRE

Ramana, Chilakamarti V.; Boldogh, Istvan; Izumi, Tadahide; Mitra, Sankar

1998-01-01

Apurinic/apyrimidinic (AP) endonuclease (APE; EC 4.2.99.18) plays a central role in repair of DNA damage due to reactive oxygen species (ROS) because its DNA 3′-phosphoesterase activity removes 3′ blocking groups in DNA that are generated by DNA glycosylase/AP-lyases during removal of oxidized bases and by direct ROS reaction with DNA. The major human APE (APE-1) gene is activated selectively by sublethal levels of a variety of ROS and ROS generators, including ionizing radiation, but not by ...

Molecular genetic and biochemical analyses of a DNA repair gene from Serratia marcescens

International Nuclear Information System (INIS)

Murphy, K.E.

1989-01-01

In Escherichia coli, the SOS response and two 3-methyladenine DNA glycosylases (TagI and TagII) are required for repair of DNA damaged by alkylating agents such as methyl methanesulfonate (MMS). Mutations of the recA gene eliminate the SOS response. TagI and TagII are encoded by the tag and alkA genes, respectively. A gene (rpr) encoding 3-methyladenine DNA glycosylase activity was isolated from the Gram-negative bacterium Serratia marcescens. The gene, localized to a 1.5-kilobase pair SmaI-HindIII restriction fragment, was cloned into plasmid pUC18. The clone complemented E. coli tag alkA and recA mutations for MMS resistance. The rpr gene did not, however, complement recA mutations for resistance to ultraviolet light or the ability to perform homologous recombination reactions, nor did it complement E. coli ada or alkB mutations. Two proteins of molecular weights 42,000 and 16,000 were produced from the rpr locus. Analysis of deletion and insertion mutants of rpr suggested that the 42kD molecule is the active protein. The 16kD protein may either be a breakdown product of the 42kD species or may be encoded by another gene overlapping the reading frame of the rpr gene. Biochemical assays showed that the rpr gene product (Rpr) possesses 3-methyladenine DNA glycosylase activity

Mechanism of error-free DNA synthesis across N1-methyl-deoxyadenosine by human DNA polymerase-ι

Energy Technology Data Exchange (ETDEWEB)

Jain, Rinku; Choudhury, Jayati Roy; Buku, Angeliki; Johnson, Robert E.; Prakash, Louise; Prakash, Satya; Aggarwal, Aneel K.

2017-03-08

N1-methyl-deoxyadenosine (1-MeA) is formed by methylation of deoxyadenosine at the N1 atom. 1-MeA presents a block to replicative DNA polymerases due to its inability to participate in Watson-Crick (W-C) base pairing. Here we determine how human DNA polymerase-ι (Polι) promotes error-free replication across 1-MeA. Steady state kinetic analyses indicate that Polι is ~100 fold more efficient in incorporating the correct nucleotide T versus the incorrect nucleotide C opposite 1-MeA. To understand the basis of this selectivity, we determined ternary structures of Polι bound to template 1-MeA and incoming dTTP or dCTP. In both structures, template 1-MeA rotates to the syn conformation but pairs differently with dTTP versus dCTP. Thus, whereas dTTP partakes in stable Hoogsteen base pairing with 1-MeA, dCTP fails to gain a “foothold” and is largely disordered. Together, our kinetic and structural studies show how Polι maintains discrimination between correct and incorrect incoming nucleotide opposite 1-MeA in preserving genome integrity.

Aag Hypoxanthine-DNA Glycosylase Is Synthesized in the Forespore Compartment and Involved in Counteracting the Genotoxic and Mutagenic Effects of Hypoxanthine and Alkylated Bases in DNA during Bacillus subtilis Sporulation.

Science.gov (United States)

Ayala-García, Víctor M; Valenzuela-García, Luz I; Setlow, Peter; Pedraza-Reyes, Mario

2016-12-15

Aag from Bacillus subtilis has been implicated in in vitro removal of hypoxanthine and alkylated bases from DNA. The regulation of expression of aag in B. subtilis and the resistance to genotoxic agents and mutagenic properties of an Aag-deficient strain were studied here. A strain with a transcriptional aag-lacZ fusion expressed low levels of β-galactosidase during growth and early sporulation but exhibited increased transcription during late stages of this developmental process. Notably, aag-lacZ expression was higher inside the forespore than in the mother cell compartment, and this expression was abolished in a sigG-deficient background, suggesting a forespore-specific mechanism of aag transcription. Two additional findings supported this suggestion: (i) expression of an aag-yfp fusion was observed in the forespore, and (ii) in vivo mapping of the aag transcription start site revealed the existence of upstream regulatory sequences possessing homology to σ G -dependent promoters. In comparison with the wild-type strain, disruption of aag significantly reduced survival of sporulating B. subtilis cells following nitrous acid or methyl methanesulfonate treatments, and the Rif r mutation frequency was significantly increased in an aag strain. These results suggest that Aag protects the genome of developing B. subtilis sporangia from the cytotoxic and genotoxic effects of base deamination and alkylation. In this study, evidence is presented revealing that aag, encoding a DNA glycosylase implicated in processing of hypoxanthine and alkylated DNA bases, exhibits a forespore-specific pattern of gene expression during B. subtilis sporulation. Consistent with this spatiotemporal mode of expression, Aag was found to protect the sporulating cells of this microorganism from the noxious and mutagenic effects of base deamination and alkylation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Characterization of cDNA for human tripeptidyl peptidase II: The N-terminal part of the enzyme is similar to subtilisin

International Nuclear Information System (INIS)

Tomkinson, B.; Jonsson, A-K

1991-01-01

Tripeptidyl peptidase II is a high molecular weight serine exopeptidase, which has been purified from rat liver and human erythrocytes. Four clones, representing 4453 bp, or 90% of the mRNA of the human enzyme, have been isolated from two different cDNA libraries. One clone, designated A2, was obtained after screening a human B-lymphocyte cDNA library with a degenerated oligonucleotide mixture. The B-lymphocyte cDNA library, obtained from human fibroblasts, were rescreened with a 147 bp fragment from the 5' part of the A2 clone, whereby three different overlapping cDNA clones could be isolated. The deduced amino acid sequence, 1196 amino acid residues, corresponding to the longest open rading frame of the assembled nucleotide sequence, was compared to sequences of current databases. This revealed a 56% similarity between the bacterial enzyme subtilisin and the N-terminal part of tripeptidyl peptidase II. The enzyme was found to be represented by two different mRNAs of 4.2 and 5.0 kilobases, respectively, which probably result from the utilziation of two different polyadenylation sites. Futhermore, cDNA corresponding to both the N-terminal and C-terminal part of tripeptidyl peptidase II hybridized with genomic DNA from mouse, horse, calf, and hen, even under fairly high stringency conditions, indicating that tripeptidyl peptidase II is highly conserved

DNA-dependent protein kinase inhibits AID-induced antibody gene conversion.

Directory of Open Access Journals (Sweden)

Adam J L Cook

2007-04-01

Full Text Available Affinity maturation and class switching of antibodies requires activation-induced cytidine deaminase (AID-dependent hypermutation of Ig V(DJ rearrangements and Ig S regions, respectively, in activated B cells. AID deaminates deoxycytidine bases in Ig genes, converting them into deoxyuridines. In V(DJ regions, subsequent excision of the deaminated bases by uracil-DNA glycosylase, or by mismatch repair, leads to further point mutation or gene conversion, depending on the species. In Ig S regions, nicking at the abasic sites produced by AID and uracil-DNA glycosylases results in staggered double-strand breaks, whose repair by nonhomologous end joining mediates Ig class switching. We have tested whether nonhomologous end joining also plays a role in V(DJ hypermutation using chicken DT40 cells deficient for Ku70 or the DNA-dependent protein kinase catalytic subunit (DNA-PKcs. Inactivation of the Ku70 or DNA-PKcs genes in DT40 cells elevated the rate of AID-induced gene conversion as much as 5-fold. Furthermore, DNA-PKcs-deficiency appeared to reduce point mutation. The data provide strong evidence that double-strand DNA ends capable of recruiting the DNA-dependent protein kinase complex are important intermediates in Ig V gene conversion.

Combustion products of 1,3-butadiene inhibit catalase activity and induce expression of oxidative DNA damage repair enzymes in human bronchial epithelial cells.

Science.gov (United States)

Kennedy, Christopher H; Catallo, W James; Wilson, Vincent L; Mitchell, James B

2009-10-01

1,3-Butadiene, an important petrochemical, is commonly burned off when excess amounts need to be destroyed. This combustion process produces butadiene soot (BDS), which is composed of a complex mixture of polycyclic aromatic hydrocarbons in particulates ranging in size from enzyme inactivation due to protein amino acid oxidation and (2) induce oxidative DNA damage in NHBE cells. Thus, our aims were to determine the effect of butadiene soot ethanol extract (BSEE) on both enzyme activity and the expression of proteins involved in the repair of oxidative DNA damage. Catalase was found to be sensitive to BDS as catalase activity was potently diminished in the presence of BSEE. Using Western analysis, both the alpha isoform of human 8-oxoguanine DNA glycosylase (alpha-hOGG1) and human apurinic/apyrimidinic endonuclease (APE-1) were shown to be significantly overexpressed as compared to untreated controls after exposure of NHBE cells to BSEE. Our results indicate that BSEE is capable of effectively inactivating the antioxidant enzyme catalase, presumably via oxidation of protein amino acids. The presence of oxidized biomolecules may partially explain the extranuclear fluorescence that is detected when NHBE cells are treated with an organic extract of BDS. Overexpression of both alpha-hOGG1 and APE-1 proteins following treatment of NHBE cells with BSEE suggests that this mixture causes oxidative DNA damage.

Perinatal transmission of human papilomavirus DNA

Directory of Open Access Journals (Sweden)

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

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

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

Inactivation by oxidation and recruitment into stress granules of hOGG1 but not APE1 in human cells exposed to sub-lethal concentrations of cadmium

International Nuclear Information System (INIS)

Bravard, Anne; Campalans, Anna; Vacher, Monique; Gouget, Barbara; Levalois, Celine; Chevillard, Sylvie; Radicella, J. Pablo

2010-01-01

The induction of mutations in mammalian cells exposed to cadmium has been associated with the oxidative stress triggered by the metal. There is increasing evidence that the mutagenic potential of Cd is not restricted to the induction of DNA lesions. Cd has been shown to inactivate several DNA repair enzymes. Here we show that exposure of human cells to sub-lethal concentrations of Cd leads to a time- and concentration-dependent decrease in hOGG1 activity, the major DNA glycosylase activity responsible for the initiation of the base excision repair (BER) of 8-oxoguanine, an abundant and mutagenic form of oxidized guanine. Although there is a slight effect on the level of hOGG1 transcripts, we show that the inhibition of the 8-oxoguanine DNA glycosylase activity is mainly associated with an oxidation of the hOGG1 protein and its disappearance from the soluble fraction of total cell extracts. Confocal microscopy analyses show that in cells exposed to Cd hOGG1-GFP is recruited to discrete structures in the cytoplasm. These structures were identified as stress granules. Removal of Cd from the medium allows the recovery of the DNA glycosylase activity and the presence of hOGG1 in a soluble form. In contrast to hOGG1, we show here that exposure to Cd does not affect the activity of the second enzyme of the pathway, the major AP endonuclease APE1.

Assessment of genotoxicity of methyl-tert-butyl ether, benzene, toluene, ethylbenzene, and xylene to human lymphocytes using comet assay

International Nuclear Information System (INIS)

Chen, Colin S.; Hseu, You C.; Liang, Shih H.; Kuo, J.-Y.; Chen, Ssu. C.

2008-01-01

Methyl-tert-butyl ether (MTBE) is a gasoline oxygenate and antiknock additive substituting for lead alkyls currently in use worldwide. Benzene, toluene, ethylbenzene, and xylene (BTEX) are volatile monoaromatic hydrocarbons which are commonly found together in crude petroleum and petroleum products such as gasoline. The aim of this study is to evaluate the genotoxic effects of these tested chemicals in human lymphocytes. Using the alkaline comet assay, we showed that all of the tested chemicals induce DNA damage in isolated human lymphocytes. This effect could follow from the induction of DNA strands breaks. The neutral version of the test revealed that MTBE, benzene, and xylenes induce DNA double-strand breaks at 200 μM. Apart from MTBE, the spin traps, 5,5-dimethyl-pyrroline-N-oxide (DMPO) and N-tert-butyl-α-phenylnitrone (PBN) can decrease the level of DNA damage in BTEX at 200 μM. This indicated that DNA damage could result from the participation of free radicals in DNA-damaging effect, which was further supported by the fact that post-treatment of formamidopyrimidine-DNA glycosylase (Fpg), enzyme recognizing oxidized DNA purines, gave rise to a significant increase in the extent of DNA damage in cells treated with benzene, and xylene at 200 μM. The results obtained suggested that MTBE and BTEX could induce a variety type of DNA damage such as single-strand breaks (SSBs), double-strand breaks (DSBs), and oxidative base modification

DNA repair is indispensable for survival after acute inflammation

Science.gov (United States)

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

2012-01-01

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

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.

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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.

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

Energy Technology Data Exchange (ETDEWEB)

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

2014-02-17

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

Unscheduled synthesis of DNA and poly(ADP-ribose) in human fibroblasts following DNA damage

International Nuclear Information System (INIS)

McCurry, L.S.; Jacobson, M.K.

1981-01-01

Unscheduled DNA synthesis has been measured in human fibroblasts under conditions of reduced rates of conversion of NAD to poly)ADP-ribose). Cells heterozygous for the xeroderma pigmentosum genotype showed normal rates of uv induced unscheduled DNA synthesis under conditions in which the rate of poly(ADP-ribose) synthesis was one-half the rate of normal cells. The addition of theophylline, a potent inhibitor of poly(ADP-ribose) polymerase, to the culture medium of normal cells blocked over 90% of the conversion of NAD to poly(ADP-ribose) following treatment with uv or N-methyl-N'-nitro-N-nitro-soguanidine but did not affect the rate of unscheduled DNA synthesis

Characterization of protein interactomes of DNA damages: application to oxidation injuries

International Nuclear Information System (INIS)

Pietras-Barbier, Ewa

2013-01-01

Cyclo-nucleosides are complex DNA damages implying both bases and sugar residues. They are generated by free radicals, in particular by the effect of ionizing radiations, and are not easily covered by cellular mechanisms. Using a protein trapping technique on probes containing these injuries, the negative influence of cyclo-nucleosides on the recognition of its target sequence by a DREF transcription factor and on the interactions of PARP1 with DNA have been identified. Interactions between Fpg bacterial glycosylase and cyclo-nucleosides have been analysed and it has been found that this enzyme has an affinity for them, without excision activity. Finally, a Thermococcus gammatolerans radiation resistant archae has been studied: the formation of simple and complex oxidation injuries at strong radiation doses has been measured and the action mechanism of two new glycosylases has been explained. (author) [fr

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

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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.

Environmental exposure to human carcinogens in teenagers and the association with DNA damage

International Nuclear Information System (INIS)

Franken, Carmen; Koppen, Gudrun; Lambrechts, Nathalie; Govarts, Eva; Bruckers, Liesbeth; Den Hond, Elly; Loots, Ilse; Nelen, Vera; Sioen, Isabelle; Nawrot, Tim S.; Baeyens, Willy; Van Larebeke, Nicolas; Boonen, Francis; Ooms, Daniëlla; Wevers, Mai; Jacobs, Griet; Covaci, Adrian; Schettgen, Thomas; Schoeters, Greet

2017-01-01

Background: We investigated whether human environmental exposure to chemicals that are labeled as (potential) carcinogens leads to increased (oxidative) damage to DNA in adolescents. Material and methods: Six hundred 14–15-year-old youngsters were recruited all over Flanders (Belgium) and in two areas with important industrial activities. DNA damage was assessed by alkaline and formamidopyrimidine DNA glycosylase (Fpg) modified comet assays in peripheral blood cells and analysis of urinary 8-hydroxydeoxyguanosine (8-OHdG) levels. Personal exposure to potentially carcinogenic compounds was measured in urine, namely: chromium, cadmium, nickel, 1-hydroxypyrene as a proxy for exposure to other carcinogenic polycyclic aromatic hydrocarbons (PAHs), t,t-muconic acid as a metabolite of benzene, 2,5-dichlorophenol (2,5-DCP), organophosphate pesticide metabolites, and di(2-ethylhexyl) phthalate (DEHP) metabolites. In blood, arsenic, polychlorinated biphenyl (PCB) congeners 118 and 156, hexachlorobenzene (HCB), dichlorodiphenyltrichloroethane (DDT) and perfluorooctanoic acid (PFOA) were analyzed. Levels of methylmercury (MeHg) were measured in hair. Multiple linear regression models were used to establish exposure-response relationships. Results: Biomarkers of exposure to PAHs and urinary chromium were associated with higher levels of both 8-OHdG in urine and DNA damage detected by the alkaline comet assay. Concentrations of 8-OHdG in urine increased in relation with increasing concentrations of urinary t,t-muconic acid, cadmium, nickel, 2,5-DCP, and DEHP metabolites. Increased concentrations of PFOA in blood were associated with higher levels of DNA damage measured by the alkaline comet assay, whereas DDT was associated in the same direction with the Fpg-modified comet assay. Inverse associations were observed between blood arsenic, hair MeHg, PCB 156 and HCB, and urinary 8-OHdG. The latter exposure biomarkers were also associated with higher fish intake. Urinary nickel

Environmental exposure to human carcinogens in teenagers and the association with DNA damage

Energy Technology Data Exchange (ETDEWEB)

Franken, Carmen, E-mail: carmen.franken@vito.be [Flemish Institute for Technological Research (VITO), Mol (Belgium); Department of Biomedical Sciences, University of Antwerp, Antwerp (Belgium); Koppen, Gudrun; Lambrechts, Nathalie; Govarts, Eva [Flemish Institute for Technological Research (VITO), Mol (Belgium); Bruckers, Liesbeth [Interuniversity Institute for Biostatistics and Statistical Bioinformatics, Hasselt University, Hasselt (Belgium); Den Hond, Elly [Flemish Institute for Technological Research (VITO), Mol (Belgium); Loots, Ilse [Political and Social Sciences, University of Antwerp, Antwerp (Belgium); Nelen, Vera [Provincial Institute for Hygiene, Antwerp (Belgium); Sioen, Isabelle [Department of Public Health, Ghent University, Ghent (Belgium); Department of Food Safety and Food Quality, Ghent University, Ghent (Belgium); Nawrot, Tim S. [Centre for Environmental Sciences, Hasselt University, Diepenbeek (Belgium); Department of Public Health & Primary Care, Leuven University, Leuven (Belgium); Baeyens, Willy [Department of Analytical and Environmental Chemistry, Vrije Universiteit Brussel, Brussels (Belgium); Van Larebeke, Nicolas [Department of Analytical and Environmental Chemistry, Vrije Universiteit Brussel, Brussels (Belgium); Department of Radiotherapy and Experimental Cancerology, Ghent University, Ghent (Belgium); Boonen, Francis; Ooms, Daniëlla; Wevers, Mai; Jacobs, Griet [Flemish Institute for Technological Research (VITO), Mol (Belgium); Covaci, Adrian [Toxicological Centre, Department of Pharmaceutical Sciences, University of Antwerp, Antwerp (Belgium); Schettgen, Thomas [Department of Occupational and Social Medicine, RWTH Aachen University, Aachen (Germany); Schoeters, Greet [Flemish Institute for Technological Research (VITO), Mol (Belgium); Department of Biomedical Sciences, University of Antwerp, Antwerp (Belgium); University of Southern Denmark, Institute of Public Health, Department of Environmental Medicine, Odense (Denmark)

2017-01-15

Background: We investigated whether human environmental exposure to chemicals that are labeled as (potential) carcinogens leads to increased (oxidative) damage to DNA in adolescents. Material and methods: Six hundred 14–15-year-old youngsters were recruited all over Flanders (Belgium) and in two areas with important industrial activities. DNA damage was assessed by alkaline and formamidopyrimidine DNA glycosylase (Fpg) modified comet assays in peripheral blood cells and analysis of urinary 8-hydroxydeoxyguanosine (8-OHdG) levels. Personal exposure to potentially carcinogenic compounds was measured in urine, namely: chromium, cadmium, nickel, 1-hydroxypyrene as a proxy for exposure to other carcinogenic polycyclic aromatic hydrocarbons (PAHs), t,t-muconic acid as a metabolite of benzene, 2,5-dichlorophenol (2,5-DCP), organophosphate pesticide metabolites, and di(2-ethylhexyl) phthalate (DEHP) metabolites. In blood, arsenic, polychlorinated biphenyl (PCB) congeners 118 and 156, hexachlorobenzene (HCB), dichlorodiphenyltrichloroethane (DDT) and perfluorooctanoic acid (PFOA) were analyzed. Levels of methylmercury (MeHg) were measured in hair. Multiple linear regression models were used to establish exposure-response relationships. Results: Biomarkers of exposure to PAHs and urinary chromium were associated with higher levels of both 8-OHdG in urine and DNA damage detected by the alkaline comet assay. Concentrations of 8-OHdG in urine increased in relation with increasing concentrations of urinary t,t-muconic acid, cadmium, nickel, 2,5-DCP, and DEHP metabolites. Increased concentrations of PFOA in blood were associated with higher levels of DNA damage measured by the alkaline comet assay, whereas DDT was associated in the same direction with the Fpg-modified comet assay. Inverse associations were observed between blood arsenic, hair MeHg, PCB 156 and HCB, and urinary 8-OHdG. The latter exposure biomarkers were also associated with higher fish intake. Urinary nickel

The effect of dietary folic acid deficiency on the cytotoxic and mutagenic responses to methyl methanesulfonate in wild-type and in 3-methyladenine DNA glycosylase-deficient Aag null mice.

Science.gov (United States)

Branda, Richard F; O'Neill, J Patrick; Brooks, Elice M; Powden, Cheryl; Naud, Shelly J; Nicklas, Janice A

2007-02-03

Folic acid deficiency (FA-) augments DNA damage caused by alkylating agents. The role of DNA repair in modulating this damage was investigated in mice. Weanling wild-type or 3-methyladenine glycosylase (Aag) null mice were maintained on a FA- diet or the same diet supplemented with folic acid (FA+) for 4 weeks. They were then treated with methyl methanesulfonate (MMS), 100mg/kg i.p. Six weeks later, spleen cells were collected for assays of non-selected and 6-thioguanine (TG) selected cloning efficiency to measure the mutant frequency at the Hprt locus. In wild-type mice, there was no significant effect of either MMS treatment or folate dietary content on splenocyte non-selected cloning efficiency. In contrast, non-selected cloning efficiency was significantly higher in MMS-treated Aag null mice than in saline treated controls (diet-gene interaction variable, p=0.04). The non-selected cloning efficiency was significantly higher in the FA+ diet than in the FA- diet group after MMS treatment of Aag null mice. Mutant frequency after MMS treatment was significantly higher in FA- wild-type and Aag null mice and in FA+ Aag null mice, but not in FA+ wild-type mice. For the Aag null mice, mutant frequency was higher in the FA+ mice than in the FA- mice after either saline or MMS treatment. These studies indicate that in wild-type mice treated with MMS, dietary folate content (FA+ or FA-) had no effect on cytotoxicity, but FA- diet increased DNA mutation frequency compared to FA+ diet. In Aag null mice, FA- diet increased the cytotoxic effects of alkylating agents but decreased the risk of DNA mutation.

Quantitative analysis of gene-specific DNA damage in human spermatozoa

International Nuclear Information System (INIS)

Sawyer, Dennis E.; Mercer, Belinda G.; Wiklendt, Agnieszka M.; Aitken, R. John

2003-01-01

Recent studies have suggested that human spermatozoa are highly susceptible to DNA damage induced by oxidative stress. However, a detailed analysis of the precise nature of this damage and the extent to which it affects the mitochondrial and nuclear genomes has not been reported. To induce DNA damage, human spermatozoa were treated in vitro with hydrogen peroxide (H 2 O 2 ; 0-5 mM) or iron (as Fe(II)SO 4 , 0-500 μM). Quantitative PCR (QPCR) was used to measure DNA damage in individual nuclear genes (hprt, β-pol and β-globin) and mitochondrial DNA. Single strand breaks were also assessed by alkaline gel electrophoresis. H 2 O 2 was found to be genotoxic toward spermatozoa at concentrations as high as 1.25 mM, but DNA damage was not detected in these cells with lower concentrations of H 2 O 2 . The mitochondrial genome of human spermatozoa was significantly (P 2 O 2 -induced DNA damage than the nuclear genome. However, both nDNA and mtDNA in human spermatozoa were significantly (P<0.001) more resistant to damage than DNA from a variety of cell lines of germ cell and myoblastoid origin. Interestingly, significant DNA damage was also not detected in human spermatozoa treated with iron. These studies report, for the first time, quantitative measurements of DNA damage in specific genes of male germ cells, and challenge the commonly held belief that human spermatozoa are particularly vulnerable to DNA damage

Ada response – a strategy for repair of alkylated DNA in bacteria

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Mielecki, Damian; Grzesiuk, Elżbieta

2014-01-01

Alkylating agents are widespread in the environment and also occur endogenously. They can be cytotoxic or mutagenic to the cells introducing alkylated bases to DNA or RNA. All organisms have evolved multiple DNA repair mechanisms to counteract the effects of DNA alkylation: the most cytotoxic lesion, N3-methyladenine (3meA), is excised by AlkA glycosylase initiating base excision repair (BER); toxic N1-methyladenine (1meA) and N3-methylcytosine (3meC), induced in DNA and RNA, are removed by AlkB dioxygenase; and mutagenic and cytotoxic O6-methylguanine (O6meG) is repaired by Ada methyltransferase. In Escherichia coli, Ada response involves the expression of four genes, ada, alkA, alkB, and aidB, encoding respective proteins Ada, AlkA, AlkB, and AidB. The Ada response is conserved among many bacterial species; however, it can be organized differently, with diverse substrate specificity of the particular proteins. Here, an overview of the organization of the Ada regulon and function of individual proteins is presented. We put special effort into the characterization of AlkB dioxygenases, their substrate specificity, and function in the repair of alkylation lesions in DNA/RNA. PMID:24810496

Synthesis of modified oligonucleotides for repair and replication studies of single and double radio-induced DNA lesions

International Nuclear Information System (INIS)

Muller, E.

2002-01-01

Several oxidative processes induce the formation of DNA lesions. In order to evaluate the biological and structural significance of such damage, several DNA lesions were inserted into synthetic oligonucleotides at defined sites. The research work aimed at describing the preparation of oligonucleotides t hat contained DNA damage and the evaluation of the biological properties of the lesions. A first part described the incorporation of radiation-induced lesions, namely (5'S,6S)-5',6-cyclo-5,6-dihydro-2'-deoxyuridine and (5'S,5S,6S)-5',6-cyclo-5-hydroxy-5,6-dihydro-2'-desoxyuridine into oligonucleotides. The modified DNA fragments were characterised by several spectroscopic and biochemical analyses including ESI MS, MALDI-TOF MS, CLHP and enzymatic digestions. During in vitro DNA synthesis by Taq DNA polymerase and Klenow exo fragment, the pyrimidine cyclo-nucleosides were found to block the progression of the enzymes. Then, repair studies by ADN N glycosylases, operating in the base excision repair pathway, have shown that the anhydro-nucleoside lesions were not recognised nor excised by Fpg, endo III, endo VIII, yNtg1 yNtg2 and yOgg1. Interestingly, the Latococcus lactis Fpg protein recognises (formation of a non covalent complex) but do not excise the damage. The incorporation into oligonucleotides of the (5R*) and (5S*) diastereoisomers of 1-[2-deoxy-β-D-erythro-pentofuranosyl]-5-hydroxy-hydantoin, generated by several oxidative processes was then described. In vitro DNA replication assays using modified oligonucleotides matrix showed a lethal potential of the latter base damage. Repair studies by ADN N-glycosylases showed that the damage was substrate for Fpg, endo III, endo VIII, Ntg1, Ntg2 and Fpg-L1. The rates of excision as inferred from the determination of the Michaelis kinetics constants were found to be affected by the presence of the damage. MALDI-TOF MS was used in order to gain insights into mechanistic aspects of oligonucleotides cleavage by the

N-terminal domains of human DNA polymerase lambda promote primer realignment during translesion DNA synthesis

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Taggart, David J.; Dayeh, Daniel M.; Fredrickson, Saul W.; Suo, Zucai

2014-01-01

The X-family DNA polymerases λ (Polλ) and β (Polβ) possess similar 5′-2-deoxyribose-5-phosphatelyase (dRPase) and polymerase domains. Besides these domains, Polλ also possesses a BRCA1 C-terminal (BRCT) domain and a proline-rich domain at its N terminus. However, it is unclear how these non-enzymatic domains contribute to the unique biological functions of Polλ. Here, we used primer extension assays and a newly developed high-throughput short oligonucleotide sequencing assay (HT-SOSA) to compare the efficiency of lesion bypass and fidelity of human Polβ, Polλ and two N-terminal deletion constructs of Polλ during the bypass of either an abasic site or a 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) lesion. We demonstrate that the BRCT domain of Polλ enhances the efficiency of abasic site bypass by approximately 1.6-fold. In contrast, deletion of the N-terminal domains of Polλ did not affect the efficiency of 8-oxodG bypass relative to nucleotide incorporations opposite undamaged dG. HT-SOSA analysis demonstrated that Polλ and Polβ preferentially generated −1 or −2 frameshift mutations when bypassing an abasic site and the single or double base deletion frequency was highly sequence dependent. Interestingly, the BRCT and proline-rich domains of Polλ cooperatively promoted the generation of −2 frameshift mutations when the abasic site was situated within a sequence context that was susceptible to homology-driven primer realignment. Furthermore, both N-terminal domains of Polλ increased the generation of −1 frameshift mutations during 8-oxodG bypass and influenced the frequency of substitution mutations produced by Polλ opposite the 8-oxodG lesion. Overall, our data support a model wherein the BRCT and proline-rich domains of Polλ act cooperatively to promote primer/template realignment between DNA strands of limited sequence homology. This function of the N-terminal domains may facilitate the role of Polλ as a gap-filling polymerase

Genetic Variability in DNA Repair Proteins in Age-Related Macular Degeneration

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Janusz Blasiak

2012-10-01

Full Text Available The pathogenesis of age-related macular degeneration (AMD is complex and involves interactions between environmental and genetic factors, with oxidative stress playing an important role inducing damage in biomolecules, including DNA. Therefore, genetic variability in the components of DNA repair systems may influence the ability of the cell to cope with oxidative stress and in this way contribute to the pathogenesis of AMD. However, few reports have been published on this subject so far. We demonstrated that the c.977C>G polymorphism (rs1052133 in the hOGG1 gene and the c.972G>C polymorphism (rs3219489 in the MUTYH gene, the products of which play important roles in the repair of oxidatively damaged DNA, might be associated with the risk of AMD. Oxidative stress may promote misincorporation of uracil into DNA, where it is targeted by several DNA glycosylases. We observed that the g.4235T>C (rs2337395 and c.−32A>G (rs3087404 polymorphisms in two genes encoding such glycosylases, UNG and SMUG1, respectively, could be associated with the occurrence of AMD. Polymorphisms in some other DNA repair genes, including XPD (ERCC2, XRCC1 and ERCC6 (CSB have also been reported to be associated with AMD. These data confirm the importance of the cellular reaction to DNA damage, and this may be influenced by variability in DNA repair genes, in AMD pathogenesis.

Duplex Interrogation by a Direct DNA Repair Protein in Search of Base Damage

Science.gov (United States)

Yi, Chengqi; Chen, Baoen; Qi, Bo; Zhang, Wen; Jia, Guifang; Zhang, Liang; Li, Charles J.; Dinner, Aaron R.; Yang, Cai-Guang; He, Chuan

2012-01-01

ALKBH2 is a direct DNA repair dioxygenase guarding mammalian genome against N1-methyladenine, N3-methylcytosine, and 1,N6-ethenoadenine damage. A prerequisite for repair is to identify these lesions in the genome. Here we present crystal structures of ALKBH2 bound to different duplex DNAs. Together with computational and biochemical analyses, our results suggest that DNA interrogation by ALKBH2 displays two novel features: i) ALKBH2 probes base-pair stability and detects base pairs with reduced stability; ii) ALKBH2 does not have nor need a “damage-checking site”, which is critical for preventing spurious base-cleavage for several glycosylases. The demethylation mechanism of ALKBH2 insures that only cognate lesions are oxidized and reversed to normal bases, and that a flipped, non-substrate base remains intact in the active site. Overall, the combination of duplex interrogation and oxidation chemistry allows ALKBH2 to detect and process diverse lesions efficiently and correctly. PMID:22659876

Does aerobic exercises induce mtDNA mutation in human blood ...

African Journals Online (AJOL)

The aim of this study was to determine the effect of eight weeks aerobic training on mitochondrial DNA (mtDNA) mutation in human blood leucocytes. Twenty untrained healthy students (training group: n =10, age = 20.7±1.5 yrs, weight = 67.7±10 kg, BF% = 17.5±7.35 & control group: n =10, age = 21±1.3 yrs, weight ...

Ada response - a strategy for repair of alkylated DNA in bacteria.

Science.gov (United States)

Mielecki, Damian; Grzesiuk, Elżbieta

2014-06-01

Alkylating agents are widespread in the environment and also occur endogenously. They can be cytotoxic or mutagenic to the cells introducing alkylated bases to DNA or RNA. All organisms have evolved multiple DNA repair mechanisms to counteract the effects of DNA alkylation: the most cytotoxic lesion, N(3)-methyladenine (3meA), is excised by AlkA glycosylase initiating base excision repair (BER); toxic N(1)-methyladenine (1meA) and N(3)-methylcytosine (3meC), induced in DNA and RNA, are removed by AlkB dioxygenase; and mutagenic and cytotoxic O(6)-methylguanine (O(6) meG) is repaired by Ada methyltransferase. In Escherichia coli, Ada response involves the expression of four genes, ada, alkA, alkB, and aidB, encoding respective proteins Ada, AlkA, AlkB, and AidB. The Ada response is conserved among many bacterial species; however, it can be organized differently, with diverse substrate specificity of the particular proteins. Here, an overview of the organization of the Ada regulon and function of individual proteins is presented. We put special effort into the characterization of AlkB dioxygenases, their substrate specificity, and function in the repair of alkylation lesions in DNA/RNA. © 2014 The Authors. FEMS Microbiology Letters published by John Wiley & Sons Ltd on behalf of Federation of European Microbiological Societies.

New insights in the removal of the hydantoins, oxidation product of pyrimidines, via the base excision and nucleotide incision repair pathways.

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Modesto Redrejo-Rodríguez

Full Text Available BACKGROUND: Oxidative damage to DNA, if not repaired, can be both miscoding and blocking. These genetic alterations can lead to mutations and/or cell death, which in turn cause cancer and aging. Oxidized DNA bases are substrates for two overlapping repair pathways: base excision (BER and nucleotide incision repair (NIR. Hydantoin derivatives such as 5-hydroxyhydantoin (5OH-Hyd and 5-methyl-5-hydroxyhydantoin (5OH-5Me-Hyd, major products of cytosine and thymine oxidative degradation pathways, respectively, have been detected in cancer cells and ancient DNA. Hydantoins are blocking lesions for DNA polymerases and excised by bacterial and yeast DNA glycosylases in the BER pathway. However little is known about repair of pyrimidine-derived hydantoins in human cells. METHODOLOGY/PRINCIPAL FINDINGS: Here, using both denaturing PAGE and MALDI-TOF MS analyses we report that the bacterial, yeast and human AP endonucleases can incise duplex DNA 5' next to 5OH-Hyd and 5OH-5Me-Hyd thus initiating the NIR pathway. We have fully reconstituted the NIR pathway for these lesions in vitro using purified human proteins. Depletion of Nfo in E. coli and APE1 in HeLa cells abolishes the NIR activity in cell-free extracts. Importantly, a number of redundant DNA glycosylase activities can excise hydantoin residues, including human NTH1, NEIL1 and NEIL2 and the former protein being a major DNA glycosylase activity in HeLa cells extracts. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that both BER and NIR pathways can compete and/or back-up each other to remove hydantoin DNA lesions in vivo.

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

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Kin Chan

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

N-Butyrate alters chromatin accessibility to DNA repair enzymes

International Nuclear Information System (INIS)

Smith, P.J.

1986-01-01

Current evidence suggests that the complex nature of mammalian chromatin can result in the concealment of DNA damage from repair enzymes and their co-factors. Recently it has been proposed that the acetylation of histone proteins in chromatin may provide a surveillance system whereby damaged regions of DNA become exposed due to changes in chromatin accessibility. This hypothesis has been tested by: (i) using n-butyrate to induce hyperacetylation in human adenocarcinoma (HT29) cells; (ii) monitoring the enzymatic accessibility of chromatin in permeabilised cells; (iii) measuring u.v. repair-associated nicking of DNA in intact cells and (iv) determining the effects of n-butyrate on cellular sensitivity to DNA damaging agents. The results indicate that the accessibility of chromatin to Micrococcus luteus u.v. endonuclease is enhanced by greater than 2-fold in n-butyrate-treated cells and that there is a corresponding increase in u.v. repair incision rates in intact cells exposed to the drug. Non-toxic levels of n-butyrate induce a block to G1 phase transit and there is a significant growth delay on removal of the drug. Resistance of HT29 cells to u.v.-radiation and adriamycin is enhanced in n-butyrate-treated cells whereas X-ray sensitivity is increased. Although changes in the responses of cells to DNA damaging agents must be considered in relation to the effects of n-butyrate on growth rate and cell-cycle distribution, the results are not inconsistent with the proposal that increased enzymatic-accessibility/repair is biologically favourable for the resistance of cells to u.v.-radiation damage. Overall the results support the suggested operation of a histone acetylation-based chromatin surveillance system in human cells

Human Genome Research: Decoding DNA

Science.gov (United States)

dropdown arrow Site Map A-Z Index Menu Synopsis Human Genome Research: Decoding DNA Resources with of the DNA double helix during April 2003. James D. Watson, Francis Crick, and Maurice Wilkins were company Celera announced the completion of a "working draft" reference DNA sequence of the human

Modular Nuclease-Responsive DNA Three-Way Junction-Based Dynamic Assembly of a DNA Device and Its Sensing Application.

Science.gov (United States)

Zhu, Jing; Wang, Lei; Xu, Xiaowen; Wei, Haiping; Jiang, Wei

2016-04-05

Here, we explored a modular strategy for rational design of nuclease-responsive three-way junctions (TWJs) and fabricated a dynamic DNA device in a "plug-and-play" fashion. First, inactivated TWJs were designed, which contained three functional domains: the inaccessible toehold and branch migration domains, the specific sites of nucleases, and the auxiliary complementary sequence. The actions of different nucleases on their specific sites in TWJs caused the close proximity of the same toehold and branch migration domains, resulting in the activation of the TWJs and the formation of a universal trigger for the subsequent dynamic assembly. Second, two hairpins (H1 and H2) were introduced, which could coexist in a metastable state, initially to act as the components for the dynamic assembly. Once the trigger initiated the opening of H1 via TWJs-driven strand displacement, the cascade hybridization of hairpins immediately switched on, resulting in the formation of the concatemers of H1/H2 complex appending numerous integrated G-quadruplexes, which were used to obtain label-free signal readout. The inherent modularity of this design allowed us to fabricate a flexible DNA dynamic device and detect multiple nucleases through altering the recognition pattern slightly. Taking uracil-DNA glycosylase and CpG methyltransferase M.SssI as models, we successfully realized the butt joint between the uracil-DNA glycosylase and M.SssI recognition events and the dynamic assembly process. Furthermore, we achieved ultrasensitive assay of nuclease activity and the inhibitor screening. The DNA device proposed here will offer an adaptive and flexible tool for clinical diagnosis and anticancer drug discovery.

Biochemical characterization and novel inhibitor identification of Mycobacterium tuberculosis Endonuclease VIII 2 (Rv3297

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Kiran Lata

2017-12-01

Full Text Available Nei2 (Rv3297 is a DNA Base Excision Repair (BER glycosylase that is essential for survival of Mycobacterium tuberculosis in primates. We show that MtbNei2 is a bifunctional glycosylase that specifically acts on oxidized pyrimidine-containing single-stranded, double-stranded, 5’/3’ fork and bubble DNA substrates. MtbNei2 possesses Uracil DNA glycosylase activity unlike E. coli Nei. Mutational studies demonstrate that Pro2 and Glu3 located in the active site are essential for glycosylase activity of MtbNei2. Mutational analysis demonstrated that an unstructured C-terminal zinc finger domain that was important for activity in E. coli Nei and Fpg, was not required for the glycosylase activity of MtbNei2. Lastly, we screened the NCI natural product compound database and identified three natural product inhibitors with IC50 values ranging between 41.8 μM-92.7 μM against MtbNei2 in in vitro inhibition assays. Surface Plasmon Resonance (SPR experiments showed that the binding affinity of the best inhibitor, NSC31867, was 74 nM. The present results set the stage for exploiting this important target in developing new therapeutic strategies that target Mycobacterial BER.

Cloning, sequencing, and expression of cDNA for human β-glucuronidase

International Nuclear Information System (INIS)

Oshima, A.; Kyle, J.W.; Miller, R.D.

1987-01-01

The authors report here the cDNA sequence for human placental β-glucuronidase (β-D-glucuronoside glucuronosohydrolase, EC 3.2.1.31) and demonstrate expression of the human enzyme in transfected COS cells. They also sequenced a partial cDNA clone from human fibroblasts that contained a 153-base-pair deletion within the coding sequence and found a second type of cDNA clone from placenta that contained the same deletion. Nuclease S1 mapping studies demonstrated two types of mRNAs in human placenta that corresponded to the two types of cDNA clones isolated. The NH 2 -terminal amino acid sequence determined for human spleen β-glucuronidase agreed with that inferred from the DNA sequence of the two placental clones, beginning at amino acid 23, suggesting a cleaved signal sequence of 22 amino acids. When transfected into COS cells, plasmids containing either placental clone expressed an immunoprecipitable protein that contained N-linked oligosaccharides as evidenced by sensitivity to endoglycosidase F. However, only transfection with the clone containing the 153-base-pair segment led to expression of human β-glucuronidase activity. These studies provide the sequence for the full-length cDNA for human β-glucuronidase, demonstrate the existence of two populations of mRNA for β-glucuronidase in human placenta, only one of which specifies a catalytically active enzyme, and illustrate the importance of expression studies in verifying that a cDNA is functionally full-length

DNA methylation in human fibroblasts following DNA damage and repair

International Nuclear Information System (INIS)

Kastan, M.B.

1984-01-01

Methylation of deoxycytidine (dCyd) incorporated by DNA excision repair synthesis in human diploid fibroblasts following damage with ultraviolet radiation (UV), N-methyl-N-nitrosourea, or N-acetoxy-2-acetylaminofluorene was studied utilizing [6- 3 H]dCyd to label repaired DNA specifically and high performance liquid chromatographic analysis to quantify the percentage of deoxycytidine converted to 5-methyldeoxycytidine (m 5 dCyd). In confluent, nondividing cells, methylation in repair patches induced by all three agents is slow and incomplete. Whereas after DNA replication a level of 3.4% m 5 dCyd is reached in less than 2 hours, following UV-stimulated repair synthesis in confluent cells it takes about 3 days to reach a level of approx.2.0% m 5 dCyd in the repair patch. This undermethylation of repair patches occurs throughout the genome. In cells from cultures in logarithmic-phase growth, m 5 dCyd formation in UV-induced repair patches occurs faster and to a greater extent, reaching a level of approx.2.7% in 10-20 hours. Pre-existing hypomethylated repair patches in confluent cells are methylated further when the cells are stimulated to divide; however, the repair patch may still not be fully methylated before cell division occurs. Thus DNA damage and repair may lead to heritable loss of methylation at some sites. The distribution within chromatin of m 5 dCyd in repair patches was also investigated. Over a wide range of extents of digestion by staphylococcal nuclease or deoxyribonuclease I, the level of hypomethylation in repaired DNA in nuclease sensitive and resistant regions of chromatin was constant relative to the genomic level of methylation in these regions. Similar conclusions were reached in experiments with isolated mononucleosomes

Locating the uracil-5-yl radical formed upon photoirradiation of 5-bromouracil-substituted DNA

Science.gov (United States)

Hashiya, Fumitaka; Saha, Abhijit; Kizaki, Seiichiro; Li, Yue; Sugiyama, Hiroshi

2014-01-01

In a previous study, we found that 2-deoxyribonolactone is effectively generated in the specific 5-bromouracil (BrU)-substituted sequence 5′-(G/C)[A]n = 1,2BrUBrU-3′ and proposed that a formed uracil-5-yl radical mainly abstracts the C1′ hydrogen from the 5′-side of BrUBrU under 302-nm irradiation condition. In the present work, we performed photoirradiation of BrU-substituted DNA in the presence of a hydrogen donor, tetrahydrofuran, to quench the uracil-5-yl radical to uracil and then subjected the sample to uracil DNA glycosylase digestion. Slab gel sequence analysis indicated that uracil residues were formed at the hot-spot sequence of 5′-(G/C)[A]n = 1,2BrUBrU-3′ in 302-nm irradiation of BrU-substituted DNA. Furthermore, we found that the uracil residue was also formed at the reverse sequence 5′-BrUBrU[A]n = 1,2(G/C)-3′, which suggests that both 5′-(G/C)[A]n = 1,2BrUBrU-3′ and 5′-BrUBrU[A]n = 1,2(G/C)-3′ are hot-spot sequences for the formation of the uracil-5-yl radical. PMID:25398904

Repair of oxidative DNA base damage in the host genome influences the HIV integration site sequence preference.

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Geoffrey R Bennett

Full Text Available Host base excision repair (BER proteins that repair oxidative damage enhance HIV infection. These proteins include the oxidative DNA damage glycosylases 8-oxo-guanine DNA glycosylase (OGG1 and mutY homolog (MYH as well as DNA polymerase beta (Polβ. While deletion of oxidative BER genes leads to decreased HIV infection and integration efficiency, the mechanism remains unknown. One hypothesis is that BER proteins repair the DNA gapped integration intermediate. An alternative hypothesis considers that the most common oxidative DNA base damages occur on guanines. The subtle consensus sequence preference at HIV integration sites includes multiple G:C base pairs surrounding the points of joining. These observations suggest a role for oxidative BER during integration targeting at the nucleotide level. We examined the hypothesis that BER repairs a gapped integration intermediate by measuring HIV infection efficiency in Polβ null cell lines complemented with active site point mutants of Polβ. A DNA synthesis defective mutant, but not a 5'dRP lyase mutant, rescued HIV infection efficiency to wild type levels; this suggested Polβ DNA synthesis activity is not necessary while 5'dRP lyase activity is required for efficient HIV infection. An alternate hypothesis that BER events in the host genome influence HIV integration site selection was examined by sequencing integration sites in OGG1 and MYH null cells. In the absence of these 8-oxo-guanine specific glycosylases the chromatin elements of HIV integration site selection remain the same as in wild type cells. However, the HIV integration site sequence preference at G:C base pairs is altered at several positions in OGG1 and MYH null cells. Inefficient HIV infection in the absence of oxidative BER proteins does not appear related to repair of the gapped integration intermediate; instead oxidative damage repair may participate in HIV integration site preference at the sequence level.

The future of human DNA vaccines.

Science.gov (United States)

Li, Lei; Saade, Fadi; Petrovsky, Nikolai

2012-12-31

DNA vaccines have evolved greatly over the last 20 years since their invention, but have yet to become a competitive alternative to conventional protein or carbohydrate based human vaccines. Whilst safety concerns were an initial barrier, the Achilles heel of DNA vaccines remains their poor immunogenicity when compared to protein vaccines. A wide variety of strategies have been developed to optimize DNA vaccine immunogenicity, including codon optimization, genetic adjuvants, electroporation and sophisticated prime-boost regimens, with each of these methods having its advantages and limitations. Whilst each of these methods has contributed to incremental improvements in DNA vaccine efficacy, more is still needed if human DNA vaccines are to succeed commercially. This review foresees a final breakthrough in human DNA vaccines will come from application of the latest cutting-edge technologies, including "epigenetics" and "omics" approaches, alongside traditional techniques to improve immunogenicity such as adjuvants and electroporation, thereby overcoming the current limitations of DNA vaccines in humans. Copyright © 2012 Elsevier B.V. All rights reserved.

Human mitochondrial DNA (mtDNA) types in Malaysia

International Nuclear Information System (INIS)

Lian, L.H.; Koh, C.L.; Lim, M.E.

2000-01-01

Each human cell contains hundreds of mitochondria and thousands of double-stranded circular mtDNA. The delineation of human mtDNA variation and genetics over the past decade has provided unique and often startling insights into human evolution, degenerative diseases, and aging. Each mtDNA of 16,569 base pairs, encodes 13 polypeptides essential to the enzymes of the mitochondrial energy generating pathway, plus the necessary tRNAs and rRNAs. The highly polymorphic noncoding D-(displacement) loop region, also called the control region, is approximately 1.2 kb long. It contains two well-characterized hypervariable (HV-) regions, HV1 and HV2. MtDNA identification is usually based on these sequence differences. According to the TWTGDAM (Technical Working Group for DNA Analysis Methods), the minimum requirement for a mtDNA database for HV1 is from positions 16024 to 16365 and for HV2, from positions 00073 to 00340. The targeted Malaysian population subgroups for this study were mainly the Malays, Chinese, Indians, and indigenous Ibans, Bidayuhs, Kadazan-Dusuns, and Bajaus. Research methodologies undertaken included DNA extraction of samples from unrelated individuals, amplification of the specific regions via the polymerase chain reaction (PCR), and preparation of template DNA for sequencing by using an automated DNA sequencer. Sufficient nucleotide sequence data were generated from the mtDNA analysis. When the sequences were analyzed, sequence variations were found to be caused by nucleotide substitutions, insertions, and deletions. Of the three causes of the sequence variations, nucleotide substitutions (86.1%) accounted for the vast majority of polymorphism. It is noted that transitions (83.5%) were predominant when compared to the significantly lower frequencies of transversions (2.6%). Insertions (0.9%) and deletions (13.0%) were rather rare and found only in HV2. The data generated will also form the basis of a Malaysian DNA sequence database of mtDNA D

Critical role of DNA intercalation in enzyme-catalyzed nucleotide flipping

Science.gov (United States)

Hendershot, Jenna M.; O'Brien, Patrick J.

2014-01-01

Nucleotide flipping is a common feature of DNA-modifying enzymes that allows access to target sites within duplex DNA. Structural studies have identified many intercalating amino acid side chains in a wide variety of enzymes, but the functional contribution of these intercalating residues is poorly understood. We used site-directed mutagenesis and transient kinetic approaches to dissect the energetic contribution of intercalation for human alkyladenine DNA glycosylase, an enzyme that initiates repair of alkylation damage. When AAG flips out a damaged nucleotide, the void in the duplex is filled by a conserved tyrosine (Y162). We find that tyrosine intercalation confers 140-fold stabilization of the extrahelical specific recognition complex, and that Y162 functions as a plug to slow the rate of unflipping by 6000-fold relative to the Y162A mutant. Surprisingly, mutation to the smaller alanine side chain increases the rate of nucleotide flipping by 50-fold relative to the wild-type enzyme. This provides evidence against the popular model that DNA intercalation accelerates nucleotide flipping. In the case of AAG, DNA intercalation contributes to the specific binding of a damaged nucleotide, but this enhanced specificity comes at the cost of reduced speed of nucleotide flipping. PMID:25324304

Nucleotide sequence of a cDNA coding for the amino-terminal region of human prepro. alpha. 1(III) collagen

Energy Technology Data Exchange (ETDEWEB)

Toman, P D; Ricca, G A [Rorer Biotechnology, Inc., Springfield, VA (USA); de Crombrugghe, B [National Institutes of Health, Bethesda, MD (USA)

1988-07-25

Type III Collagen is synthesized in a variety of tissues as a precursor macromolecule containing a leader sequence, a N-propeptide, a N-telopeptide, the triple helical region, a C-telopeptide, and C-propeptide. To further characterize the human type III collagen precursor, a human placental cDNA library was constructed in gt11 using an oligonucleotide derived from a partial cDNA sequence corresponding to the carboxy-terminal part of the 1(III) collagen. A cDNA was identified which contains the leader sequence, the N-propeptide and N-telopeptide regions. The DNA sequence of these regions are presented here. The triple helical, C-telopeptide and C-propeptide amino acid sequence for human type III collagen has been determined previously. A comparison of the human amino acid sequence with mouse, chicken, and calf sequence shows 81%, 81%, and 92% similarity, respectively. At the DNA level, the sequence similarity between human and mouse or chicken type III collagen sequences in this area is 82% and 77%, respectively.

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...

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

Science.gov (United States)

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

1999-01-01

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

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

Directory of Open Access Journals (Sweden)

Natalie R Gassman

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

Genotoxic potential of the perfluorinated chemicals PFOA, PFOS, PFBS, PFNA and PFHxA in human HepG2 cells

DEFF Research Database (Denmark)

Eriksen, Kirsten Thorup; Raaschou-Nielsen, Ole; Sørensen, Mette

2010-01-01

, the increase in ROS production was not concentration-dependent and the compounds did not generate DNA damage that could be detected by the alkaline comet assay as strand breakage and alkali-labile sites or formamidopyrimidine-DNA-glycosylase (FPG) sites. Perfluorobutane sulfonate (PFBS) and perfluorohexanoic...

The role of DNA polymerase ζ in translesion synthesis across bulky DNA adducts and cross-links in human cells

Energy Technology Data Exchange (ETDEWEB)

Suzuki, Tetsuya, E-mail: suzukite@hiroshima-u.ac.jp [Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501 (Japan); Grúz, Petr; Honma, Masamitsu [Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501 (Japan); Adachi, Noritaka [Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027 (Japan); Nohmi, Takehiko [Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501 (Japan)

2016-09-15

Highlights: • Human cells knockout (KO) and expressing catalytically dead (CD) variant of DNA polymerase ζ (Pol ζ) have been established by gene targeting techniques with Nalm-6 cells. • Both Pol ζ KO and CD cells displayed prolonged cell cycle and higher incidence of micronucleus formation than the wild-type cells in the absence of exogenous genotoxic treatments. • Pol ζ protects human cells from genotoxic stresses that induce bulky DNA lesions and cross-links. • Pol ζ plays quite limited roles in protection against strand-breaks in DNA. - Abstract: Translesion DNA synthesis (TLS) is a cellular defense mechanism against genotoxins. Defects or mutations in specialized DNA polymerases (Pols) involved in TLS are believed to result in hypersensitivity to various genotoxic stresses. Here, DNA polymerase ζ (Pol ζ)-deficient (KO: knockout) and Pol ζ catalytically dead (CD) human cells were established and their sensitivity towards cytotoxic activities of various genotoxins was examined. The CD cells were engineered by altering the DNA sequence encoding two amino acids essential for the catalytic activity of Pol ζ, i.e., D2781 and D2783, to alanines. Both Pol ζ KO and CD cells displayed a prolonged cell cycle and higher incidence of micronuclei formation than the wild-type (WT) cells in the absence of exogenous genotoxic treatments, and the order of abnormality was CD > KO > WT cells. Both KO and CD cells exhibited higher sensitivity towards the killing effects of benzo[a]pyrene diol epoxide, mitomycin C, potassium bromate, N-methyl-N′-nitro-N-nitrosoguanidine, and ultraviolet C irradiation than WT cells, and there were no differences between the sensitivities of KO and CD cells. Interestingly, neither KO nor CD cells were sensitive to the cytotoxic effects of hydrogen peroxide. Since KO and CD cells displayed similar sensitivities to the genotoxins, we employed only KO cells to further examine their sensitivity to other genotoxic agents. KO cells were

The role of DNA polymerase ζ in translesion synthesis across bulky DNA adducts and cross-links in human cells

International Nuclear Information System (INIS)

Suzuki, Tetsuya; Grúz, Petr; Honma, Masamitsu; Adachi, Noritaka; Nohmi, Takehiko

2016-01-01

Highlights: • Human cells knockout (KO) and expressing catalytically dead (CD) variant of DNA polymerase ζ (Pol ζ) have been established by gene targeting techniques with Nalm-6 cells. • Both Pol ζ KO and CD cells displayed prolonged cell cycle and higher incidence of micronucleus formation than the wild-type cells in the absence of exogenous genotoxic treatments. • Pol ζ protects human cells from genotoxic stresses that induce bulky DNA lesions and cross-links. • Pol ζ plays quite limited roles in protection against strand-breaks in DNA. - Abstract: Translesion DNA synthesis (TLS) is a cellular defense mechanism against genotoxins. Defects or mutations in specialized DNA polymerases (Pols) involved in TLS are believed to result in hypersensitivity to various genotoxic stresses. Here, DNA polymerase ζ (Pol ζ)-deficient (KO: knockout) and Pol ζ catalytically dead (CD) human cells were established and their sensitivity towards cytotoxic activities of various genotoxins was examined. The CD cells were engineered by altering the DNA sequence encoding two amino acids essential for the catalytic activity of Pol ζ, i.e., D2781 and D2783, to alanines. Both Pol ζ KO and CD cells displayed a prolonged cell cycle and higher incidence of micronuclei formation than the wild-type (WT) cells in the absence of exogenous genotoxic treatments, and the order of abnormality was CD > KO > WT cells. Both KO and CD cells exhibited higher sensitivity towards the killing effects of benzo[a]pyrene diol epoxide, mitomycin C, potassium bromate, N-methyl-N′-nitro-N-nitrosoguanidine, and ultraviolet C irradiation than WT cells, and there were no differences between the sensitivities of KO and CD cells. Interestingly, neither KO nor CD cells were sensitive to the cytotoxic effects of hydrogen peroxide. Since KO and CD cells displayed similar sensitivities to the genotoxins, we employed only KO cells to further examine their sensitivity to other genotoxic agents. KO cells were

Cockayne Syndrome group B protein stimulates NEIL2 DNA glycosylase activity

DEFF Research Database (Denmark)

Aamann, Maria Diget; Hvitby, Christina Poulsen; Popuri, Venkateswarlu

2014-01-01

Cockayne Syndrome is a segmental premature aging syndrome, which can be caused by loss of function of the CSB protein. CSB is essential for genome maintenance and has numerous interaction partners with established roles in different DNA repair pathways including transcription coupled nucleotide...... activity on a 5-hydroxyl uracil lesion in a DNA bubble structure substrate in vitro. A novel 4,6-diamino-5-formamidopyrimidine (FapyA) specific incision activity of NEIL2 was also stimulated by CSB. To further elucidate the biological role of the interaction, immunofluorescence studies were performed...

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.

Pyrrolo-dC modified duplex DNA as a novel probe for the sensitive assay of base excision repair enzyme activity.

Science.gov (United States)

Lee, Chang Yeol; Park, Ki Soo; Park, Hyun Gyu

2017-12-15

We develop a novel approach to determine formamidopyrimidine DNA glycosylase (Fpg) activity by taking advantage of the unique fluorescence property of pyrrolo-dC (PdC) positioned opposite to 8-oxoguanine (8-oxoG) in duplex DNA. In its initial state, PdC in duplex DNA undergoes the efficient stacking and collisional quenching interactions, showing the low fluorescence signal. In contrast, the presence of Fpg, which specifically removes 8-oxoG and incises resulting apurinic (AP) site, transforms duplex DNA into single-stranded (ss) DNAs. As a result, the intrinsic fluorescence signal of PdC in ssDNA is recovered to exhibit the significantly enhanced fluorescence signal. Based on this Fpg-dependent fluorescence response of PdC, we could reliably determine Fpg activity down to 1.25U/ml with a linear response from 0 to 50U/ml. In addition, the diagnostic capability of this strategy was successfully demonstrated by reliably assaying Fpg activity in human blood serum, showing its great potential in the practical applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of DNA sequence, ionic strength, and cationic DNA affinity binders on the methylation of DNA by N-methyl-N-nitrosourea

International Nuclear Information System (INIS)

Wurdeman, R.L.; Gold, B.

1988-01-01

DNA alkylation by N-alkyl-N-nitrosoureas is generally accepted to be responsible for their mutagenic, carcinogenic, and antineoplastic activities. The exact nature of the ultimate alkylating intermediate is still controversial, with a variety of species having been nominated. The sequence specificity for DNA alkylation by simple N-alkyl-N-nitrosoureas has not been reported, although such information is basic in understanding the specific point mutations induced by these compounds in oncogene targets. These two points are addressed by using N-methyl-N-nitrosourea methylation of a 576 base-pair 32 P-end-labeled DNA restriction fragment and high-resolution polyacrylamide sequencing gels. This method provides information on the formation of N 7 -methylguanine, by the generation of single-strand breaks upon exposure to piperidine

Inter-laboratory variation in DNA damage using a standard comet assay protocol

DEFF Research Database (Denmark)

Forchhammer, Lykke; Ersson, Clara; Loft, Steffen

2012-01-01

determined the baseline level of DNA strand breaks (SBs)/alkaline labile sites and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites in coded samples of mononuclear blood cells (MNBCs) from healthy volunteers. There were technical problems in seven laboratories in adopting the standard protocol...... analysed by the standard protocol. The SBs and FPG-sensitive sites were measured in the same experiment, indicating that the large spread in the latter lesions was the main reason for the reduced inter-laboratory variation. However, it remains worrying that half of the participating laboratories obtained...

Human diseases associated with defective DNA repair

International Nuclear Information System (INIS)

Friedberg, E.C.; Ehmann, U.K.; Williams, J.I.

1979-01-01

The observations on xeroderma pigmentosum (XP) cells in culture were the first indications of defective DNA repair in association with human disease. Since then, a wealth of information on DNA repair in XP, and to a lesser extent in other diseases, has accumulated in the literature. Rather than clarifying the understanding of DNA repair mechanisms in normal cells and of defective DNA repair in human disease, the literature suggests an extraordinary complexity of both of the phenomena. In this review a number of discrete human diseases are considered separately. An attempt was made to systematically describe the pertinent clinical features and cellular and biochemical defects in these diseases, with an emphasis on defects in DNA metabolism, particularly DNA repair. Wherever possible observations have been correlated and unifying hypotheses presented concerning the nature of the basic defect(s) in these diseases. Discussions of the following diseases are presented: XP, ataxia telangiectasia; Fanconi's anemia; Hutchinson-Gilford progeria syndrome; Bloom's syndrome, Cockayne's syndrome; Down's syndrome; retinoblastoma; chronic lymphocytic leukemia; and other miscellaneous human diseases with possble DNA repair defects

7-N-Acetylcysteine-pyrrole conjugate-A potent DNA reactive metabolite of pyrrolizidine alkaloids.

Science.gov (United States)

He, Xiaobo; Ma, Liang; Xia, Qingsu; Fu, Peter P

2016-10-01

Plants containing pyrrolizidine alkaloids (PAs) are widespread throughout the world and are the most common poisonous plants affecting livestock, wildlife, and humans. PAs require metabolic activation to form reactive dehydropyrrolizidine alkaloids (dehydro-PAs) that are capable of alkylating cellular DNA and proteins, form (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-DNA and DHP-protein adducts, and lead to cytotoxicity, genotoxicity, and tumorigenicity. In this study, we determined that the metabolism of riddelliine and monocrotaline by human and rat liver microsomes in the presence of N-acetylcysteine both produced 7-N-acetylcysteine-DHP (7-NAC-DHP) and DHP. Reactions of 7-NAC-DHP with 2'-deoxyguanosine (dG), 2'-deoxyadenosine (dA), and calf thymus DNA in aqueous solution followed by enzymatic hydrolysis yielded DHP-dG and/or DHP-dA adducts. These results indicate that 7-NAC-DHP is a reactive metabolite that can lead to DNA adduct formation. Copyright © 2016. Published by Elsevier B.V.

7-N-Acetylcysteine-pyrrole conjugate—A potent DNA reactive metabolite of pyrrolizidine alkaloids

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Xiaobo He

2016-10-01

Full Text Available Plants containing pyrrolizidine alkaloids (PAs are widespread throughout the world and are the most common poisonous plants affecting livestock, wildlife, and humans. PAs require metabolic activation to form reactive dehydropyrrolizidine alkaloids (dehydro-PAs that are capable of alkylating cellular DNA and proteins, form (±-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP-DNA and DHP-protein adducts, and lead to cytotoxicity, genotoxicity, and tumorigenicity. In this study, we determined that the metabolism of riddelliine and monocrotaline by human and rat liver microsomes in the presence of N-acetylcysteine both produced 7-N-acetylcysteine-DHP (7-NAC-DHP and DHP. Reactions of 7-NAC-DHP with 2′-deoxyguanosine (dG, 2′-deoxyadenosine (dA, and calf thymus DNA in aqueous solution followed by enzymatic hydrolysis yielded DHP-dG and/or DHP-dA adducts. These results indicate that 7-NAC-DHP is a reactive metabolite that can lead to DNA adduct formation.

TET-mediated oxidation of methylcytosine causes TDG or NEIL glycosylase dependent gene reactivation.

Science.gov (United States)

Müller, Udo; Bauer, Christina; Siegl, Michael; Rottach, Andrea; Leonhardt, Heinrich

2014-07-01

The discovery of hydroxymethyl-, formyl- and carboxylcytosine, generated through oxidation of methylcytosine by TET dioxygenases, raised the question how these modifications contribute to epigenetic regulation. As they are subjected to complex regulation in vivo, we dissected links to gene expression with in vitro modified reporter constructs. We used an Oct4 promoter-driven reporter gene and demonstrated that in vitro methylation causes gene silencing while subsequent oxidation with purified catalytic domain of TET1 leads to gene reactivation. To identify proteins involved in this pathway we screened for TET interacting factors and identified TDG, PARP1, XRCC1 and LIG3 that are involved in base-excision repair. Knockout and rescue experiments demonstrated that gene reactivation depended on the glycosylase TDG, but not MBD4, while NEIL1, 2 and 3 could partially rescue the loss of TDG. These results clearly show that oxidation of methylcytosine by TET dioxygenases and subsequent removal by TDG or NEIL glycosylases and the BER pathway results in reactivation of epigenetically silenced genes. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Nucleotide sequence preservation of human mitochondrial DNA

International Nuclear Information System (INIS)

Monnat, R.J. Jr.; Loeb, L.A.

1985-01-01

Recombinant DNA techniques have been used to quantitate the amount of nucleotide sequence divergence in the mitochondrial DNA population of individual normal humans. Mitochondrial DNA was isolated from the peripheral blood lymphocytes of five normal humans and cloned in M13 mp11; 49 kilobases of nucleotide sequence information was obtained from 248 independently isolated clones from the five normal donors. Both between- and within-individual differences were identified. Between-individual differences were identified in approximately = to 1/200 nucleotides. In contrast, only one within-individual difference was identified in 49 kilobases of nucleotide sequence information. This high degree of mitochondrial nucleotide sequence homogeneity in human somatic cells is in marked contrast to the rapid evolutionary divergence of human mitochondrial DNA and suggests the existence of mechanisms for the concerted preservation of mammalian mitochondrial DNA sequences in single organisms

Comparison of alkylating agent and radiation carcinogenesis: some aspects of the possible involvement of effects on DNA

International Nuclear Information System (INIS)

Lawley, P.D.

1976-01-01

A series of alkylating agents was classified in terms of increasing relative ability to react at O-atom sites in DNA, MMS 6 -alkylguanines are directly miscoding bases. This series of agents was also used in a study of comparative carcinogenicity with respect to induction of thymic lymphoma in mice, a system in which x-irradiation yields tumors. A positive correlation between ability of agents to alkylate O-6 of guanine in DNA of thymus and carcinogenic potency was found. Although MMS was not active in this system, it was noted that it can induce tumors in other systems. The relationship between repair of alkylation and radiation induced damage in DNA was briefly discussed. The methylating agents induce single-strand breaks in DNA, and the principal repair system appears to fall into the category of short repair as denoted by Regan and Setlow (1974). These single-strand breaks may result from spontaneous hydrolytic depurinations of 3- and 7-methylpurines, or from enzymatic depurinations, e.g., of 3-alkyladenine and O 6 -alkylguanine. Aralkylating agents, which are also carcinogens, can evoke an alternative repair response of the uvr type

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

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Dizdaroglu, Miral; Kirkali, Güldal; Jaruga, Pawel

2008-12-15

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

Measuring oxidative damage to DNA and its repair with the comet assay.

Science.gov (United States)

Collins, Andrew R

2014-02-01

Single cell gel electrophoresis, or the comet assay, was devised as a sensitive method for detecting DNA strand breaks, at the level of individual cells. A simple modification, incorporating a digestion of DNA with a lesion-specific endonuclease, makes it possible to measure oxidised bases. With the inclusion of formamidopyrimidine DNA glycosylase to recognise oxidised purines, or Nth (endonuclease III) to detect oxidised pyrimidines, the comet assay has been used extensively in human biomonitoring to monitor oxidative stress, usually in peripheral blood mononuclear cells. There is evidence to suggest that the enzymic approach is more accurate than chromatographic methods, when applied to low background levels of base oxidation. However, there are potential problems of over-estimation (because the enzymes are not completely specific) or under-estimation (failure to detect lesions that are close together). Attempts have been made to improve the inter-laboratory reproducibility of the comet assay. In addition to measuring DNA damage, the assay can be used to monitor the cellular or in vitro repair of strand breaks or oxidised bases. It also has applications in assessing the antioxidant status of cells. In its various forms, the comet assay is now an invaluable tool in human biomonitoring and genotoxicity testing. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn. Copyright © 2013 Elsevier B.V. All rights reserved.

MUTYH mediates the toxicity of combined DNA 6-thioguanine and UVA radiation

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De Luca, Gabriele; Leopardi, Paola; Mancuso, Maria Teresa; Casorelli, Ida; Pichierri, Pietro; Karran, Peter; Bignami, Margherita

2015-01-01

The therapeutic thiopurines, including the immunosuppressant azathioprine (Aza) cause the accumulation of the UVA photosensitizer 6-thioguanine (6-TG) in the DNA of the patients' cells. DNA 6-TG and UVA are synergistically cytotoxic and their interaction causes oxidative damage. The MUTYH DNA glycosylase participates in the base excision repair of oxidized DNA bases. Using Mutyh-nullmouse fibroblasts (MEFs) we examined whether MUTYH provides protection against the lethal effects of combined DNA 6-TG/UVA. Surprisingly, Mutyh-null MEFs were more resistant than wild-type MEFs, despite accumulating higher levels of DNA 8-oxo-7,8-dihydroguanine (8-oxoG). Their enhanced 6-TG/UVA resistance reflected the absence of the MUTYH protein and MEFs expressing enzymatically-dead human variants were as sensitive as wild-type cells. Consistent with their enhanced resistance, Mutyh-null cells sustained fewer DNA strand breaks and lower levels of chromosomal damage after 6-TG/UVA. Although 6-TG/UVA treatment caused early checkpoint activation irrespective of the MUTYH status, Mutyh-null cells failed to arrest in S-phase at late time points. MUTYH-dependent toxicity was also apparent in vivo. Mutyh−/−mice survived better than wild-type during a 12-month chronicexposure to Aza/UVA treatments that significantly increased levels of skin DNA 8-oxoG. Two squamous cell skin carcinomas arose in Aza/UVA treated Mutyh−/− mice whereas similarly treated wild-type animals remained tumor-free. PMID:25638157

Hepatic oxidative stress, genotoxicity and vascular dysfunction in lean or obese zucker rats

DEFF Research Database (Denmark)

Løhr, Mille; Folkmann, Janne Kjærsgaard; Sheykhzade, Majid

2015-01-01

Metabolic syndrome is associated with increased risk of cardiovascular disease, which could be related to oxidative stress. Here, we investigated the associations between hepatic oxidative stress and vascular function in pressurized mesenteric arteries from lean and obese Zucker rats at 14, 24 an......-generated DNA damage despite substantial hepatic steatosis.......Metabolic syndrome is associated with increased risk of cardiovascular disease, which could be related to oxidative stress. Here, we investigated the associations between hepatic oxidative stress and vascular function in pressurized mesenteric arteries from lean and obese Zucker rats at 14, 24...... and 37 weeks of age. Obese Zucker rats had more hepatic fat accumulation than their lean counterparts. Nevertheless, the obese rats had unaltered age-related level of hepatic oxidatively damaged DNA in terms of formamidopyrimidine DNA glycosylase (FPG) or human oxoguanine DNA glycosylase (hOGG1...

Repair of DNA-polypeptide crosslinks by human excision nuclease

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Reardon, Joyce T.; Sancar, Aziz

2006-03-01

DNA-protein crosslinks are relatively common DNA lesions that form during the physiological processing of DNA by replication and recombination proteins, by side reactions of base excision repair enzymes, and by cellular exposure to bifunctional DNA-damaging agents such as platinum compounds. The mechanism by which pathological DNA-protein crosslinks are repaired in humans is not known. In this study, we investigated the mechanism of recognition and repair of protein-DNA and oligopeptide-DNA crosslinks by the human excision nuclease. Under our assay conditions, the human nucleotide excision repair system did not remove a 16-kDa protein crosslinked to DNA at a detectable level. However, 4- and 12-aa-long oligopeptides crosslinked to the DNA backbone were recognized by some of the damage recognition factors of the human excision nuclease with moderate selectivity and were excised from DNA at relatively efficient rates. Our data suggest that, if coupled with proteolytic degradation of the crosslinked protein, the human excision nuclease may be the major enzyme system for eliminating protein-DNA crosslinks from the genome. damage recognition | nucleotide excision repair

Cloning of the human androgen receptor cDNA

International Nuclear Information System (INIS)

Govindan, M.V.; Burelle, M.; Cantin, C.; Kabrie, C.; Labrie, F.; Lachance, Y.; Leblanc, G.; Lefebvre, C.; Patel, P.; Simard, J.

1988-01-01

The authors discuss how in order to define the functional domains of the human androgen receptor, complementary DNA (cDNA) clones encoding the human androgen receptor (hAR) have been isolated from a human testis λgtll cDNA library using synthetic oligonnucleotide probes, homologous to segments of the human glucocorticoid, estradiol and progesterone receptors. The cDNA clones corresponding to the human glucocorticoid, estradiol and progesterone receptors were eliminated after cross-hybridization with their respective cDNA probes and/or after restriction mapping of the cDNA clones. The remaining cDNA clones were classified into different groups after analysis by restriction digestion and cross-hybridization. Two of the largest cDNA clones from each group were inserted into an expression vector in both orientations. The linearized plasmids were used as templates in in vitro transcription with T7 RNA polymerase. Subsequent in vitro translation of the purified transcripts in rabbit reticulocyte lysate followed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) permitted the characterization of the encoded polyeptides. The expressed proteins larger than 30,000 Da were analyzed for their ability to bind tritium-labelled dihydrotestosterone ([ 3 H] DHT) with high affinity and specificity

Exposure to Ultrafine Particles from Ambient Air and Oxidative Stress-Induced DNA Damage

DEFF Research Database (Denmark)

Bräuner, Elvira Vaclavik; Forchhammer, Lykke; Møller, Peter

2007-01-01

mononuclear cells (PBMCs) during controlled exposure to urban air particles with assignment of number concentration (NC) to four size modes with average diameters of 12, 23, 57, and 212 nm. DESIGN. Twenty-nine healthy adults participated in a randomized, two-factor cross-over study with or without biking...... exercise for 180 min and with exposure to particles (NC 6169-15362/cm3) or filtered air (NC 91-542/cm3) for 24 hr. METHODS: The levels of DNA strand breaks (SBs), oxidized purines as formamidopyrimidine DNA glycolase (FPG) sites, and activity of 7,8-dihydro-8-oxoguanine-DNA glycosylase (OGG1) in PBMCs were...

Human Parvovirus B19 Utilizes Cellular DNA Replication Machinery for Viral DNA Replication.

Science.gov (United States)

Zou, Wei; Wang, Zekun; Xiong, Min; Chen, Aaron Yun; Xu, Peng; Ganaie, Safder S; Badawi, Yomna; Kleiboeker, Steve; Nishimune, Hiroshi; Ye, Shui Qing; Qiu, Jianming

2018-03-01

Human parvovirus B19 (B19V) infection of human erythroid progenitor cells (EPCs) induces a DNA damage response and cell cycle arrest at late S phase, which facilitates viral DNA replication. However, it is not clear exactly which cellular factors are employed by this single-stranded DNA virus. Here, we used microarrays to systematically analyze the dynamic transcriptome of EPCs infected with B19V. We found that DNA metabolism, DNA replication, DNA repair, DNA damage response, cell cycle, and cell cycle arrest pathways were significantly regulated after B19V infection. Confocal microscopy analyses revealed that most cellular DNA replication proteins were recruited to the centers of viral DNA replication, but not the DNA repair DNA polymerases. Our results suggest that DNA replication polymerase δ and polymerase α are responsible for B19V DNA replication by knocking down its expression in EPCs. We further showed that although RPA32 is essential for B19V DNA replication and the phosphorylated forms of RPA32 colocalized with the replicating viral genomes, RPA32 phosphorylation was not necessary for B19V DNA replication. Thus, this report provides evidence that B19V uses the cellular DNA replication machinery for viral DNA replication. IMPORTANCE Human parvovirus B19 (B19V) infection can cause transient aplastic crisis, persistent viremia, and pure red cell aplasia. In fetuses, B19V infection can result in nonimmune hydrops fetalis and fetal death. These clinical manifestations of B19V infection are a direct outcome of the death of human erythroid progenitors that host B19V replication. B19V infection induces a DNA damage response that is important for cell cycle arrest at late S phase. Here, we analyzed dynamic changes in cellular gene expression and found that DNA metabolic processes are tightly regulated during B19V infection. Although genes involved in cellular DNA replication were downregulated overall, the cellular DNA replication machinery was tightly

Human circulating plasma DNA significantly decreases while lymphocyte DNA damage increases under chronic occupational exposure to low-dose gamma-neutron and tritium β-radiation

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Korzeneva, Inna B., E-mail: inna.korzeneva@molgen.vniief.ru [Russian Federal Nuclear Center – All-Russian Research Institute of Experimental Physics (RFNC-VNIIEF) 607190, Sarov, 37 Mira ave., Nizhniy Novgorod Region (Russian Federation); Kostuyk, Svetlana V.; Ershova, Liza S. [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, 115478 Moscow, 1 Moskvorechye str. (Russian Federation); Osipov, Andrian N. [Federal Medial and Biological Center named after Burnazyan of the Federal Medical and Biological Agency (FMBTz named after Burnazyan of FMBA), Moscow (Russian Federation); State Research Center - Burnasyan Federal Medical Biophysical Center of Federal Medical Biological Agency, Zhivopisnaya, 46, Moscow, 123098 (Russian Federation); Zhuravleva, Veronika F.; Pankratova, Galina V. [Russian Federal Nuclear Center – All-Russian Research Institute of Experimental Physics (RFNC-VNIIEF) 607190, Sarov, 37 Mira ave., Nizhniy Novgorod Region (Russian Federation); Porokhovnik, Lev N.; Veiko, Natalia N. [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, 115478 Moscow, 1 Moskvorechye str. (Russian Federation)

2015-09-15

Highlights: • The chronic exposure to low-dose IR induces DSBs in human lymphocytes (TM index). • Exposure to IR decreases the level of human circulating DNA (cfDNA index). • IR induces an increase of DNase1 activity (DNase1 index) in plasma. • IR induces an increase of the level of antibodies to DNA (Ab DNA index) in plasma. • The ratio cfDNA/(DNase 1 × Ab DNA × TM) is a potential marker of human exposure to IR. - Abstract: The blood plasma of healthy people contains cell-fee (circulating) DNA (cfDNA). Apoptotic cells are the main source of the cfDNA. The cfDNA concentration increases in case of the organism’s cell death rate increase, for example in case of exposure to high-dose ionizing radiation (IR). The objects of the present research are the blood plasma and blood lymphocytes of people, who contacted occupationally with the sources of external gamma/neutron radiation or internal β-radiation of tritium N = 176). As the controls (references), blood samples of people, who had never been occupationally subjected to the IR sources, were used (N = 109). With respect to the plasma samples of each donor there were defined: the cfDNA concentration (the cfDNA index), DNase1 activity (the DNase1 index) and titre of antibodies to DNA (the Ab DNA index). The general DNA damage in the cells was defined (using the Comet assay, the tail moment (TM) index). A chronic effect of the low-dose ionizing radiation on a human being is accompanied by the enhancement of the DNA damage in lymphocytes along with a considerable cfDNA content reduction, while the DNase1 content and concentration of antibodies to DNA (Ab DNA) increase. All the aforementioned changes were also observed in people, who had not worked with the IR sources for more than a year. The ratio cfDNA/(DNase1 × Ab DNA × TM) is proposed to be used as a marker of the chronic exposure of a person to the external low-dose IR. It was formulated the assumption that the joint analysis of the cfDNA, DNase1, Ab

Does a role for selenium in DNA damage repair explain apparent controversies in its use in chemoprevention?

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Diamond, Alan M.

2013-01-01

The trace element selenium is an essential micronutrient that has received considerable attention for its potential use in the prevention of cancer. In spite of this interest, the mechanism(s) by which selenium might function as a chemopreventive remain to be determined. Considerable experimental evidence indicates that one possible mechanism by which selenium supplementation may exert its benefits is by enhancing the DNA damage repair response, and this includes data obtained using cultured cells, animal models as well as in human clinical studies. In these studies, selenium supplementation has been shown to be beneficial in reducing the frequency of DNA adducts and chromosome breaks, consequentially reducing the likelihood of detrimental mutations that ultimately contribute to carcinogenesis. The benefits of selenium can be envisioned as being due, at least in part, to it being a critical constituent of selenoproteins such as glutathione peroxidases and thioredoxin reductases, proteins that play important roles in antioxidant defence and maintaining the cellular reducing environment. Selenium, therefore, may be protective by preventing DNA damage from occurring as well as by increasing the activity of repair enzymes such as DNA glycosylases and DNA damage repair pathways that involve p53, BRCA1 and Gadd45. An improved understanding of the mechanism of selenium’s impact on DNA repair processes may help to resolve the apparently contradicting data obtained from decades of animal work, human epidemiology and more recently, clinical supplementation studies. PMID:23204505

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

Science.gov (United States)

Ouzon-Shubeita, Hala; Lee, Seongmin

2014-01-01

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

Reduced DNA repair in mouse satellite DNA after treatment with methylmethanesulfonate, and N-methyl-N-nitrosourea.

Science.gov (United States)

Bodell, W J; Banerjee, M R

1976-01-01

We have measured DNA repair in mouse satellite and main band DNA as resolved by Ag+-Cs2SO4 centrifugation in response to treatment with the alkylating agents, methyl methanesulfonate, and N-methyl-N-nitrosourea. We find that there is a statistically significant lower incorporation of 3H-Tdr into the satellite DNA as compared to the main band at varying periods after treatment with the alkylating agents. This suggests a reduced repair activity in the satellite DNA. We have measured the extent of binding of 14C-methyl methanesulfonate to the satellite, and main band DNA, and no difference in binding was observed, indicating that the reduced repair activity of satellite DNA is not due to a difference in binding of alkylating agents. We believe that the reduced incorporation of 3H-Tdr into satellite DNA may be due to its location in the condensed chromatin fraction. PMID:184436

Studies on the repair of damaged DNA in bacteriophage, bacterial and mammalian systems. Comprehensive report, 1 February 1981-15 September 1983

International Nuclear Information System (INIS)

Friedberg, E.C.

1983-01-01

We have explored the molecular mechanism of the repair of DNA at a number of different levels of biological organization, by investigating bacteriophage, bacterial, yeast and mammalian (including human) cells. We have demonstrated that uv endonuclease of phage T4 not only possesses pyrimidine dimer (PD)-DNA glycosylase activity but also apyrimidinic (AP) endonuclease activity. The demonstration of both activities provided an explanation for the specific endonucleosytic cleavage of DNA at sites of pyrimidine dimers catalyzed by this small protein. A new apurinic/apyrimidinic (AP) endonuclease, specific for sites of of base loss in single stranded DNA has been isolated from E. celi and presumably recognizes these lesions in single stranded regions of duplex DNA. We have partially purified this enzyme and have carried out a preliminary characterization of the activity. We treated xeroderma pigmentosum and normal cells with sodium butyrate in the hope of restoring normal levels of excision repair to the former. Although this result was not obtained, we established that all cells treated with sodium butyrate show enhanced levels of repair synthesis, thus providing a means for increasing the sensitivity of this commonly used technique for measuring DNA repair in mammalian cells in culture

DNA damage induced by the direct effect of He ion particles

International Nuclear Information System (INIS)

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

2006-01-01

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

Melatonin Protects Human Cells from Clustered DNA Damages, Killing and Acquisition of Soft Agar Growth Induced by X-rays or 970 MeV/n Fe ions

Energy Technology Data Exchange (ETDEWEB)

Das, B.; Sutherland, B.; Bennett, P. V.; Cutter, N. C.; Sutherland, J. C.

2011-06-01

We tested the ability of melatonin (N-acetyl-5 methoxytryptamine), a highly effective radical scavenger and human hormone, to protect DNA in solution and in human cells against induction of complex DNA clusters and biological damage induced by low or high linear energy transfer radiation (100 kVp X-rays, 970 MeV/nucleon Fe ions). Plasmid DNA in solution was treated with increasing concentrations of melatonin (0.0-3.5 mM) and were irradiated with X-rays. Human cells (28SC monocytes) were also irradiated with X-rays and Fe ions with and without 2 mM melatonin. Agarose plugs containing genomic DNA were subjected to Contour Clamped Homogeneous Electrophoretic Field (CHEF) followed by imaging and clustered DNA damages were measured by using Number Average length analysis. Transformation experiments on human primary fibroblast cells using soft agar colony assay were carried out which were irradiated with Fe ions with or without 2 mM melatonin. In plasmid DNA in solution, melatonin reduced the induction of single- and double-strand breaks. Pretreatment of human 28SC cells for 24 h before irradiation with 2 mM melatonin reduced the level of X-ray induced double-strand breaks by {approx}50%, of abasic clustered damages about 40%, and of Fe ion-induced double-strand breaks (41% reduction) and abasic clusters (34% reduction). It decreased transformation to soft agar growth of human primary cells by a factor of 10, but reduced killing by Fe ions only by 20-40%. Melatonin's effective reduction of radiation-induced critical DNA damages, cell killing, and striking decrease of transformation suggest that it is an excellent candidate as a countermeasure against radiation exposure, including radiation exposure to astronaut crews in space travel.

Phosphorylation and cellular function of the human Rpa2 N-terminus in the budding yeast Saccharomyces cerevisiae.

Science.gov (United States)

Ghospurkar, Padmaja L; Wilson, Timothy M; Liu, Shengqin; Herauf, Anna; Steffes, Jenna; Mueller, Erica N; Oakley, Gregory G; Haring, Stuart J

2015-02-01

Maintenance of genome integrity is critical for proper cell growth. This occurs through accurate DNA replication and repair of DNA lesions. A key factor involved in both DNA replication and the DNA damage response is the heterotrimeric single-stranded DNA (ssDNA) binding complex Replication Protein A (RPA). Although the RPA complex appears to be structurally conserved throughout eukaryotes, the primary amino acid sequence of each subunit can vary considerably. Examination of sequence differences along with the functional interchangeability of orthologous RPA subunits or regions could provide insight into important regions and their functions. This might also allow for study in simpler systems. We determined that substitution of yeast Replication Factor A (RFA) with human RPA does not support yeast cell viability. Exchange of a single yeast RFA subunit with the corresponding human RPA subunit does not function due to lack of inter-species subunit interactions. Substitution of yeast Rfa2 with domains/regions of human Rpa2 important for Rpa2 function (i.e., the N-terminus and the loop 3-4 region) supports viability in yeast cells, and hybrid proteins containing human Rpa2 N-terminal phospho-mutations result in similar DNA damage phenotypes to analogous yeast Rfa2 N-terminal phospho-mutants. Finally, the human Rpa2 N-terminus (NT) fused to yeast Rfa2 is phosphorylated in a manner similar to human Rpa2 in human cells, indicating that conserved kinases recognize the human domain in yeast. The implication is that budding yeast represents a potential model system for studying not only human Rpa2 N-terminal phosphorylation, but also phosphorylation of Rpa2 N-termini from other eukaryotic organisms. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

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

Science.gov (United States)

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

2012-01-01

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

Brain cDNA clone for human cholinesterase

International Nuclear Information System (INIS)

McTiernan, C.; Adkins, S.; Chatonnet, A.; Vaughan, T.A.; Bartels, C.F.; Kott, M.; Rosenberry, T.L.; La Du, B.N.; Lockridge, O.

1987-01-01

A cDNA library from human basal ganglia was screened with oligonucleotide probes corresponding to portions of the amino acid sequence of human serum cholinesterase. Five overlapping clones, representing 2.4 kilobases, were isolated. The sequenced cDNA contained 207 base pairs of coding sequence 5' to the amino terminus of the mature protein in which there were four ATG translation start sites in the same reading frame as the protein. Only the ATG coding for Met-(-28) lay within a favorable consensus sequence for functional initiators. There were 1722 base pairs of coding sequence corresponding to the protein found circulating in human serum. The amino acid sequence deduced from the cDNA exactly matched the 574 amino acid sequence of human serum cholinesterase, as previously determined by Edman degradation. Therefore, our clones represented cholinesterase rather than acetylcholinesterase. It was concluded that the amino acid sequences of cholinesterase from two different tissues, human brain and human serum, were identical. Hybridization of genomic DNA blots suggested that a single gene, or very few genes coded for cholinesterase

Cloning and comparative mapping of a human chromosome 4-specific alpha satellite DNA sequence

Energy Technology Data Exchange (ETDEWEB)

D' Aiuto, L.; Marzella, R.; Archidiacono, N.; Rocchi, M. (Universita di Bari (Italy)); Antonacci, R. (Instituto Anatomia Umana Normale, Modena (Italy))

1993-11-01

The authors have isolated and characterized two human alphoid DNA clones: p4n1/4 and pZ4.1. Clone p4n1/4 identifies specifically the centromeric region of chromosome 4; pZ4.1 recognizes a subset of alphoid DNA shared by chromosomes 4 and 9. The specificity was determined using fluorescence in situ hybridization experiments on metaphase spreads and Southern blotting analysis of human-hamster somatic cell hybrids. The genomic organization of both subsets was also investigated. Comparative mapping on chimpanzee and gorilla chromosomes was performed. p4n1/4 hybridizes to chimpanzee chromosomes 11 and 13, homologs of human chromosomes 9 and 2q, respectively. On gorilla metaphase spreads, p4n1/4 hybridizes exclusively to the centromeric region of chromosome 19, partially homologous to human chromosome 17. No hybridization signal was detected on chromosome 3 of both chimpanzee and gorilla, in both species homolog of human chromosome 4. Identical comparative mapping results were obtained using pZ4.1 probe, although the latter recognizes an alphoid subset distinct from the one recognized by p4n1/4. The implications of these results in the evolution of centromeric regions of primate chromosomes are discussed. 33 refs., 4 figs.

Efficiency and Fidelity of Human DNA Polymerases λ and β during Gap-Filling DNA Synthesis

Science.gov (United States)

Brown, Jessica A.; Pack, Lindsey R.; Sanman, Laura E.; Suo, Zucai

2010-01-01

The base excision repair (BER) pathway coordinates the replacement of 1 to 10 nucleotides at sites of single-base lesions. This process generates DNA substrates with various gap sizes which can alter the catalytic efficiency and fidelity of a DNA polymerase during gap-filling DNA synthesis. Here, we quantitatively determined the substrate specificity and base substitution fidelity of human DNA polymerase λ (Pol λ), an enzyme proposed to support the known BER DNA polymerase β (Pol β), as it filled 1- to 10-nucleotide gaps at 1-nucleotide intervals. Pol λ incorporated a correct nucleotide with relatively high efficiency until the gap size exceeded 9 nucleotides. Unlike Pol λ, Pol β did not have an absolute threshold on gap size as the catalytic efficiency for a correct dNTP gradually decreased as the gap size increased from 2 to 10 nucleotides and then recovered for non-gapped DNA. Surprisingly, an increase in gap size resulted in lower polymerase fidelity for Pol λ, and this downregulation of fidelity was controlled by its non-enzymatic N-terminal domains. Overall, Pol λ was up to 160-fold more error-prone than Pol β, thereby suggesting Pol λ would be more mutagenic during long gap-filling DNA synthesis. In addition, dCTP was the preferred misincorporation for Pol λ and its N-terminal domain truncation mutants. This nucleotide preference was shown to be dependent upon the identity of the adjacent 5′-template base. Our results suggested that both Pol λ and Pol β would catalyze nucleotide incorporation with the highest combination of efficiency and accuracy when the DNA substrate contains a single-nucleotide gap. Thus, Pol λ, like Pol β, is better suited to catalyze gap-filling DNA synthesis during short-patch BER in vivo, although, Pol λ may play a role in long-patch BER. PMID:20961817

DNA amplification is rare in normal human cells

International Nuclear Information System (INIS)

Wright, J.A.; Watt, F.M.; Hudson, D.L.; Stark, G.R.; Smith, H.S.; Hancock, M.C.

1990-01-01

Three types of normal human cells were selected in tissue culture with three drugs without observing a single amplification event from a total of 5 x 10 8 cells. No drug-resistant colonies were observed when normal foreskin keratinocytes were selected with N-(phosphonacetyl)-L-aspartate or with hydroxyurea or when normal mammary epithelial cells were selected with methotrexate. Some slightly resistant colonies with limited potential for growth were obtained when normal diploid fibroblast cells derived from fetal lung were selected with methotrexate or hydroxyurea but careful copy-number analysis of the dihydrofolate reductase and ribonucleotide reductase genes revealed no evidence of amplification. The rarity of DNA amplification in normal human cells contrasts strongly with the situation in tumors and in established cell lines, where amplification of onogenes and of genes mediating drug resistance is frequent. The results suggest that tumors and cell lines have acquired the abnormal ability to amplify DNA with high frequency

Base Flip in DNA Studied by Molecular Dynamics Simulationsof Differently-Oxidized Forms of Methyl-Cytosine

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Mahdi Bagherpoor Helabad

2014-07-01

Full Text Available Distortions in the DNA sequence, such as damage or mispairs, are specifically recognized and processed by DNA repair enzymes. Many repair proteins and, in particular, glycosylases flip the target base out of the DNA helix into the enzyme’s active site. Our molecular dynamics simulations of DNA with intact and damaged (oxidized methyl-cytosine show that the probability of being flipped is similar for damaged and intact methyl-cytosine. However, the accessibility of the different 5-methyl groups allows direct discrimination of the oxidized forms. Hydrogen-bonded patterns that vary between methyl-cytosine forms carrying a carbonyl oxygen atom are likely to be detected by the repair enzymes and may thus help target site recognition.

The persistence of human DNA in soil following surface decomposition.

Science.gov (United States)

Emmons, Alexandra L; DeBruyn, Jennifer M; Mundorff, Amy Z; Cobaugh, Kelly L; Cabana, Graciela S

2017-09-01

Though recent decades have seen a marked increase in research concerning the impact of human decomposition on the grave soil environment, the fate of human DNA in grave soil has been relatively understudied. With the purpose of supplementing the growing body of literature in forensic soil taphonomy, this study assessed the relative persistence of human DNA in soil over the course of decomposition. Endpoint PCR was used to assess the presence or absence of human nuclear and mitochondrial DNA, while qPCR was used to evaluate the quantity of human DNA recovered from the soil beneath four cadavers at the University of Tennessee's Anthropology Research Facility (ARF). Human nuclear DNA from the soil was largely unrecoverable, while human mitochondrial DNA was detectable in the soil throughout all decomposition stages. Mitochondrial DNA copy abundances were not significantly different between decomposition stages and were not significantly correlated to soil edaphic parameters tested. There was, however, a significant positive correlation between mitochondrial DNA copy abundances and the human associated bacteria, Bacteroides, as estimated by 16S rRNA gene abundances. These results show that human mitochondrial DNA can persist in grave soil and be consistently detected throughout decomposition. Copyright © 2017 The Chartered Society of Forensic Sciences. Published by Elsevier B.V. All rights reserved.

Enhanced unscheduled DNA synthesis in UV-irradiated human skin explants treated with T4N5 liposomes

International Nuclear Information System (INIS)

Yarosh, D.B.; Kibitel, J.T.; Green, L.A.; Spinowitz, A.

1991-01-01

Epidermal keratinocytes cultured from explants of skin cancer patients, including biopsies from xeroderma pigmentosum patients, were ultraviolet light-irradiated and DNA repair synthesis was measured. Repair capacity was much lower in xeroderma pigmentosum patients than in normal patients. The extent of DNA repair replication did not decline with the age of the normal patient. Treatment with T4N5 liposomes containing a DNA repair enzyme enhanced repair synthesis in both normal and xeroderma pigmentosum keratinocytes in an irradiation- and liposome-dose dependent manner. These results provide no evidence that aging people or skin cancer patients are predisposed to cutaneous malignancy by a DNA repair deficiency, but do demonstrate that T4N5 liposomes enhance DNA repair in the keratinocytes of the susceptible xeroderma pigmentosum and skin cancer population

Cytotoxic and mutagenic effects of specific carcinogen-DNA adducts in diploid human fibroblasts

International Nuclear Information System (INIS)

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

1985-01-01

A comparison of the cytotoxicity and mutagenicity of a series of carcinogens in normal diploid human fibroblasts and in cells deficient in one or more DNA repair processes has provided insight into the specific DNA adduct(s) responsible for these biological effects. The carcinogens tested include ultraviolet radiation; reactive derivatives of structurally related aromatic amides; metabolites of benzo(a)pyrene; the simple alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine and N-ethyl-N-nitrosourea; and aflatoxin B 1 dichloride, a model for the reactive 2,3-epoxide of aflatoxin B 1 . Exponentially growing cells were exposed to agents and assayed for mutations and cell killing. Cells deficient in repair of particular DNA adducts or lesions proved more sensitive to the agent causing those lesions than did normally repairing cells. Many of the carcinogens were compared for their mutagenic and/or cytotoxic effect, not only as a function of dose administered, but also as a function of the initial number of adducts or photoproducts induced in DNA and the number remaining at critical times posttreatment. The results demonstrated a high correlation between the number of DNA lesions remaining unexcised at the time the DNA was replicated and frequency of mutations induced. Comparative studies of the frequency of UV-induced transformation of normal and repair-deficient cells showed this also to be true for transformation

Selenium-Mediated Dehalogenation of Halogenated Nucleosides and its Relevance to the DNA Repair Pathway.

Science.gov (United States)

Mondal, Santanu; Manna, Debasish; Mugesh, Govindasamy

2015-08-03

Halogenated nucleosides can be incorporated into the newly synthesized DNA of replicating cells and therefore are commonly used in the detection of proliferating cells in living tissues. Dehalogenation of these modified nucleosides is one of the key pathways involved in DNA repair mediated by the uracil-DNA glycosylase. Herein, we report the first example of a selenium-mediated dehalogenation of halogenated nucleosides. We also show that the mechanism for the debromination is remarkably different from that of deiodination and that the presence of a ribose or deoxyribose moiety in the nucleosides facilitates the deiodination. The results described herein should help in understanding the metabolism of halogenated nucleosides in DNA and RNA. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mercuric dichloride induces DNA damage in human salivary gland tissue cells and lymphocytes

Energy Technology Data Exchange (ETDEWEB)

Schmid, Katharina; Kroemer, Susanne [University of Regensburg, Regensburg (Germany); Sassen, Andrea [University of Regensburg, Department of Pathology, Regensburg (Germany); Staudenmaier, Rainer [Technical University of Munich, Department of Otorhinolaryngology, Head and Neck Surgery, Munich (Germany); Reichl, Franz-Xaver [University of Munich, Institute of Pharmacology and Toxicology, Munich (Germany); Harreus, Ulrich [University of Munich, Department of Otorhinolaryngology, Head and Neck Surgery, Munich (Germany); Hagen, Rudolf; Kleinsasser, Norbert [University of Wuerzburg, Department of Otorhinolaryngology, Head and Neck Surgery, Wuerzburg (Germany)

2007-11-15

Amalgam is still one of the most frequently used dental filling materials. However, the possible adverse effects especially that of the mercuric component have led to continued controversy. Considering that mercury may be released from amalgam fillings into the oral cavity and also reach the circulating blood after absorption and resorption, it eventually may contribute to tumorigenesis in a variety of target cells. The present investigation focuses on genotoxic effects below a cytotoxic dose level of mercuric dichloride (HgCl{sub 2}) in human samples of salivary glands and lymphocytes to elucidate a possible role in tumor initiation. DNA migration due to single strand breaks, alkali labile sites and incomplete excision repair was quantified with the aid of the single cell microgel electrophoresis (Comet) assay. The concepts of Olive Tail Moment, percentage of DNA in the Tail and Tail Length were used as measures of DNA damage. To control for cytotoxic effects, the trypan blue exclusion test was applied. Human samples of the parotid salivary gland and lymphocytes of ten donors were exposed to HgCl{sub 2} concentrations from 1 to 50 {mu}M. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and dimethyl sulfoxide (DMSO) served as controls. Increasing dose-dependent DNA migration could be demonstrated after exposure to HgCl{sub 2} in cells of the salivary glands and lymphocytes. In both cell types a significant increase in DNA migration could be shown starting from HgCl{sub 2} concentrations of 5 {mu}M in comparison to the negative control. The viability of the cell systems was not affected except at the highest concentration (50 {mu}M) tested. These data indicate genotoxic effects of mercuric dichloride in human salivary glands and lymphocytes at concentrations not leading to cytotoxic effects or cell death. Consequently, a contributory role in oral salivary gland tumor initiation warrants further investigation. (orig.)

DNA methylation-based variation between human populations.

Science.gov (United States)

Kader, Farzeen; Ghai, Meenu

2017-02-01

Several studies have proved that DNA methylation affects regulation of gene expression and development. Epigenome-wide studies have reported variation in methylation patterns between populations, including Caucasians, non-Caucasians (Blacks), Hispanics, Arabs, and numerous populations of the African continent. Not only has DNA methylation differences shown to impact externally visible characteristics, but is also a potential biomarker for underlying racial health disparities between human populations. Ethnicity-related methylation differences set their mark during early embryonic development. Genetic variations, such as single-nucleotide polymorphisms and environmental factors, such as age, dietary folate, socioeconomic status, and smoking, impacts DNA methylation levels, which reciprocally impacts expression of phenotypes. Studies show that it is necessary to address these external influences when attempting to differentiate between populations since the relative impacts of these factors on the human methylome remain uncertain. The present review summarises several reported attempts to establish the contribution of differential DNA methylation to natural human variation, and shows that DNA methylation could represent new opportunities for risk stratification and prevention of several diseases amongst populations world-wide. Variation of methylation patterns between human populations is an exciting prospect which inspires further valuable research to apply the concept in routine medical and forensic casework. However, trans-generational inheritance needs to be quantified to decipher the proportion of variation contributed by DNA methylation. The future holds thorough evaluation of the epigenome to understand quantification, heritability, and the effect of DNA methylation on phenotypes. In addition, methylation profiling of the same ethnic groups across geographical locations will shed light on conserved methylation differences in populations.

Cloning of the cDNA for human 12-lipoxygenase

International Nuclear Information System (INIS)

Izumi, T.; Hoshiko, S.; Radmark, O.; Samuelsson, B.

1990-01-01

A full-length cDNA clone encoding 12-lipoxygenase was isolated from a human platelet cDNA library by using a cDNA for human reticulocyte 15-lipoxygenase as probe for the initial screening. The cDNA had an open reading frame encoding 662 amino acid residues with a calculated molecular weight of 75,590. Three independent clones revealed minor heterogeneities in their DNA sequences. Thus, in three positions of the deduced amino acid sequence, there is a choice between two different amino acids. The deduced sequence from the clone plT3 showed 65% identity with human reticulocyte 15-lipoxygenase and 42% identity with human leukocyte 5-lipoxygenase. The 12-lipoxygenase cDNA recognized a 3.0-kilobase mRNA species in platelets and human erythroleukemia cells (HEL cells). Phorbol 12-tetradecanoyl 13-acetate induced megakaryocytic differentiation of HEL cells and 12-lipoxygenase activity and increased mRNA for 12-lipoxygenase. The identity of the cloned 12-lipoxygenase was assured by expression in a mammalian cell line (COS cells). Human platelet 12-lipoxygenase has been difficult to purify to homogeneity. The cloning of this cDNA will increase the possibilities to elucidate the structure and function of this enzyme

Epigenetic changes of Arabidopsis genome associated with altered DNA methyltransferase and demethylase expressions after gamma irradiation

International Nuclear Information System (INIS)

Kim, Ji Eun; Cho, Eun Ju; Kim, Ji Hong; Chung, Byung Yeoup; Kim, Jin Hong

2012-01-01

DNA methylation at carbon 5 of cytosines is a hall mark of epigenetic inactivation and heterochromatin in both plants and mammals. In Arabidopsis, DNA methylation has two roles that protect the genome from selfish DNA elements and regulate gene expression. Plant genome has three types of DNA methyltransferase, METHYLTRANSFERASE 1 (MET1), DOMAINREARRANGED METHYLASE (DRM) and CHROMOMETHYLASE 3 (CMT3) that are capable of methylating CG, CHG (where H is A, T, or C) and CHH sites, respectively. MET1 is a maintenance DNA methyltransferase that controls CG methylation. Two members of the DRM family, DRM1 and DRM2, are responsible for de novo methylation of CG, CHG, and CHH sites but show a preference for CHH sites. Finally, CMT3 principally carries out CHG methylation and is involved in both de novo methylation and maintenance. Alternatively, active DNA demethylation may occur through the glycosylase activity by removing the methylcytosines from DNA. It may have essential roles in preventing transcriptional silencing of transgenes and endogenous genes and in activating the expression of imprinted genes. DNA demetylation in Arabidopsis is mediated by the DEMETER (DME) family of bifunctional DNA glycosylase. Three targets of DME are MEA (MEDEA), FWA (FLOWERING WAGENINGEN), and FIS2 (FERTILIZATION INDEPENDENT SEED 2). The DME family contains DEMETER-LIKE 2 (DML2), DML3, and REPRESSOR OF SILENING 1 (ROS1). DNA demetylation by ROS1, DML2, and DML3 protect the hypermethylation of specific genome loci. ROS1 is necessary to suppress the promoter methylation and the silencing of endogenous genes. In contrast, the function of DML2 and DML3 has not been reported. Several recent studies have suggested that epigenetic alterations such as change in DNA methylation and histone modification should be caused in plant genomes upon exposure to ionizing radiation. However, there is a lack of data exploring the underlying mechanisms. Therefore, the present study aims to characterize and

Epigenetic changes of Arabidopsis genome associated with altered DNA methyltransferase and demethylase expressions after gamma irradiation

Energy Technology Data Exchange (ETDEWEB)

Kim, Ji Eun; Cho, Eun Ju; Kim, Ji Hong; Chung, Byung Yeoup; Kim, Jin Hong [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

2012-05-15

DNA methylation at carbon 5 of cytosines is a hall mark of epigenetic inactivation and heterochromatin in both plants and mammals. In Arabidopsis, DNA methylation has two roles that protect the genome from selfish DNA elements and regulate gene expression. Plant genome has three types of DNA methyltransferase, METHYLTRANSFERASE 1 (MET1), DOMAINREARRANGED METHYLASE (DRM) and CHROMOMETHYLASE 3 (CMT3) that are capable of methylating CG, CHG (where H is A, T, or C) and CHH sites, respectively. MET1 is a maintenance DNA methyltransferase that controls CG methylation. Two members of the DRM family, DRM1 and DRM2, are responsible for de novo methylation of CG, CHG, and CHH sites but show a preference for CHH sites. Finally, CMT3 principally carries out CHG methylation and is involved in both de novo methylation and maintenance. Alternatively, active DNA demethylation may occur through the glycosylase activity by removing the methylcytosines from DNA. It may have essential roles in preventing transcriptional silencing of transgenes and endogenous genes and in activating the expression of imprinted genes. DNA demetylation in Arabidopsis is mediated by the DEMETER (DME) family of bifunctional DNA glycosylase. Three targets of DME are MEA (MEDEA), FWA (FLOWERING WAGENINGEN), and FIS2 (FERTILIZATION INDEPENDENT SEED 2). The DME family contains DEMETER-LIKE 2 (DML2), DML3, and REPRESSOR OF SILENING 1 (ROS1). DNA demetylation by ROS1, DML2, and DML3 protect the hypermethylation of specific genome loci. ROS1 is necessary to suppress the promoter methylation and the silencing of endogenous genes. In contrast, the function of DML2 and DML3 has not been reported. Several recent studies have suggested that epigenetic alterations such as change in DNA methylation and histone modification should be caused in plant genomes upon exposure to ionizing radiation. However, there is a lack of data exploring the underlying mechanisms. Therefore, the present study aims to characterize and

Kinetics and Thermodynamics of DNA Processing by Wild Type DNA-Glycosylase Endo III and Its Catalytically Inactive Mutant Forms

Directory of Open Access Journals (Sweden)

Olga A. Kladova

2018-03-01

Full Text Available Endonuclease III (Endo III or Nth is one of the key enzymes responsible for initiating the base excision repair of oxidized or reduced pyrimidine bases in DNA. In this study, a thermodynamic analysis of structural rearrangements of the specific and nonspecific DNA-duplexes during their interaction with Endo III is performed based on stopped-flow kinetic data. 1,3-diaza-2-oxophenoxazine (tCO, a fluorescent analog of the natural nucleobase cytosine, is used to record multistep DNA binding and lesion recognition within a temperature range (5–37 °C. Standard Gibbs energy, enthalpy, and entropy of the specific steps are derived from kinetic data using Van’t Hoff plots. The data suggest that enthalpy-driven exothermic 5,6-dihydrouracil (DHU recognition and desolvation-accompanied entropy-driven adjustment of the enzyme–substrate complex into a catalytically active state play equally important parts in the overall process. The roles of catalytically significant amino acids Lys120 and Asp138 in the DNA lesion recognition and catalysis are identified. Lys120 participates not only in the catalytic steps but also in the processes of local duplex distortion, whereas substitution Asp138Ala leads to a complete loss of the ability of Endo III to distort a DNA double chain during enzyme–DNA complex formation.

Sequence of human protamine 2 cDNA

Energy Technology Data Exchange (ETDEWEB)

Domenjoud, L; Fronia, C; Uhde, F; Engel, W [Universitaet Goettingen (West Germany)

1988-08-11

The authors report the cloning and sequencing of a cDNA clone for human protamine 2 (hp2), isolated from a human testis cDNA library cloned in the vector {lambda}-gt11. A 66mer oligonucleotide, that corresponds to an amino acid sequence which is highly conserved between hp2 and mouse protamine 2 (mp2) served as hybridization probe. The homology between the amino acid sequence deduced from our cDNA and the published amino acid sequence for hp2 is 100%.

PCR-Free Enrichment of Mitochondrial DNA from Human Blood and Cell Lines for High Quality Next-Generation DNA Sequencing.

Directory of Open Access Journals (Sweden)

Meetha P Gould

Full Text Available Recent advances in sequencing technology allow for accurate detection of mitochondrial sequence variants, even those in low abundance at heteroplasmic sites. Considerable sequencing cost savings can be achieved by enriching samples for mitochondrial (relative to nuclear DNA. Reduction in nuclear DNA (nDNA content can also help to avoid false positive variants resulting from nuclear mitochondrial sequences (numts. We isolate intact mitochondrial organelles from both human cell lines and blood components using two separate methods: a magnetic bead binding protocol and differential centrifugation. DNA is extracted and further enriched for mitochondrial DNA (mtDNA by an enzyme digest. Only 1 ng of the purified DNA is necessary for library preparation and next generation sequence (NGS analysis. Enrichment methods are assessed and compared using mtDNA (versus nDNA content as a metric, measured by using real-time quantitative PCR and NGS read analysis. Among the various strategies examined, the optimal is differential centrifugation isolation followed by exonuclease digest. This strategy yields >35% mtDNA reads in blood and cell lines, which corresponds to hundreds-fold enrichment over baseline. The strategy also avoids false variant calls that, as we show, can be induced by the long-range PCR approaches that are the current standard in enrichment procedures. This optimization procedure allows mtDNA enrichment for efficient and accurate massively parallel sequencing, enabling NGS from samples with small amounts of starting material. This will decrease costs by increasing the number of samples that may be multiplexed, ultimately facilitating efforts to better understand mitochondria-related diseases.

Dihydropyridines decrease X-ray-induced DNA base damage in mammalian cells

Energy Technology Data Exchange (ETDEWEB)

Wojewodzka, M., E-mail: marylaw@ichtj.waw.pl [Center of Radiobiology and Biological Dosimetry, Institute of Nuclear Chemistry and Technology, Warszawa (Poland); Gradzka, I.; Buraczewska, I.; Brzoska, K.; Sochanowicz, B. [Center of Radiobiology and Biological Dosimetry, Institute of Nuclear Chemistry and Technology, Warszawa (Poland); Goncharova, R.; Kuzhir, T. [Institute of Genetics and Cytology, Belarussian National Academy of Sciences, Minsk (Belarus); Szumiel, I. [Center of Radiobiology and Biological Dosimetry, Institute of Nuclear Chemistry and Technology, Warszawa (Poland)

2009-12-01

Compounds with the structural motif of 1,4-dihydropyridine display a broad spectrum of biological activities, often defined as bioprotective. Among them are L-type calcium channel blockers, however, also derivatives which do not block calcium channels exert various effects at the cellular and organismal levels. We examined the effect of sodium 3,5-bis-ethoxycarbonyl-2,6-dimethyl-1,4-dihydropyridine-4-carboxylate (denoted here as DHP and previously also as AV-153) on X-ray-induced DNA damage and mutation frequency at the HGPRT (hypoxanthine-guanine phosphoribosyl transferase) locus in Chinese hamster ovary CHO-K1 cells. Using formamido-pyrimidine glycosylase (FPG) comet assay, we found that 1-h DHP (10 nM) treatment before X-irradiation considerably reduced the initial level of FPG-recognized DNA base damage, which was consistent with decreased 8-oxo-7,8-dihydro-2'-deoxyguanosine content and mutation frequency lowered by about 40%. No effect on single strand break rejoining or on cell survival was observed. Similar base damage-protective effect was observed for two calcium channel blockers: nifedipine (structurally similar to DHP) or verapamil (structurally unrelated). So far, the specificity of the DHP-caused reduction in DNA damage - practically limited to base damage - has no satisfactory explanation.

Mechanism underlying the effect of long-term exposure to low dose of pesticides on DNA integrity.

Science.gov (United States)

Alleva, Renata; Manzella, Nicola; Gaetani, Simona; Bacchetti, Tiziana; Bracci, Massimo; Ciarapica, Veronica; Monaco, Federica; Borghi, Battista; Amati, Monica; Ferretti, Gianna; Tomasetti, Marco

2018-04-01

Pesticides, including herbicides, insecticides and fungicides, are widely used in intensive agriculture. Recently, the long-term effects of pesticide exposure were found to be associated with many diseases. In this study, we evaluated the long-term effect of low-level exposure to a mixture of pesticides on DNA damage response (DDR) in relation to individual detoxifying variability. A residential population chronically exposed to pesticides was enrolled, biological/environmental pesticide levels; paroxonase 1 (PON-1) activity and 192 Q/R polymorphism and DDR were evaluated at three different periods of pesticide exposure. OGG1-dependent DNA repair activity was decreased in relation to pesticide exposure. The increase of DNA lesions and pesticide levels in the intensive pesticide-spraying period was independent on PON-1 activity. Next, human bronchial epithelial and neuronal cells were used as a model for in vitro evaluation of the mechanistic effect of pesticides. Pesticides induced mitochondrial dysfunction leading to ROS formation. ROS from mitochondria induced DNA damage, which in turn induced OGG1-dependent DNA repair activity through 8-oxoguanine DNA glycosylase 1 (OGG1) expression and activation. Even though OGG1 was overexpressed, an inhibition of its activity, associated with DNA lesion accumulation, was found at prolonged pesticide-exposure. A post-translational regulation of OGG1 by pesticide may be postulated. Taken together, long-term exposure to low-levels of pesticides affects DDR resulting in accumulation of DNA lesions that eventually may lead to cancer or neurological disorders. © 2018 Wiley Periodicals, Inc.

DNA-mediated strand displacement facilitates sensitive electronic detection of antibodies in human serums.

Science.gov (United States)

Dou, Baoting; Yang, Jianmei; Shi, Kai; Yuan, Ruo; Xiang, Yun

2016-09-15

We describe here the development of a sensitive and convenient electronic sensor for the detection of antibodies in human serums. The sensor is constructed by self-assembly formation of a mixed monolayer containing the small molecule epitope conjugated double stranded DNA probes on gold electrode. The target antibody binds the epitope on the dsDNA probe and lowers the melting temperature of the duplex, which facilitates the displacement of the antibody-linked strand of the duplex probe by an invading methylene blue-tagged single stranded DNA (MB-ssDNA) through the strand displacement reaction and leads to the capture of many MB-ssDNA on the sensor surface. Subsequent electrochemical oxidation of the methylene blue labels results in amplified current response for sensitive monitoring of the antibodies. The antibody assay conditions are optimized and the sensor exhibits a linear range between 1.0 and 25.0nM with a detection limit of 0.67nM for the target antibody. The sensor is also selective and can be employed to detect the target antibodies in human serum samples. With the advantages of using small molecule epitope as the antibody recognition element over traditional antigen, the versatile manipulability of the DNA probes and the unique properties of the electrochemical transduction technique, the developed sensor thus hold great potential for simple and sensitive detection of different antibodies and other proteins in real samples. Copyright © 2016 Elsevier B.V. All rights reserved.

O6-alkylguanine-DNA-alkyltransferase activity and nitrosourea sensitivity in human cancer cell lines.

OpenAIRE

Walker, M. C.; Masters, J. R.; Margison, G. P.

1992-01-01

The DNA repair enzyme, O6-alkylguanine-DNA-alkyltransferase (ATase), is thought to be the principal mechanism controlling resistance to nitrosoureas and related alkylating agents. We compared the sensitivities of five human testis and five bladder tumour cell lines to two nitrosoureas (N-nitroso-N-methylurea (MNU) and mitozolomide) with cellular levels of ATase. Enzyme levels ranged from 3 to 206 fmol mg-1 protein (0.1 x 10(4) to 5.1 x 10(4) molecules/cell) in the testis lines and from 11 to ...

A DNA Vaccine Protects Human Immune Cells against Zika Virus Infection in Humanized Mice

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Guohua Yi

2017-11-01

Full Text Available A DNA vaccine encoding prM and E protein has been shown to induce protection against Zika virus (ZIKV infection in mice and monkeys. However, its effectiveness in humans remains undefined. Moreover, identification of which immune cell types are specifically infected in humans is unclear. We show that human myeloid cells and B cells are primary targets of ZIKV in humanized mice. We also show that a DNA vaccine encoding full length prM and E protein protects humanized mice from ZIKV infection. Following administration of the DNA vaccine, humanized DRAG mice developed antibodies targeting ZIKV as measured by ELISA and neutralization assays. Moreover, following ZIKV challenge, vaccinated animals presented virtually no detectable virus in human cells and in serum, whereas unvaccinated animals displayed robust infection, as measured by qRT-PCR. Our results utilizing humanized mice show potential efficacy for a targeted DNA vaccine against ZIKV in humans.

Cloning of a cDNA encoding the human cation-dependent mannose 6-phosphate-specific receptor

International Nuclear Information System (INIS)

Pohlmann, R.; Nagel, G.; Schmidt, B.

1987-01-01

Complementary DNA clones for the human cation-dependent mannose 6-phosphate-specific receptor have been isolated from a human placenta library in λgt11. The nucleotide sequence of the 2463-base-pair cDNA insert includes a 145-base-pair 5' untranslated region, an open reading frame of 831 base pairs corresponding to 277 amino acids, and a 1487-base-pair 3' untranslated region. The deduced amino acid sequence is colinear with that determined by amino acid sequencing of the N-terminus peptide (41 residues) and nine tryptic peptides (93 additional residues). The receptor is synthesized as a precursor with a signal peptide of 20 amino acids. The hydrophobicity profile of the receptor indicates a single membrane-spanning domain, which separates an N-terminal region containing five potential N-glycosylation sites from a C-terminal region lacking N-glycosylation sites. Thus the N-terminal (M/sub r/ = 18,299) and C-terminal (M/sub r/ ≤ 7648) segments of the mature receptor are assumed to be exposed to the extracytosolic and cytosolic sides of the membrane, respectively. Analysis of a panel of somatic cell (mouse-human) hybrids shows that the gene for the receptor is located on human chromosome 12

Mutagenesis and repair induced by the DNA advanced glycation end product N2-1-(carboxyethyl)-2'-deoxyguanosine in human cells.

Science.gov (United States)

Tamae, Daniel; Lim, Punnajit; Wuenschell, Gerald E; Termini, John

2011-03-29

Glycation of biopolymers by glucose-derived α-oxo-aldehydes such as methylglyoxal (MG) is believed to play a major role in the complex pathologies associated with diabetes and metabolic disease. In contrast to the extensive literature detailing the formation and physiological consequences of protein glycation, there is little information about the corresponding phenomenon for DNA. To assess the potential contribution of DNA glycation to genetic instability, we prepared shuttle vectors containing defined levels of the DNA glycation adduct N(2)-(1-carboxyethyl)-2'-deoxyguanosine (CEdG) and transfected them into isogenic human fibroblasts that differed solely in the capacity to conduct nucleotide excision repair (NER). In the NER-compromised fibroblasts, the induced mutation frequencies increased up to 18-fold relative to background over a range of ∼10-1400 CEdG adducts/10(5) dG, whereas the same substrates transfected into NER-competent cells induced a response that was 5-fold over background at the highest adduct density. The positive linear correlation (R(2) = 0.998) of mutation frequency with increasing CEdG level in NER-defective cells suggested that NER was the primary if not exclusive mechanism for repair of this adduct in human fibroblasts. Consistent with predictions from biochemical studies using CEdG-substituted oligonucleotides, guanine transversions were the predominant mutation resulting from replication of MG-modified plasmids. At high CEdG levels, significant increases in the number of AT → GC transitions were observed exclusively in NER-competent cells (P involvement of an NER-dependent mutagenic process in response to critical levels of DNA damage, possibly mediated by error-prone Y-family polymerases.

RAPD analysis of alfalfa DNA mutation via N+ implantation

International Nuclear Information System (INIS)

Li Yufeng; Huang Qunce; Yu Zengliang; Liang Yunzhang

2003-01-01

Germination capacity of alfalfa seeds under low energy N + implantation manifests oscillations going down with dose strength. From analyzing alfalfa genome DNA under low energy N + implantation by RAPD (Random Amplified Polymorphous DNA), it is recommended that 30 polymorphic DNA fragments be amplified with 8 primers in total 100 primers, and fluorescence intensity of the identical DNA fragment amplified by RAPD is different between CK and treatments. Number of different polymorphic DNA fragments between treatment and CK via N + implantation manifests going up with dose strength

The dnaN gene codes for the beta subunit of DNA polymerase III holoenzyme of escherichia coli.

Science.gov (United States)

Burgers, P M; Kornberg, A; Sakakibara, Y

1981-09-01

An Escherichia coli mutant, dnaN59, stops DNA synthesis promptly upon a shift to a high temperature; the wild-type dnaN gene carried in a transducing phage encodes a polypeptide of about 41,000 daltons [Sakakibara, Y. & Mizukami, T. (1980) Mol. Gen. Genet. 178, 541-553; Yuasa, S. & Sakakibara, Y. (1980) Mol. Gen. Genet. 180, 267-273]. We now find that the product of dnaN gene is the beta subunit of DNA polymerase III holoenzyme, the principal DNA synthetic multipolypeptide complex in E. coli. The conclusion is based on the following observations: (i) Extracts from dnaN59 cells were defective in phage phi X174 and G4 DNA synthesis after the mutant cells had been exposed to the increased temperature. (ii) The enzymatic defect was overcome by addition of purified beta subunit but not by other subunits of DNA polymerase III holoenzyme or by other replication proteins required for phi X174 DNA synthesis. (iii) Partially purified beta subunit from the dnaN mutant, unlike that from the wild type, was inactive in reconstituting the holoenzyme when mixed with the other purified subunits. (iv) Increased dosage of the dnaN gene provided by a plasmid carrying the gene raised cellular levels of the beta subunit 5- to 6-fold.

Recognition of damaged DNA by Escherichia coli Fpg protein: insights from structural and kinetic data

International Nuclear Information System (INIS)

Zharkov, Dmitry O.; Ishchenko, Alexander A.; Douglas, Kenneth T.; Nevinsky, Georgy A.

2003-01-01

Formamidopyrimidine-DNA glycosylase (Fpg) excises oxidized purines from damaged DNA. The recent determination of the three-dimensional structure of the covalent complex of DNA with Escherichia coli Fpg, obtained by reducing the Schiff base intermediate formed during the reaction [Gilboa et al., J. Biol. Chem. 277 (2002) 19811] has revealed a number of potential specific and non-specific interactions between Fpg and DNA. We analyze the structural data for Fpg in the light of the kinetic and thermodynamic data obtained by the method of stepwise increase in ligand complexity to estimate relative contributions of individual nucleotide units of lesion-containing DNA to its total affinity for this enzyme [Ishchenko et al., Biochemistry 41 (2002) 7540]. Stopped-flow kinetic analysis that has allowed the dissection of Fpg catalysis in time [Fedorova et al., Biochemistry 41 (2002) 1520] is also correlated with the structural data

Benchmarking BarraCUDA on Epigenetic DNA and nVidia Pascal GPUs

OpenAIRE

Langdon, W

2016-01-01

Typically BarraCUDA uses CUDA graphics cards to map DNA reads to the human genome. Previously its software source code was genetically improved for short paired end next generation sequences. On longer, 150 base paired end noisy Cambridge Epigenetix's data, a Pascal GTX 1080 processes about 10000 strings per second, comparable with twin nVidia Tesla K40.

No evidence of Neandertal mtDNA contribution to early modern humans.

Directory of Open Access Journals (Sweden)

David Serre

2004-03-01

Full Text Available The retrieval of mitochondrial DNA (mtDNA sequences from four Neandertal fossils from Germany, Russia, and Croatia has demonstrated that these individuals carried closely related mtDNAs that are not found among current humans. However, these results do not definitively resolve the question of a possible Neandertal contribution to the gene pool of modern humans since such a contribution might have been erased by genetic drift or by the continuous influx of modern human DNA into the Neandertal gene pool. A further concern is that if some Neandertals carried mtDNA sequences similar to contemporaneous humans, such sequences may be erroneously regarded as modern contaminations when retrieved from fossils. Here we address these issues by the analysis of 24 Neandertal and 40 early modern human remains. The biomolecular preservation of four Neandertals and of five early modern humans was good enough to suggest the preservation of DNA. All four Neandertals yielded mtDNA sequences similar to those previously determined from Neandertal individuals, whereas none of the five early modern humans contained such mtDNA sequences. In combination with current mtDNA data, this excludes any large genetic contribution by Neandertals to early modern humans, but does not rule out the possibility of a smaller contribution.

A nucleotide-analogue-induced gain of function corrects the error-prone nature of human DNA polymerase iota.

Science.gov (United States)

Ketkar, Amit; Zafar, Maroof K; Banerjee, Surajit; Marquez, Victor E; Egli, Martin; Eoff, Robert L

2012-06-27

Y-family DNA polymerases participate in replication stress and DNA damage tolerance mechanisms. The properties that allow these enzymes to copy past bulky adducts or distorted template DNA can result in a greater propensity for them to make mistakes. Of the four human Y-family members, human DNA polymerase iota (hpol ι) is the most error-prone. In the current study, we elucidate the molecular basis for improving the fidelity of hpol ι through use of the fixed-conformation nucleotide North-methanocarba-2'-deoxyadenosine triphosphate (N-MC-dATP). Three crystal structures were solved of hpol ι in complex with DNA containing a template 2'-deoxythymidine (dT) paired with an incoming dNTP or modified nucleotide triphosphate. The ternary complex of hpol ι inserting N-MC-dATP opposite dT reveals that the adenine ring is stabilized in the anti orientation about the pseudo-glycosyl torsion angle, which mimics precisely the mutagenic arrangement of dGTP:dT normally preferred by hpol ι. The stabilized anti conformation occurs without notable contacts from the protein but likely results from constraints imposed by the bicyclo[3.1.0]hexane scaffold of the modified nucleotide. Unmodified dATP and South-MC-dATP each adopt syn glycosyl orientations to form Hoogsteen base pairs with dT. The Hoogsteen orientation exhibits weaker base-stacking interactions and is less catalytically favorable than anti N-MC-dATP. Thus, N-MC-dATP corrects the error-prone nature of hpol ι by preventing the Hoogsteen base-pairing mode normally observed for hpol ι-catalyzed insertion of dATP opposite dT. These results provide a previously unrecognized means of altering the efficiency and the fidelity of a human translesion DNA polymerase.

A nucleotide analogue induced gain of function corrects the error-prone nature of human DNA polymerase iota

Science.gov (United States)

Ketkar, Amit; Zafar, Maroof K.; Banerjee, Surajit; Marquez, Victor E.; Egli, Martin; Eoff, Robert L

2012-01-01

Y-family DNA polymerases participate in replication stress and DNA damage tolerance mechanisms. The properties that allow these enzymes to copy past bulky adducts or distorted template DNA can result in a greater propensity for them to make mistakes. Of the four human Y-family members, human DNA polymerase iota (hpol ι) is the most error-prone. In the current study, we elucidate the molecular basis for improving the fidelity of hpol ι through use of the fixed-conformation nucleotide North-methanocarba-2′-deoxyadenosine triphosphate (N-MC-dATP). Three crystal structures were solved of hpol ι in complex with DNA containing a template 2′-deoxythymidine (dT) paired with an incoming dNTP or modified nucleotide triphosphate. The ternary complex of hpol ι inserting N-MC-dATP opposite dT reveals that the adenine ring is stabilized in the anti orientation about the pseudo-glycosyl torsion angle (χ), which mimics precisely the mutagenic arrangement of dGTP:dT normally preferred by hpol ι. The stabilized anti conformation occurs without notable contacts from the protein but likely results from constraints imposed by the bicyclo[3.1.0]hexane scaffold of the modified nucleotide. Unmodified dATP and South-MC-dATP each adopt syn glycosyl orientations to form Hoogsteen base pairs with dT. The Hoogsteen orientation exhibits weaker base stacking interactions and is less catalytically favorable than anti N-MC-dATP. Thus, N-MC-dATP corrects the error-prone nature of hpol ι by preventing the Hoogsteen base-pairing mode normally observed for hpol ι-catalyzed insertion of dATP opposite dT. These results provide a previously unrecognized means of altering the efficiency and the fidelity of a human translesion DNA polymerase. PMID:22632140

FXR silencing in human colon cancer by DNA methylation and KRAS signaling.

Science.gov (United States)

Bailey, Ann M; Zhan, Le; Maru, Dipen; Shureiqi, Imad; Pickering, Curtis R; Kiriakova, Galina; Izzo, Julie; He, Nan; Wei, Caimiao; Baladandayuthapani, Veerabhadran; Liang, Han; Kopetz, Scott; Powis, Garth; Guo, Grace L

2014-01-01

Farnesoid X receptor (FXR) is a bile acid nuclear receptor described through mouse knockout studies as a tumor suppressor for the development of colon adenocarcinomas. This study investigates the regulation of FXR in the development of human colon cancer. We used immunohistochemistry of FXR in normal tissue (n = 238), polyps (n = 32), and adenocarcinomas, staged I-IV (n = 43, 39, 68, and 9), of the colon; RT-quantitative PCR, reverse-phase protein array, and Western blot analysis in 15 colon cancer cell lines; NR1H4 promoter methylation and mRNA expression in colon cancer samples from The Cancer Genome Atlas; DNA methyltransferase inhibition; methyl-DNA immunoprecipitation (MeDIP); bisulfite sequencing; and V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) knockdown assessment to investigate FXR regulation in colon cancer development. Immunohistochemistry and quantitative RT-PCR revealed that expression and function of FXR was reduced in precancerous lesions and silenced in a majority of stage I-IV tumors. FXR expression negatively correlated with phosphatidylinositol-4, 5-bisphosphate 3 kinase signaling and the epithelial-to-mesenchymal transition. The NR1H4 promoter is methylated in ~12% colon cancer The Cancer Genome Atlas samples, and methylation patterns segregate with tumor subtypes. Inhibition of DNA methylation and KRAS silencing both increased FXR expression. FXR expression is decreased early in human colon cancer progression, and both DNA methylation and KRAS signaling may be contributing factors to FXR silencing. FXR potentially suppresses epithelial-to-mesenchymal transition and other oncogenic signaling cascades, and restoration of FXR activity, by blocking silencing mechanisms or increasing residual FXR activity, represents promising therapeutic options for the treatment of colon cancer.

Amplification of a transcriptionally active DNA sequence in the human brain

International Nuclear Information System (INIS)

Yakovlev, A.G.; Sazonov, A.E.; Spunde, A.Ya.; Gindilis, V.M.

1986-01-01

The authors present their findings of tissue-specific amplification of a DNA fragment actively transcribed in the human brain. This genome fragment was found in the library complement of cDNA of the human brain and evidently belongs to a new class of moderate repetitions of DNA with an unstable copying capacity in the human genome. The authors isolated total cell RNA from various human tissues (brain, placenta), and rat tissues (brain, liver), by the method of hot phenol extraction with guanidine thiocynate. The poly(A + ) RNA fraction was isolated by chromatography. Synthesis of cDNA was done on a matrix of poly(A + ) RNA of human brain. The cDNA obtained was cloned in plasmid pBR322 for the PstI site using (dC/dG) sequences synthesized on the 3' ends of the vector molecule and cDNA respectively. In cloning 75 ng cDNA, the authors obtained approximately 10 5 recombinant. This library was analyzed by the hybridization method on columns with two radioactive ( 32 P) probes: the total cDNA preparation and the total nuclear DNA from the human brain. The number of copies of the cloned DNA fragment in the genome was determined by dot hybridization. Restricting fragments of human and rat DNA genomes homologous to the cloned cDNA were identified on radio-autographs. In each case, 10 micrograms of EcoRI DNA hydrolyzate was fractionated in 1% agarose gel. The probe was also readied with RNA samples fractionated in agarose gel with formaldehyde and transferred to a nitrocellulose filter under weak vacuum. The filter was hybridized with 0.1 micrograms DNA pAG 02, labeled with ( 32 P) to a specific activity of 0.5-1 x 10 9 counts/min x microgram. The autograph was exposed with amplifying screens at -70 0 C for 2 days

Cloning human DNA repair genes

International Nuclear Information System (INIS)

Jeggo, P.A.; Carr, A.M.; Lehmann, A.R.

1994-01-01

Many human genes involved in the repair of UV damage have been cloned using different procedures and they have been of great value in assisting the understanding of the mechanism of nucleotide excision-repair. Genes involved in repair of ionizing radiation damage have proved more difficult to isolate. Positional cloning has localized the XRCC5 gene to a small region of chromosome 2q33-35, and a series of yeast artificial chromosomes covering this region have been isolated. Very recent work has shown that the XRCC5 gene encodes the 80 kDa subunit of the Ku DNA-binding protein. The Ku80 gene also maps to this region. Studies with fission yeast have shown that radiation sensitivity can result not only from defective DNA repair but also from abnormal cell cycle control following DNA damage. Several genes involved in this 'check-point' control in fission yeast have been isolated and characterized in detail. It is likely that a similar checkpoint control mechanism exists in human cells. (author)

Variation of DNA damage levels in peripheral blood mononuclear cells isolated in different laboratories

DEFF Research Database (Denmark)

Godschalk, Roger W L; Ersson, Clara; Stępnik, Maciej

2014-01-01

This study investigated the levels of DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG) sensitive sites, as assessed by the comet assay, in peripheral blood mononuclear cells (PBMC) from healthy women from five different countries in Europe. The laboratory in each country (referred...... to as 'centre') collected and cryopreserved PBMC samples from three donors, using a standardised cell isolation protocol. The samples were analysed in 13 different laboratories for DNA damage, which is measured by the comet assay. The study aim was to assess variation in DNA damage in PBMC samples that were......%) by standardisation of the primary comet assay endpoint with calibration curve samples. The level of DNA strand breaks in the samples from two of the centres (0.56-0.61 lesions/10(6) bp) was significantly higher compared with the other three centres (0.41-0.45 lesions/10(6) bp). In contrast, there was no difference...

PFGE analysis of DNA double-strand breaks and DNA repair process in human osteosarcoma cells irradiated by X-ray

International Nuclear Information System (INIS)

Cao Jianping; Majima, H.; Yamaguchi, C.

2000-01-01

Objective: To study the induction of DNA double-strand breaks (DSBs) in human osteosarcoma cells irradiated by X-ray, the DNA DSBs repair process and the tumour cell radiosensitivity. Methods: Two cell lines of human osteosarcoma, Rho0 and 143. B were used. Initial DNA damage of DSBs by X-ray irradiation was measured using clamped homogeneous electrical field (CHEF) electrophoresis. Results: X-ray-induced DNA DSBs of human osteosarcoma cells after CHEF-electrophoresis increased linearly with the irradiation dose between 0 and 50 Gy. The repair of DNA DSBs in human osteosarcoma cells increased with the post-irradiation incubation time. In contrast to 14.3B cell line at the same dose point, much more DNA DSBs were induced in Rho0 cell line after X-ray irradiation. Conclusion: CHEF pulsed-field gel electrophoresis (PEGE) is a sensitive method for the determination of radiation-induced DNA DSBs in high molecular weight DNA of human osteosarcoma cells. Radiation-induced DNA DSBs of osteosarcoma increase with the dose in a linear manner. After incubation, both Rho0 cell line and 143. B cell line can repair the DNA DSBs. Between two cell lines of human osteosarcoma, Rho0 and 143.B, Rho0 cell line is more sensitive to ionizing radiation than 143.B line

DNA Sequences Proximal to Human Mitochondrial DNA Deletion Breakpoints Prevalent in Human Disease Form G-quadruplexes, a Class of DNA Structures Inefficiently Unwound by the Mitochondrial Replicative Twinkle Helicase*

Science.gov (United States)

Bharti, Sanjay Kumar; Sommers, Joshua A.; Zhou, Jun; Kaplan, Daniel L.; Spelbrink, Johannes N.; Mergny, Jean-Louis; Brosh, Robert M.

2014-01-01

Mitochondrial DNA deletions are prominent in human genetic disorders, cancer, and aging. It is thought that stalling of the mitochondrial replication machinery during DNA synthesis is a prominent source of mitochondrial genome instability; however, the precise molecular determinants of defective mitochondrial replication are not well understood. In this work, we performed a computational analysis of the human mitochondrial genome using the “Pattern Finder” G-quadruplex (G4) predictor algorithm to assess whether G4-forming sequences reside in close proximity (within 20 base pairs) to known mitochondrial DNA deletion breakpoints. We then used this information to map G4P sequences with deletions characteristic of representative mitochondrial genetic disorders and also those identified in various cancers and aging. Circular dichroism and UV spectral analysis demonstrated that mitochondrial G-rich sequences near deletion breakpoints prevalent in human disease form G-quadruplex DNA structures. A biochemical analysis of purified recombinant human Twinkle protein (gene product of c10orf2) showed that the mitochondrial replicative helicase inefficiently unwinds well characterized intermolecular and intramolecular G-quadruplex DNA substrates, as well as a unimolecular G4 substrate derived from a mitochondrial sequence that nests a deletion breakpoint described in human renal cell carcinoma. Although G4 has been implicated in the initiation of mitochondrial DNA replication, our current findings suggest that mitochondrial G-quadruplexes are also likely to be a source of instability for the mitochondrial genome by perturbing the normal progression of the mitochondrial replication machinery, including DNA unwinding by Twinkle helicase. PMID:25193669

A potential impact of DNA repair on ageing and lifespan in the ageing model organism Podospora anserina

DEFF Research Database (Denmark)

Soerensen, Mette; Gredilla, Ricardo; Müller-Ohldach, Mathis

2009-01-01

and hence contribute to ageing and lifespan control in this ageing model. Additionally, we find low DNA glycosylase activities in the long-lived mutants grisea and DeltaPaCox17::ble, which are characterized by low mitochondrial ROS generation. Overall, our data identify a potential role of mtDNA repair......The free radical theory of ageing states that ROS play a key role in age-related decrease in mitochondrial function via the damage of mitochondrial DNA (mtDNA), proteins and lipids. In the sexually reproducing ascomycete Podospora anserina ageing is, as in other eukaryotes, associated with mtDNA...... instability and mitochondrial dysfunction. Part of the mtDNA instabilities may arise due to accumulation of ROS induced mtDNA lesions, which, as previously suggested for mammals, may be caused by an age-related decrease in base excision repair (BER). Alignments of known BER protein sequences with the P...

DNA repair of UV photoproducts and mutagenesis in human mitochondrial DNA

International Nuclear Information System (INIS)

Pascucci, B.; Dogliotti, E.; Versteegh, A.; Hoffen, A. van; Zeeland, A.A. van; Mullenders, L.H.F.

1997-01-01

The induction and repair of DNA photolesions and mutations in the mitochondrial (mt) DNA of human cells in culture were analysed after cell exposure to UV-C light. The level of induction of cyclobutane pyrimidine dimers (CPD) in mitochondrial and nuclear DNA was comparable, while a higher frequency of pyrimidine (6-4) pyrimidone photoproducts (6-4 PP) was detected in mitochondrial than in nuclear DNA. Besides the known defect in CPD removal, mitochondria were shown to be deficient also in the excision of 6-4 PP. The effects of repair-defective conditions for the two major UV photolesions on mutagensis was assessed by analysing the frequency and spectrum of spontaneous and UV-induced mutations by restriction site mutation (RSM) method in a restriction endonuclease site, NciI (5'CCCGG3') located within the coding sequence of the mitochondrial gene for tRNA Leu . The spontaneous mutation frequency and spectrum at the NciI site of mitochondrial DNA was very similar to the RSM background mutation frequency (approximately 10 -5 ) and type (predominantly GC > AT transitions at GL 1 ) of the NciI site). Conversely, an approximately tenfold increase over background mutation frequency was recorded after cell exposure to 20 J/m 2 . In this case, the majority of mutations were C > T transitions preferentially located on the non-transcribed DNA strand at C 1 and C 2 of the NciI site. This mutation spectrum is expected by UV mutagenesis. This is the first evidence of induction of mutations in mitochondrial DNA by treatment of human cells with a carcinogen. (author)

DNA repair in PHA stimulated human lymphocytes

International Nuclear Information System (INIS)

Catena, C.; Mattoni, A.

1984-01-01

Damage an repair of radiation induced DNA strand breaks were measured by alkaline lysis and hydroxyapatite chromatography. PHA stimulated human lymphocytes show that the rejoining process is complete within the first 50 min., afterwords secondary DNA damage and chromatid aberration. DNA repair, in synchronized culture, allows to evaluate individual repair capacity and this in turn can contribute to the discovery of individual who, although they do not demonstrate apparent clinical signs, are carriers of DNA repair deficiency. Being evident that a correlation exists between DNA repair capacity and carcinogenesis, the possibility of evaluating the existent relationship between DNA repair and survival in tumor cells comes therefore into discussion

Oxidative DNA damage and its repair in rat spleen following subchronic exposure to aniline

International Nuclear Information System (INIS)

Ma Huaxian; Wang Jianling; Abdel-Rahman, Sherif Z.; Boor, Paul J.; Khan, M. Firoze

2008-01-01

The mechanisms by which aniline exposure elicits splenotoxic response, especially the tumorigenic response, are not well-understood. Splenotoxicity of aniline is associated with iron overload and generation of reactive oxygen species (ROS) which can cause oxidative damage to DNA, proteins and lipids (oxidative stress). 8-Hydroxy-2'-deoxyguanosine (8-OHdG) is one of the most abundant oxidative DNA lesions resulting from ROS, and 8-oxoguanine glycosylase 1 (OGG1), a specific DNA glycosylase/lyase enzyme, plays a key role in the removal of 8-OHdG adducts. This study focused on examining DNA damage (8-OHdG) and repair (OGG1) in the spleen in an experimental condition preceding a tumorigenic response. To achieve that, male Sprague-Dawley rats were subchronically exposed to aniline (0.5 mmol/kg/day via drinking water for 30 days), while controls received drinking water only. Aniline treatment led to a significant increase in splenic oxidative DNA damage, manifested as a 2.8-fold increase in 8-OHdG levels. DNA repair activity, measured as OGG1 base excision repair (BER) activity, increased by ∼ 1.3 fold in the nuclear protein extracts (NE) and ∼ 1.2 fold in the mitochondrial protein extracts (ME) of spleens from aniline-treated rats as compared to the controls. Real-time PCR analysis for OGG1 mRNA expression in the spleen revealed a 2-fold increase in expression in aniline-treated rats than the controls. Likewise, OGG1 protein expression in the NEs of spleens from aniline-treated rats was ∼ 1.5 fold higher, whereas in the MEs it was ∼ 1.3 fold higher than the controls. Aniline treatment also led to stronger immunostaining for both 8-OHdG and OGG1 in the spleens, confined to the red pulp areas. It is thus evident from our studies that aniline-induced oxidative stress is associated with increased oxidative DNA damage. The BER pathway was also activated, but not enough to prevent the accumulation of oxidative DNA damage (8-OHdG). Accumulation of mutagenic oxidative

Protective effects of folic acid on DNA damage and DNA methylation levels induced by N-methyl- N'-nitro- N-nitrosoguanidine in Kazakh esophageal epithelial cells.

Science.gov (United States)

Chen, Y; Feng, H; Chen, D; Abuduwaili, K; Li, X; Zhang, H

2018-01-01

The protective effects of folic acid on DNA damage and DNA methylation induced by N-methyl- N'-nitro- N-nitrosoguanidine (MNNG) in Kazakh esophageal epithelial cells were investigated using a 3 × 3 factorial design trial. The cells were cultured in vitro and exposed to media containing different concentrations of folic acid and MNNG, after which growth indices were detected. DNA damage levels were measured using comet assays, and genome-wide DNA methylation levels (MLs) were measured using high-performance liquid chromatography. The DNA methylation of methylenetetrahydrofolate reductase (MTHFR) and folate receptor- α (FR α) genes was detected by bisulfite sequencing polymerase chain reaction (PCR). The results showed significant increases in tail DNA concentration, tail length, and Olive tail moment ( p methylation frequencies of MTHFR and FR α genes. In particular, significant differences were observed in the promoter regions of both genes ( p methylation in Kazakh esophageal epithelial cells upon MNNG exposure. Thus, sufficient folic acid levels could play a protective role against the damage induced by this compound.

Human circulating plasma DNA significantly decreases while lymphocyte DNA damage increases under chronic occupational exposure to low-dose gamma-neutron and tritium β-radiation.

Science.gov (United States)

Korzeneva, Inna B; Kostuyk, Svetlana V; Ershova, Liza S; Osipov, Andrian N; Zhuravleva, Veronika F; Pankratova, Galina V; Porokhovnik, Lev N; Veiko, Natalia N

2015-09-01

The blood plasma of healthy people contains cell-fee (circulating) DNA (cfDNA). Apoptotic cells are the main source of the cfDNA. The cfDNA concentration increases in case of the organism's cell death rate increase, for example in case of exposure to high-dose ionizing radiation (IR). The objects of the present research are the blood plasma and blood lymphocytes of people, who contacted occupationally with the sources of external gamma/neutron radiation or internal β-radiation of tritium N = 176). As the controls (references), blood samples of people, who had never been occupationally subjected to the IR sources, were used (N = 109). With respect to the plasma samples of each donor there were defined: the cfDNA concentration (the cfDNA index), DNase1 activity (the DNase1 index) and titre of antibodies to DNA (the Ab DNA index). The general DNA damage in the cells was defined (using the Comet assay, the tail moment (TM) index). A chronic effect of the low-dose ionizing radiation on a human being is accompanied by the enhancement of the DNA damage in lymphocytes along with a considerable cfDNA content reduction, while the DNase1 content and concentration of antibodies to DNA (Ab DNA) increase. All the aforementioned changes were also observed in people, who had not worked with the IR sources for more than a year. The ratio cfDNA/(DNase1×Ab DNA × TM) is proposed to be used as a marker of the chronic exposure of a person to the external low-dose IR. It was formulated the assumption that the joint analysis of the cfDNA, DNase1, Ab DNA and TM values may provide the information about the human organism's cell resistivity to chronic exposure to the low-dose IR and about the development of the adaptive response in the organism that is aimed, firstly, at the effective cfDNA elimination from the blood circulation, and, secondly - at survival of the cells, including the cells with the damaged DNA. Copyright © 2015. Published by Elsevier B.V.

Pioglitazone retrieves hepatic antioxidant DNA repair in a mice model of high fat diet

Directory of Open Access Journals (Sweden)

Yang Ching-Hsiu

2008-09-01

Full Text Available Abstract Background Pioglitazone was reported to improve hepatic steatosis and necroinflammation in human studies. To investigate whether the hepato-protective effect of pioglitazone was associated with an improvement of antioxidant defense mechanism, oxidative DNA damage and repair activity were determined in a high fat diet model. Male C57BL/6 mice were respectively fed with a 30% fat diet, the same diet with pioglitazone 100 mg/kg/day, or a chow diet as control for 8 weeks. Tissue oxidative stress was indicated by malondialdehyde concentration. Oxidative DNA damage was detected by immunohistochemical 8-oxoG staining. Enzymatic antioxidant defense was detected by the real-time PCR of superoxide dismutase (Sod1, Sod2 and DNA glycosylase (Ogg1, MutY. Oxidative DNA repair was detected by immunohistochemical staining and western blotting of OGG1 expression. Results Our results show that hepatic steatosis was induced by a high-fat diet and improved by adding pioglitazone. Malondialdehyde concentration and 8-oxoG staining were strongly increased in the high-fat diet group, but attenuated by pioglitazone. Gene expressions of antioxidant defense mechanism: Sod1, Sod2, Ogg1 and MutY significantly decreased in the high-fat diet group but reversed by pioglitazone co-administration. Conclusion The attenuation of hepatic oxidative DNA damage by pioglitazone in a high-fat diet may be mediated by up-regulation of the antioxidant defense mechanism and oxidative DNA repair activity. The diminution of oxidative damage may explain the clinical benefit of pioglitazone treatment in patients with non-alcoholic fatty liver disease.

Pioglitazone retrieves hepatic antioxidant DNA repair in a mice model of high fat diet

Science.gov (United States)

Hsiao, Pi-Jung; Hsieh, Tusty-Jiuan; Kuo, Kung-Kai; Hung, Wei-Wen; Tsai, Kun-Bow; Yang, Ching-Hsiu; Yu, Ming-Lung; Shin, Shyi-Jang

2008-01-01

Background Pioglitazone was reported to improve hepatic steatosis and necroinflammation in human studies. To investigate whether the hepato-protective effect of pioglitazone was associated with an improvement of antioxidant defense mechanism, oxidative DNA damage and repair activity were determined in a high fat diet model. Male C57BL/6 mice were respectively fed with a 30% fat diet, the same diet with pioglitazone 100 mg/kg/day, or a chow diet as control for 8 weeks. Tissue oxidative stress was indicated by malondialdehyde concentration. Oxidative DNA damage was detected by immunohistochemical 8-oxoG staining. Enzymatic antioxidant defense was detected by the real-time PCR of superoxide dismutase (Sod1, Sod2) and DNA glycosylase (Ogg1, MutY). Oxidative DNA repair was detected by immunohistochemical staining and western blotting of OGG1 expression. Results Our results show that hepatic steatosis was induced by a high-fat diet and improved by adding pioglitazone. Malondialdehyde concentration and 8-oxoG staining were strongly increased in the high-fat diet group, but attenuated by pioglitazone. Gene expressions of antioxidant defense mechanism: Sod1, Sod2, Ogg1 and MutY significantly decreased in the high-fat diet group but reversed by pioglitazone co-administration. Conclusion The attenuation of hepatic oxidative DNA damage by pioglitazone in a high-fat diet may be mediated by up-regulation of the antioxidant defense mechanism and oxidative DNA repair activity. The diminution of oxidative damage may explain the clinical benefit of pioglitazone treatment in patients with non-alcoholic fatty liver disease. PMID:18822121

Cell Type-Specific Chromatin Signatures Underline Regulatory DNA Elements in Human Induced Pluripotent Stem Cells and Somatic Cells.

Science.gov (United States)

Zhao, Ming-Tao; Shao, Ning-Yi; Hu, Shijun; Ma, Ning; Srinivasan, Rajini; Jahanbani, Fereshteh; Lee, Jaecheol; Zhang, Sophia L; Snyder, Michael P; Wu, Joseph C

2017-11-10

Regulatory DNA elements in the human genome play important roles in determining the transcriptional abundance and spatiotemporal gene expression during embryonic heart development and somatic cell reprogramming. It is not well known how chromatin marks in regulatory DNA elements are modulated to establish cell type-specific gene expression in the human heart. We aimed to decipher the cell type-specific epigenetic signatures in regulatory DNA elements and how they modulate heart-specific gene expression. We profiled genome-wide transcriptional activity and a variety of epigenetic marks in the regulatory DNA elements using massive RNA-seq (n=12) and ChIP-seq (chromatin immunoprecipitation combined with high-throughput sequencing; n=84) in human endothelial cells (CD31 + CD144 + ), cardiac progenitor cells (Sca-1 + ), fibroblasts (DDR2 + ), and their respective induced pluripotent stem cells. We uncovered 2 classes of regulatory DNA elements: class I was identified with ubiquitous enhancer (H3K4me1) and promoter (H3K4me3) marks in all cell types, whereas class II was enriched with H3K4me1 and H3K4me3 in a cell type-specific manner. Both class I and class II regulatory elements exhibited stimulatory roles in nearby gene expression in a given cell type. However, class I promoters displayed more dominant regulatory effects on transcriptional abundance regardless of distal enhancers. Transcription factor network analysis indicated that human induced pluripotent stem cells and somatic cells from the heart selected their preferential regulatory elements to maintain cell type-specific gene expression. In addition, we validated the function of these enhancer elements in transgenic mouse embryos and human cells and identified a few enhancers that could possibly regulate the cardiac-specific gene expression. Given that a large number of genetic variants associated with human diseases are located in regulatory DNA elements, our study provides valuable resources for deciphering

Effects of Olive Metabolites on DNA Cleavage Mediated by Human Type II Topoisomerases

Science.gov (United States)

2016-01-01

Several naturally occurring dietary polyphenols with chemopreventive or anticancer properties are topoisomerase II poisons. To identify additional phytochemicals that enhance topoisomerase II-mediated DNA cleavage, a library of 341 Mediterranean plant extracts was screened for activity against human topoisomerase IIα. An extract from Phillyrea latifolia L., a member of the olive tree family, displayed high activity against the human enzyme. On the basis of previous metabolomics studies, we identified several polyphenols (hydroxytyrosol, oleuropein, verbascoside, tyrosol, and caffeic acid) as potential candidates for topoisomerase II poisons. Of these, hydroxytyrosol, oleuropein, and verbascoside enhanced topoisomerase II-mediated DNA cleavage. The potency of these olive metabolites increased 10–100-fold in the presence of an oxidant. Hydroxytyrosol, oleuropein, and verbascoside displayed hallmark characteristics of covalent topoisomerase II poisons. (1) The activity of the metabolites was abrogated by a reducing agent. (2) Compounds inhibited topoisomerase II activity when they were incubated with the enzyme prior to the addition of DNA. (3) Compounds were unable to poison a topoisomerase IIα construct that lacked the N-terminal domain. Because hydroxytyrosol, oleuropein, and verbascoside are broadly distributed across the olive family, extracts from the leaves, bark, and fruit of 11 olive tree species were tested for activity against human topoisomerase IIα. Several of the extracts enhanced enzyme-mediated DNA cleavage. Finally, a commercial olive leaf supplement and extra virgin olive oils pressed from a variety of Olea europea subspecies enhanced DNA cleavage mediated by topoisomerase IIα. Thus, olive metabolites appear to act as topoisomerase II poisons in complex formulations intended for human dietary consumption. PMID:26132160

DNA Methylation Landscapes of Human Fetal Development

NARCIS (Netherlands)

Slieker, Roderick C.; Roost, Matthias S.; van Iperen, Liesbeth; Suchiman, H. Eka D; Tobi, Elmar W.; Carlotti, Françoise; de Koning, Eelco J P; Slagboom, P. Eline; Heijmans, Bastiaan T.; Chuva de Sousa Lopes, Susana M.

2015-01-01

Remodelling the methylome is a hallmark of mammalian development and cell differentiation. However, current knowledge of DNA methylation dynamics in human tissue specification and organ development largely stems from the extrapolation of studies in vitro and animal models. Here, we report on the DNA

Human Chromosome 7: DNA Sequence and Biology

OpenAIRE

Scherer, Stephen W.; Cheung, Joseph; MacDonald, Jeffrey R.; Osborne, Lucy R.; Nakabayashi, Kazuhiko; Herbrick, Jo-Anne; Carson, Andrew R.; Parker-Katiraee, Layla; Skaug, Jennifer; Khaja, Razi; Zhang, Junjun; Hudek, Alexander K.; Li, Martin; Haddad, May; Duggan, Gavin E.

2003-01-01

DNA sequence and annotation of the entire human chromosome 7, encompassing nearly 158 million nucleotides of DNA and 1917 gene structures, are presented. To generate a higher order description, additional structural features such as imprinted genes, fragile sites, and segmental duplications were integrated at the level of the DNA sequence with medical genetic data, including 440 chromosome rearrangement breakpoints associated with disease. This approach enabled the discovery of candidate gene...

The single-strand DNA binding activity of human PC4 preventsmutagenesis and killing by oxidative DNA damage

Energy Technology Data Exchange (ETDEWEB)

Wang, Jen-Yeu; Sarker, Altaf Hossain; Cooper, Priscilla K.; Volkert, Michael R.

2004-02-01

Human positive cofactor 4 (PC4) is a transcriptional coactivator with a highly conserved single-strand DNA (ssDNA) binding domain of unknown function. We identified PC4 as a suppressor of the oxidative mutator phenotype of the Escherichia coli fpg mutY mutant and demonstrate that this suppression requires its ssDNA binding activity. Yeast mutants lacking their PC4 ortholog Sub1 are sensitive to hydrogen peroxide and exhibit spontaneous and peroxide induced hypermutability. PC4 expression suppresses the peroxide sensitivity of the yeast sub l{Delta} mutant, suggesting that the human protein has a similar function. A role for yeast and human proteins in DNA repair is suggested by the demonstration that Sub1 acts in a peroxide-resistance pathway involving Rad2 and by the physical interaction of PC4 with the human Rad2 homolog XPG. We show XPG recruits PC4 to a bubble-containing DNA substrate with resulting displacement of XPG and formation of a PC4-DNA complex. We discuss the possible requirement for PC4 in either global or transcription-coupled repair of oxidative DNA damage to mediate the release of XPG bound to its substrate.

Effects of ionizing radiations on DNA-protein complexes

International Nuclear Information System (INIS)

Gillard, N.

2005-11-01

The radio-induced destruction of DNA-protein complexes may have serious consequences for systems implicated in important cellular functions. The first system which has been studied is the lactose operon system, that regulates gene expression in Escherichia coli. First of all, the repressor-operator complex is destroyed after irradiation of the complex or of the protein alone. The damaging of the domain of repressor binding to DNA (headpiece) has been demonstrated and studied from the point of view of peptide chain integrity, conformation and amino acids damages. Secondly, dysfunctions of the in vitro induction of an irradiated repressor-unirradiated DNA complex have been observed. These perturbations, due to a decrease of the number of inducer binding sites, are correlated to the damaging of tryptophan residues. Moreover, the inducer protects the repressor when they are irradiated together, both by acting as a scavenger in the bulk, and by the masking of its binding site on the protein. The second studied system is formed by Fpg (for Formamido pyrimidine glycosylase), a DNA repair protein and a DNA with an oxidative lesion. The results show that irradiation disturbs the repair both by decreasing its efficiency of DNA lesion recognition and binding, and by altering its enzymatic activity. (author)

Characterization and immunological identification of cDNA clones encoding two human DNA topoisomerase II isozymes

International Nuclear Information System (INIS)

Chung, T.D.Y.; Drake, F.H.; Tan, K.B.; Per, S.R.; Crooke, S.T.; Mirabelli, C.K.

1989-01-01

Several DNA topoisomerase II partial cDNA clones obtained from a human Raji-HN2 cDNA library were sequenced and two classes of nucleotide sequences were found. One member of the first class, SP1, was identical to an internal fragment of human HeLa cell Topo II cDNA described earlier. A member of the second class, SP11, shared extensive nucleotide (75%) and predicted peptide (92%) sequence similarities with the first two-thirds of HeLa Topo II. Each class of cDNAs hybridized to unique, nonoverlapping restriction enzyme fragments of genomic DNA from several human cell lines. Synthetic 24-mer oligonucleotide probes specific for each cDNA class hybridized to 6.5-kilobase mRNAs; furthermore, hybridization of probe specific for one class was not blocked by probe specific for the other. Antibodies raised against a synthetic SP1-encoded dodecapeptide specifically recognized the 170-kDa form of Topo II, while antibodies raised against the corresponding SP11-encoded dodecapeptide, or a second unique SP11-encoded tridecapeptide, selectively recognized the 180-kDa form of Topo II. These data provide genetic and immunochemical evidence for two Topo II isozymes

DNA damage and repair activity after broccoli intake in young healthy smokers

DEFF Research Database (Denmark)

Riso, Patrizia; Martini, Daniela; Møller, Peter

2010-01-01

compounds, including smokers. The aim of the study was to evaluate the effect of broccoli intake on biomarkers of DNA damage and repair. Twenty-seven young healthy smokers consumed a portion of steamed broccoli (250 g/day) or a control diet for 10 days each within a crossover design with a washout period...... mRNA expression levels of repair and defence enzymes: 8-oxoguanine DNA glycosylase (OGG1), nucleoside diphosphate linked moiety X-type motif 1 (NUDT1) and heme oxygenase 1 (HO-1). After broccoli consumption, the level of oxidised DNA lesions decreased by 41% (95% confidence interval: 10%, 72......%) and the resistance to H(2)O(2)-induced DNA strand breaks increased by 23% (95% CI: 13%, 34%). Following broccoli intake, a higher protection was observed in subjects with glutathione S-transferase (GST) M1-null genotype. The expression level and activity of repair enzymes was unaltered. In conclusion, broccoli...

Increased oxidative DNA damage in mononuclear leukocytes in vitiligo

Energy Technology Data Exchange (ETDEWEB)

Giovannelli, Lisa [Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy)]. E-mail: lisag@pharm.unifi.it; Bellandi, Serena [Department of Dermatological Sciences, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy); Pitozzi, Vanessa [Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy); Fabbri, Paolo [Department of Dermatological Sciences, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy); Dolara, Piero [Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy); Moretti, Silvia [Department of Dermatological Sciences, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy)

2004-11-22

Vitiligo is an acquired pigmentary disorder of the skin of unknown aetiology. The autocytotoxic hypothesis suggests that melanocyte impairment could be related to increased oxidative stress. Evidences have been reported that in vitiligo oxidative stress might also be present systemically. We used the comet assay (single cell alkaline gel electrophoresis) to evaluate DNA strand breaks and DNA base oxidation, measured as formamidopyrimidine DNA glycosylase (FPG)-sensitive sites, in peripheral blood cells from patients with active vitiligo and healthy controls. The basal level of oxidative DNA damage in mononuclear leukocytes was increased in vitiligo compared to normal subjects, whereas DNA strand breaks (SBs) were not changed. This alteration was not accompanied by a different capability to respond to in vitro oxidative challenge. No differences in the basal levels of DNA damage in polymorphonuclear leukocytes were found between patients and healthy subjects. Thus, this study supports the hypothesis that in vitiligo a systemic oxidative stress exists, and demonstrates for the first time the presence of oxidative alterations at the nuclear level. The increase in oxidative DNA damage shown in the mononuclear component of peripheral blood leukocytes from vitiligo patients was not particularly severe. However, these findings support an adjuvant role of antioxidant treatment in vitiligo.

Increased oxidative DNA damage in mononuclear leukocytes in vitiligo

International Nuclear Information System (INIS)

Giovannelli, Lisa; Bellandi, Serena; Pitozzi, Vanessa; Fabbri, Paolo; Dolara, Piero; Moretti, Silvia

2004-01-01

Vitiligo is an acquired pigmentary disorder of the skin of unknown aetiology. The autocytotoxic hypothesis suggests that melanocyte impairment could be related to increased oxidative stress. Evidences have been reported that in vitiligo oxidative stress might also be present systemically. We used the comet assay (single cell alkaline gel electrophoresis) to evaluate DNA strand breaks and DNA base oxidation, measured as formamidopyrimidine DNA glycosylase (FPG)-sensitive sites, in peripheral blood cells from patients with active vitiligo and healthy controls. The basal level of oxidative DNA damage in mononuclear leukocytes was increased in vitiligo compared to normal subjects, whereas DNA strand breaks (SBs) were not changed. This alteration was not accompanied by a different capability to respond to in vitro oxidative challenge. No differences in the basal levels of DNA damage in polymorphonuclear leukocytes were found between patients and healthy subjects. Thus, this study supports the hypothesis that in vitiligo a systemic oxidative stress exists, and demonstrates for the first time the presence of oxidative alterations at the nuclear level. The increase in oxidative DNA damage shown in the mononuclear component of peripheral blood leukocytes from vitiligo patients was not particularly severe. However, these findings support an adjuvant role of antioxidant treatment in vitiligo

Dichromatic laser radiation effects on DNA of Escherichia coli and plasmids

Science.gov (United States)

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

2015-04-01

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

GC-Rich Extracellular DNA Induces Oxidative Stress, Double-Strand DNA Breaks, and DNA Damage Response in Human Adipose-Derived Mesenchymal Stem Cells.

Science.gov (United States)

Kostyuk, Svetlana; Smirnova, Tatiana; Kameneva, Larisa; Porokhovnik, Lev; Speranskij, Anatolij; Ershova, Elizaveta; Stukalov, Sergey; Izevskaya, Vera; Veiko, Natalia

2015-01-01

Cell free DNA (cfDNA) circulates throughout the bloodstream of both healthy people and patients with various diseases. CfDNA is substantially enriched in its GC-content as compared with human genomic DNA. Exposure of haMSCs to GC-DNA induces short-term oxidative stress (determined with H2DCFH-DA) and results in both single- and double-strand DNA breaks (comet assay and γH2AX, foci). As a result in the cells significantly increases the expression of repair genes (BRCA1 (RT-PCR), PCNA (FACS)) and antiapoptotic genes (BCL2 (RT-PCR and FACS), BCL2A1, BCL2L1, BIRC3, and BIRC2 (RT-PCR)). Under the action of GC-DNA the potential of mitochondria was increased. Here we show that GC-rich extracellular DNA stimulates adipocyte differentiation of human adipose-derived mesenchymal stem cells (haMSCs). Exposure to GC-DNA leads to an increase in the level of RNAPPARG2 and LPL (RT-PCR), in the level of fatty acid binding protein FABP4 (FACS analysis) and in the level of fat (Oil Red O). GC-rich fragments in the pool of cfDNA can potentially induce oxidative stress and DNA damage response and affect the direction of mesenchymal stem cells differentiation in human adipose-derived mesenchymal stem cells. Such a response may be one of the causes of obesity or osteoporosis.

Adenovirus 36 DNA in human adipose tissue.

Science.gov (United States)

Ponterio, E; Cangemi, R; Mariani, S; Casella, G; De Cesare, A; Trovato, F M; Garozzo, A; Gnessi, L

2015-12-01

Recent studies have suggested a possible correlation between obesity and adenovirus 36 (Adv36) infection in humans. As information on adenoviral DNA presence in human adipose tissue are limited, we evaluated the presence of Adv36 DNA in adipose tissue of 21 adult overweight or obese patients. Total DNA was extracted from adipose tissue biopsies. Virus detection was performed using PCR protocols with primers against specific Adv36 fiber protein and the viral oncogenic E4orf1 protein nucleotide sequences. Sequences were aligned with the NCBI database and phylogenetic analyses were carried out with MEGA6 software. Adv36 DNA was found in four samples (19%). This study indicates that some individuals carry Adv36 in the visceral adipose tissue. Further studies are needed to determine the specific effect of Adv36 infection on adipocytes, the prevalence of Adv36 infection and its relationship with obesity in the perspective of developing a vaccine that could potentially prevent or mitigate infection.

Isolation and characterization of full-length cDNA clones coding for cholinesterase from fetal human tissues

International Nuclear Information System (INIS)

Prody, C.A.; Zevin-Sonkin, D.; Gnatt, A.; Goldberg, O.; Soreq, H.

1987-01-01

To study the primary structure and regulation of human cholinesterases, oligodeoxynucleotide probes were prepared according to a consensus peptide sequence present in the active site of both human serum pseudocholinesterase and Torpedo electric organ true acetylcholinesterase. Using these probes, the authors isolated several cDNA clones from λgt10 libraries of fetal brain and liver origins. These include 2.4-kilobase cDNA clones that code for a polypeptide containing a putative signal peptide and the N-terminal, active site, and C-terminal peptides of human BtChoEase, suggesting that they code either for BtChoEase itself or for a very similar but distinct fetal form of cholinesterase. In RNA blots of poly(A) + RNA from the cholinesterase-producing fetal brain and liver, these cDNAs hybridized with a single 2.5-kilobase band. Blot hybridization to human genomic DNA revealed that these fetal BtChoEase cDNA clones hybridize with DNA fragments of the total length of 17.5 kilobases, and signal intensities indicated that these sequences are not present in many copies. Both the cDNA-encoded protein and its nucleotide sequence display striking homology to parallel sequences published for Torpedo AcChoEase. These finding demonstrate extensive homologies between the fetal BtChoEase encoded by these clones and other cholinesterases of various forms and species

Ancient pathogen DNA in human teeth and petrous bones

DEFF Research Database (Denmark)

Margaryan, Ashot; Hansen, Henrik B.; Rasmussen, Simon

2018-01-01

Recent ancient DNA (aDNA) studies of human pathogens have provided invaluable insights into their evolutionary history and prevalence in space and time. Most of these studies were based on DNA extracted from teeth or postcranial bones. In contrast, no pathogen DNA has been reported from the petro...

3-Nitrobenzanthrone (3-NBA) induced micronucleus formation and DNA damage in human hepatoma (HepG2) cells.

Science.gov (United States)

Lamy, Evelyn; Kassie, Fekadu; Gminski, Richard; Schmeiser, Heinz H; Mersch-Sundermann, Volker

2004-01-15

3-Nitrobenzanthrone (3-NBA), identified in diesel exhaust and in airborne particulate matter, is a potent mutagen in Salmonella, induces micronuclei formation in mice and in human cells and DNA adducts in rats. In the present study, we investigated the genotoxic potency of 3-NBA in human HepG2 cells using the micronucleus (MN) assay and the single cell gel electrophoresis (SCGE). 3-NBA caused a genotoxic effect at concentrations > or =12 nM in both assays. In the micronucleus assay, we found 98.7+/-10.3 MN/1000 BNC at a concentration of 100 nM 3-NBA in comparison to 27.3+/-0.6 MN/1000 BNC with the negative control. At the same concentration, the DNA-migration (SCGE) showed an Olive tail moment (OTM) of 2.7+/-0.45 and %DNA in the tail of 8.28+/-0.76; OTM and %DNA in the tail of cells treated with the negative control were 0.73+/-0.08 and 2.81+/-0.30, respectively. The results are discussed under consideration of former studies.

DNA repair by MGMT, but not AAG, causes a threshold in alkylation-induced colorectal carcinogenesis.

Science.gov (United States)

Fahrer, Jörg; Frisch, Janina; Nagel, Georg; Kraus, Alexander; Dörsam, Bastian; Thomas, Adam D; Reißig, Sonja; Waisman, Ari; Kaina, Bernd

2015-10-01

Epidemiological studies indicate that N-nitroso compounds (NOC) are causally linked to colorectal cancer (CRC). NOC induce DNA alkylations, including O (6)-methylguanine (O (6)-MeG) and N-methylated purines, which are repaired by O (6)-MeG-DNA methyltransferase (MGMT) and N-alkyladenine-DNA glycosylase (AAG)-initiated base excision repair, respectively. In view of recent evidence of nonlinear mutagenicity for NOC-like compounds, the question arises as to the existence of threshold doses in CRC formation. Here, we set out to determine the impact of DNA repair on the dose-response of alkylation-induced CRC. DNA repair proficient (WT) and deficient (Mgmt (-/-), Aag (-/-) and Mgmt (-/-)/Aag (-/-)) mice were treated with azoxymethane (AOM) and dextran sodium sulfate to trigger CRC. Tumors were quantified by non-invasive mini-endoscopy. A non-linear increase in CRC formation was observed in WT and Aag (-/-) mice. In contrast, a linear dose-dependent increase in tumor frequency was found in Mgmt (-/-) and Mgmt (-/-)/Aag (-/-) mice. The data were corroborated by hockey stick modeling, yielding similar carcinogenic thresholds for WT and Aag (-/-) and no threshold for MGMT lacking mice. O (6)-MeG levels and depletion of MGMT correlated well with the observed dose-response in CRC formation. AOM induced dose-dependently DNA double-strand breaks in colon crypts including Lgr5-positive colon stem cells, which coincided with ATR-Chk1-p53 signaling. Intriguingly, Mgmt (-/-) mice displayed significantly enhanced levels of γ-H2AX, suggesting the usefulness of γ-H2AX as an early genotoxicity marker in the colorectum. This study demonstrates for the first time a non-linear dose-response for alkylation-induced colorectal carcinogenesis and reveals DNA repair by MGMT, but not AAG, as a key node in determining a carcinogenic threshold. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Structure of human DNA polymerase iota and the mechanism of DNA synthesis.

Science.gov (United States)

Makarova, A V; Kulbachinskiy, A V

2012-06-01

Cellular DNA polymerases belong to several families and carry out different functions. Highly accurate replicative DNA polymerases play the major role in cell genome replication. A number of new specialized DNA polymerases were discovered at the turn of XX-XXI centuries and have been intensively studied during the last decade. Due to the special structure of the active site, these enzymes efficiently perform synthesis on damaged DNA but are characterized by low fidelity. Human DNA polymerase iota (Pol ι) belongs to the Y-family of specialized DNA polymerases and is one of the most error-prone enzymes involved in DNA synthesis. In contrast to other DNA polymerases, Pol ι is able to use noncanonical Hoogsteen interactions for nucleotide base pairing. This allows it to incorporate nucleotides opposite various lesions in the DNA template that impair Watson-Crick interactions. Based on the data of X-ray structural analysis of Pol ι in complexes with various DNA templates and dNTP substrates, we consider the structural peculiarities of the Pol ι active site and discuss possible mechanisms that ensure the unique behavior of the enzyme on damaged and undamaged DNA.

DNA and bone structure preservation in medieval human skeletons.

Science.gov (United States)

Coulson-Thomas, Yvette M; Norton, Andrew L; Coulson-Thomas, Vivien J; Florencio-Silva, Rinaldo; Ali, Nadir; Elmrghni, Samir; Gil, Cristiane D; Sasso, Gisela R S; Dixon, Ronald A; Nader, Helena B

2015-06-01

Morphological and ultrastructural data from archaeological human bones are scarce, particularly data that have been correlated with information on the preservation of molecules such as DNA. Here we examine the bone structure of macroscopically well-preserved medieval human skeletons by transmission electron microscopy and immunohistochemistry, and the quantity and quality of DNA extracted from these skeletons. DNA technology has been increasingly used for analyzing physical evidence in archaeological forensics; however, the isolation of ancient DNA is difficult since it is highly degraded, extraction yields are low and the co-extraction of PCR inhibitors is a problem. We adapted and optimised a method that is frequently used for isolating DNA from modern samples, Chelex(®) 100 (Bio-Rad) extraction, for isolating DNA from archaeological human bones and teeth. The isolated DNA was analysed by real-time PCR using primers targeting the sex determining region on the Y chromosome (SRY) and STR typing using the AmpFlSTR(®) Identifiler PCR Amplification kit. Our results clearly show the preservation of bone matrix in medieval bones and the presence of intact osteocytes with well preserved encapsulated nuclei. In addition, we show how effective Chelex(®) 100 is for isolating ancient DNA from archaeological bones and teeth. This optimised method is suitable for STR typing using kits aimed specifically at degraded and difficult DNA templates since amplicons of up to 250bp were successfully amplified. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Stability of the human sperm DNA methylome to folic acid fortification and short-term supplementation.

Science.gov (United States)

Chan, D; McGraw, S; Klein, K; Wallock, L M; Konermann, C; Plass, C; Chan, P; Robaire, B; Jacob, R A; Greenwood, C M T; Trasler, J M

2017-02-01

Do short-term and long-term exposures to low-dose folic acid supplementation alter DNA methylation in sperm? No alterations in sperm DNA methylation patterns were found following the administration of low-dose folic acid supplements of 400 μg/day for 90 days (short-term exposure) or when pre-fortification of food with folic acid and post-fortification sperm samples (long-term exposure) were compared. Excess dietary folate may be detrimental to health and DNA methylation profiles due to folate's role in one-carbon metabolism and the formation of S-adenosyl methionine, the universal methyl donor. DNA methylation patterns are established in developing male germ cells and have been suggested to be affected by high-dose (5 mg/day) folic acid supplementation. This is a control versus treatment study where genome-wide sperm DNA methylation patterns were examined prior to fortification of food (1996-1997) in men with no history of infertility at baseline and following 90-day exposure to placebo (n = 9) or supplement containing 400 μg folic acid/day (n = 10). Additionally, pre-fortification sperm DNA methylation profiles (n = 19) were compared with those of a group of post-fortification (post-2004) men (n = 8) who had been exposed for several years to dietary folic acid fortification. Blood and seminal plasma folate levels were measured in participants before and following the 90-day treatment with placebo or supplement. Sperm DNA methylation was assessed using the whole-genome and genome-wide techniques, MassArray epityper, restriction landmark genomic scanning, methyl-CpG immunoprecipitation and Illumina HumanMethylation450 Bead Array. Following treatment, supplemented individuals had significantly higher levels of blood and seminal plasma folates compared to placebo. Initial first-generation genome-wide analyses of sperm DNA methylation showed little evidence of changes when comparing pre- and post-treatment samples. With Illumina HumanMethylation450 BeadChip arrays

Structure and mechanism of human DNA polymerase [eta

Energy Technology Data Exchange (ETDEWEB)

Biertümpfel, Christian; Zhao, Ye; Kondo, Yuji; Ramón-Maiques, Santiago; Gregory, Mark; Lee, Jae Young; Masutani, Chikahide; Lehmann, Alan R.; Hanaoka, Fumio; Yang, Wei (Sussex); (NIH); (Gakushuin); (Osaka)

2010-11-03

The variant form of the human syndrome xeroderma pigmentosum (XPV) is caused by a deficiency in DNA polymerase {eta} (Pol{eta}), a DNA polymerase that enables replication through ultraviolet-induced pyrimidine dimers. Here we report high-resolution crystal structures of human Pol{eta} at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. Pol{eta} acts like a 'molecular splint' to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for two-metal ion catalysis. Two residues conserved among Pol{eta} orthologues form specific hydrogen bonds with the lesion and the incoming nucleotide to assist translesion synthesis. On the basis of the structures, eight Pol{eta} missense mutations causing XPV can be rationalized as undermining the molecular splint or perturbing the active-site alignment. The structures also provide an insight into the role of Pol{eta} in replicating through D loop and DNA fragile sites.

Human DNA polymerase delta double-mutant D316A;E318A interferes with DNA mismatch repair in vitro

DEFF Research Database (Denmark)

Liu, Dekang; Frederiksen, Jane H.; Liberti, Sascha Emilie

2017-01-01

DNA mismatch repair (MMR) is a highly-conserved DNA repair mechanism, whose primary role is to remove DNA replication errors preventing them from manifesting as mutations, thereby increasing the overall genome stability. Defects in MMR are associated with increased cancer risk in humans and other...... organisms. Here, we characterize the interaction between MMR and a proofreading-deficient allele of the human replicative DNA polymerase delta, PolδD316A;E318A, which has a higher capacity for strand displacement DNA synthesis than wild type Polδ. Human cell lines overexpressing PolδD316A;E318A display...

Smoking cessation reverses DNA double-strand breaks in human mononuclear cells.

Directory of Open Access Journals (Sweden)

Mari Ishida

Full Text Available OBJECTIVE: Cigarette smoking is a major risk factor for atherosclerotic cardiovascular disease, which is responsible for a significant proportion of smoking-related deaths. However, the precise mechanism whereby smoking induces this pathology has not been fully delineated. Based on observation of DNA double-strand breaks (DSBs, the most harmful type of DNA damage, in atherosclerotic lesions, we hypothesized that there is a direct association between smoking and DSBs. The goal of this study was to investigate whether smoking induces DSBs and smoking cessation reverses DSBs in vivo through examination of peripheral mononuclear cells (MNCs. APPROACH AND RESULTS: Immunoreactivity of oxidative modification of DNA and DSBs were increased in human atherosclerotic lesions but not in the adjacent normal area. DSBs in human MNCs isolated from the blood of volunteers can be detected as cytologically visible "foci" using an antibody against the phosphorylated form of the histone H2AX (γ-H2AX. Young healthy active smokers (n = 15 showed increased γ-H2AX foci number when compared with non-smokers (n = 12 (foci number/cell: median, 0.37/cell; interquartile range [IQR], 0.31-0.58 vs. 4.36/cell; IQR, 3.09-7.39, p<0.0001. Smoking cessation for 1 month reduced the γ-H2AX foci number (median, 4.44/cell; IQR, 4.36-5.24 to 0.28/cell; IQR, 0.12-0.53, p<0.05. A positive correlation was noted between γ-H2AX foci number and exhaled carbon monoxide levels (r = 0.75, p<0.01. CONCLUSIONS: Smoking induces DSBs in human MNCs in vivo, and importantly, smoking cessation for 1 month resulted in a decrease in DSBs to a level comparable to that seen in non-smokers. These data reinforce the notion that the cigarette smoking induces DSBs and highlight the importance of smoking cessation.

The DnaA N-terminal domain interacts with Hda to facilitate replicase clamp-mediated inactivation of DnaA.

Science.gov (United States)

Su'etsugu, Masayuki; Harada, Yuji; Keyamura, Kenji; Matsunaga, Chika; Kasho, Kazutoshi; Abe, Yoshito; Ueda, Tadashi; Katayama, Tsutomu

2013-12-01

DnaA activity for replication initiation of the Escherichia coli chromosome is negatively regulated by feedback from the DNA-loaded form of the replicase clamp. In this process, called RIDA (regulatory inactivation of DnaA), ATP-bound DnaA transiently assembles into a complex consisting of Hda and the DNA-clamp, which promotes inter-AAA+ domain association between Hda and DnaA and stimulates hydrolysis of DnaA-bound ATP, producing inactive ADP-DnaA. Using a truncated DnaA mutant, we previously demonstrated that the DnaA N-terminal domain is involved in RIDA. However, the precise role of the N-terminal domain in RIDA has remained largely unclear. Here, we used an in vitro reconstituted system to demonstrate that the Asn-44 residue in the N-terminal domain of DnaA is crucial for RIDA but not for replication initiation. Moreover, an assay termed PDAX (pull-down after cross-linking) revealed an unstable interaction between a DnaA-N44A mutant and Hda. In vivo, this mutant exhibited an increase in the cellular level of ATP-bound DnaA. These results establish a model in which interaction between DnaA Asn-44 and Hda stabilizes the association between the AAA+ domains of DnaA and Hda to facilitate DnaA-ATP hydrolysis during RIDA. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

GC-Rich Extracellular DNA Induces Oxidative Stress, Double-Strand DNA Breaks, and DNA Damage Response in Human Adipose-Derived Mesenchymal Stem Cells

Directory of Open Access Journals (Sweden)

Svetlana Kostyuk

2015-01-01

Full Text Available Background. Cell free DNA (cfDNA circulates throughout the bloodstream of both healthy people and patients with various diseases. CfDNA is substantially enriched in its GC-content as compared with human genomic DNA. Principal Findings. Exposure of haMSCs to GC-DNA induces short-term oxidative stress (determined with H2DCFH-DA and results in both single- and double-strand DNA breaks (comet assay and γH2AX, foci. As a result in the cells significantly increases the expression of repair genes (BRCA1 (RT-PCR, PCNA (FACS and antiapoptotic genes (BCL2 (RT-PCR and FACS, BCL2A1, BCL2L1, BIRC3, and BIRC2 (RT-PCR. Under the action of GC-DNA the potential of mitochondria was increased. Here we show that GC-rich extracellular DNA stimulates adipocyte differentiation of human adipose-derived mesenchymal stem cells (haMSCs. Exposure to GC-DNA leads to an increase in the level of RNAPPARG2 and LPL (RT-PCR, in the level of fatty acid binding protein FABP4 (FACS analysis and in the level of fat (Oil Red O. Conclusions. GC-rich fragments in the pool of cfDNA can potentially induce oxidative stress and DNA damage response and affect the direction of mesenchymal stem cells differentiation in human adipose—derived mesenchymal stem cells. Such a response may be one of the causes of obesity or osteoporosis.

DNA damage and repair in human skin in situ

International Nuclear Information System (INIS)

Sutherland, B.M.; Gange, R.W.; Freeman, S.E.; Sutherland, J.C.

1987-01-01

Understanding the molecular and cellular origins of sunlight-induced skin cancers in man requires knowledge of the damages inflicted on human skin during sunlight exposure, as well as the ability of cells in skin to repair or circumvent such damage. Although repair has been studied extensively in procaryotic and eucaryotic cells - including human cells in culture - there are important differences between repair by human skin cells in culture and human skin in situ: quantitative differences in rates of repair, as well as qualitative differences, including the presence or absence of repair mechanisms. Quantitation of DNA damage and repair in human skin required the development of new approaches for measuring damage at low levels in nanogram quantities of non-radioactive DNA. The method allows for analysis of multiple samples and the resulting data should be related to behavior of the DNA molecules by analytic expressions. Furthermore, it should be possible to assay a variety of lesions using the same methodology. The development of new analysis methods, new technology, and new biochemical probes for the study of DNA damage and repair are described. 28 refs., 4 figs

DNA damage and repair in human skin in situ

Energy Technology Data Exchange (ETDEWEB)

Sutherland, B.M.; Gange, R.W.; Freeman, S.E.; Sutherland, J.C.

1987-01-01

Understanding the molecular and cellular origins of sunlight-induced skin cancers in man requires knowledge of the damages inflicted on human skin during sunlight exposure, as well as the ability of cells in skin to repair or circumvent such damage. Although repair has been studied extensively in procaryotic and eucaryotic cells - including human cells in culture - there are important differences between repair by human skin cells in culture and human skin in situ: quantitative differences in rates of repair, as well as qualitative differences, including the presence or absence of repair mechanisms. Quantitation of DNA damage and repair in human skin required the development of new approaches for measuring damage at low levels in nanogram quantities of non-radioactive DNA. The method allows for analysis of multiple samples and the resulting data should be related to behavior of the DNA molecules by analytic expressions. Furthermore, it should be possible to assay a variety of lesions using the same methodology. The development of new analysis methods, new technology, and new biochemical probes for the study of DNA damage and repair are described. 28 refs., 4 figs.

Spontaneous unscheduled DNA synthesis in human lymphocytes

International Nuclear Information System (INIS)

Forell, B.; Myers, L.S. Jr.; Norman, A.

1979-01-01

The rate of spontaneous unscheduled DNA synthesis in human lymphocytes was estimated from measurements of tritiated thymidine incorporation into double-stranded DNA (ds-DNA) during incubation of cells in vitro. The contribution of scheduled DNA synthesis to the observed incorporation was reduced by inhibiting replication with hydroxyurea and by separating freshly replicated single-stranded DNA (ss-DNA) from repaired ds-DNA by column chromatography. The residual contribution of scheduled DNA synthesis was estimated by observing effects on thymidine incorporation of: (a) increasing the rate of production of apurinic sites, and alternatively, (b) increasing the number of cells in S-phase. Corrections based on estimates of endogenous pool size were also made. The rate of spontaneous unscheduled DNA synthesis is estimated to be 490 +- 120 thymidine molecules incorporated per cell per hour. These results compare favorably with estimates made from rates of depurination and depyrimidination of DNA, measured in molecular systems if we assume thymidine is incorporated by a short patch mechanism which incorporates an average of four bases per lesion

Screening Test for Shed Skin Cells by Measuring the Ratio of Human DNA to Staphylococcus epidermidis DNA.

Science.gov (United States)

Nakanishi, Hiroaki; Ohmori, Takeshi; Hara, Masaaki; Takahashi, Shirushi; Kurosu, Akira; Takada, Aya; Saito, Kazuyuki

2016-05-01

A novel screening method for shed skin cells by detecting Staphylococcus epidermidis (S. epidermidis), which is a resident bacterium on skin, was developed. Staphylococcus epidermidis was detected using real-time PCR. Staphylococcus epidermidis was detected in all 20 human skin surface samples. Although not present in blood and urine samples, S. epidermidis was detected in 6 of 20 saliva samples, and 5 of 18 semen samples. The ratio of human DNA to S. epidermidisDNA was significantly smaller in human skin surface samples than in saliva and semen samples in which S. epidermidis was detected. Therefore, although skin cells could not be identified by detecting only S. epidermidis, they could be distinguished by measuring the S. epidermidis to human DNA ratio. This method could be applied to casework touch samples, which suggests that it is useful for screening whether skin cells and human DNA are present on potential evidentiary touch samples. © 2016 American Academy of Forensic Sciences.

Endangered species: mitochondrial DNA loss as a mechanism of human disease.

Science.gov (United States)

Herrera, Alan; Garcia, Iraselia; Gaytan, Norma; Jones, Edith; Maldonado, Alicia; Gilkerson, Robert

2015-06-01

Human mitochondrial DNA (mtDNA) is a small maternally inherited DNA, typically present in hundreds of copies in a single human cell. Thus, despite its small size, the mitochondrial genome plays a crucial role in the metabolic homeostasis of the cell. Our understanding of mtDNA genotype-phenotype relationships is derived largely from studies of the classical mitochondrial neuromuscular diseases, in which mutations of mtDNA lead to compromised mitochondrial bioenergetic function, with devastating pathological consequences. Emerging research suggests that loss, rather than mutation, of mtDNA plays a major role across a range of prevalent human diseases, including diabetes mellitus, cardiovascular disease, and aging. Here, we examine the 'rules' of mitochondrial genetics and function, the clinical settings in which loss of mtDNA is an emerging pathogenic mechanism, and explore mtDNA damage and its consequences for the organellar network and cell at large. As extranuclear genetic material arrayed throughout the cell to support metabolism, mtDNA is increasingly implicated in a host of disease conditions, opening a range of exciting questions regarding mtDNA and its role in cellular homeostasis.

hSSB1 (NABP2/OBFC2B) is regulated by oxidative stress

OpenAIRE

Nicolas Paquet; Mark N. Adams; Nicholas W. Ashton; Christine Touma; Roland Gamsjaeger; Liza Cubeddu; Vincent Leong; Sam Beard; Emma Bolderson; Catherine H. Botting; Kenneth J. O’Byrne; Derek J. Richard

2016-01-01

The maintenance of genome stability is an essential cellular process to prevent the development of diseases including cancer. hSSB1 (NABP2/ OBFC2A) is a critical component of the DNA damage response where it participates in the repair of double-strand DNA breaks and in base excision repair of oxidized guanine residues (8-oxoguanine) by aiding the localization of the human 8-oxoguanine glycosylase (hOGG1) to damaged DNA. Here we demonstrate that following oxidative stress, hSSB1 is stabilized ...

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

Science.gov (United States)

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

2016-05-20

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

The nucleotide sequence of human transition protein 1 cDNA

Energy Technology Data Exchange (ETDEWEB)

Luerssen, H; Hoyer-Fender, S; Engel, W [Universitaet Goettingen (West Germany)

1988-08-11

The authors have screened a human testis cDNA library with an oligonucleotide of 81 mer prepared according to a part of the published nucleotide sequence of the rat transition protein TP 1. They have isolated a cDNA clone with the length of 441 bp containing the coding region of 162 bp for human transition protein 1. There is about 84% homology in the coding region of the sequence compared to rat. The human cDNA-clone encodes a polypeptide of 54 amino acids of which 7 are different to that of rat.

Replication stress and oxidative damage contribute to aberrant constitutive activation of DNA damage signalling in human gliomas

DEFF Research Database (Denmark)

Bartkova, J; Hamerlik, P; Stockhausen, Marie

2010-01-01

damage signalling in low- and high-grade human gliomas, and analyze the sources of such endogenous genotoxic stress. Based on analyses of human glioblastoma multiforme (GBM) cell lines, normal astrocytes and clinical specimens from grade II astrocytomas (n=41) and grade IV GBM (n=60), we conclude...... that the DDR machinery is constitutively activated in gliomas, as documented by phosphorylated histone H2AX (gammaH2AX), activation of the ATM-Chk2-p53 pathway, 53BP1 foci and other markers. Oxidative DNA damage (8-oxoguanine) was high in some GBM cell lines and many GBM tumors, while it was low in normal...... brain and grade II astrocytomas, despite the degree of DDR activation was higher in grade II tumors. Markers indicative of ongoing DNA replication stress (Chk1 activation, Rad17 phosphorylation, replication protein A foci and single-stranded DNA) were present in GBM cells under high- or low...

Inaccurate DNA synthesis in cell extracts of yeast producing active human DNA polymerase iota.

Science.gov (United States)

Makarova, Alena V; Grabow, Corinn; Gening, Leonid V; Tarantul, Vyacheslav Z; Tahirov, Tahir H; Bessho, Tadayoshi; Pavlov, Youri I

2011-01-31

Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn(2+) ions, can bypass some DNA lesions and misincorporates "G" opposite template "T" more frequently than incorporates the correct "A." We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of "G" versus "A" method of Gening, abbreviated as "misGvA"). We provide unambiguous proof of the "misGvA" approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The "misGvA" activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.

Inaccurate DNA synthesis in cell extracts of yeast producing active human DNA polymerase iota.

Directory of Open Access Journals (Sweden)

Alena V Makarova

2011-01-01

Full Text Available Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn(2+ ions, can bypass some DNA lesions and misincorporates "G" opposite template "T" more frequently than incorporates the correct "A." We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of "G" versus "A" method of Gening, abbreviated as "misGvA". We provide unambiguous proof of the "misGvA" approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The "misGvA" activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.

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.

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

Continued colonization of the human genome by mitochondrial DNA.

Directory of Open Access Journals (Sweden)

Miria Ricchetti

2004-09-01

Full Text Available Integration of mitochondrial DNA fragments into nuclear chromosomes (giving rise to nuclear DNA sequences of mitochondrial origin, or NUMTs is an ongoing process that shapes nuclear genomes. In yeast this process depends on double-strand-break repair. Since NUMTs lack amplification and specific integration mechanisms, they represent the prototype of exogenous insertions in the nucleus. From sequence analysis of the genome of Homo sapiens, followed by sampling humans from different ethnic backgrounds, and chimpanzees, we have identified 27 NUMTs that are specific to humans and must have colonized human chromosomes in the last 4-6 million years. Thus, we measured the fixation rate of NUMTs in the human genome. Six such NUMTs show insertion polymorphism and provide a useful set of DNA markers for human population genetics. We also found that during recent human evolution, Chromosomes 18 and Y have been more susceptible to colonization by NUMTs. Surprisingly, 23 out of 27 human-specific NUMTs are inserted in known or predicted genes, mainly in introns. Some individuals carry a NUMT insertion in a tumor-suppressor gene and in a putative angiogenesis inhibitor. Therefore in humans, but not in yeast, NUMT integrations preferentially target coding or regulatory sequences. This is indeed the case for novel insertions associated with human diseases and those driven by environmental insults. We thus propose a mutagenic phenomenon that may be responsible for a variety of genetic diseases in humans and suggest that genetic or environmental factors that increase the frequency of chromosome breaks provide the impetus for the continued colonization of the human genome by mitochondrial DNA.

DNA repair genes

International Nuclear Information System (INIS)

Morimyo, Mitsuoki

1995-01-01

Fission yeast S. pombe is assumed to be a good model for cloning of human DNA repair genes, because human gene is normally expressed in S. pombe and has a very similar protein sequence to yeast protein. We have tried to elucidate the DNA repair mechanisms of S. pombe as a model system for those of mammals. (J.P.N.)

Characterization of the cDNA encoding human nucleophosmin and studies of its role in normal and abnormal growth

International Nuclear Information System (INIS)

Chan, Waiyee; Liu, Qingrong; Borjigin, J.; Busch, H.; Rennert, O.M.; Tease, L.A.; Chan, Puikwong

1989-01-01

A cDNA encoding human nucleophosmin (protein B23) was obtained by screening a human placental cDNA library in δgtll first with monoclonal antibody to rat nucleophosmin and then with confirmed partial cDNA of human nucleophosmin as probes. The cDNA had 1,311 bp with a coding sequence encoding a protein of 294 amino acids. The identity of the cDNA was confirmed by the presence of encoded amino acid sequences identical with those determined by sequencing pure rat nucleophosmin (a total of 138 amino acids). The most striking feature of the sequence is an acidic cluster located in the middle of the molecule. The cluster consists of 26 Asp/Glu and 1 Phe and Ala. Comparison of human nucleophosmin and Xenopus nucleolar protein NO38 shows 64.3% sequence identity. The N-terminal 130 amino acids of human nucleophosmin also bear 50% identity with that of Xenopus nucleoplasmin. Northern blot analysis of rat liver total RNA with a partial nucleophosmin cDNA as probe demonstrated a homogeneous mRNA band of about 1.6 kb. Similar observations were made in hypertrophic rat liver and Novikoff hepatoma. When the protein levels were compared with Western blot immunoassays, Navikoff hepatoma showed 20 times more nucleophosmin, while only about 5 times more nucleophosmin was observed in hypertrophic rat liver than in unstimulated normal liver

Sperm DNA fragmentation affects epigenetic feature in human male pronucleus.

Science.gov (United States)

Rajabi, H; Mohseni-Kouchesfehani, H; Eslami-Arshaghi, T; Salehi, M

2018-02-01

To evaluate whether the sperm DNA fragmentation affects male pronucleus epigenetic factors, semen analysis was performed and DNA fragmentation was assessed by the method of sperm chromatin structure assay (SCSA). Human-mouse interspecies fertilisation was used to create human male pronucleus. Male pronucleus DNA methylation and H4K12 acetylation were evaluated by immunostaining. Results showed a significant positive correlation between the level of sperm DNA fragmentation and DNA methylation in male pronuclei. In other words, an increase in DNA damage caused an upsurge in DNA methylation. In the case of H4K12 acetylation, no correlation was detected between DNA damage and the level of histone acetylation in the normal group, but results for the group in which male pronuclei were derived from sperm cells with DNA fragmentation, increased DNA damage led to a decreased acetylation level. Sperm DNA fragmentation interferes with the active demethylation process and disrupts the insertion of histones into the male chromatin in the male pronucleus, following fertilisation. © 2017 Blackwell Verlag GmbH.

Activation of apurinic/apyrimidinic endonuclease in human cells by reactive oxygen species and its correlation with their adaptive response to genotoxicity of free radicals

Science.gov (United States)

Ramana, Chilakamarti V.; Boldogh, Istvan; Izumi, Tadahide; Mitra, Sankar

1998-01-01

Apurinic/apyrimidinic (AP) endonuclease (APE; EC 4.2.99.18) plays a central role in repair of DNA damage due to reactive oxygen species (ROS) because its DNA 3′-phosphoesterase activity removes 3′ blocking groups in DNA that are generated by DNA glycosylase/AP-lyases during removal of oxidized bases and by direct ROS reaction with DNA. The major human APE (APE-1) gene is activated selectively by sublethal levels of a variety of ROS and ROS generators, including ionizing radiation, but not by other genotoxicants—e.g., UV light and alkylating agents. Increased expression of APE mRNA and protein was observed both in the HeLa S3 tumor line and in WI 38 primary fibroblasts, and it was accompanied by translocation of the endonuclease to the nucleus. ROS-treated cells showed a significant increase in resistance to the cytotoxicity of such ROS generators as H2O2 and bleomycin, but not to UV light. This “adaptive response” appears to result from enhanced repair of cytotoxic DNA lesions due to an increased activity of APE-1, which may be limiting in the base excision repair process for ROS-induced toxic lesions. PMID:9560228

Genome-Wide Prediction of DNA Methylation Using DNA Composition and Sequence Complexity in Human.

Science.gov (United States)

Wu, Chengchao; Yao, Shixin; Li, Xinghao; Chen, Chujia; Hu, Xuehai

2017-02-16

DNA methylation plays a significant role in transcriptional regulation by repressing activity. Change of the DNA methylation level is an important factor affecting the expression of target genes and downstream phenotypes. Because current experimental technologies can only assay a small proportion of CpG sites in the human genome, it is urgent to develop reliable computational models for predicting genome-wide DNA methylation. Here, we proposed a novel algorithm that accurately extracted sequence complexity features (seven features) and developed a support-vector-machine-based prediction model with integration of the reported DNA composition features (trinucleotide frequency and GC content, 65 features) by utilizing the methylation profiles of embryonic stem cells in human. The prediction results from 22 human chromosomes with size-varied windows showed that the 600-bp window achieved the best average accuracy of 94.7%. Moreover, comparisons with two existing methods further showed the superiority of our model, and cross-species predictions on mouse data also demonstrated that our model has certain generalization ability. Finally, a statistical test of the experimental data and the predicted data on functional regions annotated by ChromHMM found that six out of 10 regions were consistent, which implies reliable prediction of unassayed CpG sites. Accordingly, we believe that our novel model will be useful and reliable in predicting DNA methylation.

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

Base excision repair in Archaea: back to the future in DNA repair.

Science.gov (United States)

Grasso, Stefano; Tell, Gianluca

2014-09-01

Together with Bacteria and Eukarya, Archaea represents one of the three domain of life. In contrast with the morphological difference existing between Archaea and Eukarya, these two domains are closely related. Phylogenetic analyses confirm this evolutionary relationship showing that most of the proteins involved in DNA transcription and replication are highly conserved. On the contrary, information is scanty about DNA repair pathways and their mechanisms. In the present review the most important proteins involved in base excision repair, namely glycosylases, AP lyases, AP endonucleases, polymerases, sliding clamps, flap endonucleases, and ligases, will be discussed and compared with bacterial and eukaryotic ones. Finally, possible applications and future perspectives derived from studies on Archaea and their repair pathways, will be taken into account. Copyright © 2014 Elsevier B.V. All rights reserved.

Cellular radiosensitivity and DNA damage in primary human fibroblasts

International Nuclear Information System (INIS)

Wurm, R.; Burnet, N.G.; Duggal, N.

1994-01-01

To evaluate the relationship between radiation-induced cell survival and DNA damage in primary human fibroblasts to decide whether the initial or residual DNA damage levels are more predictive of normal tissue cellular radiosensitivity. Five primary human nonsyndromic and two primary ataxia telangiectasia fibroblast strains grown in monolayer were studied. Cell survival was assessed by clonogenic assay. Irradiation was given at high dose rate (HDR) 1-2 Gy/min. DNA damage was measured in stationary phase cells and expressed as fraction released from the well by pulsed-field gel electrophoresis (PFGE). For initial damage, cells were embedded in agarose and irradiated at HDR on ice. Residual DNA damage was measured in monolayer by allowing a 4-h repair period after HDR irradiation. Following HDR irradiation, cell survival varied between SF 2 0.025 to 0.23. Measurement of initial DNA damage demonstrated linear induction up to 30 Gy, with small differences in the slope of the dose-response curve between strains. No correlation between cell survival and initial damage was found. Residual damage increased linearly up to 80 Gy with a variation in slope by a factor of 3.2. Cell survival correlated with the slope of the dose-response curves for residual damage of the different strains (p = 0.003). The relationship between radiation-induced cell survival and DNA damage in primary human fibroblasts of differing radiosensitivity is closest with the amount of DNA damage remaining after repair. If assays of DNA damage are to be used as predictors of normal tissue response to radiation, residual DNA damage provides the most likely correlation with cell survival. 52 refs., 5 figs., 2 tabs

Human DNA quantification and sample quality assessment: Developmental validation of the PowerQuant(®) system.

Science.gov (United States)

Ewing, Margaret M; Thompson, Jonelle M; McLaren, Robert S; Purpero, Vincent M; Thomas, Kelli J; Dobrowski, Patricia A; DeGroot, Gretchen A; Romsos, Erica L; Storts, Douglas R

2016-07-01

Quantification of the total amount of human DNA isolated from a forensic evidence item is crucial for DNA normalization prior to short tandem repeat (STR) DNA analysis and a federal quality assurance standard requirement. Previous commercial quantification methods determine the total human DNA and total human male DNA concentrations, but provide limited information about the condition of the DNA sample. The PowerQuant(®) System includes targets for quantification of total human and total human male DNA as well as targets for evaluating whether the human DNA is degraded and/or PCR inhibitors are present in the sample. A developmental validation of the PowerQuant(®) System was completed, following SWGDAM Validation Guidelines, to evaluate the assay's specificity, sensitivity, precision and accuracy, as well as the ability to detect degraded DNA or PCR inhibitors. In addition to the total human DNA and total human male DNA concentrations in a sample, data from the degradation target and internal PCR control (IPC) provide a forensic DNA analyst meaningful information about the quality of the isolated human DNA and the presence of PCR inhibitors in the sample that can be used to determine the most effective workflow and assist downstream interpretation. Copyright © 2016 The Author(s). Published by Elsevier Ireland Ltd.. All rights reserved.

In situ enzymology of DNA replication and ultraviolet-induced DNA repair synthesis in permeable human cells

International Nuclear Information System (INIS)

Dresler, S.; Frattini, M.G.; Robinson-Hill, R.M.

1988-01-01

Using permeable diploid human fibroblasts, the authors have studied the deoxyribonucleoside triphosphate concentration dependences of ultraviolet- (UV-) induced DNA repair synthesis and semiconservative DNA replication. In both cell types (AG1518 and IMR-90) examined, the apparent K m values for dCTP, dGTP, and dTTP for DNA replication were between 1.2 and 2.9 μM. For UV-induced DNA repair synthesis, the apparent K m values were substantially lower, ranging from 0.11 to 0.44 μM for AG1518 cells and from 0.06 to 0.24 μM for IMR-90 cells. Recent data implicate DNA polymerase δ in UV-induced repair synthesis and suggest that DNA polymerases α and δ are both involved in semiconservative replication. They measured K m values for dGTP and dTTP for polymerases α and δ, for comparison with the values for replication and repair synthesis. The deoxyribonucleotide K m values for DNA polymerase δ are much greater than the K m values for UV-induced repair synthesis, suggesting that when polymerase δ functions in DNA repair, its characteristics are altered substantially either by association with accessory proteins or by direct posttranslational modification. In contrast, the deoxyribonucleotide binding characteristics of the DNA replication machinery differ little from those of the isolated DNA polymerases. The K m values for UV-induced repair synthesis are 5-80-fold lower than deoxyribonucleotide concentrations that have been reported for intact cultured diploid human fibroblasts. For replication, however, the K m for dGTP is only slightly lower than the average cellular dGTP concentration that has been reported for exponentially growing human fibroblasts. This finding is consistent with the concept that nucleotide compartmentation is required for the attainment of high rates of DNA replication in vivo

Human uracil DNA N-glycosidase: studies in normal and repair defective cultured fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Kuhnlein, U; Lee, B; Linn, S

1978-01-01

Uracil DNA N-glycosidase, an enzyme which participates in the excision of uracil from DNA, was measured in extracts from fibroblast lines cultured from normal subjects, from several subjects with the genetic disease xeroderma pigmentosum, and from a subject with ataxia telangiectasia. The cell lines representative of complementation groups A and D of xeroderma pigmentosum and of ataxia telangiectasia had roughly the same level of activity as did the normal cells. On the other hand, cells from two xeroderma pigmentosum variants (XP4BE and XP13BE) had roughly half the normal level of activity, and cells from the heterozygous mother of XP4BE had an intermediate level of activity. In spite of these quantitative differences, no systematic alterations in reaction characteristics, apparent K/sub m/ for substrate, or purification characteristics were noted for enzyme from any of the lines. Thus a causal relationship, if any, between levels of activity and the disease symptoms is equivocal.

Oxidized DNA induces an adaptive response in human fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Kostyuk, Svetlana V., E-mail: svet.kostyuk@gmail.com [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation); Tabakov, Viacheslav J.; Chestkov, Valerij V.; Konkova, Marina S.; Glebova, Kristina V.; Baydakova, Galina V.; Ershova, Elizaveta S.; Izhevskaya, Vera L. [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation); Baranova, Ancha, E-mail: abaranov@gmu.edu [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation); Center for the Study of Chronic Metabolic Diseases, School of System Biology, George Mason University, Fairfax, VA 22030 (United States); Veiko, Natalia N. [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation)

2013-07-15

Highlights: • We describe the effects of gDNAOX on human fibroblasts cultivated in serum withdrawal conditions. • gDNAOX evokes an adaptive response in human fibroblasts. • gDNAOX increases the survival rates in serum starving cell populations. • gDNAOX enhances the survival rates in cell populations irradiated at 1.2 Gy dose. • gDNAOX up-regulates NRF2 and inhibits NF-kappaB-signaling. - Abstract: Cell-free DNA (cfDNA) released from dying cells contains a substantial proportion of oxidized nucleotides, thus, forming cfDNA{sup OX}. The levels of cfDNA{sup OX} are increased in the serum of patients with chronic diseases. Oxidation of DNA turns it into a stress signal. The samples of genomic DNA (gDNA) oxidized by H{sub 2}O{sub 2}in vitro (gDNA{sup OX}) induce effects similar to that of DNA released from damaged cells. Here we describe the effects of gDNA{sup OX} on human fibroblasts cultivated in the stressful conditions of serum withdrawal. In these cells, gDNA{sup OX} evokes an adaptive response that leads to an increase in the rates of survival in serum starving cell populations as well as in populations irradiated at the dose of 1.2 Gy. These effects are not seen in control populations of fibroblasts treated with non-modified gDNA. In particular, the exposure to gDNA{sup OX} leads to a decrease in the expression of the proliferation marker Ki-67 and an increase in levels of PSNA, a decrease in the proportion of subG1- and G2/M cells, a decrease in proportion of cells with double strand breaks (DSBs). Both gDNA{sup OX} and gDNA suppress the expression of DNA sensors TLR9 and AIM2 and up-regulate nuclear factor-erythroid 2 p45-related factor 2 (NRF2), while only gDNA{sup OX} inhibits NF-κB signaling. gDNA{sup OX} is a model for oxidized cfDNA{sup OX} that is released from the dying tumor cells and being carried to the distant organs. The systemic effects of oxidized DNA have to be taken into account when treating tumors. In particular, the damaged DNA

Extrachromosomal circles of satellite repeats and 5S ribosomal DNA in human cells

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Cohen Sarit

2010-03-01

Full Text Available Abstract Background Extrachomosomal circular DNA (eccDNA is ubiquitous in eukaryotic organisms and was detected in every organism tested, including in humans. A two-dimensional gel electrophoresis facilitates the detection of eccDNA in preparations of genomic DNA. Using this technique we have previously demonstrated that most of eccDNA consists of exact multiples of chromosomal tandemly repeated DNA, including both coding genes and satellite DNA. Results Here we report the occurrence of eccDNA in every tested human cell line. It has heterogeneous mass ranging from less than 2 kb to over 20 kb. We describe eccDNA homologous to human alpha satellite and the SstI mega satellite. Moreover, we show, for the first time, circular multimers of the human 5S ribosomal DNA (rDNA, similar to previous findings in Drosophila and plants. We further demonstrate structures that correspond to intermediates of rolling circle replication, which emerge from the circular multimers of 5S rDNA and SstI satellite. Conclusions These findings, and previous reports, support the general notion that every chromosomal tandem repeat is prone to generate eccDNA in eukryoric organisms including humans. They suggest the possible involvement of eccDNA in the length variability observed in arrays of tandem repeats. The implications of eccDNA on genome biology may include mechanisms of centromere evolution, concerted evolution and homogenization of tandem repeats and genomic plasticity.

Preliminary perspectives on DNA collection in anti-human trafficking efforts.

Science.gov (United States)

Katsanis, Sara H; Kim, Joyce; Minear, Mollie A; Chandrasekharan, Subhashini; Wagner, Jennifer K

2014-01-01

Forensic DNA methodologies have potential applications in the investigation of human trafficking cases. DNA and relationship testing may be useful for confirmation of biological relationship claims in immigration, identification of trafficked individuals who are missing persons, and family reunification of displaced individuals after mass disasters and conflicts. As these applications rely on the collection of DNA from non-criminals and potentially vulnerable individuals, questions arise as to how to address the ethical challenges of collection, security, and privacy of collected samples and DNA profiles. We administered a survey targeted to victims' advocates to gain preliminary understanding of perspectives regarding human trafficking definitions, DNA and sex workers, and perceived trust of authorities potentially involved in DNA collection. We asked respondents to consider the use of DNA for investigating adoption fraud, sex trafficking, and post-conflict child soldier cases. We found some key differences in perspectives on defining what qualifies as "trafficking." When we varied terminology between "sex worker" and "sex trafficking victim" we detected differences in perception on which authorities can be trusted. Respondents were supportive of the hypothetical models proposed to collect DNA. Most were favorable of DNA specimens being controlled by an authority outside of law enforcement. Participants voiced concerns focused on privacy, misuse of DNA samples and data, unintentional harms, data security, and infrastructure. These preliminary data indicate that while there is perceived value in programs to use DNA for investigating cases of human trafficking, these programs may need to consider levels of trust in authorities as their logistics are developed and implemented.

Cloning and sequencing of cDNA encoding human DNA topoisomerase II and localization of the gene to chromosome region 17q21-22

International Nuclear Information System (INIS)

Tsai-Pflugfelder, M.; Liu, L.F.; Liu, A.A.; Tewey, K.M.; Whang-Peng, J.; Knutsen, T.; Huebner, K.; Croce, C.M.; Wang, J.C.

1988-01-01

Two overlapping cDNA clones encoding human DNA topoisomerase II were identified by two independent methods. In one, a human cDNA library in phage λ was screened by hybridization with a mixed oligonucleotide probe encoding a stretch of seven amino acids found in yeast and Drosophila DNA topoisomerase II; in the other, a different human cDNA library in a λgt11 expression vector was screened for the expression of antigenic determinants that are recognized by rabbit antibodies specific to human DNA topoisomerase II. The entire coding sequences of the human DNA topoisomerase II gene were determined from these and several additional clones, identified through the use of the cloned human TOP2 gene sequences as probes. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is encoded by a single-copy gene. The location of the gene was mapped to chromosome 17q21-22 by in situ hybridization of a cloned fragment to metaphase chromosomes and by hybridization analysis with a panel of mouse-human hybrid cell lines, each retaining a subset of human chromosomes

Expression of a Human Cytochrome P450 in Yeast Permits Analysis of Pathways for Response to and Repair of Aflatoxin-Induced DNA Damage†

OpenAIRE

Guo, Yingying; Breeden, Linda L.; Zarbl, Helmut; Preston, Bradley D.; Eaton, David L.

2005-01-01

Aflatoxin B1 (AFB1) is a human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In humans, AFB1 is primarily bioactivated by cytochrome P450 1A2 (CYP1A2) and 3A4 to a genotoxic epoxide that forms N7-guanine DNA adducts. A series of yeast haploid mutants defective in DNA repair and cell cycle checkpoints were transformed with human CYP1A2 to investigate how these DNA adducts are repaired. Cell survival and mutagenesis following aflatoxin B1 treatment was assayed in str...

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

Science.gov (United States)

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

2013-11-01

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

Detection of extracellular genomic DNA scaffold in human thrombus

DEFF Research Database (Denmark)

Oklu, Rahmi; Albadawi, Hassan; Watkins, Michael T

2012-01-01

into thrombus remodeling. MATERIALS AND METHODS: Ten human thrombus samples were collected during cases of thrombectomy and open surgical repair of abdominal aortic aneurysms (five samples 1 y old). Additionally, an acute murine hindlimb ischemia model was created to evaluate...... thrombus samples in mice. Human sections were immunostained for the H2A/H2B/DNA complex, myeloperoxidase, fibrinogen, and von Willebrand factor. Mouse sections were immunostained with the H2A antibody. All samples were further evaluated after hematoxylin and eosin and Masson trichrome staining. RESULTS......: An extensive network of extracellular histone/DNA complex was demonstrated in the matrix of human ex vivo thrombus. This network is present throughout the highly cellular acute thrombus. However, in chronic thrombi, detection of the histone/DNA network was predominantly in regions of low collagen content...

Quantification and presence of human ancient DNA in burial place ...

African Journals Online (AJOL)

Quantification and presence of human ancient DNA in burial place remains of Turkey using real time polymerase chain reaction. ... A published real-time PCR assay, which allows for the combined analysis of nuclear or ancient DNA and mitochondrial DNA, was modified. This approach can be used for recovering DNA from ...

DNA repair in human cells

International Nuclear Information System (INIS)

Regan, J.D.; Carrier, W.L.; Kusano, I.; Furuno-Fukushi, I.; Dunn, W.C. Jr.; Francis, A.A.; Lee, W.H.

1982-01-01

Our primary objective is to elucidate the molecular events in human cells when cellular macromolecules such as DNA are damaged by radiation or chemical agents. We study and characterize (i) the sequence of DNA repair events, (ii) the various modalities of repair, (iii) the genetic inhibition of repair due to mutation, (iv) the physiological inhibition of repair due to mutation, (v) the physiological inhibition of repair due to biochemical inhibitors, and (vi) the genetic basis of repair. Our ultimate goals are to (i) isolate and analyze the repair component of the mutagenic and/or carcinogenic event in human cells, and (ii) elucidate the magnitude and significance of this repair component as it impinges on the practical problems of human irradiation or exposure to actual or potential chemical mutagens and carcinogens. The significance of these studies lies in (i) the ubiquitousness of repair (most organisms, including man, have several complex repair systems), (ii) the belief that mutagenic and carcinogenic events may arise only from residual (nonrepaired) lesions or that error-prone repair systems may be the major induction mechanisms of the mutagenic or carcinogenic event, and (iii) the clear association of repair defects and highly carcinogenic disease states in man [xeroderma pigmentosum (XP)

DNA methylation and healthy human aging.

Science.gov (United States)

Jones, Meaghan J; Goodman, Sarah J; Kobor, Michael S

2015-12-01

The process of aging results in a host of changes at the cellular and molecular levels, which include senescence, telomere shortening, and changes in gene expression. Epigenetic patterns also change over the lifespan, suggesting that epigenetic changes may constitute an important component of the aging process. The epigenetic mark that has been most highly studied is DNA methylation, the presence of methyl groups at CpG dinucleotides. These dinucleotides are often located near gene promoters and associate with gene expression levels. Early studies indicated that global levels of DNA methylation increase over the first few years of life and then decrease beginning in late adulthood. Recently, with the advent of microarray and next-generation sequencing technologies, increases in variability of DNA methylation with age have been observed, and a number of site-specific patterns have been identified. It has also been shown that certain CpG sites are highly associated with age, to the extent that prediction models using a small number of these sites can accurately predict the chronological age of the donor. Together, these observations point to the existence of two phenomena that both contribute to age-related DNA methylation changes: epigenetic drift and the epigenetic clock. In this review, we focus on healthy human aging throughout the lifetime and discuss the dynamics of DNA methylation as well as how interactions between the genome, environment, and the epigenome influence aging rates. We also discuss the impact of determining 'epigenetic age' for human health and outline some important caveats to existing and future studies. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

High quality DNA from human papillomavirus (HPV for PCR/RFLPs

Directory of Open Access Journals (Sweden)

Denise Wanderlei-Silva

2005-01-01

Full Text Available The analysis of DNA in clinical samples for a secure diagnostic has become indispensable nowadays. Techniques approaching isolation of high molecular weigth DNA of HPV could lead to efficient amplification and early clinical diagnosis of the virus DNA by PCR (polymerase chain reaction. We describe a fast, non-toxical, efficient and cheap method for DNA isolation of human papilloma virus (HPV from cervical smears using guanidine (DNAzol solution. A 450 bp DNA band correponding to the late region (L1 of the virus genome was detected by PCR, showing that the DNAzol extraction soluction generated a good viral DNA yield. The electrophoretic pattern after digestion with restriction endonucleases (RFLPs/PCR revealed the predominance of HPV-16 and HPV-33 in the samples from the State of Alagoas, Brazil.A detecção de DNA em amostras clínicas visando um diagnóstico mais seguro vem se tornando uma prática comum em laboratórios de análise clínica. Metodologias que objetivem o isolamento de DNA de alto peso molecular de HPV podem levar a uma amplificação precisa e diagnose precoce do DNA do vírus por PCR (reação de polimerase em cadeia. Nós descrevemos um método para o isolamento do DNA do vírus do papiloma humano de amostras cervicais utilizando o detergente guanidina (solução DNAzol. O método foi rápido, não-tóxico e eficiente. Uma banda de DNA de 450 pb correspondente à região tardia (L1 do genoma viral foi detectada por PCR, mostrando que a extração com DNAzol gerou quantidade suficiente de DNA para análise. O padrão eletroforético, após digestão com endonucleases de restrição (RFLPs/PCR, revelou predominância de HPV 16 e HPV-33 nas amostras no Estado de Alagoas, Brasil.

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...

Fidelity and mutational spectrum of Pfu DNA polymerase on a human mitochondrial DNA sequence.

Science.gov (United States)

André, P; Kim, A; Khrapko, K; Thilly, W G

1997-08-01

The study of rare genetic changes in human tissues requires specialized techniques. Point mutations at fractions at or below 10(-6) must be observed to discover even the most prominent features of the point mutational spectrum. PCR permits the increase in number of mutant copies but does so at the expense of creating many additional mutations or "PCR noise". Thus, each DNA sequence studied must be characterized with regard to the DNA polymerase and conditions used to avoid interpreting a PCR-generated mutation as one arising in human tissue. The thermostable DNA polymerase derived from Pyrococcus furiosus designated Pfu has the highest fidelity of any DNA thermostable polymerase studied to date, and this property recommends it for analyses of tissue mutational spectra. Here, we apply constant denaturant capillary electrophoresis (CDCE) to separate and isolate the products of DNA amplification. This new strategy permitted direct enumeration and identification of point mutations created by Pfu DNA polymerase in a 96-bp low melting domain of a human mitochondrial sequence despite the very low mutant fractions generated in the PCR process. This sequence, containing part of the tRNA glycine and NADH dehydrogenase subunit 3 genes, is the target of our studies of mitochondrial mutagenesis in human cells and tissues. Incorrectly synthesized sequences were separated from the wild type as mutant/wild-type heteroduplexes by sequential enrichment on CDCE. An artificially constructed mutant was used as an internal standard to permit calculation of the mutant fraction. Our study found that the average error rate (mutations per base pair duplication) of Pfu was 6.5 x 10(-7), and five of its more frequent mutations (hot spots) consisted of three transversions (GC-->TA, AT-->TA, and AT-->CG), one transition (AT-->GC), and one 1-bp deletion (in an AAAAAA sequence). To achieve an even higher sensitivity, the amount of Pfu-induced mutants must be reduced.

Cloning and characterization of the human colipase cDNA

International Nuclear Information System (INIS)

Lowe, M.E.; Rosenblum, J.L.; McEwen, P.; Strauss, A.W.

1990-01-01

Pancreatic lipase hydrolyzes dietary triglycerides to monoglycerides and fatty acids. In the presence of bile salts, the activity of pancreatic lipase is markedly decreased. The activity can be restored by the addition of colipase, a low molecular weight protein secreted by the pancreas. The action of pancreatic lipase in the gut lumen is dependent upon its interaction with colipase. As a first step in elucidating the molecular events governing the interaction of lipase and colipase with each other and with fatty acids, a cDNA encoding human colipase was isolated from a λgt11 cDNA library with a rabbit polyclonal anti-human colipase antibody. The full-length 525 bp cDNA contained an open reading frame encoding 112 amino acids, including a 17 amino acid signal peptide. The predicted sequence contains 100% of the published protein sequence for human colipase determined by chemical methods, but predicts the presence of five additional NH 2 -terminal amino acids and four additional COOH-terminal amino acids. Comparison of the predicted protein sequence with the known sequences of colipase from other species reveals regions of extensive identity. The authors report, for the first time, a cDNA for colipase. The cDNA predicts a human procolipase an suggests that there may also be processing at the COOH-terminus. The regions of identity with colipase from other species will aid in defining the interaction with lipase and lipids through site-specific mutagenesis

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

Science.gov (United States)

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

Directory of Open Access Journals (Sweden)

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.

Production of glycosylated physiologically normal human α1-antitrypsin by mouse fibroblasts modified by insertion of a human α1-antitrypsin cDNA using a retroviral vector

International Nuclear Information System (INIS)

Garver, R.I. Jr.; Chytil, A.; Karlsson, S.

1987-01-01

α 2 -Antitrypsin (α 1 AT) deficiency is a hereditary disorder characterized by reduced serum levels of α 1 AT, resulting in destruction of the lower respiratory tract by neutrophil elastase. As an approach to augment α 1 AT levels in this disorder with physiologically normal human α 1 AT, the authors have integrated a full-length normal human α 1 AT cDNA into the genome of mouse fibroblasts. To accomplish this, the retroviral vector N2 was modified by inserting the simian virus 40 early promoter followed by the α 1 AT cDNA. Southern analysis demonstrated that the intact cDNA was present in the genome of selected clones of the transfected murine fibroblasts psi2 and infected NIH 3T3. The clones produced three mRNA transcripts containing human α 1 AT sequences, secreted an α 1 AT molecule recognized by an anti-human α 1 AT antibody, with the same molecular mass as normal human α 1 AT and that complexed with and inhibited human neutrophil elastase. The psi2 produced α 1 AT was glycosylated, and when infused intravenously into mice, it had a serum half-life similar to normal α 1 AT purified from human plasma and markedly longer than that of nonglycosylated human α 1 AT cDNA-directed yeast-produced α 1 AT. These studies demonstrate the feasibility of using a retroviral vector to insert the normal human α 1 AT cDNA into non-α 1 AT-producing cells, resulting in the synthesis and secretion of physiologically normal α 1 AT

Organometallic DNA-B12 Conjugates as Potential Oligonucleotide Vectors: Synthesis and Structural and Binding Studies with Human Cobalamin-Transport Proteins.

Science.gov (United States)

Mutti, Elena; Hunger, Miriam; Fedosov, Sergey; Nexo, Ebba; Kräutler, Bernhard

2017-11-16

The synthesis and structural characterization of Co-(dN) 25 -Cbl (Cbl: cobalamin; dN: deoxynucleotide) and Co-(dN) 39 -Cbl, which are organometallic DNA-B 12 conjugates with single DNA strands consisting of 25 and 39 deoxynucleotides, respectively, and binding studies of these two DNA-Cbl conjugates to three homologous human Cbl transporting proteins, transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are reported. This investigation tests the suitability of such DNA-Cbls for the task of eventual in vivo oligonucleotide delivery. The binding of DNA-Cbl to TC, IF, and HC was investigated in competition with either a fluorescent Cbl derivative and Co-(dN) 25 -Cbl, or radiolabeled vitamin B 12 ( 57 Co-CNCbl) and Co-(dN) 25 -Cbl or Co-(dN) 39 -Cbl. Binding of the new DNA-Cbl conjugates was fast and tight with TC, but poorer with HC and IF, which extends a similar original finding with the simpler DNA-Cbl, Co-(dN) 18 -Cbl. The contrasting affinities of TC versus IF and HC for the DNA-Cbl conjugates are rationalized herein by a stepwise mechanism of Cbl binding. Critical contributions to overall affinity result from gradual conformational adaptations of the Cbl-binding proteins to the DNA-Cbl, which is first bound to the respective β domains. This transition is fast with TC, but slow with IF and HC, with which weaker binding results. The invariably tight interaction of the DNA-Cbl conjugates with TC makes the Cbl moiety a potential natural vector for the specific delivery of oligonucleotide loads from the blood into cells. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Capacity of ultraviolet-induced DNA repair in human glioma cells

Energy Technology Data Exchange (ETDEWEB)

Itoh, Hiroji

1987-04-01

A DNA repair abnormality is likely related to an increased incidence of neoplasms in several autosomal recessive diseases such as xeroderma pigmentosum, Fanconi's anemia, Bloom's syndrome and ataxia telangiectasia. In human glioma cells, however, there are only a few reports on DNA repair. In this study, an ultraviolet (UV)-induced DNA repair was examined systematically in many human glioma cells. Two human malignant glioma cell lines (MMG-851, U-251-MG) and 7 human glioma cell strains (4, benign; 3, malignant) of short term culture, in which glial fibrillary acidic protein (GFAP) staining were positive, were used. To investigate the capacity of DNA repair, UV sensitivity was determined by colony formation; excision repair by autoradiography and Cytosine Arabinoside (Ara-C) assay; and post-replication repair by the joining rate of newly synthesized DNA. As a result, the colony-forming abilities of malignant glioma cell lines were lower than those of normal human fibroblasts, but no difference was found between two malignant glioma cell lines. The excision repair of the malignant group (2 cell lines and 3 cell strains) was apparently lower than that of the benign group (4 cell strains). In two malignant glioma cell lines, the excision repair of MMG-851 was lower than that of U-251-MG, and the post-replication repair of MMG-851 was higher than that of U-251-MG. These results were considered to correspond well with colony-forming ability. The results indicate that there are some differences in each human malignant glioma cell in its UV-induced DNA repair mechanism, and that the excision repair of the malignant glioma cells is apparently lower than that of the benign glioma cells. These findings may be useful for diagnosis and treatment.

Identification of DNA repair genes in the human genome

International Nuclear Information System (INIS)

Hoeijmakers, J.H.J.; van Duin, M.; Westerveld, A.; Yasui, A.; Bootsma, D.

1986-01-01

To identify human DNA repair genes we have transfected human genomic DNA ligated to a dominant marker to excision repair deficient xeroderma pigmentosum (XP) and CHO cells. This resulted in the cloning of a human gene, ERCC-1, that complements the defect of a UV- and mitomycin-C sensitive CHO mutant 43-3B. The ERCC-1 gene has a size of 15 kb, consists of 10 exons and is located in the region 19q13.2-q13.3. Its primary transcript is processed into two mRNAs by alternative splicing of an internal coding exon. One of these transcripts encodes a polypeptide of 297 aminoacids. A putative DNA binding protein domain and nuclear location signal could be identified. Significant AA-homology is found between ERCC-1 and the yeast excision repair gene RAD10. 58 references, 6 figures, 1 table

Sequence of a cloned cDNA encoding human ribosomal protein S11

Energy Technology Data Exchange (ETDEWEB)

Lott, J B; Mackie, G A

1988-02-11

The authors have isolated a cloned cDNA that encodes human ribosomal protein (rp) S11 by screening a human fibroblast cDNA library with a labelled 204 bp DNA fragment encompassing residues 212-416 of pRS11, a rat rp Sll cDNA clone. The human rp S11 cloned cDNA consists of 15 residues of the 5' leader, the entire coding sequence and all 51 residues of the 3' untranslated region. The predicted amino acid sequence of 158 residues is identical to rat rpS11. The nucleotide sequence in the coding region differs, however, from that in rat in the first position in two codons and in the third position in 44 codons.

mtDNA variation predicts population size in humans and reveals a major Southern Asian chapter in human prehistory.

Science.gov (United States)

Atkinson, Quentin D; Gray, Russell D; Drummond, Alexei J

2008-02-01

The relative timing and size of regional human population growth following our expansion from Africa remain unknown. Human mitochondrial DNA (mtDNA) diversity carries a legacy of our population history. Given a set of sequences, we can use coalescent theory to estimate past population size through time and draw inferences about human population history. However, recent work has challenged the validity of using mtDNA diversity to infer species population sizes. Here we use Bayesian coalescent inference methods, together with a global data set of 357 human mtDNA coding-region sequences, to infer human population sizes through time across 8 major geographic regions. Our estimates of relative population sizes show remarkable concordance with the contemporary regional distribution of humans across Africa, Eurasia, and the Americas, indicating that mtDNA diversity is a good predictor of population size in humans. Plots of population size through time show slow growth in sub-Saharan Africa beginning 143-193 kya, followed by a rapid expansion into Eurasia after the emergence of the first non-African mtDNA lineages 50-70 kya. Outside Africa, the earliest and fastest growth is inferred in Southern Asia approximately 52 kya, followed by a succession of growth phases in Northern and Central Asia (approximately 49 kya), Australia (approximately 48 kya), Europe (approximately 42 kya), the Middle East and North Africa (approximately 40 kya), New Guinea (approximately 39 kya), the Americas (approximately 18 kya), and a second expansion in Europe (approximately 10-15 kya). Comparisons of relative regional population sizes through time suggest that between approximately 45 and 20 kya most of humanity lived in Southern Asia. These findings not only support the use of mtDNA data for estimating human population size but also provide a unique picture of human prehistory and demonstrate the importance of Southern Asia to our recent evolutionary past.

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

International Nuclear Information System (INIS)

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

1986-01-01

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

Identification of person and quantification of human DNA recovered from mosquitoes (Culicidae).

Science.gov (United States)

Curic, Goran; Hercog, Rajna; Vrselja, Zvonimir; Wagner, Jasenka

2014-01-01

Mosquitoes (Culicidae) are widespread insects and can be important in forensic context as a source of human DNA. In order to establish the quantity of human DNA in mosquitoes' gut after different post-feeding interval and for how long after taking a bloodmeal the human donor could be identified, 174 blood-engorged mosquitoes (subfamily Anophelinae and Culicinae) were captured, kept alive and sacrificed at 8h intervals. Human DNA was amplified using forensic PCR kits (Identifiler, MiniFiler, and Quantifiler). A full DNA profiles were obtained from all Culicinae mosquitoes (74/74) up to 48 h and profiling was successful up to 88 h after a bloodmeal. Duration of post-feeding interval had a significant negative effect on the possibility of obtaining a full profile (pfeeding interval. Culicinae mosquitoes are a suitable source of human DNA for forensic STR kits more than three days after a bloodmeal. Human DNA recovered from mosquito can be used for matching purposes and could be useful in revealing spatial and temporal relation of events that took place at the crime scene. Therefore, mosquitoes at the crime scene, dead or alive, could be a valuable piece of forensic evidence. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Identification of a mammalian nuclear factor and human cDNA-encoded proteins that recognize DNA containing apurinic sites

International Nuclear Information System (INIS)

Lenz, J.; Okenquist, S.A.; LoSardo, J.E.; Hamilton, K.K.; Doetsch, P.W.

1990-01-01

Damage to DNA can have lethal or mutagenic consequences for cells unless it is detected and repaired by cellular proteins. Repair depends on the ability of cellular factors to distinguish the damaged sites. Electrophoretic binding assays were used to identify a factor from the nuclei of mammalian cells that bound to DNA containing apurinic sites. A binding assay based on the use of β-galactosidase fusion proteins was subsequently used to isolate recombinant clones of human cDNAs that encoded apurinic DNA-binding proteins. Two distinct human cDNAs were identified that encoded proteins that bound apurinic DNA preferentially over undamaged, methylated, or UV-irradiated DNA. These approaches may offer a general method for the detection of proteins that recognize various types of DNA damage and for the cloning of genes encoding such proteins

Space Radiation Effects on Human Cells: Modeling DNA Breakage, DNA Damage Foci Distribution, Chromosomal Aberrations and Tissue Effects

Science.gov (United States)

Ponomarev, A. L.; Huff, J. L.; Cucinotta, F. A.

2011-01-01

Future long-tem space travel will face challenges from radiation concerns as the space environment poses health risk to humans in space from radiations with high biological efficiency and adverse post-flight long-term effects. Solar particles events may dramatically affect the crew performance, while Galactic Cosmic Rays will induce a chronic exposure to high-linear-energy-transfer (LET) particles. These types of radiation, not present on the ground level, can increase the probability of a fatal cancer later in astronaut life. No feasible shielding is possible from radiation in space, especially for the heavy ion component, as suggested solutions will require a dramatic increase in the mass of the mission. Our research group focuses on fundamental research and strategic analysis leading to better shielding design and to better understanding of the biological mechanisms of radiation damage. We present our recent effort to model DNA damage and tissue damage using computational models based on the physics of heavy ion radiation, DNA structure and DNA damage and repair in human cells. Our particular area of expertise include the clustered DNA damage from high-LET radiation, the visualization of DSBs (DNA double strand breaks) via DNA damage foci, image analysis and the statistics of the foci for different experimental situations, chromosomal aberration formation through DSB misrepair, the kinetics of DSB repair leading to a model-derived spectrum of chromosomal aberrations, and, finally, the simulation of human tissue and the pattern of apoptotic cell damage. This compendium of theoretical and experimental data sheds light on the complex nature of radiation interacting with human DNA, cells and tissues, which can lead to mutagenesis and carcinogenesis later in human life after the space mission.

An Improved Methodology to Overcome Key Issues in Human Fecal Metagenomic DNA Extraction

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Jitendra Kumar

2016-12-01

Full Text Available Microbes are ubiquitously distributed in nature, and recent culture-independent studies have highlighted the significance of gut microbiota in human health and disease. Fecal DNA is the primary source for the majority of human gut microbiome studies. However, further improvement is needed to obtain fecal metagenomic DNA with sufficient amount and good quality but low host genomic DNA contamination. In the current study, we demonstrate a quick, robust, unbiased, and cost-effective method for the isolation of high molecular weight (>23 kb metagenomic DNA (260/280 ratio >1.8 with a good yield (55.8 ± 3.8 ng/mg of feces. We also confirm that there is very low human genomic DNA contamination (eubacterial: human genomic DNA marker genes = 227.9:1 in the human feces. The newly-developed method robustly performs for fresh as well as stored fecal samples as demonstrated by 16S rRNA gene sequencing using 454 FLX+. Moreover, 16S rRNA gene analysis indicated that compared to other DNA extraction methods tested, the fecal metagenomic DNA isolated with current methodology retains species richness and does not show microbial diversity biases, which is further confirmed by qPCR with a known quantity of spike-in genomes. Overall, our data highlight a protocol with a balance between quality, amount, user-friendliness, and cost effectiveness for its suitability toward usage for culture-independent analysis of the human gut microbiome, which provides a robust solution to overcome key issues associated with fecal metagenomic DNA isolation in human gut microbiome studies.

Evaluating droplet digital PCR for the quantification of human genomic DNA: converting copies per nanoliter to nanograms nuclear DNA per microliter.

Science.gov (United States)

Duewer, David L; Kline, Margaret C; Romsos, Erica L; Toman, Blaza

2018-05-01

The highly multiplexed polymerase chain reaction (PCR) assays used for forensic human identification perform best when used with an accurately determined quantity of input DNA. To help ensure the reliable performance of these assays, we are developing a certified reference material (CRM) for calibrating human genomic DNA working standards. To enable sharing information over time and place, CRMs must provide accurate and stable values that are metrologically traceable to a common reference. We have shown that droplet digital PCR (ddPCR) limiting dilution end-point measurements of the concentration of DNA copies per volume of sample can be traceably linked to the International System of Units (SI). Unlike values assigned using conventional relationships between ultraviolet absorbance and DNA mass concentration, entity-based ddPCR measurements are expected to be stable over time. However, the forensic community expects DNA quantity to be stated in terms of mass concentration rather than entity concentration. The transformation can be accomplished given SI-traceable values and uncertainties for the number of nucleotide bases per human haploid genome equivalent (HHGE) and the average molar mass of a nucleotide monomer in the DNA polymer. This report presents the considerations required to establish the metrological traceability of ddPCR-based mass concentration estimates of human nuclear DNA. Graphical abstract The roots of metrological traceability for human nuclear DNA mass concentration results. Values for the factors in blue must be established experimentally. Values for the factors in red have been established from authoritative source materials. HHGE stands for "haploid human genome equivalent"; there are two HHGE per diploid human genome.

Repair of U/G and U/A in DNA by UNG2-associated repair complexes takes place predominantly by short-patch repair both in proliferating and growth-arrested cells

DEFF Research Database (Denmark)

Akbari, Mansour; Otterlei, Marit; Pena Diaz, Javier

2004-01-01

Nuclear uracil-DNA glycosylase UNG2 has an established role in repair of U/A pairs resulting from misincorporation of dUMP during replication. In antigen-stimulated B-lymphocytes UNG2 removes uracil from U/G mispairs as part of somatic hypermutation and class switch recombination processes. Using......, PCNA and DNA ligase, the latter detected as activity. Short-patch repair was the predominant mechanism both in extracts and UNG2-ARC from proliferating and less BER-proficient growth-arrested cells. Repair of U/G mispairs and U/A pairs was completely inhibited by neutralizing UNG...

Human RAD50 makes a functional DNA-binding complex.

Science.gov (United States)

Kinoshita, Eri; van Rossum-Fikkert, Sari; Sanchez, Humberto; Kertokalio, Aryandi; Wyman, Claire

2015-06-01

The MRE11-RAD50-NBS1 (MRN) complex has several distinct functions in DNA repair including important roles in both non-homologous end-joining (NHEJ) and homologous recombination (HR). The biochemical activities of MR(N) have been well characterized implying specific functional roles for the components. The arrangement of proteins in the complex implies interdependence of their biochemical activities making it difficult to separate specific functions. We obtained purified human RAD50 and observed that it binds ATP, undergoes ATP-dependent conformational changes as well as having ATPase activity. Scanning force microscopy analysis clearly showed that RAD50 binds DNA although not as oligomers. RAD50 alone was not functional in tethering DNA molecules. ATP increased formation of RAD50 multimers which were however globular lacking extended coiled coils, in contrast to the MR complex where ATP induced oligomers have obvious coiled coils protruding from a central domain. These results suggest that MRE11 is important in maintaining the structural arrangement of RAD50 in the protein complex and perhaps has a role in reinforcing proper alignment of the coiled coils in the ATP-bound state. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Caffeine and human DNA metabolism: the magic and the mystery

International Nuclear Information System (INIS)

Kaufmann, William K.; Heffernan, Timothy P.; Beaulieu, Lea M.; Doherty, Sharon; Frank, Alexandra R.; Zhou Yingchun; Bryant, Miriam F.; Zhou Tong; Luche, Douglas D.; Nikolaishvili-Feinberg, Nana; Simpson, Dennis A.; Cordeiro-Stone, Marila

2003-01-01

The ability of caffeine to reverse cell cycle checkpoint function and enhance genotoxicity after DNA damage was examined in telomerase-expressing human fibroblasts. Caffeine reversed the ATM-dependent S and G2 checkpoint responses to DNA damage induced by ionizing radiation (IR), as well as the ATR- and Chk1-dependent S checkpoint response to ultraviolet radiation (UVC). Remarkably, under conditions in which IR-induced G2 delay was reversed by caffeine, IR-induced G1 arrest was not. Incubation in caffeine did not increase the percentage of cells entering the S phase 6-8 h after irradiation; ATM-dependent phosphorylation of p53 and transactivation of p21 Cip1/Waf1 post-IR were resistant to caffeine. Caffeine alone induced a concentration- and time-dependent inhibition of DNA synthesis. It inhibited the entry of human fibroblasts into S phase by 70-80% regardless of the presence or absence of wildtype ATM or p53. Caffeine also enhanced the inhibition of cell proliferation induced by UVC in XP variant fibroblasts. This effect was reversed by expression of DNA polymerase η, indicating that translesion synthesis of UVC-induced pyrimidine dimers by DNA pol η protects human fibroblasts against UVC genotoxic effects even when other DNA repair functions are compromised by caffeine

Caffeine and human DNA metabolism: the magic and the mystery

Energy Technology Data Exchange (ETDEWEB)

Kaufmann, William K.; Heffernan, Timothy P.; Beaulieu, Lea M.; Doherty, Sharon; Frank, Alexandra R.; Zhou Yingchun; Bryant, Miriam F.; Zhou Tong; Luche, Douglas D.; Nikolaishvili-Feinberg, Nana; Simpson, Dennis A.; Cordeiro-Stone, Marila

2003-11-27

The ability of caffeine to reverse cell cycle checkpoint function and enhance genotoxicity after DNA damage was examined in telomerase-expressing human fibroblasts. Caffeine reversed the ATM-dependent S and G2 checkpoint responses to DNA damage induced by ionizing radiation (IR), as well as the ATR- and Chk1-dependent S checkpoint response to ultraviolet radiation (UVC). Remarkably, under conditions in which IR-induced G2 delay was reversed by caffeine, IR-induced G1 arrest was not. Incubation in caffeine did not increase the percentage of cells entering the S phase 6-8 h after irradiation; ATM-dependent phosphorylation of p53 and transactivation of p21{sup Cip1/Waf1} post-IR were resistant to caffeine. Caffeine alone induced a concentration- and time-dependent inhibition of DNA synthesis. It inhibited the entry of human fibroblasts into S phase by 70-80% regardless of the presence or absence of wildtype ATM or p53. Caffeine also enhanced the inhibition of cell proliferation induced by UVC in XP variant fibroblasts. This effect was reversed by expression of DNA polymerase {eta}, indicating that translesion synthesis of UVC-induced pyrimidine dimers by DNA pol {eta} protects human fibroblasts against UVC genotoxic effects even when other DNA repair functions are compromised by caffeine.

Understanding human DNA sequence variation.

Science.gov (United States)

Kidd, K K; Pakstis, A J; Speed, W C; Kidd, J R

2004-01-01

Over the past century researchers have identified normal genetic variation and studied that variation in diverse human populations to determine the amounts and distributions of that variation. That information is being used to develop an understanding of the demographic histories of the different populations and the species as a whole, among other studies. With the advent of DNA-based markers in the last quarter century, these studies have accelerated. One of the challenges for the next century is to understand that variation. One component of that understanding will be population genetics. We present here examples of many of the ways these new data can be analyzed from a population perspective using results from our laboratory on multiple individual DNA-based polymorphisms, many clustered in haplotypes, studied in multiple populations representing all major geographic regions of the world. These data support an "out of Africa" hypothesis for human dispersal around the world and begin to refine the understanding of population structures and genetic relationships. We are also developing baseline information against which we can compare findings at different loci to aid in the identification of loci subject, now and in the past, to selection (directional or balancing). We do not yet have a comprehensive understanding of the extensive variation in the human genome, but some of that understanding is coming from population genetics.

The N-terminus of RPA large subunit and its spatial position are important for the 5'->3' resection of DNA double-strand breaks.

Science.gov (United States)

Tammaro, Margaret; Liao, Shuren; McCane, Jill; Yan, Hong

2015-10-15

The first step of homology-dependent repair of DNA double-strand breaks (DSBs) is the resection of the 5' strand to generate 3' ss-DNA. Of the two major nucleases responsible for resection, EXO1 has intrinsic 5'->3' directionality, but DNA2 does not. DNA2 acts with RecQ helicases such as the Werner syndrome protein (WRN) and the heterotrimeric eukaryotic ss-DNA binding protein RPA. We have found that the N-terminus of the RPA large subunit (RPA1N) interacts with both WRN and DNA2 and is essential for stimulating WRN's 3'->5' helicase activity and DNA2's 5'->3' ss-DNA exonuclease activity. A mutant RPA complex that lacks RPA1N is unable to support resection in Xenopus egg extracts and human cells. Furthermore, relocating RPA1N to the middle subunit but not to the small subunit causes severe defects in stimulating DNA2 and WRN and in supporting resection. Together, these findings suggest that RPA1N and its spatial position are critical for restricting the directionality of the WRN-DNA2 resection pathway. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

The Conjugative Relaxase TrwC Promotes Integration of Foreign DNA in the Human Genome.

Science.gov (United States)

González-Prieto, Coral; Gabriel, Richard; Dehio, Christoph; Schmidt, Manfred; Llosa, Matxalen

2017-06-15

Bacterial conjugation is a mechanism of horizontal DNA transfer. The relaxase TrwC of the conjugative plasmid R388 cleaves one strand of the transferred DNA at the oriT gene, covalently attaches to it, and leads the single-stranded DNA (ssDNA) into the recipient cell. In addition, TrwC catalyzes site-specific integration of the transferred DNA into its target sequence present in the genome of the recipient bacterium. Here, we report the analysis of the efficiency and specificity of the integrase activity of TrwC in human cells, using the type IV secretion system of the human pathogen Bartonella henselae to introduce relaxase-DNA complexes. Compared to Mob relaxase from plasmid pBGR1, we found that TrwC mediated a 10-fold increase in the rate of plasmid DNA transfer to human cells and a 100-fold increase in the rate of chromosomal integration of the transferred DNA. We used linear amplification-mediated PCR and plasmid rescue to characterize the integration pattern in the human genome. DNA sequence analysis revealed mostly reconstituted oriT sequences, indicating that TrwC is active and recircularizes transferred DNA in human cells. One TrwC-mediated site-specific integration event was detected, proving that TrwC is capable of mediating site-specific integration in the human genome, albeit with very low efficiency compared to the rate of random integration. Our results suggest that TrwC may stabilize the plasmid DNA molecules in the nucleus of the human cell, probably by recircularization of the transferred DNA strand. This stabilization would increase the opportunities for integration of the DNA by the host machinery. IMPORTANCE Different biotechnological applications, including gene therapy strategies, require permanent modification of target cells. Long-term expression is achieved either by extrachromosomal persistence or by integration of the introduced DNA. Here, we studied the utility of conjugative relaxase TrwC, a bacterial protein with site

N-acetyl cysteine protects against ionizing radiation-induced DNA damage but not against cell killing in yeast and mammals

Energy Technology Data Exchange (ETDEWEB)

Reliene, Ramune [Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Department of Medicine, Center for Human Nutrition, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Pollard, Julianne M. [Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Biomedical Physics Interdepartmental Program, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Sobol, Zhanna; Trouiller, Benedicte [Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Gatti, Richard A. [Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Department of Human Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Schiestl, Robert H., E-mail: rschiestl@mednet.ucla.edu [Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Biomedical Physics Interdepartmental Program, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Department of Radiation Oncology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Department of Environmental Health Sciences, School of Public Health, University of California Los Angeles, Los Angeles, CA 90095 (United States)

2009-06-01

Ionizing radiation (IR) induces DNA strand breaks leading to cell death or deleterious genome rearrangements. In the present study, we examined the role of N-acetyl-L-cysteine (NAC), a clinically proven safe agent, for it's ability to protect against {gamma}-ray-induced DNA strand breaks and/or DNA deletions in yeast and mammals. In the yeast Saccharomyces cerevisiae, DNA deletions were scored by reversion to histidine prototrophy. Human lymphoblastoid cells were examined for the frequency of {gamma}-H2AX foci formation, indicative of DNA double strand break formation. DNA strand breaks were also measured in mouse peripheral blood by the alkaline comet assay. In yeast, NAC reduced the frequency of IR-induced DNA deletions. However, NAC did not protect against cell death. NAC also reduced {gamma}-H2AX foci formation in human lymphoblastoid cells but had no protective effect in the colony survival assay. NAC administration via drinking water fully protected against DNA strand breaks in mice whole-body irradiated with 1 Gy but not with 4 Gy. NAC treatment in the absence of irradiation was not genotoxic. These data suggest that, given the safety and efficacy of NAC in humans, NAC may be useful in radiation therapy to prevent radiation-mediated genotoxicity, but does not interfere with efficient cancer cell killing.

Enhanced capacity of DNA repair in human cytomegalovirus-infected cells

International Nuclear Information System (INIS)

Nishiyama, Y.; Rapp, F.

1981-01-01

Plaque formation in Vero cells by UV-irradiated herpes simplex virus was enhanced by infection with human cytomegalovirus (HCMV), UV irradiation, or treatment with methylmethanesulfonate. Preinfection of Vero cells with HCMV enhanced reactivation of UV-irradiated herpes simplex virus more significantly than did treatment with UV or methylmethanesulfonate alone. A similar enhancement by HCMV was observed in human embryonic fibroblasts, but not in xeroderma pigmentosum (XP12BE) cells. It was also found that HCMV infection enhanced hydroxyurea-resistant DNA synthesis induced by UV light or methylmethanesulfonate. Alkaline sucrose gradient sedimentation analysis revealed an enhanced rate of synthesis of all size classes of DNA in UV-irradiated HCMV-infected Vero cells. However, HCMV infection did not induce repairable lesions in cellular DNA and did not significantly inhibit host cell DNA synthesis, unlike UV or methylmethanesulfonate. These results indicate that HCMV enhanced DNA repair capacity in the host cells without producing detectable lesions in cellular DNA and without inhibiting DNA synthesis. This repair appeared to be error proof for UV-damaged herpes simplex virus DNA when tested with herpes simplex virus thymidine kinase-negative mutants

DNA-mediated gene transfer into human diploid fibroblasts derived from normal and ataxia-telangiectasia donors: parameters for DNA transfer and properties of DNA transformants

International Nuclear Information System (INIS)

Debenham, P.G.; Webb, M.B.T.; Masson, W.K.; Cox, R.

1984-01-01

An investigation was made of the feasibility of DNA-mediated gene transfer into human diploid fibroblasts derived from patients with the radiation sensitive syndrome ataxia-telangiectasia (A-T) and from a normal donor. Although they are markedly different in their growth characteristics, both normal and A-T strains give similar frequencies for DNA transfer in a model system using the recombinant plasmid pSV2-gpt. pSV2-gpt DNA transformants arise with a frequency between 10 -5 and 10 -4 per viable cell. Analysis of such transformants, although possible, is severely handicapped by the limited clonal life span of diploid human cells. Despite these problems it may be concluded that diploid human fibroblasts are competent recipients for DNA-mediated gene transfer and the putative repair deficiency of A-T does not markedly effect the efficiency of this process. (author)

DNA polymerases beta and lambda mediate overlapping and independent roles in base excision repair in mouse embryonic fibroblasts.

Directory of Open Access Journals (Sweden)

Elena K Braithwaite

2010-08-01

Full Text Available Base excision repair (BER is a DNA repair pathway designed to correct small base lesions in genomic DNA. While DNA polymerase beta (pol beta is known to be the main polymerase in the BER pathway, various studies have implicated other DNA polymerases in back-up roles. One such polymerase, DNA polymerase lambda (pol lambda, was shown to be important in BER of oxidative DNA damage. To further explore roles of the X-family DNA polymerases lambda and beta in BER, we prepared a mouse embryonic fibroblast cell line with deletions in the genes for both pol beta and pol lambda. Neutral red viability assays demonstrated that pol lambda and pol beta double null cells were hypersensitive to alkylating and oxidizing DNA damaging agents. In vitro BER assays revealed a modest contribution of pol lambda to single-nucleotide BER of base lesions. Additionally, using co-immunoprecipitation experiments with purified enzymes and whole cell extracts, we found that both pol lambda and pol beta interact with the upstream DNA glycosylases for repair of alkylated and oxidized DNA bases. Such interactions could be important in coordinating roles of these polymerases during BER.

Lack of evidence from HPLC 32P-post-labelling for tamoxifen-DNA adducts in the human endometrium.

Science.gov (United States)

Carmichael, P L; Sardar, S; Crooks, N; Neven, P; Van Hoof, I; Ugwumadu, A; Bourne, T; Tomas, E; Hellberg, P; Hewer, A J; Phillips, D H

1999-02-01

Tamoxifen is associated with an increased incidence of endometrial cancer in women. It is also a potent carcinogen in rat liver and forms covalent DNA adducts in this tissue. A previous study exploring DNA adducts in human endometria, utilizing thin layer chromatography 32P-postlabelling, found no evidence for adducts in tamoxifen-treated women [Carmichael,P.L., Ugwumadu,A.H.N., Neven,P., Hewer,A.J., Poon,G.K. and Phillips,D.H. (1996) Cancer Res., 56, 1475-1479]. However, subsequent work utilizing HPLC 32P-post-labelling [Hemminki,K., Ranjaniemi,H., Lindahl,B. and Moberger,B. (1996) Cancer Res., 56, 4374-4377] suggested that very low levels could be detected. We have sought to investigate this question further by reproducing the HPLC methodology at two centres, and analysing endometrial DNA from 20 patients treated with 20 mg/day tamoxifen for between 22 and 65 months. Liver DNA isolated from tamoxifen-treated rats was used as a positive control. We found no convincing evidence for tamoxifen-derived DNA adducts in human endometrium. HPLC elution profiles of post-labelled DNA from tamoxifen-treated women were indistinguishable from those obtained with DNA from 14 untreated women and from six women taking toremifene, an analogue of tamoxifen.

Preservation and rapid purification of DNA from decomposing human tissue samples.

Science.gov (United States)

Sorensen, Amy; Rahman, Elizabeth; Canela, Cassandra; Gangitano, David; Hughes-Stamm, Sheree

2016-11-01

One of the key features to be considered in a mass disaster is victim identification. However, the recovery and identification of human remains are sometimes complicated by harsh environmental conditions, limited facilities, loss of electricity and lack of refrigeration. If human remains cannot be collected, stored, or identified immediately, bodies decompose and DNA degrades making genotyping more difficult and ultimately decreasing DNA profiling success. In order to prevent further DNA damage and degradation after collection, tissue preservatives may be used. The goal of this study was to evaluate three customized (modified TENT, DESS, LST) and two commercial DNA preservatives (RNAlater and DNAgard ® ) on fresh and decomposed human skin and muscle samples stored in hot (35°C) and humid (60-70% relative humidity) conditions for up to three months. Skin and muscle samples were harvested from the thigh of three human cadavers placed outdoors for up to two weeks. In addition, the possibility of purifying DNA directly from the preservative solutions ("free DNA") was investigated in order to eliminate lengthy tissue digestion processes and increase throughput. The efficiency of each preservative was evaluated based on the quantity of DNA recovered from both the "free DNA" in solution and the tissue sample itself in conjunction with the quality and completeness of downstream STR profiles. As expected, DNA quantity and STR success decreased with time of decomposition. However, a marked decrease in DNA quantity and STR quality was observed in all samples after the bodies entered the bloat stage (approximately six days of decomposition in this study). Similar amounts of DNA were retrieved from skin and muscle samples over time, but slightly more complete STR profiles were obtained from muscle tissue. Although higher amounts of DNA were recovered from tissue samples than from the surrounding preservative, the average number of reportable alleles from the "free DNA" was

Assessment of okadaic acid effects on cytotoxicity, DNA damage and DNA repair in human cells.

Science.gov (United States)

Valdiglesias, Vanessa; Méndez, Josefina; Pásaro, Eduardo; Cemeli, Eduardo; Anderson, Diana; Laffon, Blanca

2010-07-07

Okadaic acid (OA) is a phycotoxin produced by several types of dinoflagellates causing diarrheic shellfish poisoning (DSP) in humans. Symptoms induced by DSP toxins are mainly gastrointestinal, but the intoxication does not appear to be fatal. Despite this, this toxin presents a potential threat to human health even at concentrations too low to induce acute toxicity, since previous animal studies have shown that OA has very potent tumour promoting activity. However, its concrete action mechanism has not been described yet and the results reported with regard to OA cytotoxicity and genotoxicity are often contradictory. In the present study, the genotoxic and cytotoxic effects of OA on three different types of human cells (peripheral blood leukocytes, HepG2 hepatoma cells, and SHSY5Y neuroblastoma cells) were evaluated. Cells were treated with a range of OA concentrations in the presence and absence of S9 fraction, and MTT test and Comet assay were performed in order to evaluate cytotoxicity and genotoxicity, respectively. The possible effects of OA on DNA repair were also studied by means of the DNA repair competence assay, using bleomycin as DNA damage inductor. Treatment with OA in absence of S9 fraction induced not statistically significant decrease in cell viability and significant increase in DNA damage in all cell types at the highest concentrations investigated. However, only SHSY5Y cells showed OA induced genotoxic and cytotoxic effects in presence of S9 fraction. Furthermore, we found that OA can induce modulations in DNA repair processes when exposure was performed prior to BLM treatment, in co-exposure, or during the subsequent DNA repair process. Copyright 2010 Elsevier B.V. All rights reserved.

Formation of covalent complexes between human O sup 6 -alkylguanine-DNA alkyltransferase and BCNU-treated defined length synthetic oligodeoxynucleotides

Energy Technology Data Exchange (ETDEWEB)

Brent, T.P.; Remack, J.S. (St. Jude Children' s Research Hospital, Memphis, TN (USA))

1988-07-25

Repair of chloroethylnitrosourea (CENU)-induced precursors of DNA interstrand cross-links by O{sup 6}-alkylguanine-DNA alkyltransferase (GAT or GATase) appears to be a factor in tumor resistance to therapy with this class of antineoplastic drugs. Since human GAT is highly specific for O{sup 6}-guanine, yet the probably cross-link structure is N{prime}-Guanine N{sup 3}cytosine ethane, rearrangement of the initial O{sup 6}-guanine adduct via O{sup 6},N{sup 1}ethanoguanine has been proposed. The authors suggested that GAT reaction with this intermediate would produce DNA covalently linked to protein through an ethane link from N{sup 1}-guanine to the alkylacceptor site on GAT. In preliminary studies they demonstrated a covalent complex between GAT and carmustine (BCNU)-treated DNA by a precipitation assay method. They have now developed a method for isolating the reaction product of BCNU-treated synthetic 14-mer ({sup 32}P)-labeled oligodeoxynucleotide and GAT using polyacrylamide gel electrophoresis. This approach can be used to characterize the adducts induced by CENUs that lead to complex formation with GAT.

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