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

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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

High-resolution analysis of cytosine methylation in ancient DNA.

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Bastien Llamas

Full Text Available Epigenetic changes to gene expression can result in heritable phenotypic characteristics that are not encoded in the DNA itself, but rather by biochemical modifications to the DNA or associated chromatin proteins. Interposed between genes and environment, these epigenetic modifications can be influenced by environmental factors to affect phenotype for multiple generations. This raises the possibility that epigenetic states provide a substrate for natural selection, with the potential to participate in the rapid adaptation of species to changes in environment. Any direct test of this hypothesis would require the ability to measure epigenetic states over evolutionary timescales. Here we describe the first single-base resolution of cytosine methylation patterns in an ancient mammalian genome, by bisulphite allelic sequencing of loci from late Pleistocene Bison priscus remains. Retrotransposons and the differentially methylated regions of imprinted loci displayed methylation patterns identical to those derived from fresh bovine tissue, indicating that methylation patterns are preserved in the ancient DNA. Our findings establish the biochemical stability of methylated cytosines over extensive time frames, and provide the first direct evidence that cytosine methylation patterns are retained in DNA from ancient specimens. The ability to resolve cytosine methylation in ancient DNA provides a powerful means to study the role of epigenetics in evolution.

Roles of DNA methyltransferases in Arabidopsis development ...

African Journals Online (AJOL)

Mutations that cause severe loss of DNA methylation often leads to abnormal development. In the present review, we summarized recent findings of the three major DNA methyltransferases mutants playing vital role in development of Arabidopsis thaliana. Keywords: DNA methylation, epigenetics, methyltransferase, mutant ...

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.

Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication.

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Haruta, Mayumi; Shimada, Midori; Nishiyama, Atsuya; Johmura, Yoshikazu; Le Tallec, Benoît; Debatisse, Michelle; Nakanishi, Makoto

2016-01-22

The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Interactions within the mammalian DNA methyltransferase family

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Ehrenhofer-Murray Ann E

2003-05-01

Full Text Available Abstract Background In mammals, epigenetic information is established and maintained via the postreplicative methylation of cytosine residues by the DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Dnmt1 is required for maintenance methylation whereas Dnmt3a and Dnmt3b are responsible for de novo methylation. Contrary to Dnmt3a or Dnmt3b, the isolated C-terminal region of Dnmt1 is catalytically inactive, despite the presence of the sequence motifs typical of active DNA methyltransferases. Deletion analysis has revealed that a large part of the N-terminal domain is required for enzymatic activity. Results The role played by the N-terminal domain in this regulation has been investigated using the yeast two-hybrid system. We show here the presence of an intra-molecular interaction in Dnmt1 but not in Dnmt3a or Dnmt3b. This interaction was confirmed by immunoprecipitation and was localized by deletion mapping. Furthermore, a systematic analysis of interactions among the Dnmt family members has revealed that DNMT3L interacts with the C-terminal domain of Dnmt3a and Dnmt3b. Conclusions The lack of methylating ability of the isolated C-terminal domain of Dnmt1 could be explained in part by a physical interaction between N- and C-terminal domains that apparently is required for activation of the catalytic domain. Our deletion analysis suggests that the tertiary structure of Dnmt1 is important in this process rather than a particular sequence motif. Furthermore, the interaction between DNMT3L and the C-terminal domains of Dnmt3a and Dnmt3b suggests a mechanism whereby the enzymatically inactive DNMT3L brings about the methylation of its substrate by recruiting an active methylase.

Interactions within the mammalian DNA methyltransferase family

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Margot, Jean B; Ehrenhofer-Murray, Ann E; Leonhardt, Heinrich

2003-01-01

Background In mammals, epigenetic information is established and maintained via the postreplicative methylation of cytosine residues by the DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Dnmt1 is required for maintenance methylation whereas Dnmt3a and Dnmt3b are responsible for de novo methylation. Contrary to Dnmt3a or Dnmt3b, the isolated C-terminal region of Dnmt1 is catalytically inactive, despite the presence of the sequence motifs typical of active DNA methyltransferases. Deletion analysis has revealed that a large part of the N-terminal domain is required for enzymatic activity. Results The role played by the N-terminal domain in this regulation has been investigated using the yeast two-hybrid system. We show here the presence of an intra-molecular interaction in Dnmt1 but not in Dnmt3a or Dnmt3b. This interaction was confirmed by immunoprecipitation and was localized by deletion mapping. Furthermore, a systematic analysis of interactions among the Dnmt family members has revealed that DNMT3L interacts with the C-terminal domain of Dnmt3a and Dnmt3b. Conclusions The lack of methylating ability of the isolated C-terminal domain of Dnmt1 could be explained in part by a physical interaction between N- and C-terminal domains that apparently is required for activation of the catalytic domain. Our deletion analysis suggests that the tertiary structure of Dnmt1 is important in this process rather than a particular sequence motif. Furthermore, the interaction between DNMT3L and the C-terminal domains of Dnmt3a and Dnmt3b suggests a mechanism whereby the enzymatically inactive DNMT3L brings about the methylation of its substrate by recruiting an active methylase. PMID:12777184

DNA methyltransferases and stress-related genes expression in zebrafish larvae after exposure to heat and copper during reprogramming of DNA methylation.

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Dorts, Jennifer; Falisse, Elodie; Schoofs, Emilie; Flamion, Enora; Kestemont, Patrick; Silvestre, Frédéric

2016-10-12

DNA methylation, a well-studied epigenetic mark, is important for gene regulation in adulthood and for development. Using genetic and epigenetic approaches, the present study aimed at evaluating the effects of heat stress and copper exposure during zebrafish early embryogenesis when patterns of DNA methylation are being established, a process called reprogramming. Embryos were exposed to 325 μg Cu/L from fertilization (<1 h post fertilization - hpf) to 4 hpf at either 26.5 °C or 34 °C, followed by incubation in clean water at 26.5 °C till 96 hpf. Significant increased mortality rates and delayed hatching were observed following exposure to combined high temperature and Cu. Secondly, both stressors, alone or in combination, significantly upregulated the expression of de novo DNA methyltransferase genes (dnmt3) along with no differences in global cytosine methylation level. Finally, Cu exposure significantly increased the expression of metallothionein (mt2) and heat shock protein (hsp70), the latter being also increased following exposure to high temperature. These results highlighted the sensitivity of early embryogenesis and more precisely of the reprogramming period to environmental challenges, in a realistic situation of combined stressors.

Increased cytosine DNA-methyltransferase activity in A/J mouse lung cells following carcinogen exposure and during tumor progression

International Nuclear Information System (INIS)

Belinsky, S.A.; Issa, J.-P.J.; Baylin, S.B.

1994-01-01

Considerable evidence has accumulated that 5-methylcytosine modification of mammalian DNA, both in exons and CpG rich islands located in promoter regions, is important in gene regulation. For example, a decrease of 5-methylcytosine in 5' flanking regions or exons of genes has been associated with increased gene transcription. In addition, hypermethylation at specific regions of chromosomes 17p and 3p have also been observed in lung and colon cancer. During colon cancer development, these hypermethylation changes precede allelic loss. In addition, the activity of the enzyme which maintains the methylation status at CpG dinucleotides, DNA methyltransferase (MT), has been shown to increase during colon cancer progression. These observations suggest changes in methylation patterns within specific genes could result in either inappropriate gene expression or gene deletion, both of which would contribute to the establishment of the malignant phenotype. The purpose of this investigation was to determine if DNA MT activity is elevated in target (alveolar type II), but not in nontarget (Clara, endothelial, macrophage) lung cells isolated from the A/J mouse following exposure to nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK). In addition, the activity of this enzyme during tumor progression was examined

Cytosine modifications after gamma irradiation in aerated aqueous solution of Escherichia coli DNA

International Nuclear Information System (INIS)

Polverelli, M.

1983-04-01

After gamma irradiation of cytosine in aerated aqueous solution and utilization of various spectrometric methods (mass spectrometry, proton nuclear magnetic resonance and infrared spectrometry) about ten new radiolysis products were identified. The formation of N-glycolylbiuret in H 2 18 O aqueous solution of irradiated cytosine at pH 4,5 indicated that the preferred 18 OH hydroxyl radical attack was at C-5. The formation of trans 1-carbamoyl-4,5 dihydroxyimidazolidin-2 oxo which is the major product after cytosine pyrimidine ring rearrangement took place preferentially at neutral pH, while N-glycolylbiuret predominated at pH 4,5. The deamination pathway was predominant when cytosine was irradiated at acidic pH values (pH 2 ) or in copper complexes. The development of a new acid hydrolysis method using fluorhydric acid stabilized in pyridine made easier the evaluation of cytosine modifications after gamma irradiation in aerated aqueous solution of E. Coli DNA- 14 C-2 cytosine. This hydrolytic agent removed the modified bases from the polynucleotidic chain. A difference was found between the proportion of radiolytic products removed by acid hydrolysis and by irradiation of the free base in solution [fr

Induction of epigenetic variation in Arabidopsis by over-expression of DNA METHYLTRANSFERASE1 (MET1.

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Samuel Brocklehurst

Full Text Available Epigenetic marks such as DNA methylation and histone modification can vary among plant accessions creating epi-alleles with different levels of expression competence. Mutations in epigenetic pathway functions are powerful tools to induce epigenetic variation. As an alternative approach, we investigated the potential of over-expressing an epigenetic function, using DNA METHYLTRANSFERASE1 (MET1 for proof-of-concept. In Arabidopsis thaliana, MET1 controls maintenance of cytosine methylation at symmetrical CG positions. At some loci, which contain dense DNA methylation in CG- and non-CG context, loss of MET1 causes joint loss of all cytosines methylation marks. We find that over-expression of both catalytically active and inactive versions of MET1 stochastically generates new epi-alleles at loci encoding transposable elements, non-coding RNAs and proteins, which results for most loci in an increase in expression. Individual transformants share some common phenotypes and genes with altered gene expression. Altered expression states can be transmitted to the next generation, which does not require the continuous presence of the MET1 transgene. Long-term stability and epigenetic features differ for individual loci. Our data show that over-expression of MET1, and potentially of other genes encoding epigenetic factors, offers an alternative strategy to identify epigenetic target genes and to create novel epi-alleles.

NSun2-Mediated Cytosine-5 Methylation of Vault Noncoding RNA Determines Its Processing into Regulatory Small RNAs

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Shobbir Hussain

2013-07-01

Full Text Available Autosomal-recessive loss of the NSUN2 gene has been identified as a causative link to intellectual disability disorders in humans. NSun2 is an RNA methyltransferase modifying cytosine-5 in transfer RNAs (tRNAs, yet the identification of cytosine methylation in other RNA species has been hampered by the lack of sensitive and reliable molecular techniques. Here, we describe miCLIP as an additional approach for identifying RNA methylation sites in transcriptomes. miCLIP is a customized version of the individual-nucleotide-resolution crosslinking and immunoprecipitation (iCLIP method. We confirm site-specific methylation in tRNAs and additional messenger and noncoding RNAs (ncRNAs. Among these, vault ncRNAs contained six NSun2-methylated cytosines, three of which were confirmed by RNA bisulfite sequencing. Using patient cells lacking the NSun2 protein, we further show that loss of cytosine-5 methylation in vault RNAs causes aberrant processing into Argonaute-associated small RNA fragments that can function as microRNAs. Thus, impaired processing of vault ncRNA may contribute to the etiology of NSun2-deficiency human disorders.

Structural insight into maintenance methylation by mouse DNA methyltransferase 1 (Dnmt1)

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Takeshita, Kohei; Suetake, Isao; Yamashita, Eiki; Suga, Michihiro; Narita, Hirotaka; Nakagawa, Atsushi; Tajima, Shoji

2011-01-01

Methylation of cytosine in DNA plays a crucial role in development through inheritable gene silencing. The DNA methyltransferase Dnmt1 is responsible for the propagation of methylation patterns to the next generation via its preferential methylation of hemimethylated CpG sites in the genome; however, how Dnmt1 maintains methylation patterns is not fully understood. Here we report the crystal structure of the large fragment (291–1620) of mouse Dnmt1 and its complexes with cofactor S-adenosyl-L-methionine and its product S-adenosyl-L-homocystein. Notably, in the absence of DNA, the N-terminal domain responsible for targeting Dnmt1 to replication foci is inserted into the DNA-binding pocket, indicating that this domain must be removed for methylation to occur. Upon binding of S-adenosyl-L-methionine, the catalytic cysteine residue undergoes a conformation transition to a catalytically competent position. For the recognition of hemimethylated DNA, Dnmt1 is expected to utilize a target recognition domain that overhangs the putative DNA-binding pocket. Taking into considerations the recent report of a shorter fragment structure of Dnmt1 that the CXXC motif positions itself in the catalytic pocket and prevents aberrant de novo methylation, we propose that maintenance methylation is a multistep process accompanied by structural changes. PMID:21518897

Crystal structure of MboIIA methyltransferase.

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Osipiuk, Jerzy; Walsh, Martin A; Joachimiak, Andrzej

2003-09-15

DNA methyltransferases (MTases) are sequence-specific enzymes which transfer a methyl group from S-adenosyl-L-methionine (AdoMet) to the amino group of either cytosine or adenine within a recognized DNA sequence. Methylation of a base in a specific DNA sequence protects DNA from nucleolytic cleavage by restriction enzymes recognizing the same DNA sequence. We have determined at 1.74 A resolution the crystal structure of a beta-class DNA MTase MboIIA (M.MboIIA) from the bacterium Moraxella bovis, the smallest DNA MTase determined to date. M.MboIIA methylates the 3' adenine of the pentanucleotide sequence 5'-GAAGA-3'. The protein crystallizes with two molecules in the asymmetric unit which we propose to resemble the dimer when M.MboIIA is not bound to DNA. The overall structure of the enzyme closely resembles that of M.RsrI. However, the cofactor-binding pocket in M.MboIIA forms a closed structure which is in contrast to the open-form structures of other known MTases.

The role of cytosine methylation on charge transport through a DNA strand

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Qi, Jianqing, E-mail: jqqi@uw.edu; Anantram, M. P., E-mail: anantmp@uw.edu [Department of Electrical Engineering, University of Washington, Seattle, Washington 98195-2500 (United States); Govind, Niranjan, E-mail: niri.govind@pnnl.gov [William R. Wiley Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352 (United States)

2015-09-07

Cytosine methylation has been found to play a crucial role in various biological processes, including a number of human diseases. The detection of this small modification remains challenging. In this work, we computationally explore the possibility of detecting methylated DNA strands through direct electrical conductance measurements. Using density functional theory and the Landauer-Büttiker method, we study the electronic properties and charge transport through an eight base-pair methylated DNA strand and its native counterpart. We first analyze the effect of cytosine methylation on the tight-binding parameters of two DNA strands and then model the transmission of the electrons and conductance through the strands both with and without decoherence. We find that the main difference of the tight-binding parameters between the native DNA and the methylated DNA lies in the on-site energies of (methylated) cytosine bases. The intra- and inter-strand hopping integrals between two nearest neighboring guanine base and (methylated) cytosine base also change with the addition of the methyl groups. Our calculations show that in the phase-coherent limit, the transmission of the methylated strand is close to the native strand when the energy is nearby the highest occupied molecular orbital level and larger than the native strand by 5 times in the bandgap. The trend in transmission also holds in the presence of the decoherence with the same rate. The lower conductance for the methylated strand in the experiment is suggested to be caused by the more stable structure due to the introduction of the methyl groups. We also study the role of the exchange-correlation functional and the effect of contact coupling by choosing coupling strengths ranging from weak to strong coupling limit.

Aberrant DNA methylation in 5'regions of DNA methyltransferase genes in aborted bovine clones

Institute of Scientific and Technical Information of China (English)

2008-01-01

High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning.It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation.DNA methylation is established and maintained by DNA methyltransferases(DNMTs),therefore,it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs.Since DNA methylation can strongly inhibit gene expression,aberrant DNA methylation of DNMT genes may disturb gene expression.But presently,it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos.In our study,we analyzed methylation patterns of the 5' regions of four DNMT genes including Dnmt3a,Dnmt3b,Dnmtl and Dnmt2 in four aborted bovine clones.Using bisulfite sequencing method,we found that 3 out of 4 aborted bovine clones(AF1,AF2 and AF3)showed either hypermethylation or hypomethylation in the 5' regions of Dnmt3a and Dnmt3b.indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed.However,the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized (IVF)fetuses.Besides,we found that tle 5'regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults.IVF fetuses,sperm and aborted clones.Together,our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5' regions is probably associated with the high abortion of bovine clones.

Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry

DEFF Research Database (Denmark)

Vranken, Charlotte; Deen, Jochem; Dirix, Lieve

2014-01-01

We demonstrate an approach to optical DNA mapping, which enables near single-molecule characterization of whole bacteriophage genomes. Our approach uses a DNA methyltransferase enzyme to target labelling to specific sites and copper-catalysed azide-alkyne cycloaddition to couple a fluorophore...... to the DNA. We achieve a labelling efficiency of ∼70% with an average labelling density approaching one site every 500 bp. Such labelling density bridges the gap between the output of a typical DNA sequencing experiment and the long-range information derived from traditional optical DNA mapping. We lay...... the foundations for a wider-scale adoption of DNA mapping by screening 11 methyltransferases for their ability to direct sequence-specific DNA transalkylation; the first step of the DNA labelling process and by optimizing reaction conditions for fluorophore coupling via a click reaction. Three of 11 enzymes...

Recruitment of DNA methyltransferase I to DNA repair sites

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

2005-01-01

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

Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication

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Haruta, Mayumi [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Shimada, Midori, E-mail: midorism@med.nagoya-cu.ac.jp [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Nishiyama, Atsuya; Johmura, Yoshikazu [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Le Tallec, Benoît; Debatisse, Michelle [Institut Curie, Centre de Recherche, 26 rue d’Ulm, CNRS UMR 3244, 75248 ParisCedex 05 (France); Nakanishi, Makoto, E-mail: mkt-naka@med.nagoya-cu.ac.jp [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan)

2016-01-22

The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells. - Highlights: • DNMT1 depletion results in an abnormal DNA replication program. • Aberrant DNA replication is independent of the DNA damage checkpoint in DNMT1cKO. • DNMT1 catalytic activity and RFT domain are required for proper DNA replication. • DNMT1 catalytic activity and RFT domain are required for cell proliferation.

Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication

International Nuclear Information System (INIS)

Haruta, Mayumi; Shimada, Midori; Nishiyama, Atsuya; Johmura, Yoshikazu; Le Tallec, Benoît; Debatisse, Michelle; Nakanishi, Makoto

2016-01-01

The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells. - Highlights: • DNMT1 depletion results in an abnormal DNA replication program. • Aberrant DNA replication is independent of the DNA damage checkpoint in DNMT1cKO. • DNMT1 catalytic activity and RFT domain are required for proper DNA replication. • DNMT1 catalytic activity and RFT domain are required for cell proliferation.

HPLC-UV, MALDI-TOF-MS and ESI-MS/MS analysis of the mechlorethamine DNA crosslink at a cytosine-cytosine mismatch pair.

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Pornchai Rojsitthisak

Full Text Available Mechlorethamine [ClCH(2CH(2N(CH(3CH(2CH(2Cl], a nitrogen mustard alkylating agent, has been proven to form a DNA interstrand crosslink at a cytosine-cytosine (C-C mismatch pair using gel electrophoresis. However, the atomic connectivity of this unusual crosslink is unknown.HPLC-UV, MALDI-TOF-MS, and ESI-MS/MS were used to determine the atomic connectivity of the DNA C-C crosslink formed by mechlorethamine, MALDI-TOF-MS of the HPLC-purified reaction product of mechlorethamine with the DNA duplex d[CTCACACCGTGGTTC]•d[GAACCACCGTGTGAG] (underlined bases are a C-C mismatch pair indicated formation of an interstrand crosslink at m/z 9222.088 [M-2H+Na](+. Following enzymatic digestion of the crosslinked duplex by snake venom phosphodiesterase and calf intestinal phosphatase, ESI-MS/MS indicated the presence of dC-mech-dC [mech = CH(2CH(2N(CH(3CH(2CH(2] at m/z 269.2 [M](2+ (expected m/z 269.6, exact mass 539.27 and its hydrolytic product dC-mech-OH at m/z 329.6 [M](+ (expected m/z 329.2. Fragmentation of dC-mech-dC gave product ions at m/z 294.3 and 236.9 [M](+, which are both due to loss of the 4-amino group of cytosine (as ammonia, in addition to dC and dC+HN(CH(3CH = CH(2, respectively. The presence of m/z 269.2 [M](2+ and loss of ammonia exclude crosslink formation at cytosine N(4 or O(2 and indicate crosslinking through cytosine N(3 with formation of two quaternary ammonium ions.Our results provide an important addition to the literature, as the first example of the use of HPLC and MS for analysis of a DNA adduct at the N(3 position of cytosine.

Hybrid DNA i-motif: Aminoethylprolyl-PNA (pC5) enhance the stability of DNA (dC5) i-motif structure.

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Gade, Chandrasekhar Reddy; Sharma, Nagendra K

2017-12-15

This report describes the synthesis of C-rich sequence, cytosine pentamer, of aep-PNA and its biophysical studies for the formation of hybrid DNA:aep-PNAi-motif structure with DNA cytosine pentamer (dC 5 ) under acidic pH conditions. Herein, the CD/UV/NMR/ESI-Mass studies strongly support the formation of stable hybrid DNA i-motif structure with aep-PNA even near acidic conditions. Hence aep-PNA C-rich sequence cytosine could be considered as potential DNA i-motif stabilizing agents in vivo conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Information Thermodynamics of Cytosine DNA Methylation.

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Robersy Sanchez

Full Text Available Cytosine DNA methylation (CDM is a stable epigenetic modification to the genome and a widespread regulatory process in living organisms that involves multicomponent molecular machines. Genome-wide cytosine methylation patterning participates in the epigenetic reprogramming of a cell, suggesting that the biological information contained within methylation positions may be amenable to decoding. Adaptation to a new cellular or organismal environment also implies the potential for genome-wide redistribution of CDM changes that will ensure the stability of DNA molecules. This raises the question of whether or not we would be able to sort out the regulatory methylation signals from the CDM background ("noise" induced by thermal fluctuations. Here, we propose a novel statistical and information thermodynamic description of the CDM changes to address the last question. The physical basis of our statistical mechanical model was evaluated in two respects: 1 the adherence to Landauer's principle, according to which molecular machines must dissipate a minimum energy ε = kBT ln2 at each logic operation, where kB is the Boltzmann constant, and T is the absolute temperature and 2 whether or not the binary stretch of methylation marks on the DNA molecule comprise a language of sorts, properly constrained by thermodynamic principles. The study was performed for genome-wide methylation data from 152 ecotypes and 40 trans-generational variations of Arabidopsis thaliana and 93 human tissues. The DNA persistence length, a basic mechanical property altered by CDM, was estimated with values from 39 to 66.9 nm. Classical methylome analysis can be retrieved by applying information thermodynamic modelling, which is able to discriminate signal from noise. Our finding suggests that the CDM signal comprises a language scheme properly constrained by molecular thermodynamic principles, which is part of an epigenomic communication system that obeys the same thermodynamic

Microhydration of cytosine and its radical anion: Cytosine.(H2O)n (n=1-5)

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Kim, Sunghwan; Schaefer, Henry F.

2007-02-01

Microhydration effects on cytosine and its radical anion have been investigated theoretically, by explicitly considering various structures of cytosine complexes with up to five water molecules. Each successive water molecule (through n =5) is bound by 7-10kcalmol-1 to the relevant cytosine complex. The hydration energies are uniformly higher for the analogous anion systems. While the predicted vertical detachment energy (VDE) of the isolated cytosine is only 0.48eV, it is predicted to increase to 1.27eV for the lowest-lying pentahydrate of cytosine. The adiabatic electron affinity (AEA) of cytosine was also found to increase from 0.03to0.61eV for the pentahydrate, implying that the cytosine anion, while questionable in the gas phase, is bound in aqueous solution. Both the VDE and AEA values for cytosine are smaller than those of uracil and thymine for a given hydration number. These results are in qualitative agreement with available experimental results from photodetachment-photoelectron spectroscopy studies of Schiedt et al. [Chem. Phys. 239, 511 (1998)].

In vivo control of CpG and non-CpG DNA methylation by DNA methyltransferases.

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Julia Arand

2012-06-01

Full Text Available The enzymatic control of the setting and maintenance of symmetric and non-symmetric DNA methylation patterns in a particular genome context is not well understood. Here, we describe a comprehensive analysis of DNA methylation patterns generated by high resolution sequencing of hairpin-bisulfite amplicons of selected single copy genes and repetitive elements (LINE1, B1, IAP-LTR-retrotransposons, and major satellites. The analysis unambiguously identifies a substantial amount of regional incomplete methylation maintenance, i.e. hemimethylated CpG positions, with variant degrees among cell types. Moreover, non-CpG cytosine methylation is confined to ESCs and exclusively catalysed by Dnmt3a and Dnmt3b. This sequence position-, cell type-, and region-dependent non-CpG methylation is strongly linked to neighboring CpG methylation and requires the presence of Dnmt3L. The generation of a comprehensive data set of 146,000 CpG dyads was used to apply and develop parameter estimated hidden Markov models (HMM to calculate the relative contribution of DNA methyltransferases (Dnmts for de novo and maintenance DNA methylation. The comparative modelling included wild-type ESCs and mutant ESCs deficient for Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3a/3b, respectively. The HMM analysis identifies a considerable de novo methylation activity for Dnmt1 at certain repetitive elements and single copy sequences. Dnmt3a and Dnmt3b contribute de novo function. However, both enzymes are also essential to maintain symmetrical CpG methylation at distinct repetitive and single copy sequences in ESCs.

Syntheses of [5-2H]-uracil, [5-2H]-cytosine, [6-2H]-uracil and [6-2H]-cytosine

International Nuclear Information System (INIS)

Kiritani, Reiko; Asano, Takeyoshi; Fujita, Shin-ichi; Dohmaru, Takaaki; Kawanishi, Tetsuro

1986-01-01

Syntheses of [5- 2 H]-, [6- 2 H]-uracil and [5- 2 H]-, [6- 2 H]-cytosine were investigated. The catalytic reaction of uracil or cytosine with 2 H 2 gas in alkaline media gave rise to [6- 2 H]-compounds almost exclusively. On the other hand, the reaction of 5-bromouracil or 5-bromocytosine with 2 H 2 gas gave rise to a mixture of [5- 2 H]-, [6- 2 H]- and [5- 2 H, 6- 2 H]-compounds depending on the experimental conditions. By controlling the temperature, the pressure of 2 H 2 gas and the amount of catalyst, [5- 2 H]-uracil and [5- 2 H]-cytosine were obtained. The isotopic distribution in each product was measured by 1 H NMR spectroscopy combined with an HPLC method. (author)

Magnetic immunoassay using CdSe/ZnS quantum dots as fluorescent probes to detect the level of DNA methyltransferase 1 in human serum sample

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Yu F

2018-01-01

Full Text Available Fei Yu,1,* Ya-min Xiong,1,* Song-cheng Yu,1 Lei-liang He,1 Shan-shan Niu,1 Yu-ming Wu,1 Jie Liu,1 Ling-bo Qu,2 Li-e Liu,1 Yong-jun Wu1 1College of Public Health, 2College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou, Henan, People’s Republic of China *These authors contributed equally to this work Background: DNA methyltransferase 1 (DNMT1, a dominant enzyme responsible for the transfer of a methyl group from the universal methyl donor to the 5-position of cytosine residues in DNA, is essential for mammalian development and closely related to cancer and a variety of age-related chronic diseases. DNMT1 has become a useful biomarker in early disease diagnosis and a potential therapeutic target in cancer therapy and drug development. However, till now, most of the studies on DNA methyltransferase (MTase detection have focused on the prokaryote MTase and its activity.Methods: A magnetic fluorescence-linked immunosorbent assay (FLISA using CdSe/ZnS quantum dots as fluorescent probes was proposed for the rapid and sensitive detection of the DNMT1 level in this study. Key factors that affect the precision and accuracy of the determination of DNMT1 were optimized.Results: Under the optimal conditions, the limit of detection was 0.1 ng/mL, the linear range was 0.1–1,500 ng/mL, the recovery was 91.67%–106.50%, and the relative standard deviations of intra- and inter-assays were respectively 5.45%–11.29% and 7.03%–11.25%. The cross-reactivity rates with DNA methyltransferases 3a and 3b were only 4.0% and 9.4%, respectively. Furthermore, FLISA was successfully used to detect the levels of DNMT1 in human serum samples, and compared with commercial enzyme-linked immunosorbent assay (ELISA kits. The results revealed that there was a good correlation between FLISA and commercial ELISA kits (correlation coefficient r=0.866, p=0.001. The linear scope of FLISA was broader than ELISA, and the measurement time was much shorter

Genomic DNA sequence and cytosine methylation changes of adult rice leaves after seeds space flight

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Shi, Jinming

In this study, cytosine methylation on CCGG site and genomic DNA sequence changes of adult leaves of rice after seeds space flight were detected by methylation-sensitive amplification polymorphism (MSAP) and Amplified fragment length polymorphism (AFLP) technique respectively. Rice seeds were planted in the trial field after 4 days space flight on the shenzhou-6 Spaceship of China. Adult leaves of space-treated rice including 8 plants chosen randomly and 2 plants with phenotypic mutation were used for AFLP and MSAP analysis. Polymorphism of both DNA sequence and cytosine methylation were detected. For MSAP analysis, the average polymorphic frequency of the on-ground controls, space-treated plants and mutants are 1.3%, 3.1% and 11% respectively. For AFLP analysis, the average polymorphic frequencies are 1.4%, 2.9%and 8%respectively. Total 27 and 22 polymorphic fragments were cloned sequenced from MSAP and AFLP analysis respectively. Nine of the 27 fragments from MSAP analysis show homology to coding sequence. For the 22 polymorphic fragments from AFLP analysis, no one shows homology to mRNA sequence and eight fragments show homology to repeat region or retrotransposon sequence. These results suggest that although both genomic DNA sequence and cytosine methylation status can be effected by space flight, the genomic region homology to the fragments from genome DNA and cytosine methylation analysis were different.

Hydration properties of natural and synthetic DNA sequences with methylated adenine or cytosine bases in the R.DpnI target and BDNF promoter studied by molecular dynamics simulations

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Shanak, Siba; Helms, Volkhard

2014-12-01

Adenine and cytosine methylation are two important epigenetic modifications of DNA sequences at the levels of the genome and transcriptome. To characterize the differential roles of methylating adenine or cytosine with respect to their hydration properties, we performed conventional MD simulations and free energy perturbation calculations for two particular DNA sequences, namely the brain-derived neurotrophic factor (BDNF) promoter and the R.DpnI-bound DNA that are known to undergo methylation of C5-methyl cytosine and N6-methyl adenine, respectively. We found that a single methylated cytosine has a clearly favorable hydration free energy over cytosine since the attached methyl group has a slightly polar character. In contrast, capping the strongly polar N6 of adenine with a methyl group gives a slightly unfavorable contribution to its free energy of solvation. Performing the same demethylation in the context of a DNA double-strand gave quite similar results for the more solvent-accessible cytosine but much more unfavorable results for the rather buried adenine. Interestingly, the same demethylation reactions are far more unfavorable when performed in the context of the opposite (BDNF or R.DpnI target) sequence. This suggests a natural preference for methylation in a specific sequence context. In addition, free energy calculations for demethylating adenine or cytosine in the context of B-DNA vs. Z-DNA suggest that the conformational B-Z transition of DNA transition is rather a property of cytosine methylated sequences but is not preferable for the adenine-methylated sequences investigated here.

Cytosine methylation is a conserved epigenetic feature found throughout the phylum Platyhelminthes

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2013-01-01

Background The phylum Platyhelminthes (flatworms) contains an important group of bilaterian organisms responsible for many debilitating and chronic infectious diseases of human and animal populations inhabiting the planet today. In addition to their biomedical and veterinary relevance, some platyhelminths are also frequently used models for understanding tissue regeneration and stem cell biology. Therefore, the molecular (genetic and epigenetic) characteristics that underlie trophic specialism, pathogenicity or developmental maturation are likely to be pivotal in our continued studies of this important metazoan group. Indeed, in contrast to earlier studies that failed to detect evidence of cytosine or adenine methylation in parasitic flatworm taxa, our laboratory has recently defined a critical role for cytosine methylation in Schistosoma mansoni oviposition, egg maturation and ovarian development. Thus, in order to identify whether this epigenetic modification features in other platyhelminth species or is a novelty of S. mansoni, we conducted a study simultaneously surveying for DNA methylation machinery components and DNA methylation marks throughout the phylum using both parasitic and non-parasitic representatives. Results Firstly, using both S. mansoni DNA methyltransferase 2 (SmDNMT2) and methyl-CpG binding domain protein (SmMBD) as query sequences, we illustrate that essential DNA methylation machinery components are well conserved throughout the phylum. Secondly, using both molecular (methylation specific amplification polymorphism, MSAP) and immunological (enzyme-linked immunoabsorbent assay, ELISA) methodologies, we demonstrate that representative species (Echinococcus multilocularis, Protopolystoma xenopodis, Schistosoma haematobium, Schistosoma japonicum, Fasciola hepatica and Polycelis nigra) within all four platyhelminth classes (Cestoda, Monogenea, Trematoda and ‘Turbellaria’) contain methylated cytosines within their genome compartments

Increased susceptibility to chemotherapeutic alkylating agents of mice deficient in DNA repair methyltransferase.

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Shiraishi, A; Sakumi, K; Sekiguchi, M

2000-10-01

O(6)-methylguanine-DNA methyltransferase plays vital roles in preventing induction of mutations and cancer as well as cell death related to alkylating agents. Mice defective in the MGMT: gene, encoding the methyltransferase, were used to evaluate cell death-inducing and tumorigenic activities of therapeutic agents which have alkylation potential. MGMT(-/-) mice were considerably more sensitive to dacarbazine, a monofunctional triazene, than were wild-type mice, in terms of survival. When dacarbazine was administered i.p. to 6-week-old mice and survival at 30 days was enumerated, LD(50) values of MGMT(-/-) and MGMT(+/+) mice were 20 and 450 mg/kg body wt, respectively. Increased sensitivity of MGMT(-/-) mice to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosou rea (ACNU), a bifunctional nitrosourea, was also noted. On the other hand, there was no difference in survival of MGMT(+/+) and MGMT(-/-) mice exposed to cyclophosphamide, a bifunctional nitrogen mustard. It appears that dacarbazine and ACNU produce O(6)-alkylguanine as a major toxic lesion, while cyclophosphamide yields other types of modifications in DNA which are not subjected to the action of the methyltransferase. MGMT(-/-) mice seem to be less refractory to the tumor-inducing effect of dacarbazine than are MGMT(+/+) mice. Thus, the level of O(6)-methylguanine-DNA methyltransferase activity is an important factor when determining susceptibility to drugs with the potential for alkylation.

Localization of DNA methyltransferase-1 during oocyte differentiation, in vitro maturation and early embryonic development in cow

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A. M. Luciano

2009-12-01

Full Text Available DNA methyltransferase-1 (Dnmt1 is involved in the maintenance of DNA methylation patterns and is crucial for normal mammalian development. The aim of the present study was to assess the localization of Dnmt1 in cow, during the latest phases of oocyte differentiation and during the early stages of segmentation. Dnmt1 expression and localization were assessed in oocytes according to the chromatin configuration, which in turn provides an important epigenetic mechanism for the control of global gene expression and represents a morphological marker of oocyte differentiation.We found that the initial chromatin condensation was accompanied by a slight increase in the level of global DNA methylation, as assessed by 5-methyl-cytosine immunostaining followed by laser scanning confocal microscopy analysis (LSCM. RT-PCR confirmed the presence of Dnmt1 transcripts throughout this phase of oocyte differentiation. Analogously, Dnmt1 immunodetection and LSCM indicated that the protein was always present and localized in the cytoplasm, regardless the chromatin configuration and the level of global DNA methylation. Moreover, our data indicate that while Dnmt1 is retained in the cytoplasm in metaphase II stage oocytes and zygotes, it enters the nuclei of 8-16 cell stage embryos. As suggested in mouse, the functional meaning of the presence of Dnmt1 in the bovine embryo nuclei could be the maintainement of the methylation pattern of imprinted genes. In conclusion, the present work provides useful elements for the study of Dnmt1 function during the late stage of oocyte differentiation, maturation and early embryonic development in mammals.

Isolation of DNA methyltransferase from plants

International Nuclear Information System (INIS)

Ehrlich, K.; Malbroue, C.

1987-01-01

DNA methyltransferases (DMT) were isolated from nuclei of cauliflower, soybean, and pea by extraction with 0.35 M NaCl. Assays were performed on hemimethylated Micrococcus luteus DNA or on M. luteus DNA to test for maintenance or de novo methylase activity, respectively. Fully methylated DNA was used as a substrate to determine background levels of methylation. Based on these tests, yields of maintenance DMT activity in the crude extract from pea hypocotyl, soybean hypocotyl, and cauliflower inflorescence were 2.8, 0.9, and 1.6 units per g wet tissue (one unit equals 1 pmol of methyl from [ 3 H]AdoMet incorporated into acid precipitable material per h at 30 0 ). Two peaks of DMT activity were detected in the soybean nuclear extract following phosphocellulose chromatography. One eluted at 0.4 M and the other at 0.8 M KCl. With both fractions maintenance activity was approximately 2 times that of the de novo activity. Using gel filtration the DMT eluted at 220,000 Daltons. The optimal pH for activity was between 6.5 and 7.0, and the optimal temperature was 30 0

An N-Glycosidase from Escherichia coli That Releases Free Uracil from DNA Containing Deaminated Cytosine Residues

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Lindahl, Tomas

1974-01-01

An enzyme that liberates uracil from single-stranded and double-stranded DNA containing deaminated cytosine residues and from deoxycytidylate-deoxyuridylate copolymers in the absence of Mg++ has been purified 30-fold from cell extracts of E. coli. The enzyme does not release uracil from deoxyuridine, dUMP, uridine, or RNA, nor does it liberate the normally occurring pyrimidine bases, cytosine and thymine, from DNA. The enzymatic cleavage of N-glycosidic bonds in DNA occurs without concomitant cleavage of phosphodiester bonds, resulting in the formation of free uracil and DNA strands of unaltered chain length that contain apyrimidinic sites as reaction products. The enzyme may be active in DNA repair, converting deaminated dCMP residues to an easily repairable form. PMID:4610583

Antineoplastic activity of the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine in anaplastic large cell lymphoma

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Hassler, Melanie R.; Klisaroska, Aleksandra; Kollmann, Karoline; Steiner, Irene; Bilban, Martin; Schiefer, Ana-Iris; Sexl, Veronika; Egger, Gerda

2012-01-01

DNA methylation is an epigenetic mechanism establishing long-term gene silencing during development and cell commitment, which is maintained in subsequent cell generations. Aberrant DNA methylation is found at gene promoters in most cancers and can lead to silencing of tumor suppressor genes. The DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza-CdR) is able to reactivate genes silenced by DNA methylation and has been shown to be a very potent epigenetic drug in several hematological malignancies. In this report, we demonstrate that 5-aza-CdR exhibits high antineoplastic activity against anaplastic large cell lymphoma (ALCL), a rare CD30 positive non-Hodgkin lymphoma of T-cell origin. Low dose treatment of ALCL cell lines and xenografted tumors causes apoptosis and cell cycle arrest in vitro and in vivo. This is also reflected in genome-wide expression analyses, where genes related to apoptosis and cell death are amongst the most affected targets of 5-aza-CdR. Furthermore, we observed demethylation and re-expression of p16INK4A after drug administration and senescence associated β-galactosidase activity. Thus, our data provide evidence that 5-aza-CdR is highly efficient against ALCL and warrants further clinical evaluation for future therapeutic use. PMID:22687603

Fluorescent strategy based on cationic conjugated polymer fluorescence resonance energy transfer for the quantification of 5-(hydroxymethyl)cytosine in genomic DNA.

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Hong, Tingting; Wang, Tianlu; Guo, Pu; Xing, Xiwen; Ding, Fei; Chen, Yuqi; Wu, Jinjun; Ma, Jingwei; Wu, Fan; Zhou, Xiang

2013-11-19

DNA methylation is dynamically reprogrammed during early embryonic development in mammals. It can be explained partially by the discovery of 5-(hydroxymethyl)cytosine (5-hmC), 5-formylcytosine (5-fC), and 5-carboxylcytosine (5-caC), which are identified as key players involved in both active and passive demethylation pathways. As one of the ten-eleven translocation oxidation products, 5-hmC was found relatively abundant in neuron cells and embryonic stem cells. Herein we report a new method for 5-hmC quantification in genomic DNA based on CCP-FRET (cationic conjugated polymers act as the energy donor and induce fluorescence resonance energy transfer) assay combined with KRuO4 oxidation. 5-hmC in genomic DNA can be selectively transformed into 5-fC by the oxidation of KRuO4 and then labeled with hydroxylamine-BODIPY (BODIPY = 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophore through the reaction between 5-fC and hydroxylamine-BODIPY. After the fluorescently labeled DNA was captured by CCP through electrostatic interactions, a significant FRET between CCP and hydroxylamine-BODIPY fluorophore was observed. This CCP-FRET-based assay benefits from light-harvesting, large Stokes shift, and optical signal amplification properties of the CCP. Furthermore, this CCP-FRET-based assay was quite successfully demonstrated for the 5-hmC quantification in three types of cells (mESc, HeLa, HEK 293T), providing a much more convenient choice for 5-hmC quantification in genomic DNA.

Self-assembling of calcium salt of the new DNA base 5-carboxylcytosine

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Irrera, Simona; Ruiz-Hernandez, Sergio E.; Reggente, Melania; Passeri, Daniele; Natali, Marco; Gala, Fabrizio; Zollo, Giuseppe; Rossi, Marco; Portalone, Gustavo

2017-06-01

Supramolecular architectures involving DNA bases can have a strong impact in several fields such as nanomedicine and nanodevice manufacturing. To date, in addition to the four canonical nucleobases (adenine, thymine, guanine and cytosine), four other forms of cytosine modified at the 5 position have been identified in DNA. Among these four new cytosine derivatives, 5-carboxylcytosine has been recently discovered in mammalian stem cell DNA, and proposed as the final product of the oxidative epigenetic demethylation pathway on the 5 position of cytosine. In this work, a calcium salt of 5-carboxylcytosine has been synthesized and deposited on graphite surface, where it forms self-assembled features as long range monolayers and up to one micron long filaments. These structures have been analyzed in details combining different theoretical and experimental approaches: X-ray single-crystal diffraction data were used to simulate the molecule-graphite interaction, first using molecular dynamics and then refining the results using density functional theory (DFT); finally, data obtained with DFT were used to rationalize atomic force microscopy (AFM) results.

DNA cytosine methylation in the bovine leukemia virus promoter is associated with latency in a lymphoma-derived B-cell line: potential involvement of direct inhibition of cAMP-responsive element (CRE)-binding protein/CRE modulator/activation transcription factor binding.

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Pierard, Valérie; Guiguen, Allan; Colin, Laurence; Wijmeersch, Gaëlle; Vanhulle, Caroline; Van Driessche, Benoît; Dekoninck, Ann; Blazkova, Jana; Cardona, Christelle; Merimi, Makram; Vierendeel, Valérie; Calomme, Claire; Nguyên, Thi Liên-Anh; Nuttinck, Michèle; Twizere, Jean-Claude; Kettmann, Richard; Portetelle, Daniel; Burny, Arsène; Hirsch, Ivan; Rohr, Olivier; Van Lint, Carine

2010-06-18

Bovine leukemia virus (BLV) proviral latency represents a viral strategy to escape the host immune system and allow tumor development. Besides the previously demonstrated role of histone deacetylation in the epigenetic repression of BLV expression, we showed here that BLV promoter activity was induced by several DNA methylation inhibitors (such as 5-aza-2'-deoxycytidine) and that overexpressed DNMT1 and DNMT3A, but not DNMT3B, down-regulated BLV promoter activity. Importantly, cytosine hypermethylation in the 5'-long terminal repeat (LTR) U3 and R regions was associated with true latency in the lymphoma-derived B-cell line L267 but not with defective latency in YR2 cells. Moreover, the virus-encoded transactivator Tax(BLV) decreased DNA methyltransferase expression levels, which could explain the lower level of cytosine methylation observed in the L267(LTaxSN) 5'-LTR compared with the L267 5'-LTR. Interestingly, DNA methylation inhibitors and Tax(BLV) synergistically activated BLV promoter transcriptional activity in a cAMP-responsive element (CRE)-dependent manner. Mechanistically, methylation at the -154 or -129 CpG position (relative to the transcription start site) impaired in vitro binding of CRE-binding protein (CREB) transcription factors to their respective CRE sites. Methylation at -129 CpG alone was sufficient to decrease BLV promoter-driven reporter gene expression by 2-fold. We demonstrated in vivo the recruitment of CREB/CRE modulator (CREM) and to a lesser extent activating transcription factor-1 (ATF-1) to the hypomethylated CRE region of the YR2 5'-LTR, whereas we detected no CREB/CREM/ATF recruitment to the hypermethylated corresponding region in the L267 cells. Altogether, these findings suggest that site-specific DNA methylation of the BLV promoter represses viral transcription by directly inhibiting transcription factor binding, thereby contributing to true proviral latency.

Neuronal DNA Methyltransferases: Epigenetic Mediators between Synaptic Activity and Gene Expression?

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Bayraktar, Gonca; Kreutz, Michael R

2018-04-01

DNMT3A and 3B are the main de novo DNA methyltransferases (DNMTs) in the brain that introduce new methylation marks to non-methylated DNA in postmitotic neurons. DNA methylation is a key epigenetic mark that is known to regulate important cellular processes in neuronal development and brain plasticity. Accumulating evidence disclosed rapid and dynamic changes in DNA methylation of plasticity-relevant genes that are important for learning and memory formation. To understand how DNMTs contribute to brain function and how they are regulated by neuronal activity is a prerequisite for a deeper appreciation of activity-dependent gene expression in health and disease. This review discusses the functional role of de novo methyltransferases and in particular DNMT3A1 in the adult brain with special emphasis on synaptic plasticity, memory formation, and brain disorders.

Development of fluorescent methods for DNA methyltransferase assay

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Li, Yueying; Zou, Xiaoran; Ma, Fei; Tang, Bo; Zhang, Chun-yang

2017-03-01

DNA methylation modified by DNA methyltransferase (MTase) plays an important role in regulating gene transcription, cell growth and proliferation. The aberrant DNA MTase activity may lead to a variety of human diseases including cancers. Therefore, accurate and sensitive detection of DNA MTase activity is crucial to biomedical research, clinical diagnostics and therapy. However, conventional DNA MTase assays often suffer from labor-intensive operations and time-consuming procedures. Alternatively, fluorescent methods have significant advantages of simplicity and high sensitivity, and have been widely applied for DNA MTase assay. In this review, we summarize the recent advances in the development of fluorescent methods for DNA MTase assay. These emerging methods include amplification-free and the amplification-assisted assays. Moreover, we discuss the challenges and future directions of this area.

Fusion of GFP to the M.EcoKI DNA methyltransferase produces a new probe of Type I DNA restriction and modification enzymes

International Nuclear Information System (INIS)

Chen, Kai; Roberts, Gareth A.; Stephanou, Augoustinos S.; Cooper, Laurie P.; White, John H.; Dryden, David T.F.

2010-01-01

Research highlights: → Successful fusion of GFP to M.EcoKI DNA methyltransferase. → GFP located at C-terminal of sequence specificity subunit does not later enzyme activity. → FRET confirms structural model of M.EcoKI bound to DNA. -- Abstract: We describe the fusion of enhanced green fluorescent protein to the C-terminus of the HsdS DNA sequence-specificity subunit of the Type I DNA modification methyltransferase M.EcoKI. The fusion expresses well in vivo and assembles with the two HsdM modification subunits. The fusion protein functions as a sequence-specific DNA methyltransferase protecting DNA against digestion by the EcoKI restriction endonuclease. The purified enzyme shows Foerster resonance energy transfer to fluorescently-labelled DNA duplexes containing the target sequence and to fluorescently-labelled ocr protein, a DNA mimic that binds to the M.EcoKI enzyme. Distances determined from the energy transfer experiments corroborate the structural model of M.EcoKI.

Self-assembling of calcium salt of the new DNA base 5-carboxylcytosine

Energy Technology Data Exchange (ETDEWEB)

Irrera, Simona [Department of Chemistry, SAPIENZA University of Rome, Piazzale A. Moro 5, 00185 Rome (Italy); Department of Chemistry, University College London, 20 Grodon Street, WC1H0AJ London (United Kingdom); Ruiz-Hernandez, Sergio E. [School of Chemistry, Cardiff University Main Building, Park Place, CF103AT Cardiff (United Kingdom); Reggente, Melania [Department of Basic and Applied Sciences for Engineering, SAPIENZA University of Rome, Via A. Scarpa 16, 00161 Rome (Italy); Passeri, Daniele, E-mail: daniele.passeri@uniroma1.it [Department of Basic and Applied Sciences for Engineering, SAPIENZA University of Rome, Via A. Scarpa 16, 00161 Rome (Italy); Natali, Marco [Department of Basic and Applied Sciences for Engineering, SAPIENZA University of Rome, Via A. Scarpa 16, 00161 Rome (Italy); Gala, Fabrizio [Department of Basic and Applied Sciences for Engineering, SAPIENZA University of Rome, Via A. Scarpa 16, 00161 Rome (Italy); Department of Medical-Surgical, Techno-Biomedical Sciences and Translational Medicine of SAPIENZA University of Rome, Sant’Andrea Hospital, Rome (Italy); Zollo, Giuseppe [Department of Basic and Applied Sciences for Engineering, SAPIENZA University of Rome, Via A. Scarpa 16, 00161 Rome (Italy); Rossi, Marco [Department of Basic and Applied Sciences for Engineering, SAPIENZA University of Rome, Via A. Scarpa 16, 00161 Rome (Italy); Research Center for Nanotechnology applied to Engineering of SAPIENZA University of Rome (CNIS), Piazzale A. Moro 5, 00185 Rome (Italy); Portalone, Gustavo, E-mail: gustavo.portalone@uniroma1.it [Department of Chemistry, SAPIENZA University of Rome, Piazzale A. Moro 5, 00185 Rome (Italy)

2017-06-15

Highlights: • Ca salt of 5-carboxylcytosine has been deposited on HOPG substrate. • Molecules self-assembled in monolayers and filaments. • Height of the features were measured by atomic force microscopy. • Ab-initio calculations confirmed the AFM results. - Abstract: Supramolecular architectures involving DNA bases can have a strong impact in several fields such as nanomedicine and nanodevice manufacturing. To date, in addition to the four canonical nucleobases (adenine, thymine, guanine and cytosine), four other forms of cytosine modified at the 5 position have been identified in DNA. Among these four new cytosine derivatives, 5-carboxylcytosine has been recently discovered in mammalian stem cell DNA, and proposed as the final product of the oxidative epigenetic demethylation pathway on the 5 position of cytosine. In this work, a calcium salt of 5-carboxylcytosine has been synthesized and deposited on graphite surface, where it forms self-assembled features as long range monolayers and up to one micron long filaments. These structures have been analyzed in details combining different theoretical and experimental approaches: X-ray single-crystal diffraction data were used to simulate the molecule-graphite interaction, first using molecular dynamics and then refining the results using density functional theory (DFT); finally, data obtained with DFT were used to rationalize atomic force microscopy (AFM) results.

Arabidopsis DNA methyltransferase AtDNMT2 associates with histone deacetylase AtHD2s activity

International Nuclear Information System (INIS)

Song, Yuan; Wu, Keqiang; Dhaubhadel, Sangeeta; An, Lizhe; Tian, Lining

2010-01-01

DNA methyltransferase2 (DNMT2) is always deemed to be enigmatic, because it contains highly conserved DNA methyltransferase motifs but lacks the DNA methylation catalytic capability. Here we show that Arabidopsis DNA methyltransferase2 (AtDNMT2) is localized in nucleus and associates with histone deacetylation. Bimolecular fluorescence complementation and pull-down assays show AtDNMT2 interacts with type-2 histone deacetylases (AtHD2s), a unique type of histone deacetylase family in plants. Through analyzing the expression of AtDNMT2: ss-glucuronidase (GUS) fusion protein, we demonstrate that AtDNMT2 has the ability to repress gene expression at transcription level. Meanwhile, the expression of AtDNMT2 gene is altered in athd2c mutant plants. We propose that AtDNMT2 possibly involves in the activity of histone deacetylation and plant epigenetic regulatory network.

Arabidopsis DNA methyltransferase AtDNMT2 associates with histone deacetylase AtHD2s activity

Energy Technology Data Exchange (ETDEWEB)

Song, Yuan [Key Laboratory of Arid and Grassland Agroecology, Ministry of Education, School of Life Science, Lanzhou University, Lanzhou 730000 (China); Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3 (Canada); Wu, Keqiang [Institute of Plant Biology, National Taiwan University, Taipei 106, Taiwan (China); Dhaubhadel, Sangeeta [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3 (Canada); An, Lizhe, E-mail: lizhean@lzu.edu.cn [Key Laboratory of Arid and Grassland Agroecology, Ministry of Education, School of Life Science, Lanzhou University, Lanzhou 730000 (China); Tian, Lining, E-mail: tianl@agr.gc.ca [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3 (Canada)

2010-05-28

DNA methyltransferase2 (DNMT2) is always deemed to be enigmatic, because it contains highly conserved DNA methyltransferase motifs but lacks the DNA methylation catalytic capability. Here we show that Arabidopsis DNA methyltransferase2 (AtDNMT2) is localized in nucleus and associates with histone deacetylation. Bimolecular fluorescence complementation and pull-down assays show AtDNMT2 interacts with type-2 histone deacetylases (AtHD2s), a unique type of histone deacetylase family in plants. Through analyzing the expression of AtDNMT2: ss-glucuronidase (GUS) fusion protein, we demonstrate that AtDNMT2 has the ability to repress gene expression at transcription level. Meanwhile, the expression of AtDNMT2 gene is altered in athd2c mutant plants. We propose that AtDNMT2 possibly involves in the activity of histone deacetylation and plant epigenetic regulatory network.

Genomic survey, gene expression analysis and structural modeling suggest diverse roles of DNA methyltransferases in legumes.

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Rohini Garg

Full Text Available DNA methylation plays a crucial role in development through inheritable gene silencing. Plants possess three types of DNA methyltransferases (MTases, namely Methyltransferase (MET, Chromomethylase (CMT and Domains Rearranged Methyltransferase (DRM, which maintain methylation at CG, CHG and CHH sites. DNA MTases have not been studied in legumes so far. Here, we report the identification and analysis of putative DNA MTases in five legumes, including chickpea, soybean, pigeonpea, Medicago and Lotus. MTases in legumes could be classified in known MET, CMT, DRM and DNA nucleotide methyltransferases (DNMT2 subfamilies based on their domain organization. First three MTases represent DNA MTases, whereas DNMT2 represents a transfer RNA (tRNA MTase. Structural comparison of all the MTases in plants with known MTases in mammalian and plant systems have been reported to assign structural features in context of biological functions of these proteins. The structure analysis clearly specified regions crucial for protein-protein interactions and regions important for nucleosome binding in various domains of CMT and MET proteins. In addition, structural model of DRM suggested that circular permutation of motifs does not have any effect on overall structure of DNA methyltransferase domain. These results provide valuable insights into role of various domains in molecular recognition and should facilitate mechanistic understanding of their function in mediating specific methylation patterns. Further, the comprehensive gene expression analyses of MTases in legumes provided evidence of their role in various developmental processes throughout the plant life cycle and response to various abiotic stresses. Overall, our study will be very helpful in establishing the specific functions of DNA MTases in legumes.

Crystal structure of Mycobacterium tuberculosis O-6-methylguanine-DNA methyltransferase protein clusters assembled on to damaged DNA

Czech Academy of Sciences Publication Activity Database

Miggiano, R.; Perugino, G.; Ciaramella, M.; Serpe, M.; Rejman, Dominik; Páv, Ondřej; Pohl, Radek; Garavaglia, S.; Lahiri, S.; Rizzi, M.; Rossi, F.

2016-01-01

Roč. 473, č. 2 (2016), s. 123-133 ISSN 0264-6021 EU Projects: European Commission(XE) 241587 - SYSTEMTB Institutional support: RVO:61388963 Keywords : DNA repair * DNA-binding protein * Mycobacterium tuberculosis * O-6-methylguanine-DNA methyltransferase * co-operativity * crystal structure Subject RIV: CE - Biochemistry Impact factor: 3.797, year: 2016

Quantification of 5-methyl-2'-deoxycytidine in the DNA.

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Giel-Pietraszuk, Małgorzata; Insińska-Rak, Małgorzata; Golczak, Anna; Sikorski, Marek; Barciszewska, Mirosława; Barciszewski, Jan

2015-01-01

Methylation at position 5 of cytosine (Cyt) at the CpG sequences leading to formation of 5-methyl-cytosine (m(5)Cyt) is an important element of epigenetic regulation of gene expression. Modification of the normal methylation pattern, unique to each organism, leads to the development of pathological processes and diseases, including cancer. Therefore, quantification of the DNA methylation and analysis of changes in the methylation pattern is very important from a practical point of view and can be used for diagnostic purposes, as well as monitoring of the treatment progress. In this paper we present a new method for quantification of 5-methyl-2'deoxycytidine (m(5)C) in the DNA. The technique is based on conversion of m(5)C into fluorescent 3,N(4)-etheno-5-methyl-2'deoxycytidine (εm(5)C) and its identification by reversed-phase high-performance liquid chromatography (RP-HPLC). The assay was used to evaluate m(5)C concentration in DNA of calf thymus and peripheral blood of cows bred under different conditions. This approach can be applied for measuring of 5-methylcytosine in cellular DNA from different cells and tissues.

5-aza-2′-deoxycytidine impairs mouse spermatogenesis at multiple stages through different usage of DNA methyltransferases

International Nuclear Information System (INIS)

Song, Ning; Endo, Daisuke; Song, Bin; Shibata, Yasuaki; Koji, Takehiko

2016-01-01

Mammalian spermatogenesis is a progressive process comprising spermatogonial proliferation, spermatocytic meiosis, and later spermiogenesis, which is considered to be under the regulation of epigenetic parameters. To gain insights into the significance of DNA methylation in early spermatogenesis, 5-azadC was used as a molecular biological tool to mimic the level of DNA methylation in vivo. Since the drug is incorporated into DNA during the S-phase, spermatogonia and spermatocytes would be affected primarily in mouse spermatogenesis. Adult male ICR mice were intraperitoneally injected with 5-azadC at a dose of 0.25 mg/kg/day for 10 consecutive days, allowing us to examine its maximum effect on the kinetics of spermatogonia and spermatocytes. In this short-term protocol, 5-azadC induced significant histological abnormalities, such as a marked increase in apoptosis of spermatogonia and spermatocytes, followed by severe loss of spermatids, while after termination of 5-azadC treatment, normal histology was restored in the testis within 35 days. Quantification of the methylation level of CCGG sites as well as whole DNA showed spermatogonial hypomethylation, which correlated with increased apoptosis of spermatogonia. Interestingly, the hypomethylated cells were simultaneously positive for tri-methylated histone H3 at K4. On the other hand, no changes in methylation level were found in spermatocytes, but PCNA staining clearly showed disordered accumulation of S-phase spermatocytes, which increased their apoptosis in stage XII. In addition, different immunohistochemical staining pattern was found for DNA methyltransferases (DNMTs); DNMT1was expressed in the majority of all germ cells, but DNMT3a and b were only expressed in spermatogonia. Our results indicate that 5-azadC caused DNA hypomethylation in spermatogonia, but induced prolongation of S-phase in spermatocytes, resulting in the induction of apoptosis in both cases. Thus, 5-azadC affects spermatogenesis at more than

Dysregulated DNA Methyltransferase 3A Upregulates IGFBP5 to Suppress Trophoblast Cell Migration and Invasion in Preeclampsia.

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Jia, Yuanhui; Li, Ting; Huang, Xiaojie; Xu, Xianghong; Zhou, Xinyao; Jia, Linyan; Zhu, Jingping; Xie, Dandan; Wang, Kai; Zhou, Qian; Jin, Liping; Zhang, Jiqin; Duan, Tao

2017-02-01

Preeclampsia is a unique multiple system disorder during human pregnancy, which affects ≈5% to 8% of pregnancies. Its risks and complications have become the major causes of maternal and fetal morbidity and mortality. Although abnormal placentation to which DNA methylation dysregulation is always linked is speculated to be one of the reasons causing preeclampsia, the underlying mechanisms still remain elusive to date. Here we revealed that aberrant DNA methyltransferase 3A (DNMT3A) plays a critical role in preeclampsia. Our results show that the expression and localization of DNMT3A are dysregulated in preeclamptic placenta. Moreover, knockdown of DNMT3A obviously inhibits trophoblast cell migration and invasion. Mechanistically, IGFBP5 (insulin-like growth factor-binding protein 5), known as a suppressor, is upregulated by decreased DNMT3A because of promoter hypomethylation. Importantly, IGFBP5 downregulation can rescue the defects caused by DNMT3A knockdown, thereby, consolidating the significance of IGFBP5 in the downstream of DNMT3A in trophoblast. Furthermore, we detected low promoter methylation and high protein expression of IGFBP5 in the clinical samples of preeclamptic placenta. Collectively, our study suggests that dysregulation of DNMT3A and IGFBP5 is relevant to preeclampsia. Thus, we propose that DNMT3A and IGFBP5 can serve as potential markers and targets for the clinical diagnosis and therapy of preeclampsia. © 2017 American Heart Association, Inc.

Cytosine deamination and the precipitous decline of spontaneous mutation during Earth's history.

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Lewis, Charles A; Crayle, Jesse; Zhou, Shuntai; Swanstrom, Ronald; Wolfenden, Richard

2016-07-19

The hydrolytic deamination of cytosine and 5-methylcytosine residues in DNA appears to contribute significantly to the appearance of spontaneous mutations in microorganisms and in human disease. In the present work, we examined the mechanism of cytosine deamination and the response of the uncatalyzed reaction to changing temperature. The positively charged 1,3-dimethylcytosinium ion was hydrolyzed at a rate similar to the rate of acid-catalyzed hydrolysis of 1-methylcytosine, for which it furnishes a satisfactory kinetic model and a probable mechanism. In agreement with earlier reports, uncatalyzed deamination was found to proceed at very similar rates for cytosine, 1-methylcytosine, cytidine, and cytidine 5'-phosphate, and also for cytosine residues in single-stranded DNA generated from a phagemid, in which we sequenced an insert representing the gene of the HIV-1 protease. Arrhenius plots for the uncatalyzed deamination of cytosine were linear over the temperature range from 90 °C to 200 °C and indicated a heat of activation (ΔH(‡)) of 23.4 ± 0.5 kcal/mol at pH 7. Recent evidence indicates that the surface of the earth has been cool enough to support life for more than 4 billion years and that life has been present for almost as long. If the temperature at Earth's surface is assumed to have followed Newton's law of cooling, declining exponentially from 100 °C to 25 °C during that period, then half of the cytosine-deaminating events per unit biomass would have taken place during the first 0.2 billion years, and <99.4% would have occurred during the first 2 billion years.

Critical threshold levels of DNA methyltransferase 1 are required to maintain DNA methylation across the genome in human cancer cells.

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Cai, Yi; Tsai, Hsing-Chen; Yen, Ray-Whay Chiu; Zhang, Yang W; Kong, Xiangqian; Wang, Wei; Xia, Limin; Baylin, Stephen B

2017-04-01

Reversing DNA methylation abnormalities and associated gene silencing, through inhibiting DNA methyltransferases (DNMTs) is an important potential cancer therapy paradigm. Maximizing this potential requires defining precisely how these enzymes maintain genome-wide, cancer-specific DNA methylation. To date, there is incomplete understanding of precisely how the three DNMTs, 1, 3A, and 3B, interact for maintaining DNA methylation abnormalities in cancer. By combining genetic and shRNA depletion strategies, we define not only a dominant role for DNA methyltransferase 1 (DNMT1) but also distinct roles of 3A and 3B in genome-wide DNA methylation maintenance. Lowering DNMT1 below a threshold level is required for maximal loss of DNA methylation at all genomic regions, including gene body and enhancer regions, and for maximally reversing abnormal promoter DNA hypermethylation and associated gene silencing to reexpress key genes. It is difficult to reach this threshold with patient-tolerable doses of current DNMT inhibitors (DNMTIs). We show that new approaches, like decreasing the DNMT targeting protein, UHRF1, can augment the DNA demethylation capacities of existing DNA methylation inhibitors for fully realizing their therapeutic potential. © 2017 Cai et al.; Published by Cold Spring Harbor Laboratory Press.

Caulobacter crescentus Cell Cycle-Regulated DNA Methyltransferase Uses a Novel Mechanism for Substrate Recognition.

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Woodcock, Clayton B; Yakubov, Aziz B; Reich, Norbert O

2017-08-01

Caulobacter crescentus relies on DNA methylation by the cell cycle-regulated methyltransferase (CcrM) in addition to key transcription factors to control the cell cycle and direct cellular differentiation. CcrM is shown here to efficiently methylate its cognate recognition site 5'-GANTC-3' in single-stranded and hemimethylated double-stranded DNA. We report the K m , k cat , k methylation , and K d for single-stranded and hemimethylated substrates, revealing discrimination of 10 7 -fold for noncognate sequences. The enzyme also shows a similar discrimination against single-stranded RNA. Two independent assays clearly show that CcrM is highly processive with single-stranded and hemimethylated DNA. Collectively, the data provide evidence that CcrM and other DNA-modifying enzymes may use a new mechanism to recognize DNA in a key epigenetic process.

DNMT (DNA methyltransferase) inhibitors radiosensitize human cancer cells by suppressing DNA repair activity

International Nuclear Information System (INIS)

Kim, Hak Jae; Kim, Jin Ho; Chie, Eui Kyu; Da Young, Park; Kim, In Ah; Kim, Il Han

2012-01-01

Histone modifications and DNA methylation are two major factors in epigenetic phenomenon. Unlike the histone deacetylase inhibitors, which are known to exert radiosensitizing effects, there have only been a few studies thus far concerning the role of DNA methyltransferase (DNMT) inhibitors as radiosensitizers. The principal objective of this study was to evaluate the effects of DNMT inhibitors on the radiosensitivity of human cancer cell lines, and to elucidate the mechanisms relevant to that process. A549 (lung cancer) and U373MG (glioblastoma) cells were exposed to radiation with or without six DNMT inhibitors (5-azacytidine, 5-aza-2'-deoxycytidine, zebularine, hydralazine, epigallocatechin gallate, and psammaplin A) for 18 hours prior to radiation, after which cell survival was evaluated via clonogenic assays. Cell cycle and apoptosis were analyzed via flow cytometry. Expressions of DNMT1, 3A/3B, and cleaved caspase-3 were detected via Western blotting. Expression of γH2AX, a marker of radiation-induced DNA double-strand break, was examined by immunocytochemistry. Pretreatment with psammaplin A, 5-aza-2'-deoxycytidine, and zebularine radiosensitized both A549 and U373MG cells. Pretreatment with psammaplin A increased the sub-G1 fraction of A549 cells, as compared to cells exposed to radiation alone. Prolongation of γH2AX expression was observed in the cells treated with DNMT inhibitors prior to radiation as compared with those treated by radiation alone. Psammaplin A, 5-aza-2'-deoxycytidine, and zebularine induce radiosensitivity in both A549 and U373MG cell lines, and suggest that this effect might be associated with the inhibition of DNA repair

Crystal structure of Mycobacterium tuberculosis O6-methylguanine-DNA methyltransferase protein clusters assembled on to damaged DNA.

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Miggiano, Riccardo; Perugino, Giuseppe; Ciaramella, Maria; Serpe, Mario; Rejman, Dominik; Páv, Ondřej; Pohl, Radek; Garavaglia, Silvia; Lahiri, Samarpita; Rizzi, Menico; Rossi, Franca

2016-01-15

Mycobacterium tuberculosis O(6)-methylguanine-DNA methyltransferase (MtOGT) contributes to protect the bacterial GC-rich genome against the pro-mutagenic potential of O(6)-methylated guanine in DNA. Several strains of M. tuberculosis found worldwide encode a point-mutated O(6)-methylguanine-DNA methyltransferase (OGT) variant (MtOGT-R37L), which displays an arginine-to-leucine substitution at position 37 of the poorly functionally characterized N-terminal domain of the protein. Although the impact of this mutation on the MtOGT activity has not yet been proved in vivo, we previously demonstrated that a recombinant MtOGT-R37L variant performs a suboptimal alkylated-DNA repair in vitro, suggesting a direct role for the Arg(37)-bearing region in catalysis. The crystal structure of MtOGT complexed with modified DNA solved in the present study reveals details of the protein-protein and protein-DNA interactions occurring during alkylated-DNA binding, and the protein capability also to host unmodified bases inside the active site, in a fully extrahelical conformation. Our data provide the first experimental picture at the atomic level of a possible mode of assembling three adjacent MtOGT monomers on the same monoalkylated dsDNA molecule, and disclose the conformational flexibility of discrete regions of MtOGT, including the Arg(37)-bearing random coil. This peculiar structural plasticity of MtOGT could be instrumental to proper protein clustering at damaged DNA sites, as well as to protein-DNA complexes disassembling on repair. © 2016 Authors; published by Portland Press Limited.

Identification of a highly conserved domain in the EcoRII methyltransferase which can be photolabeled with S-adenosyl-L-[methyl-3H]methionine. Evidence for UV-induced transmethylation of cysteine 186

International Nuclear Information System (INIS)

Som, S.; Friedman, S.

1991-01-01

DNA methyltransferases can be photolabeled with S-adenosyl-L-methionine (AdoMet). Specific incorporation of radioactivity has been demonstrated after photolabeling with either [methyl-3H]AdoMet or [35S]AdoMet. The labeling is believed to occur at the AdoMet binding site. With the purpose of localizing the site responsible for [methyl-3H]AdoMet photolabeling, we cleaved the labeled EcoRII methyltransferase by chemical and enzymatic reactions and isolated the radiolabeled peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high pressure liquid chromatography. The labeled peptides were identified by amino-terminal sequencing. A common region was localized which accounted for 65-70% of the total label. This region includes a highly conserved core sequence present in all DNA (cytosine 5)-methyltransferases. One such fragment was digested further with chymotrypsin, and amino acid analysis of the resulting 3H-labeled peptide was consistent with the sequence Ala-Gly-Phe-Pro-(Cys)-Gln-Pro-Phe-Ser-Leu. However, the cysteine residue was not recovered as carboxymethylcysteine. The Pro-Cys bond was found to be protected from cleavage at cysteine residues after cyanylation. These results suggest that the cysteine residue is modified by the labeling reaction. The chymotryptic fragment was hydrolyzed enzymatically to single amino acids, and the labeled amino acid was identified as S-methylcysteine by thin layer chromatography. These results indicate that the cysteine residue is located at or close to the AdoMet binding site of EcoRII methyltransferase

The orphan nuclear receptor GCNF recruits DNA methyltransferase for Oct-3/4 silencing

International Nuclear Information System (INIS)

Sato, Noriko; Kondo, Mitsumasa; Arai, Ken-ichi

2006-01-01

Somatic DNA methylation patterns are determined in part by the de novo methylation that occurs after early embryonic demethylation. Oct-3/4, a pluripotency gene, is unmethylated in the blastocyst, but undergoes de novo methylation and silencing during gastrulation. Here we show that the transcriptional repressor GCNF recruits DNA methyltransferase to the Oct-3/4 promoter and facilitates its methylation. Although acetylation of histone H3 at lysine 9 (K9) and/or 14 (K14) and methylation of H3 at lysine 4 (K4) decrease during this period, as do Oct-3/4 transcript levels, H3K9 and H3K27 methylation levels remain constant, indicating that DNA methylation does not require repressive histone modifications. We found that GCNF interacts directly with Dnmt3 molecule(s) and verified that this interaction induces the methylation of the Oct-3/4 promoter. Our finding suggests a model in which differentiation-induced GCNF recruits de novo DNA methyltransferase and facilitates the silencing of a pluripotency gene

O6-methylguanine-DNA methyltransferase in wild-type and ada mutants of Escherichia coli

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Mitra, S.; Pal, B.C.; Foote, R.S.

1982-01-01

O 6 -Methylguanine-DNA methyltransferase is induced in Escherichia coli during growth in low levels of N-methyl-N'-nitro-N-nitrosoguanidine. We have developed a sensitive assay for quantitating low levels of this activity with a synthetic DNA substrate containing 3 H-labeled O 6 -methylguanine as the only modified base. Although both wild-type and adaptation-deficient (ada) mutants of E. coli contained low but comparable numbers (from 13 to 60) of the enzyme molecules per cell, adaptation treatment caused a significant increase of the enzyme in the wild type but not in the ada mutants, suggesting that the ada mutation is in a regulatory locus and not in the structural gene for the methyltransferase

Dynamic DNA cytosine methylation in the Populus trichocarpa genome: tissue-level variation and relationship to gene expression

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Vining Kelly J

2012-01-01

Full Text Available Abstract Background DNA cytosine methylation is an epigenetic modification that has been implicated in many biological processes. However, large-scale epigenomic studies have been applied to very few plant species, and variability in methylation among specialized tissues and its relationship to gene expression is poorly understood. Results We surveyed DNA methylation from seven distinct tissue types (vegetative bud, male inflorescence [catkin], female catkin, leaf, root, xylem, phloem in the reference tree species black cottonwood (Populus trichocarpa. Using 5-methyl-cytosine DNA immunoprecipitation followed by Illumina sequencing (MeDIP-seq, we mapped a total of 129,360,151 36- or 32-mer reads to the P. trichocarpa reference genome. We validated MeDIP-seq results by bisulfite sequencing, and compared methylation and gene expression using published microarray data. Qualitative DNA methylation differences among tissues were obvious on a chromosome scale. Methylated genes had lower expression than unmethylated genes, but genes with methylation in transcribed regions ("gene body methylation" had even lower expression than genes with promoter methylation. Promoter methylation was more frequent than gene body methylation in all tissues except male catkins. Male catkins differed in demethylation of particular transposable element categories, in level of gene body methylation, and in expression range of genes with methylated transcribed regions. Tissue-specific gene expression patterns were correlated with both gene body and promoter methylation. Conclusions We found striking differences among tissues in methylation, which were apparent at the chromosomal scale and when genes and transposable elements were examined. In contrast to other studies in plants, gene body methylation had a more repressive effect on transcription than promoter methylation.

Hydroxyl radical induced cross-linking of cytosine and tyrosine in nucleohistone

International Nuclear Information System (INIS)

Gajewski, E.; Dizdaroglu, M.

1990-01-01

Hydroxyl radical induced formation of a DNA-protein cross-link involving cytosine and tyrosine in nucleohistone in buffered aqueous solution is reported. The technique of gas chromatography-mass spectrometry was used for this investigation. A γ-irradiated aqueous mixture of cytosine and tyrosine was first investigated in order to obtain gas chromatographic-mass spectrometric properties of possible cytosine-tyrosine cross-links. One cross-link was observed, and its structure was identified as the product from the formation of a covalent bond between carbon 6 of cytosine and carbon 3 of tyrosine. With the use of gas chromatography-mass spectrometry with selected-ion monitoring, this cytosine-tyrosine cross-link was identified in acidic hydrolysates of calf thymus nucleohistone γ-irradiated in N 2 O-saturated aqueous solution. The yield of this DNA-protein cross-link in nucleohistone was found to be a linear function of the radiation dose in the range of 100-500 Gy (J·kg -1 ). This yield amounted to 0.05 nmol·J -1 . Mechanisms underlying the formation of the cytosine-tyrosine cross-link in nucleohistone were proposed to involve radical-radical and/or radical addition reactions of hydroxyl adduct radicals of cytosine and tyrosine moieties, forming a covalent bond between carbon 6 of cytosine and carbon 3 of tyrosine. When oxygen was present in irradiated solutions, no cytosine-tyrosine cross-links were observed

O6-Methylguanine DNA Methyltransferase Status Does Not Predict Response or Resistance to Alkylating Agents in Well-Differentiated Pancreatic Neuroendocrine Tumors.

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Raj, Nitya; Klimstra, David S; Horvat, Natally; Zhang, Liying; Chou, Joanne F; Capanu, Marinela; Basturk, Olca; Do, Richard Kinh Gian; Allen, Peter J; Reidy-Lagunes, Diane

2017-07-01

Alkylating agents have activity in well-differentiated pancreatic neuroendocrine tumors (WD panNETs). In glioblastoma multiforme, decreased activity of O-methylguanine DNA methyltransferase (MGMT) predicts response; in panNETs, MGMT relevance is unknown. We identified patients with WD panNETs treated with alkylating agents, determined best overall response by Response Evaluation Criteria In Solid Tumors (RECIST) 1.1, and performed MGMT activity testing. Fifty-six patients were identified; 26 (46%) of the 56 patients experienced partial response, 24 (43%) of 56 experienced stable disease, and 6 (11%) of 56 experienced progression of disease. O-methylguanine DNA methyltransferase status was available for 36 tumors. For tumors with partial response, 10 (67%) of 15 were MGMT deficient, and 5 (33%) of 15 were MGMT intact. For tumors with stable disease, 7 (47%) of 15 were MGMT deficient, and 8 (53%) of 15 were MGMT intact. For tumors with progression of disease, 3 (50%) of 6 were MGMT deficient, and 3 (50%) of 6 were MGMT intact. We observed response and resistance to alkylating agents in MGMT-deficient and MGMT-intact tumors. O-methylguanine DNA methyltransferase status should not guide alkylating agent therapy in WD panNETs.

Reasons of carcinogenesis indicate a big-bang inside: a hypothesis for the aberration of DNA methylation.

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Roy, A; Roy Chattopadhyay, N

2013-07-01

Cancer involves various sets of altered gene functions which embrace all the three basic mechanisms of regulation of gene expression. However, no common mechanism is inferred till date for this versatile disease and thus no full proof remedy can be offered. Here we show that the basic mechanisms are interlinked and indicate towards one of those mechanisms as being the superior one; the methylation of cytosines in specific DNA sequences, for the initiation and maintenance of carcinogenesis. The analyses of the previous reports and the nucleotide sequences of the DNA methyltransferases strongly support the assumption that the mutation(s) in the DNA-binding site(s) of DNA-methyltransferases acts as a master regulator; though it continues the cycle from mutation to repair to methylation. We anticipate that our hypothesis will start a line of study for the proposal of a treatment regime for cancers by introducing wild type methyltransferases in the diseased cells and/or germ cells, and/or by targeting ligands to the altered binding domain(s) where a mutation in the concerned enzyme(s) is seen. Copyright © 2013. Published by Elsevier Ltd.

Potential advantages of DNA methyltransferase 1 (DNMT1)-targeted inhibition for cancer therapy.

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Jung, Yeonjoo; Park, Jinah; Kim, Tai Young; Park, Jung-Hyun; Jong, Hyun-Soon; Im, Seock-Ah; Robertson, Keith D; Bang, Yung-Jue; Kim, Tae-You

2007-10-01

The deoxyribonucleic acid (DNA) methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) has been used as a drug in a part of cancer therapy. However, because of its incorporation into DNA during DNA synthesis, 5-aza-dC can cause DNA damage, mutagenesis, and cytotoxicity. In view of the adverse effects of 5-aza-dC, DNMT-targeted inhibition may be a more effective approach than treatment with 5-aza-dC. To address the possibility of DNMT-targeted cancer therapy, we compared the effects of treatment with small interfering ribonucleic acids (siRNAs) specific for DNMT1 or DNMT3b and treatment with 5-aza-dC on transcription, cell growth, and DNA damage in gastric cancer cells. We found that DNMT1-targeted inhibition induced the re-expression and reversed DNA methylation of five (CDKN2A, RASSF1A, HTLF, RUNX3, and AKAP12B) out of seven genes examined, and 5-aza-dC reactivated and demethylated all seven genes. In contrast, DNMT3b siRNAs did not show any effect. Furthermore, the double knockdown of DNMT1 and DNMT3b did not show a synergistic effect on gene re-expression and demethylation. In addition, DNMT1 siRNAs showed an inhibitory effect of cell proliferation in the cancer cells and the induction of cell death without evidence of DNA damage, whereas treatment with 5-aza-dC caused DNA damage as demonstrated by the comet assay. These results provide a rationale for the development of a DNMT1-targeted strategy as an effective epigenetic cancer therapy.

Expression of DNA methyltransferases is influenced by growth hormone in the long-living Ames dwarf mouse in vivo and in vitro.

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Armstrong, Vanessa L; Rakoczy, Sharlene; Rojanathammanee, Lalida; Brown-Borg, Holly M

2014-08-01

Methyltransferase expression and DNA methylation are linked to aging and age-related disease. We utilized 3-, 12-, and 24-month-old Ames dwarf and their wild-type siblings to examine the genotype and age-related differences in the expression of methyltransferase enzymes related to DNA methylation in the liver, glycine-N-methyltransferase and DNA methyltransferase (DNMT). We found that DNMT proteins and transcripts are differentially expressed in dwarf mice compared with wild-type siblings that can be attributed to age and/or genotype. However, DNMT1 protein expression is drastically reduced compared with wild-type controls at every age. DNMT3a protein levels coincide with differences observed in DNMT activity. Growth hormone appears to modulate expression of DNMT1 and 3a in dwarf liver tissue and primary hepatocytes. Therefore, growth hormone may contribute to age-related processes, DNA methylation, and, ultimately, longevity. © The Author 2013. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The histone H3 lysine 9 methyltransferase DIM-5 modifies chromatin at frequency and represses light-activated gene expression.

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Ruesch, Catherine E; Ramakrishnan, Mukund; Park, Jinhee; Li, Na; Chong, Hin S; Zaman, Riasat; Joska, Tammy M; Belden, William J

2014-11-25

The transcriptional program controlling the circadian rhythm requires coordinated regulation of chromatin. Characterization of the chromodomain helicase DNA-binding enzyme CHD1 revealed DNA methylation in the promoter of the central clock gene frequency (frq) in Neurospora crassa. In this report, we show that the DNA methylation at frq is not only dependent on the DNA methyltransferase DIM-2 but also on the H3K9 methyltransferase DIM-5 and HP1. Histone H3 lysine 9 trimethylation (H3K9me3) occurs at frq and is most prominent 30 min after light-activated expression. Strains lacking dim-5 have an increase in light-induced transcription, and more White Collar-2 is found associated with the frq promoter. Consistent with the notion that DNA methylation assists in establishing the proper circadian phase, loss of H3K9 methylation results in a phase advance suggesting it delays the onset of frq expression. The dim-5 deletion strain displays an increase in circadian-regulated conidia formation on race tubes and there is a synthetic genetic interaction between dim-5 and ras-1(bd). These results indicate DIM-5 has a regulatory role in muting circadian output. Overall, the data support a model where facultative heterochromatic at frq serves to establish the appropriate phase, mute the light response, and repress circadian output. Copyright © 2015 Ruesch et al.

Ultraviolet and chemical induced DNA repair in human cells assayed by bromodeoxyuridine photolysis or cytosine arabinoside arrest

International Nuclear Information System (INIS)

Regan, J.D.; Dunn, W.C.

1979-01-01

The bromodeoxyuridine photolysis assay of DNA damage in human cells permits an estimate of both the number of repaired regions in the DNA and the size of the average repaired region - the patch size. The antineoplastic agent arabinofuranosyl cytosine (ara-C) can also be employed to assay the magnitude of repair since this agent appears to block rejoining of single-strand incisions made in the DNA during the initial step of repair. Thus, the number of incisions can be accumulated. The ara-C effect is dependent on the presence of hydroxyurea. Both assays can be employed for the study of physical or chemical DNA damages. Results comparing these assays are presented

Kinetic analysis of Yersinia pestis DNA adenine methyltransferase activity using a hemimethylated molecular break light oligonucleotide.

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Robert J Wood

Full Text Available BACKGROUND: DNA adenine methylation plays an important role in several critical bacterial processes including mismatch repair, the timing of DNA replication and the transcriptional control of gene expression. The dependence of bacterial virulence on DNA adenine methyltransferase (Dam has led to the proposal that selective Dam inhibitors might function as broad spectrum antibiotics. METHODOLOGY/PRINCIPAL FINDINGS: Herein we report the expression and purification of Yersinia pestis Dam and the development of a continuous fluorescence based assay for DNA adenine methyltransferase activity that is suitable for determining the kinetic parameters of the enzyme and for high throughput screening against potential Dam inhibitors. The assay utilised a hemimethylated break light oligonucleotide substrate containing a GATC methylation site. When this substrate was fully methylated by Dam, it became a substrate for the restriction enzyme DpnI, resulting in separation of fluorophore (fluorescein and quencher (dabcyl and therefore an increase in fluorescence. The assays were monitored in real time using a fluorescence microplate reader in 96 well format and were used for the kinetic characterisation of Yersinia pestis Dam, its substrates and the known Dam inhibitor, S-adenosylhomocysteine. The assay has been validated for high throughput screening, giving a Z-factor of 0.71+/-0.07 indicating that it is a sensitive assay for the identification of inhibitors. CONCLUSIONS/SIGNIFICANCE: The assay is therefore suitable for high throughput screening for inhibitors of DNA adenine methyltransferases and the kinetic characterisation of the inhibition.

The Impact of DNA Topology and Guide Length on Target Selection by a Cytosine-Specific Cas9.

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Tsui, Tsz Kin Martin; Hand, Travis H; Duboy, Emily C; Li, Hong

2017-06-16

Cas9 is an RNA-guided DNA cleavage enzyme being actively developed for genome editing and gene regulation. To be cleaved by Cas9, a double stranded DNA, or the protospacer, must be complementary to the guide region, typically 20-nucleotides in length, of the Cas9-bound guide RNA, and adjacent to a short Cas9-specific element called Protospacer Adjacent Motif (PAM). Understanding the correct juxtaposition of the protospacer- and PAM-interaction with Cas9 will enable development of versatile and safe Cas9-based technology. We report identification and biochemical characterization of Cas9 from Acidothermus cellulolyticus (AceCas9). AceCas9 depends on a 5'-NNNCC-3' PAM and is more efficient in cleaving negative supercoils than relaxed DNA. Kinetic as well as in vivo activity assays reveal that AceCas9 achieves optimal activity when combined with a guide RNA containing a 24-nucleotide complementarity region. The cytosine-specific, DNA topology-sensitive, and extended guide-dependent properties of AceCas9 may be explored for specific genome editing applications.

Protein arginine methyltransferase 5 (PRMT5) is a novel coactivator of constitutive androstane receptor (CAR)

International Nuclear Information System (INIS)

Kanno, Yuichiro; Inajima, Jun; Kato, Sayaka; Matsumoto, Maika; Tokumoto, Chikako; Kure, Yuki; Inouye, Yoshio

2015-01-01

The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that protein arginine methyltransferase 5 (PRMT5) is a novel CAR-interacting protein. Furthermore, the PRMT-dependent induction of a CAR reporter gene, which was independent of methyltransferase activity, was enhanced in the presence of steroid receptor coactivator 1 (SRC1), peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) or DEAD box DNA/RNA helicase DP97. Using tetracycline inducible-hCAR system in HepG2 cells, we showed that knockdown of PRMT5 with small interfering RNA suppressed tetracycline -induced mRNA expression of CYP2B6 but not of CYP2C9 or CYP3A4. PRMT5 enhanced phenobarbital-mediated transactivation of a phenobarbital-responsive enhancer module (PBREM)-driven reporter gene in co-operation with PGC-1α in rat primary hepatocytes. Based on these findings, we suggest PRMT5 to be a gene (or promoter)-selective coactivator of CAR by mediating the formation of complexes between hCAR and appropriate coactivators. - Highlights: • Nuclear receptor CAR interact with PRMT5. • PRMT5 enhances transcriptional activity of CAR. • PRMT5 synergistically enhances transactivity of CAR by the co-expression of SRC-1, DP97 or PGC1α. • PRMT5 is a gene-selective co-activator for hCAR

Protein arginine methyltransferase 5 (PRMT5) is a novel coactivator of constitutive androstane receptor (CAR)

Energy Technology Data Exchange (ETDEWEB)

Kanno, Yuichiro, E-mail: ykanno@phar.toho-u.ac.jp; Inajima, Jun; Kato, Sayaka; Matsumoto, Maika; Tokumoto, Chikako; Kure, Yuki; Inouye, Yoshio

2015-03-27

The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that protein arginine methyltransferase 5 (PRMT5) is a novel CAR-interacting protein. Furthermore, the PRMT-dependent induction of a CAR reporter gene, which was independent of methyltransferase activity, was enhanced in the presence of steroid receptor coactivator 1 (SRC1), peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) or DEAD box DNA/RNA helicase DP97. Using tetracycline inducible-hCAR system in HepG2 cells, we showed that knockdown of PRMT5 with small interfering RNA suppressed tetracycline -induced mRNA expression of CYP2B6 but not of CYP2C9 or CYP3A4. PRMT5 enhanced phenobarbital-mediated transactivation of a phenobarbital-responsive enhancer module (PBREM)-driven reporter gene in co-operation with PGC-1α in rat primary hepatocytes. Based on these findings, we suggest PRMT5 to be a gene (or promoter)-selective coactivator of CAR by mediating the formation of complexes between hCAR and appropriate coactivators. - Highlights: • Nuclear receptor CAR interact with PRMT5. • PRMT5 enhances transcriptional activity of CAR. • PRMT5 synergistically enhances transactivity of CAR by the co-expression of SRC-1, DP97 or PGC1α. • PRMT5 is a gene-selective co-activator for hCAR.

An integrated epigenetic and genetic analysis of DNA methyltransferase genes (DNMTs) in tumor resistant and susceptible chicken lines

Science.gov (United States)

Both epigenetic alterations and genetic variations play essential roles in tumorigenesis. The epigenetic modification of DNA methylation is catalyzed and maintained by the DNA methyltransferases (DNMT3a, DNMT3b and DNMT1). DNA mutations and DNA methylation profiles of DNMTs themselves and their rela...

DNA repair methyltransferase (Mgmt) knockout mice are sensitive to the lethal effects of chemotherapeutic alkylating agents.

NARCIS (Netherlands)

B.J. Glassner (Brian); G. Weeda (Geert); J.M. Allan (James); J.L.M. Broekhof (Jose'); N.H.E. Carls (Nick); I. Donker (Ingrid); B.P. Engelward (Bevin); R.J. Hampson (Richard); R. Hersmus (Remko); M.J. Hickman (Mark); R.B. Roth (Richard); H.B. Warren (Henry); M.M. Wu (Mavis); J.H.J. Hoeijmakers (Jan); L.D. Samson (Leona)

1999-01-01

textabstractWe have generated mice deficient in O6-methylguanine DNA methyltransferase activity encoded by the murine Mgmt gene using homologous recombination to delete the region encoding the Mgmt active site cysteine. Tissues from Mgmt null mice displayed very low O6-methylguanine DNA

5-Hydroxymethylcytosine is a predominantly stable DNA modification

Science.gov (United States)

Bachman, Martin; Uribe-Lewis, Santiago; Yang, Xiaoping; Williams, Michael; Murrell, Adele; Balasubramanian, Shankar

2014-12-01

5-Hydroxymethylcytosine (hmC) is an oxidation product of 5-methylcytosine which is present in the deoxyribonucleic acid (DNA) of most mammalian cells. Reduction of hmC levels in DNA is a hallmark of cancers. Elucidating the dynamics of this oxidation reaction and the lifetime of hmC in DNA is fundamental to understanding hmC function. Using stable isotope labelling of cytosine derivatives in the DNA of mammalian cells and ultrasensitive tandem liquid-chromatography mass spectrometry, we show that the majority of hmC is a stable modification, as opposed to a transient intermediate. In contrast with DNA methylation, which occurs immediately during replication, hmC forms slowly during the first 30 hours following DNA synthesis. Isotopic labelling of DNA in mouse tissues confirmed the stability of hmC in vivo and demonstrated a relationship between global levels of hmC and cell proliferation. These insights have important implications for understanding the states of chemically modified DNA bases in health and disease.

Cytosine methylation does not affect binding of transcription factor Sp1

International Nuclear Information System (INIS)

Harrington, M.A.; Jones, P.A.; Imagawa, M.; Karin, M.

1988-01-01

DNA methylation may be a component of a multilevel control mechanism that regulates eukaryotic gene expression. The authors used synthetic oligonucleotides to investigate the effect of cytosine methylation on the binding of the transcription factor Sp1 to its target sequence (a G+C-rich sequence known as a GC box). Concatemers of double-stranded 14-mers containing a GC box successfully competed with the human metallothionein IIA promoter for binding to Sp1 in DNase I protection experiments. The presence of 5-methylcytosine in the CpG sequence of the GC box did not influence Sp1 binding. The result was confirmed using double-stranded 20-mers containing 16 base pairs of complementary sequence. Electrophoretic gel retardation analysis of annealed 28-mers containing a GC box incubated with an Sp1-containing HeLa cell nuclear extract demonstrated the formation of DNA-protein complexes; formation of these complexes was not inhibited when an oligomer without a GC box was used as a competitor. Once again, the presence of a 5-methylcytosine residue in the GC box did not influence the binding of the protein to DNA. The results therefore preclude a direct effect of cytosine methylation on Sp1-DNA interactions

Spectroscopic (UV/VIS, Raman) and Electrophoresis Study of Cytosine-Guanine Oligonucleotide DNA Influenced by Magnetic Field.

Science.gov (United States)

Banihashemian, Seyedeh Maryam; Periasamy, Vengadesh; Boon Tong, Goh; Abdul Rahman, Saadah

2016-01-01

Studying the effect of a magnetic field on oligonucleotide DNA can provide a novel DNA manipulation technique for potential application in bioengineering and medicine. In this work, the optical and electrochemical response of a 100 bases oligonucleotides DNA, cytosine-guanine (CG100), is investigated via exposure to different magnetic fields (250, 500, 750, and 1000 mT). As a result of the optical response of CG100 to the magnetic field, the ultra-violet-visible spectrum indicated a slight variation in the band gap of CG100 of about 0.3 eV. Raman spectroscopy showed a significant deviation in hydrogen and phosphate bonds' vibration after exposure to the magnetic field. Oligonucleotide DNA mobility was investigated in the external electric field using the gel electrophoresis technique, which revealed a small decrease in the migration of CG100 after exposure to the magnetic field.

New Insights into 5hmC DNA Modification: Generation, Distribution and Function

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Dong-Qiao Shi

2017-07-01

Full Text Available Dynamic DNA modifications, such as methylation/demethylation on cytosine, are major epigenetic mechanisms to modulate gene expression in both eukaryotes and prokaryotes. In addition to the common methylation on the 5th position of the pyrimidine ring of cytosine (5mC, other types of modifications at the same position, such as 5-hydroxymethyl (5hmC, 5-formyl (5fC, and 5-carboxyl (5caC, are also important. Recently, 5hmC, a product of 5mC demethylation by the Ten-Eleven Translocation family proteins, was shown to regulate many cellular and developmental processes, including the pluripotency of embryonic stem cells, neuron development, and tumorigenesis in mammals. Here, we review recent advances on the generation, distribution, and function of 5hmC modification in mammals and discuss its potential roles in plants.

The inhibition of DNA repair by aphidicolin or cytosine arabinoside in X-irradiated normal and xeroderma pigmentosum fibroblasts

International Nuclear Information System (INIS)

Waters, R.; Crocombe, K.; Mirzayans, R.

1981-01-01

Normal and excision-deficient xeroderma pigmentosum fibroblasts were X-irradiated and the influence on DNA repair of either the repair inhibitor cytosine arabinoside or the specific inhibitor of DNA polymerase α, aphidicolin, investigated. The data indicated that the repair of a certain fraction of X-ray-induced lesions can be inhibited in both cell lines by both compounds. Thus, as aphidicolin blocks the operation of polymerase α, this enzyme must be involved in an excision repair pathway operating in both normal and excision-deficient xeroderma pigmentosum cells. (orig.)

Epigenetic regulation during fetal femur development: DNA methylation matters.

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María C de Andrés

Full Text Available Epigenetic modifications are heritable changes in gene expression without changes in DNA sequence. DNA methylation has been implicated in the control of several cellular processes including differentiation, gene regulation, development, genomic imprinting and X-chromosome inactivation. Methylated cytosine residues at CpG dinucleotides are commonly associated with gene repression; conversely, strategic loss of methylation during development could lead to activation of lineage-specific genes. Evidence is emerging that bone development and growth are programmed; although, interestingly, bone is constantly remodelled throughout life. Using human embryonic stem cells, human fetal bone cells (HFBCs, adult chondrocytes and STRO-1(+ marrow stromal cells from human bone marrow, we have examined a spectrum of developmental stages of femur development and the role of DNA methylation therein. Using pyrosequencing methodology we analysed the status of methylation of genes implicated in bone biology; furthermore, we correlated these methylation levels with gene expression levels using qRT-PCR and protein distribution during fetal development evaluated using immunohistochemistry. We found that during fetal femur development DNA methylation inversely correlates with expression of genes including iNOS (NOS2 and COL9A1, but not catabolic genes including MMP13 and IL1B. Furthermore, significant demethylation was evident in the osteocalcin promoter between the fetal and adult developmental stages. Increased TET1 expression and decreased expression of DNA (cytosine-5--methyltransferase 1 (DNMT1 in adult chondrocytes compared to HFBCs could contribute to the loss of methylation observed during fetal development. HFBC multipotency confirms these cells to be an ideal developmental system for investigation of DNA methylation regulation. In conclusion, these findings demonstrate the role of epigenetic regulation, specifically DNA methylation, in bone development

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

Spectroscopic (UV/VIS, Raman and Electrophoresis Study of Cytosine-Guanine Oligonucleotide DNA Influenced by Magnetic Field.

Directory of Open Access Journals (Sweden)

Seyedeh Maryam Banihashemian

Full Text Available Studying the effect of a magnetic field on oligonucleotide DNA can provide a novel DNA manipulation technique for potential application in bioengineering and medicine. In this work, the optical and electrochemical response of a 100 bases oligonucleotides DNA, cytosine-guanine (CG100, is investigated via exposure to different magnetic fields (250, 500, 750, and 1000 mT. As a result of the optical response of CG100 to the magnetic field, the ultra-violet-visible spectrum indicated a slight variation in the band gap of CG100 of about 0.3 eV. Raman spectroscopy showed a significant deviation in hydrogen and phosphate bonds' vibration after exposure to the magnetic field. Oligonucleotide DNA mobility was investigated in the external electric field using the gel electrophoresis technique, which revealed a small decrease in the migration of CG100 after exposure to the magnetic field.

Labelling of the thymidine and deoxyctidine bases of DNA by (2-14C) deoxycytidine in cultured L1210 cells

International Nuclear Information System (INIS)

Karle, J.M.; Hoeraut, R.M.; Cysyk, R.L.

1983-01-01

Exposure of cultured L1210 cells to (2- 14 C) deoxycytidine and analysis of radioactivity incorporated into DNA-pyrimidines revealed that 2.7-5.5-fold more radioactivity is incorporated into DNA-thymine than into cytosine bases. Thus, the pathway involving deamination of deoxycytidylate to deoxyuridylate and methylation to thymidylate is highly favoured over successive phosphorlation to dCTP. Several modified and endogenous pyrimidines altered the labelling of DNA-thymine and DNA-cytosine with (2- 14 C)-deoxycytidine. 3-deazauridine at 0.1 mM caused a 56% increase in the labelling of DNA-thymine. Both thymidine and 3-deazauridine (>=10 μM) increased the specific activity to DNA-cytosine by 4-fold. Cytosine arabinoside (ara-C) (>= 10 μM) reduced the labelling of both DNA-cytosine and DNA-thymine. Excess cytidine (0.1 mM) reduced the labelling of DNA-cytosine by 40%. Tetrahydrouridine at concentrations up to 1 mM had no effect. (author)

Effects of cytosine methylation on transcription factor binding sites

KAUST Repository

Medvedeva, Yulia A

2014-03-26

Background: DNA methylation in promoters is closely linked to downstream gene repression. However, whether DNA methylation is a cause or a consequence of gene repression remains an open question. If it is a cause, then DNA methylation may affect the affinity of transcription factors (TFs) for their binding sites (TFBSs). If it is a consequence, then gene repression caused by chromatin modification may be stabilized by DNA methylation. Until now, these two possibilities have been supported only by non-systematic evidence and they have not been tested on a wide range of TFs. An average promoter methylation is usually used in studies, whereas recent results suggested that methylation of individual cytosines can also be important.Results: We found that the methylation profiles of 16.6% of cytosines and the expression profiles of neighboring transcriptional start sites (TSSs) were significantly negatively correlated. We called the CpGs corresponding to such cytosines " traffic lights" We observed a strong selection against CpG " traffic lights" within TFBSs. The negative selection was stronger for transcriptional repressors as compared with transcriptional activators or multifunctional TFs as well as for core TFBS positions as compared with flanking TFBS positions.Conclusions: Our results indicate that direct and selective methylation of certain TFBS that prevents TF binding is restricted to special cases and cannot be considered as a general regulatory mechanism of transcription. 2013 Medvedeva et al.; licensee BioMed Central Ltd.

Clinical implications of cytosine deletion of exon 5 of P53 gene in non small cell lung cancer patients

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Rashid Mir

2016-01-01

Full Text Available Aim: Lung cancer is considered to be the most common cancer in the world. In humans, about 50% or more cancers have a mutated tumor suppressor p53 gene thereby resulting in accumulation of p53 protein and losing its function to activate the target genes that regulate the cell cycle and apoptosis. Extensive research conducted in murine cancer models with activated p53, loss of p53, or p53 missense mutations have facilitated researchers to understand the role of this key protein. Our study was aimed to evaluate the frequency of cytosine deletion in nonsmall cell lung cancer (NSCLC patients. Methods: One hundred NSCLC patients were genotyped for P53 (exon5, codon168 cytosine deletion leading to loss of its function and activate the target genes by allele-specific polymerase chain reaction. The P53 cytosine deletion was correlated with all the clinicopathological parameters of the patients. Results and Analysis: 59% cases were carrying P53 cytosine deletion. Similarly, the significantly higher incidence of cytosine deletion was reported in current smokers (75% in comparison to exsmoker and nonsmoker. Significantly higher frequency of cytosine deletion was reported in adenocarcinoma (68.08% than squamous cell carcinoma (52.83%. Also, a significant difference was reported between p53 cytosine deletion and metastasis (64.28%. Further, the majority of the cases assessed for response carrying P53 cytosine deletion were found to show faster disease progression. Conclusion: The data suggests that there is a significant association of the P53 exon 5 deletion of cytosine in codon 168 with metastasis and staging of the disease.

Spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA.

Science.gov (United States)

Shahal, Tamar; Gilat, Noa; Michaeli, Yael; Redy-Keisar, Orit; Shabat, Doron; Ebenstein, Yuval

2014-08-19

5-Hydroxymethylcytosine (5hmC), a modified form of the DNA base cytosine, is an important epigenetic mark linked to regulation of gene expression in development, and tumorigenesis. We have developed a spectroscopic method for a global quantification of 5hmC in genomic DNA. The assay is performed within a multiwell plate, which allows simultaneous recording of up to 350 samples. Our quantification procedure of 5hmC is direct, simple, and rapid. It relies on a two-step protocol that consists of enzymatic glucosylation of 5hmC with an azide-modified glucose, followed by a "click reaction" with an alkyne-fluorescent tag. The fluorescence intensity recorded from the DNA sample is proportional to its 5hmC content and can be quantified by a simple plate reader measurement. This labeling technique is specific and highly sensitive, allowing detection of 5hmC down to 0.002% of the total nucleotides. Our results reveal significant variations in the 5hmC content obtained from different mouse tissues, in agreement with previously reported data.

Ultrasensitive Direct Quantification of Nucleobase Modifications in DNA by Surface-Enhanced Raman Scattering: The Case of Cytosine.

Science.gov (United States)

Morla-Folch, Judit; Xie, Hai-nan; Gisbert-Quilis, Patricia; Gómez-de Pedro, Sara; Pazos-Perez, Nicolas; Alvarez-Puebla, Ramon A; Guerrini, Luca

2015-11-09

Recognition of chemical modifications in canonical nucleobases of nucleic acids is of key importance since such modified variants act as different genetic encoders, introducing variability in the biological information contained in DNA. Herein, we demonstrate the feasibility of direct SERS in combination with chemometrics and microfluidics for the identification and relative quantification of 4 different cytosine modifications in both single- and double-stranded DNA. The minute amount of DNA required per measurement, in the sub-nanogram regime, removes the necessity of pre-amplification or enrichment steps (which are also potential sources of artificial DNA damages). These findings show great potentials for the development of fast, low-cost and high-throughput screening analytical devices capable of detecting known and unknown modifications in nucleic acids (DNA and RNA) opening new windows of activity in several fields such as biology, medicine and forensic sciences. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Epigenetic silencing of the DNA mismatch repair gene, MLH1, induced by hypoxic stress in a pathway dependent on the histone demethylase, LSD1

Science.gov (United States)

Lu, Yuhong; Wajapeyee, Narendra; Turker, Mitchell S.; Glazer, Peter M.

2014-01-01

SUMMARY Silencing of the MLH1 gene is frequently seen in sporadic cancers. We report that hypoxia causes decreased H3K4 methylation at the MLH1 promoter via the H3K4 demethylases, LSD1 and PLU-1, and promotes long-term silencing of the promoter in a pathway that requires LSD1. Knockdown of LSD1 or its co-repressor, CoREST, also prevents the re-silencing (and cytosine DNA methylation) of the endogenous MLH1 promoter in RKO colon cancer cells following transient reactivation by the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza-dC). The results demonstrate that hypoxia is a critical driving force for silencing of MLH1 through chromatin modification and indicate that the LSD1/CoREST complex is essential for MLH1 silencing. PMID:25043185

Protein Arginine Methyltransferase 5 Inhibition Upregulates Foxp3+ Regulatory T Cells Frequency and Function during the Ulcerative Colitis

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Yingxia Zheng

2017-05-01

Full Text Available Ulcerative colitis (UC pathogenesis is related to imbalance of immune responses, and the equilibrium between inflammatory T cells and Foxp3+ regulatory T cells (Tregs plays an important role in the intestinal homeostasis. Protein arginine methyltransferases (PRMTs regulate chromatin remodeling and gene expression. Here, we investigated whether inhibition of PRMTs affects colitis pathogenesis in mice and inflammatory bowel disease patients and further explored the underlying mechanisms. In this study, we found that protein arginine N-methyltransferase inhibitor 1 (AMI-1 treatments increased Tregs frequency, function, and reduced colitis incidence. Adoptive transfer of AMI-1-treated Tregs could reduce the colitis incidence. Colitis was associated with increased local PRMT5 expression, which was inhibited by AMI-1 treatment. Additionally, PRMT5 knockdown T cells produced a better response to TGFβ and promoted Tregs differentiation through decreased DNA methyltransferase 1 (DNMT1 expression. PRMT5 also enhanced H3K27me3 and DNMT1 binding to Foxp3 promoter, which restricted Tregs differentiation. Furthermore, PRMT5 knockdown led to decreased Foxp3 promoter methylation during Tregs induction. PRMT5 expression had a negative relationship with Tregs in UC patients, knockdown of PRMT5 expression increased Tregs frequency and decreased TNFα, IL-6, and IL-13 levels. Our study outlines a novel regulation of PRMT5 on Tregs development and function. Strategies to decrease PRMT5 expression might have therapeutic potential to control UC.

Effects of TET2 mutations on DNA methylation in chronic myelomonocytic leukemia

Science.gov (United States)

TET2 enzymatically converts 5-methyl-cytosine to 5-hydroxymethyl-cytosine, possibly leading to loss of DNA methylation. TET2 mutations are common in myeloid leukemia and were proposed to contribute to leukemogenesis through DNA methylation. To expand on this concept, we studied chronic myelomonocyti...

Structure Determination of an Ag-I-Mediated Cytosine-Cytosine Base Pair within DNA Duplex in Solution with H-1/N-15/Ag-109 NMR Spectroscopy

Czech Academy of Sciences Publication Activity Database

Dairaku, T.; Furuita, K.; Sato, H.; Šebera, Jakub; Nakashima, K.; Kondo, J.; Yamanaka, D.; Kondo, Y.; Okamoto, I.; Ono, A.; Sychrovský, Vladimír; Kojima, C.; Tanaka, Y.

2016-01-01

Roč. 22, č. 37 (2016), s. 13028-13031 ISSN 0947-6539 R&D Projects: GA ČR GA13-27676S Institutional support: RVO:61388963 Keywords : NMR * Ag * cytosine * DFT Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 5.317, year: 2016

The use of permanganate as a sequencing reagent for identification of 5-methylcytosine residues in DNA.

OpenAIRE

Fritzsche, E; Hayatsu, H; Igloi, G L; Iida, S; Kössel, H

1987-01-01

The use of permanganate as a reagent for DNA sequencing by chemical degradation has been studied with respect to its specificity for 5-methylcytosine residues. At weakly acidic pH and room temperature, 0.2 mM potassium permanganate reacts preferentially with thymine, 5-methylcytosine, and to a lesser extent with purine residues, while cytosine remains essentially intact. Permanganate oxidation is, therefore, a suitable DNA sequencing reaction for positive discrimination between 5-methylcytosi...

PRMT5 restricts hepatitis B virus replication through epigenetic repression of covalently closed circular DNA transcription and interference with pregenomic RNA encapsidation.

Science.gov (United States)

Zhang, Wen; Chen, Jieliang; Wu, Min; Zhang, Xiaonan; Zhang, Min; Yue, Lei; Li, Yaming; Liu, Jiangxia; Li, Baocun; Shen, Fang; Wang, Yang; Bai, Lu; Protzer, Ulrike; Levrero, Massimo; Yuan, Zhenghong

2017-08-01

Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. The covalently closed circular DNA (cccDNA) minichromosome, which serves as the template for the transcription of viral RNAs, plays a key role in viral persistence. While accumulating evidence suggests that cccDNA transcription is regulated by epigenetic machinery, particularly the acetylation of cccDNA-bound histone 3 (H3) and H4, the potential contributions of histone methylation and related host factors remain obscure. Here, by screening a series of methyltransferases and demethylases, we identified protein arginine methyltransferase 5 (PRMT5) as an effective restrictor of HBV transcription and replication. In cell culture-based models for HBV infection and in liver tissues of patients with chronic HBV infection, we found that symmetric dimethylation of arginine 3 on H4 on cccDNA was a repressive marker of cccDNA transcription and was regulated by PRMT5 depending on its methyltransferase domain. Moreover, PRMT5-triggered symmetric dimethylation of arginine 3 on H4 on the cccDNA minichromosome involved an interaction with the HBV core protein and the Brg1-based human SWI/SNF chromatin remodeler, which resulted in down-regulation of the binding of RNA polymerase II to cccDNA. In addition to the inhibitory effect on cccDNA transcription, PRMT5 inhibited HBV core particle DNA production independently of its methyltransferase activity. Further study revealed that PRMT5 interfered with pregenomic RNA encapsidation by preventing its interaction with viral polymerase protein through binding to the reverse transcriptase-ribonuclease H region of polymerase, which is crucial for the polymerase-pregenomic RNA interaction. PRMT5 restricts HBV replication through a two-part mechanism including epigenetic suppression of cccDNA transcription and interference with pregenomic RNA encapsidation; these findings improve the understanding of epigenetic regulation of HBV transcription and host

Methyltransferases mediate cell memory of a genotoxic insult.

Science.gov (United States)

Rugo, R E; Mutamba, J T; Mohan, K N; Yee, T; Chaillet, J R; Greenberger, J S; Engelward, B P

2011-02-10

Characterization of the direct effects of DNA-damaging agents shows how DNA lesions lead to specific mutations. Yet, serum from Hiroshima survivors, Chernobyl liquidators and radiotherapy patients can induce a clastogenic effect on naive cells, showing indirect induction of genomic instability that persists years after exposure. Such indirect effects are not restricted to ionizing radiation, as chemical genotoxins also induce heritable and transmissible genomic instability phenotypes. Although such indirect induction of genomic instability is well described, the underlying mechanism has remained enigmatic. Here, we show that mouse embryonic stem cells exposed to γ-radiation bear the effects of the insult for weeks. Specifically, conditioned media from the progeny of exposed cells can induce DNA damage and homologous recombination in naive cells. Notably, cells exposed to conditioned media also elicit a genome-destabilizing effect on their neighbouring cells, thus demonstrating transmission of genomic instability. Moreover, we show that the underlying basis for the memory of an insult is completely dependent on two of the major DNA cytosine methyltransferases, Dnmt1 and Dnmt3a. Targeted disruption of these genes in exposed cells completely eliminates transmission of genomic instability. Furthermore, transient inactivation of Dnmt1, using a tet-suppressible allele, clears the memory of the insult, thus protecting neighbouring cells from indirect induction of genomic instability. We have thus demonstrated that a single exposure can lead to long-term, genome-destabilizing effects that spread from cell to cell, and we provide a specific molecular mechanism for these persistent bystander effects. Collectively, our results impact the current understanding of risks from toxin exposures and suggest modes of intervention for suppressing genomic instability in people exposed to carcinogenic genotoxins.

MiR-29c regulates the expression of miR-34c and miR-449a by targeting DNA methyltransferase 3a and 3b in nasopharyngeal carcinoma

International Nuclear Information System (INIS)

Niu, Man; Gao, Dan; Wen, Qiuyuan; Wei, Pingpin; Pan, Suming; Shuai, Cijun; Ma, Huiling; Xiang, Juanjuan; Li, Zheng; Fan, Songqing; Li, Guiyuan; Peng, Shuping

2016-01-01

Nasopharyngeal carcinoma (NPC) is prevalent in South East Asia and Southern China particularly, despite the reported 5-year survival ratio is relative higher than other deadly cancers such as liver, renal, pancreas cancer, the lethality is characterized by high metastatic potential in the early stage and high recurrence rate after radiation treatment. MicroRNA-29c was found to be down-regulated in the serum as well as in the tissue of nasopharyngeal carcinoma tissue. In this study, we found accidentally that the transfection of pre-miR-29c or miR-29c mimics significantly increases the expression level of miR-34c and miR-449a but doesn’t affect that of miR-222 using real-time quantitative PCR in nasopharyngeal carcinoma cell lines. To explore the molecular mechanism of the regulatory role, the cells are treated with 5-Aza-2-deoxycytidine (5-Aza-CdR) treatment and the level of miR-34c and miR-449a but not miR-222 accumulated by the treatment. DNA methyltransferase 3a, 3b were down-regulated by the 5-Aza-CdR treatment with western blot and real-time quantitative PCR. We found that pre-miR-29c or miR-29c mimics significantly increases the expression level of miR-34c and miR-449a. We further found DNA methyltransferase 3a and 3b are the target gene of miR-29c. Restoration of miR-29c in NPC cells down-regulated DNA methyltransferase 3a, 3b, but not DNA methyltransferase T1. The regulation of miR-29c/DNMTs/miR-34c/449a is an important molecular axis of NPC development and targeting DNMTs or restoring of miR-29c might be a promising therapy strategy for the prevention of NPC

Mutation Processes in 293-Based Clones Overexpressing the DNA Cytosine Deaminase APOBEC3B.

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Monica K Akre

Full Text Available Molecular, cellular, and clinical studies have combined to demonstrate a contribution from the DNA cytosine deaminase APOBEC3B (A3B to the overall mutation load in breast, head/neck, lung, bladder, cervical, ovarian, and other cancer types. However, the complete landscape of mutations attributable to this enzyme has yet to be determined in a controlled human cell system. We report a conditional and isogenic system for A3B induction, genomic DNA deamination, and mutagenesis. Human 293-derived cells were engineered to express doxycycline-inducible A3B-eGFP or eGFP constructs. Cells were subjected to 10 rounds of A3B-eGFP exposure that each caused 80-90% cell death. Control pools were subjected to parallel rounds of non-toxic eGFP exposure, and dilutions were done each round to mimic A3B-eGFP induced population fluctuations. Targeted sequencing of portions of TP53 and MYC demonstrated greater mutation accumulation in the A3B-eGFP exposed pools. Clones were generated and microarray analyses were used to identify those with the greatest number of SNP alterations for whole genome sequencing. A3B-eGFP exposed clones showed global increases in C-to-T transition mutations, enrichments for cytosine mutations within A3B-preferred trinucleotide motifs, and more copy number aberrations. Surprisingly, both control and A3B-eGFP clones also elicited strong mutator phenotypes characteristic of defective mismatch repair. Despite this additional mutational process, the 293-based system characterized here still yielded a genome-wide view of A3B-catalyzed mutagenesis in human cells and a system for additional studies on the compounded effects of simultaneous mutation mechanisms in cancer cells.

DNA Electrochemistry Shows DNMT1 Methyltransferase Hyperactivity in Colorectal Tumors.

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Furst, Ariel L; Barton, Jacqueline K

2015-07-23

DNMT1, the most abundant human methyltransferase, is responsible for translating the correct methylation pattern during DNA replication, and aberrant methylation by DNMT1 has been linked to tumorigenesis. We have developed a sensitive signal-on electrochemical assay for the measurement of DNMT1 activity in crude tissue lysates. We have further analyzed ten tumor sets and have found a direct correlation between DNMT1 hyperactivity and tumorous tissue. In the majority of samples analyzed, the tumorous tissue has significantly higher DNMT1 activity than the healthy adjacent tissue. No such correlation is observed in measurements of DNMT1 expression by qPCR, DNMT1 protein abundance by western blotting, or DNMT1 activity using a radiometric DNA labeling assay. DNMT1 hyperactivity can result from both protein overexpression and enzyme hyperactivity. DNMT1 activity measured electrochemically provides a direct measure of activity in cell lysates and, as a result, provides a sensitive and early indication of cancerous transformation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is associated with G to A mutations in K-ras in colorectal tumorigenesis.

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Esteller, M; Toyota, M; Sanchez-Cespedes, M; Capella, G; Peinado, M A; Watkins, D N; Issa, J P; Sidransky, D; Baylin, S B; Herman, J G

2000-05-01

O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair protein that removes mutagenic and cytotoxic adducts from the O6 position of guanine. O6-methylguanine mispairs with thymine during replication, and if the adduct is not removed, this results in conversion from a guanine-cytosine pair to an adenine-thymine pair. In vitro assays show that MGMT expression avoids G to A mutations and MGMT transgenic mice are protected against G to A transitions at ras genes. We have recently demonstrated that the MGMT gene is silenced by promoter methylation in many human tumors, including colorectal carcinomas. To study the relevance of defective MGMT function by aberrant methylation in relation to the presence of K-ras mutations, we studied 244 colorectal tumor samples for MGMT promoter hypermethylation and K-ras mutational status. Our results show a clear association between the inactivation of MGMT by promoter hypermethylation and the appearance of G to A mutations at K-ras: 71% (36 of 51) of the tumors displaying this particular type of mutation had abnormal MGMT methylation, whereas only 32% (12 of 37) of those with other K-ras mutations not involving G to A transitions and 35% (55 of 156) of the tumors without K-ras mutations demonstrated MGMT methylation (P = 0.002). In addition, MGMT loss associated with hypermethylation was observed in the small adenomas, including those that do not yet contain K-ras mutations. Hypermethylation of other genes such as p16INK4a and p14ARF was not associated with either MGMT hypermethylation or K-ras mutation. Our data suggest that epigenetic silencing of MGMT by promoter hypermethylation may lead to a particular genetic change in human cancer, specifically G to A transitions in the K-ras oncogene.

Elastic electron scattering from the DNA bases: cytosine and thymine

International Nuclear Information System (INIS)

Colyer, C J; Bellm, S M; Lohmanny, B; Blanco, F; Garcia, G

2012-01-01

Relative elastic differential cross sections for elastic scattering from cytosine and thymine have been measured using the crossed beam method. The experimental data are compared with theoretical cross sections calculated by the screen corrected additivity rule method.

Impact of DNA3'pp5'G capping on repair reactions at DNA 3' ends.

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Das, Ushati; Chauleau, Mathieu; Ordonez, Heather; Shuman, Stewart

2014-08-05

Many biological scenarios generate "dirty" DNA 3'-PO4 ends that cannot be sealed by classic DNA ligases or extended by DNA polymerases. The noncanonical ligase RtcB can "cap" these ends via a unique chemical mechanism entailing transfer of GMP from a covalent RtcB-GMP intermediate to a DNA 3'-PO4 to form DNA3'pp5'G. Here, we show that capping protects DNA 3' ends from resection by Escherichia coli exonucleases I and III and from end-healing by T4 polynucleotide 3' phosphatase. By contrast, the cap is an effective primer for DNA synthesis. E. coli DNA polymerase I and Mycobacterium DinB1 extend the DNAppG primer to form an alkali-labile DNApp(rG)pDNA product. The addition of dNTP depends on pairing of the cap guanine with an opposing cytosine in the template strand. Aprataxin, an enzyme implicated in repair of A5'pp5'DNA ends formed during abortive ligation by classic ligases, is highly effective as a DNA 3' decapping enzyme, converting DNAppG to DNA3'p and GMP. We conclude that the biochemical impact of DNA capping is to prevent resection and healing of a 3'-PO4 end, while permitting DNA synthesis, at the price of embedding a ribonucleotide and a pyrophosphate linkage in the repaired strand. Aprataxin affords a means to counter the impact of DNA capping.

Accurate CpG and non-CpG cytosine methylation analysis by high-throughput locus-specific pyrosequencing in plants.

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How-Kit, Alexandre; Daunay, Antoine; Mazaleyrat, Nicolas; Busato, Florence; Daviaud, Christian; Teyssier, Emeline; Deleuze, Jean-François; Gallusci, Philippe; Tost, Jörg

2015-07-01

Pyrosequencing permits accurate quantification of DNA methylation of specific regions where the proportions of the C/T polymorphism induced by sodium bisulfite treatment of DNA reflects the DNA methylation level. The commercially available high-throughput locus-specific pyrosequencing instruments allow for the simultaneous analysis of 96 samples, but restrict the DNA methylation analysis to CpG dinucleotide sites, which can be limiting in many biological systems. In contrast to mammals where DNA methylation occurs nearly exclusively on CpG dinucleotides, plants genomes harbor DNA methylation also in other sequence contexts including CHG and CHH motives, which cannot be evaluated by these pyrosequencing instruments due to software limitations. Here, we present a complete pipeline for accurate CpG and non-CpG cytosine methylation analysis at single base-resolution using high-throughput locus-specific pyrosequencing. The devised approach includes the design and validation of PCR amplification on bisulfite-treated DNA and pyrosequencing assays as well as the quantification of the methylation level at every cytosine from the raw peak intensities of the Pyrograms by two newly developed Visual Basic Applications. Our method presents accurate and reproducible results as exemplified by the cytosine methylation analysis of the promoter regions of two Tomato genes (NOR and CNR) encoding transcription regulators of fruit ripening during different stages of fruit development. Our results confirmed a significant and temporally coordinated loss of DNA methylation on specific cytosines during the early stages of fruit development in both promoters as previously shown by WGBS. The manuscript describes thus the first high-throughput locus-specific DNA methylation analysis in plants using pyrosequencing.

The BsaHI restriction-modification system: Cloning, sequencing and analysis of conserved motifs

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Roberts Richard J

2008-05-01

Full Text Available Abstract Background Restriction and modification enzymes typically recognise short DNA sequences of between two and eight bases in length. Understanding the mechanism of this recognition represents a significant challenge that we begin to address for the BsaHI restriction-modification system, which recognises the six base sequence GRCGYC. Results The DNA sequences of the genes for the BsaHI methyltransferase, bsaHIM, and restriction endonuclease, bsaHIR, have been determined (GenBank accession #EU386360, cloned and expressed in E. coli. Both the restriction endonuclease and methyltransferase enzymes share significant similarity with a group of 6 other enzymes comprising the restriction-modification systems HgiDI and HgiGI and the putative HindVP, NlaCORFDP, NpuORFC228P and SplZORFNP restriction-modification systems. A sequence alignment of these homologues shows that their amino acid sequences are largely conserved and highlights several motifs of interest. We target one such conserved motif, reading SPERRFD, at the C-terminal end of the bsaHIR gene. A mutational analysis of these amino acids indicates that the motif is crucial for enzymatic activity. Sequence alignment of the methyltransferase gene reveals a short motif within the target recognition domain that is conserved among enzymes recognising the same sequences. Thus, this motif may be used as a diagnostic tool to define the recognition sequences of the cytosine C5 methyltransferases. Conclusion We have cloned and sequenced the BsaHI restriction and modification enzymes. We have identified a region of the R. BsaHI enzyme that is crucial for its activity. Analysis of the amino acid sequence of the BsaHI methyltransferase enzyme led us to propose two new motifs that can be used in the diagnosis of the recognition sequence of the cytosine C5-methyltransferases.

[Gene cloning and bioinformatics analysis of SABATH methyltransferase in Lonicera japonica var. chinensis].

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Yu, Xiao-Dan; Jiang, Chao; Huang, Lu-Qi; Qin, Shuang-Shuang; Zeng, Xiang-Mei; Chen, Ping; Yuan, Yuan

2013-08-01

To clone SABATH methyltransferase (rLjSABATHMT) gene in Lonicera japonica var. chinensis, and compare the gene expression and intron sequence of SABATH methyltransferase orthologous in L. japonica with L. japonica var. chinensis. It provide a basis for gene regulate the formation of L. japonica floral scents. The cDNA and genome sequences of LjSABATHMT from L. japonica var. chinensis were cloned according to the gene fragments in cDNA library. The LjSABATHMT protein was characterized by bioinformatics analysis. SABATH family phylogenetic tree were built by MEGA 5.0. The transcripted level of SABATHMT orthologous were analyzed in different organs and different flower periods of L. japonica and L. japonica var. chinensis using RT-PCR analysis. Intron sequences of SABATHMT orthologous were also analyzied. The cDNA of LjSABATHMT was 1 251 bp, had a complete coding frame with 365 amino acids. The protein had the conservative SABATHMT domain, and phylogenetic tree showed that it may be a salicylic acid/benzoic acid methyltransferase. Higher expression of SABATH methyltransferase orthologous was found in flower. The intron sequence of L. japonica and L. japonica var. chinensis had rich polymorphism, and two SNP are unique genotype of L. japonica var. chinensis. The motif elements in two orthologous genes were significant differences. The intron difference of SABATH methyltransferase orthologous could be inducing to difference of gene expression between L. japonica and L. japonica var. chinensis. These results will provide important base on regulating active compounds of L. japonica.

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.

The Chemical Decomposition of 5-aza-2′-deoxycytidine (Decitabine): Kinetic Analyses and Identification of Products by NMR, HPLC, and Mass Spectrometry

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Rogstad, Daniel K.; Herring, Jason L.; Theruvathu, Jacob A.; Burdzy, Artur; Perry, Christopher C.; Neidigh, Jonathan W.; Sowers, Lawrence C.

2014-01-01

The nucleoside analog 5-aza-2′-deoxycytidine (Decitabine, DAC) is one of several drugs in clinical use that inhibit DNA methyltransferases, leading to a decrease of 5-methylcytosine in newly replicated DNA and subsequent transcriptional activation of genes silenced by cytosine methylation. In addition to methyltransferase inhibition, DAC has demonstrated toxicity and potential mutagenicity, and can induce a DNA-repair response. The mechanisms accounting for these events are not well understood. DAC is chemically unstable in aqueous solutions, but there is little consensus between previous reports as to its half-life and corresponding products of decomposition at physiological temperature and pH, potentially confounding studies on its mechanism of action and long-term use in humans. Here we have employed a battery of analytical methods to estimate kinetic rates and to characterize DAC decomposition products under conditions of physiological temperature and pH. Our results indicate that DAC decomposes into a plethora of products, formed by hydrolytic opening and deformylation of the triazine ring, in addition to anomerization and possibly other changes in the sugar ring structure. We also discuss the advantages and problems associated with each analytical method used. The results reported here will facilitate ongoing studies and clinical trials aimed at understanding the mechanisms of action, toxicity, and possible mutagenicity of DAC and related analogs. PMID:19480391

Concerted bis-alkylating reactivity of clerocidin towards unpaired cytosine residues in DNA

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Richter, Sara N.; Menegazzo, Ileana; Fabris, Daniele; Palumbo, Manlio

2004-01-01

Clerocidin (CL) is a topoisomerase II poison, which cleaves DNA irreversibly at guanines (G) and reversibly at cytosines (C). Furthermore, the drug can induce enzyme-independent strand breaks at the G and C level. It has been previously shown that G-damage is induced by alkylation of the guanine N7, followed by spontaneous depurination and nucleic acid cleavage, whereas scission at C is obtained only after treatment with hot alkali, and no information is available to explain the nature of this damage. We present here a systematic study on the reactivity of CL towards C both in the DNA environment and in solution. Selected synthetic derivatives were employed to evaluate the role of each chemical group of the drug. The structure of CL–dC adduct was then characterized by tandem mass spectrometry and NMR: the adduct is a stable condensed ring system resulting from a concerted electrophilic attack of the adjacent carbonyl and epoxide groups of CL towards the exposed NH2 and N3, respectively. This reaction mechanism, shown here for the first time, is characterized by faster kinetic rates than alkylation at G, due to the fact that the rate-determining step, alkylation at the epoxide, is an intramolecular process, provided a Schiff base linking CL and C can rapidly form, whereas the corresponding reaction of G N7 is intermolecular. These results provide helpful hints to explain the reversible/irreversible nature of topoisomerase II mediated DNA damage produced by CL at C/G steps. PMID:15494453

Silver(I)-Mediated Base Pairs in DNA Sequences Containing 7-Deazaguanine/Cytosine: towards DNA with Entirely Metallated Watson-Crick Base Pairs.

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Méndez-Arriaga, José M; Maldonado, Carmen R; Dobado, José A; Galindo, Miguel A

2018-03-26

DNA sequences comprising noncanonical 7-deazaguanine ( 7C G) and canonical cytosine (C) are capable of forming Watson-Crick base pairs via hydrogen bonds as well as silver(I)-mediated base pairs by coordination to central silver(I) ions. Duplexes I and II containing 7C G and C have been synthesized and characterized. The incorporation of silver(I) ions into these duplexes has been studied by means of temperature-dependent UV spectroscopy, circular dichroism, and DFT calculations. The results suggest the formation of DNA molecules comprising contiguous metallated 7C G-Ag I -C Watson-Crick base pairs that preserve the original B-type conformation. Furthermore, additional studies performed on duplex III indicated that, in the presence of Ag I ions, 7C G-C and 7C A-T Watson-Crick base pairs ( 7C A, 7-deazadenine; T, thymine) can be converted to metallated 7C G-Ag I -C and 7C A-Ag I -T base pairs inside the same DNA molecule whilst maintaining its initial double helix conformation. These findings are very important for the development of customized silver-DNA nanostructures based on a Watson-Crick complementarity pattern. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pathogenic mechanisms of intracellular bacteria.

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Niller, Hans Helmut; Masa, Roland; Venkei, Annamária; Mészáros, Sándor; Minarovits, Janos

2017-06-01

We wished to overview recent data on a subset of epigenetic changes elicited by intracellular bacteria in human cells. Reprogramming the gene expression pattern of various host cells may facilitate bacterial growth, survival, and spread. DNA-(cytosine C5)-methyltransferases of Mycoplasma hyorhinis targeting cytosine-phosphate-guanine (CpG) dinucleotides and a Mycobacterium tuberculosis methyltransferase targeting non-CpG sites methylated the host cell DNA and altered the pattern of gene expression. Gene silencing by CpG methylation and histone deacetylation, mediated by cellular enzymes, also occurred in M. tuberculosis-infected macrophages. M. tuberculosis elicited cell type-specific epigenetic changes: it caused increased DNA methylation in macrophages, but induced demethylation, deposition of euchromatic histone marks and activation of immune-related genes in dendritic cells. A secreted transposase of Acinetobacter baumannii silenced a cellular gene, whereas Mycobacterium leprae altered the epigenotype, phenotype, and fate of infected Schwann cells. The 'keystone pathogen' oral bacterium Porphyromonas gingivalis induced local DNA methylation and increased the level of histone acetylation in host cells. These epigenetic changes at the biofilm-gingiva interface may contribute to the development of periodontitis. Epigenetic regulators produced by intracellular bacteria alter the epigenotype and gene expression pattern of host cells and play an important role in pathogenesis.

Cloning and analysis of the genes encoding the type IIS restriction-modification system HphI from Haemophilus parahaemolyticus.

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Lubys, A; Lubienè, J; Kulakauskas, S; Stankevicius, K; Timinskas, A; Janulaitis, A

1996-07-15

The genomic region encoding the type IIS restriction-modification (R-M) system HphI (enzymes recognizing the asymmetric sequence 5'-GGTGA-3'/5'-TCACC-3') from Haemophilus parahaemolyticus were cloned into Escherichia coli and sequenced. Sequence analysis of the R-M HphI system revealed three adjacent genes aligned in the same orientation: a cytosine 5 methyltransferase (gene hphIMC), an adenine N6 methyltransferase (hphIMA) and the HphI restriction endonuclease (gene hphIR). Either methyltransferase is capable of protecting plasmid DNA in vivo against the action of the cognate restriction endonuclease. hphIMA methylation renders plasmid DNA resistant to R.Hindill at overlapping sites, suggesting that the adenine methyltransferase modifies the 3'-terminal A residue on the GGTGA strand. Strong homology was found between the N-terminal part of the m6A methyltransferasease and an unidentified reading frame interrupted by an incomplete gaIE gene of Neisseria meningitidis. The HphI R-M genes are flanked by a copy of a 56 bp direct nucleotide repeat on each side. Similar sequences have also been identified in the non-coding regions of H.influenzae Rd DNA. Possible involvement of the repeat sequences in the mobility of the HphI R-M system is discussed.

An engineered split M.HhaI-zinc finger fusion lacks the intended methyltransferase specificity

International Nuclear Information System (INIS)

Meister, Glenna E.; Chandrasegaran, Srinivasan; Ostermeier, Marc

2008-01-01

The ability to site-specifically methylate DNA in vivo would have wide applicability to the study of basic biomedical problems as well as enable studies on the potential of site-specific DNA methylation as a therapeutic strategy for the treatment of diseases. Natural DNA methyltransferases lack the specificity required for these applications. Nomura and Barbas [W. Nomura, C.F. Barbas 3rd, In vivo site-specific DNA methylation with a designed sequence-enabled DNA methylase, J. Am. Chem. Soc. 129 (2007) 8676-8677] have reported that an engineered DNA methyltransferase comprised of fragments of M.HhaI methyltransferase and zinc finger proteins has very high specificity for the chosen target site. Our analysis of this engineered enzyme shows that the fusion protein methylates target and non-target sites with similar efficiency

DNA methyltransferase mediates dose-dependent stimulation of neural stem cell proliferation by folate.

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Li, Wen; Yu, Min; Luo, Suhui; Liu, Huan; Gao, Yuxia; Wilson, John X; Huang, Guowei

2013-07-01

The proliferative response of neural stem cells (NSCs) to folate may play a critical role in the development, function and repair of the central nervous system. It is important to determine the dose-dependent effects of folate in NSC cultures that are potential sources of transplantable cells for therapies for neurodegenerative diseases. To determine the optimal concentration and mechanism of action of folate for stimulation of NSC proliferation in vitro, NSCs were exposed to folic acid or 5-methyltetrahydrofolate (5-MTHF) (0-200 μmol/L) for 24, 48 or 72 h. Immunocytochemistry and methyl thiazolyl tetrazolium assay showed that the optimal concentration of folic acid for NSC proliferation was 20-40 μmol/L. Stimulation of NSC proliferation by folic acid was associated with DNA methyltransferase (DNMT) activation and was attenuated by the DNMT inhibitor zebularine, which implies that folate dose-dependently stimulates NSC proliferation through a DNMT-dependent mechanism. Based on these new findings and previously published evidence, we have identified a mechanism by which folate stimulates NSC growth. Copyright © 2013 Elsevier Inc. All rights reserved.

Superimposed Code Theoretic Analysis of Deoxyribonucleic Acid (DNA) Codes and DNA Computing

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2010-01-01

DNA strand and its Watson - Crick complement can be used to perform mathematical computation. This research addresses how the...Acid dsDNA double stranded DNA MOSAIC Mobile Stream Processing Cluster PCR Polymerase Chain Reaction RAM Random Access Memory ssDNA single stranded DNA WC Watson – Crick A Adenine C Cytosine G Guanine T Thymine ...are 5′→3′ and strands with strikethrough are 3′→5′. A dsDNA duplex formed between a strand and its reverse complement is called a

Inhibition of DNA methyltransferase induces G2 cell cycle arrest and apoptosis in human colorectal cancer cells via inhibition of JAK2/STAT3/STAT5 signalling.

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Xiong, Hua; Chen, Zhao-Fei; Liang, Qin-Chuan; Du, Wan; Chen, Hui-Min; Su, Wen-Yu; Chen, Guo-Qiang; Han, Ze-Guang; Fang, Jing-Yuan

2009-09-01

DNA methyltransferase inhibitors (MTIs) have recently emerged as promising chemotherapeutic or preventive agents for cancer, despite their poorly characterized mechanisms of action. The present study shows that DNA methylation is integral to the regulation of SH2-containing protein tyrosine phosphatase 1 (SHP1) expression, but not for regulation of suppressors of cytokine signalling (SOCS)1 or SOCS3 in colorectal cancer (CRC) cells. SHP1 expression correlates with down-regulation of Janus kinase/signal transducers and activators of transcription (JAK2/STAT3/STAT5) signalling, which is mediated in part by tyrosine dephosphorylation events and modulation of the proteasome pathway. Up-regulation of SHP1 expression was achieved using a DNA MTI, 5-aza-2'-deoxycytidine (5-aza-dc), which also generated significant down-regulation of JAK2/STAT3/STAT5 signalling. We demonstrate that 5-aza-dc suppresses growth of CRC cells, and induces G2 cell cycle arrest and apoptosis through regulation of downstream targets of JAK2/STAT3/STAT5 signalling including Bcl-2, p16(ink4a), p21(waf1/cip1) and p27(kip1). Although 5-aza-dc did not significantly inhibit cell invasion, 5-aza-dc did down-regulate expression of focal adhesion kinase and vascular endothelial growth factor in CRC cells. Our results demonstrate that 5-aza-dc can induce SHP1 expression and inhibit JAK2/STAT3/STAT5 signalling. This study represents the first evidence towards establishing a mechanistic link between inhibition of JAK2/STAT3/STAT5 signalling and the anticancer action of 5-aza-dc in CRC cells that may lead to the use of MTIs as a therapeutic intervention for human colorectal cancer.

O6-methylguanine DNA methyltransferase in human fetal tissues: fetal and maternal factors

International Nuclear Information System (INIS)

D'Ambrosio, S.M.; Samuel, M.J.; Dutta-Choudhury, T.A.; Wani, A.A.

1986-01-01

O 6 -Methylguanine methyltransferase (O 6 -MT) was measured and compared in extracts of 7 human fetal tissues obtained from 21 different fetal specimens as a function of fetal age and race, and maternal smoking and drug usage. Activity was determined from the proteinase-K solubilized radioactivity transferred from the DNA to the O 6 -MT. S9 homogenates were incubated with a heat depurinated [ 3 H]-methylnitrosourea alkylated DNA. Liver exhibited the highest activity followed by kidney, lung, small intestine, large intestine, skin and brain. Each of the tissues exhibited a 3- to 5-fold level of interindividual variation of O 6 -MT. There did not appear to be any significant difference of O 6 -MT in the tissues obtained from mothers who smoked cigarettes during pregnancy. Also, fetal race and age did not appear to account for the level of variation of O 6 -MT. The fetal tissues obtained from an individual using phenobarbital and smoking exhibited 4-fold increases in O 6 -MT activity. The tissues obtained from another individual on kidney dialysis were 2- to 3-fold higher than the normal population. These data suggest that the variation in human O 6 -MT can not be explained by racial or smoking factors, but may be modulated by certain drugs

Transgenerational epigenetics: Inheritance of global cytosine methylation and methylation-related epigenetic markers in the shrub Lavandula latifolia.

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Herrera, Carlos M; Alonso, Conchita; Medrano, Mónica; Pérez, Ricardo; Bazaga, Pilar

2018-04-01

The ecological and evolutionary significance of natural epigenetic variation (i.e., not based on DNA sequence variants) variation will depend critically on whether epigenetic states are transmitted from parents to offspring, but little is known on epigenetic inheritance in nonmodel plants. We present a quantitative analysis of transgenerational transmission of global DNA cytosine methylation (= proportion of all genomic cytosines that are methylated) and individual epigenetic markers (= methylation status of anonymous MSAP markers) in the shrub Lavandula latifolia. Methods based on parent-offspring correlations and parental variance component estimation were applied to epigenetic features of field-growing plants ('maternal parents') and greenhouse-grown progenies. Transmission of genetic markers (AFLP) was also assessed for reference. Maternal parents differed significantly in global DNA cytosine methylation (range = 21.7-36.7%). Greenhouse-grown maternal families differed significantly in global methylation, and their differences were significantly related to maternal origin. Methylation-sensitive amplified polymorphism (MSAP) markers exhibited significant transgenerational transmission, as denoted by significant maternal variance component of marker scores in greenhouse families and significant mother-offspring correlations of marker scores. Although transmission-related measurements for global methylation and MSAP markers were quantitatively lower than those for AFLP markers taken as reference, this study has revealed extensive transgenerational transmission of genome-wide global cytosine methylation and anonymous epigenetic markers in L. latifolia. Similarity of results for global cytosine methylation and epigenetic markers lends robustness to this conclusion, and stresses the value of considering both types of information in epigenetic studies of nonmodel plants. © 2018 Botanical Society of America.

Chilling-Mediated DNA Methylation Changes during Dormancy and Its Release Reveal the Importance of Epigenetic Regulation during Winter Dormancy in Apple (Malus x domestica Borkh.).

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Kumar, Gulshan; Rattan, Usha Kumari; Singh, Anil Kumar

2016-01-01

Winter dormancy is a well known mechanism adopted by temperate plants, to mitigate the chilling temperature of winters. However, acquisition of sufficient chilling during winter dormancy ensures the normal phenological traits in subsequent growing period. Thus, low temperature appears to play crucial roles in growth and development of temperate plants. Apple, being an important temperate fruit crop, also requires sufficient chilling to release winter dormancy and normal phenological traits, which are often associated with yield and quality of fruits. DNA cytosine methylation is one of the important epigenetic modifications which remarkably affect the gene expression during various developmental and adaptive processes. In present study, methylation sensitive amplified polymorphism was employed to assess the changes in cytosine methylation during dormancy, active growth and fruit set in apple, under differential chilling conditions. Under high chill conditions, total methylation was decreased from 27.2% in dormant bud to 21.0% in fruit set stage, while no significant reduction was found under low chill conditions. Moreover, the demethylation was found to be decreased, while methylation increased from dormant bud to fruit set stage under low chill as compared to high chill conditions. In addition, RNA-Seq analysis showed high expression of DNA methyltransferases and histone methyltransferases during dormancy and fruit set, and low expression of DNA glcosylases during active growth under low chill conditions, which was in accordance with changes in methylation patterns. The RNA-Seq data of 47 genes associated with MSAP fragments involved in cellular metabolism, stress response, antioxidant system and transcriptional regulation showed correlation between methylation and their expression. Similarly, bisulfite sequencing and qRT-PCR analysis of selected genes also showed correlation between gene body methylation and gene expression. Moreover, significant association

Chilling-Mediated DNA Methylation Changes during Dormancy and Its Release Reveal the Importance of Epigenetic Regulation during Winter Dormancy in Apple (Malus x domestica Borkh..

Directory of Open Access Journals (Sweden)

Gulshan Kumar

Full Text Available Winter dormancy is a well known mechanism adopted by temperate plants, to mitigate the chilling temperature of winters. However, acquisition of sufficient chilling during winter dormancy ensures the normal phenological traits in subsequent growing period. Thus, low temperature appears to play crucial roles in growth and development of temperate plants. Apple, being an important temperate fruit crop, also requires sufficient chilling to release winter dormancy and normal phenological traits, which are often associated with yield and quality of fruits. DNA cytosine methylation is one of the important epigenetic modifications which remarkably affect the gene expression during various developmental and adaptive processes. In present study, methylation sensitive amplified polymorphism was employed to assess the changes in cytosine methylation during dormancy, active growth and fruit set in apple, under differential chilling conditions. Under high chill conditions, total methylation was decreased from 27.2% in dormant bud to 21.0% in fruit set stage, while no significant reduction was found under low chill conditions. Moreover, the demethylation was found to be decreased, while methylation increased from dormant bud to fruit set stage under low chill as compared to high chill conditions. In addition, RNA-Seq analysis showed high expression of DNA methyltransferases and histone methyltransferases during dormancy and fruit set, and low expression of DNA glcosylases during active growth under low chill conditions, which was in accordance with changes in methylation patterns. The RNA-Seq data of 47 genes associated with MSAP fragments involved in cellular metabolism, stress response, antioxidant system and transcriptional regulation showed correlation between methylation and their expression. Similarly, bisulfite sequencing and qRT-PCR analysis of selected genes also showed correlation between gene body methylation and gene expression. Moreover

Characterization of a Vitis vinifera cv. Cabernet Sauvignon 3',5'-O-methyltransferase showing strong preference for anthocyanins and glycosylated flavonols.

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Lücker, Joost; Martens, Stefan; Lund, Steven T

2010-09-01

At ripening initiation in red grapevine (Vitis vinifera) berries, the exocarp turns color from green to red and then to purple due to the accumulation and extent of methylation of anthocyanins. The accumulation of transcripts encoding an O-methyltransferase was recently shown to be closely correlated with the onset of ripening and the degree of blue/purple pigmentation in grapevine berries; however, the biochemical function of this gene has remained uncharacterized. In this study, an O-methyltransferase cDNA that showed a distinct expression pattern when compared to closely related sequences was expressed in Escherichia coli and enzyme assays were carried out with a broad array of anthocyanin and other flavonoid substrates. We demonstrate that this enzyme carries out 3',5'-O-methylation of anthocyanins and flavonol compounds in vitro, which are known to be present in grape berries, with a preference for glycosylated substrates. The highest relative specific activity for the enzyme was found with delphinidin 3-O-glucoside as substrate. The enzyme is not able to methylate flavan type skeletons with chiral centers, such as either catechins or dihydroquercetin. The enzyme showed negligible specific activity for caffeoyl-CoA, compared to flavonol and anthocyanin substrates. Phylogenetic analysis of the O-methyltransferase suggests that it may be a member of a distinct subclass of Type 2 bivalent metal-dependent S-adenosyl-methionine O-methyltransferases. (c) 2010. Published by Elsevier Ltd. All rights reserved.

Electron Attachment to the Gas Phase DNA Bases Cytosine and Thymine

Czech Academy of Sciences Publication Activity Database

Denifl, S.; Ptasiňska, S.; Probst, M.; Hrušák, Jan; Scheier, P.; Märk, T. D.

2004-01-01

Roč. 108, č. 31 (2004), s. 6562-6569 ISSN 1089-5639 R&D Projects: GA ČR GA203/02/0737 Institutional research plan: CEZ:AV0Z4040901 Keywords : gas-phase * cytosine * thymine Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.639, year: 2004

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

Science.gov (United States)

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

2018-04-06

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

Studies on N5-methyltetrahydrofolate-homocystein methyltransferase in normal and leukemia leukocytes.

Science.gov (United States)

Peytremann, R; Thorndike, J; Beck, W S

1975-11-01

A cobalamin-dependent N5-methyltetra-hydrofolate-homocysteine methyltransferase (methyl-transferase) was demonstrated in unfractioned extracts of human normal and leukemia leukocytes. Activity was substantially reduced in the absence of an added cobalamin derivative. Presumably, this residual activity reflects the endogeneous level of holoenzyme. Enzyme activity was notably higher in lymphoid cells than in myeloid cells. Thus, mean specific activities (+/-SD) were: chronic lymphocytic leukemia lymphocytes, 2.15+/-1.16; normal lymphocytes, 0.91+/-0.59; normal mature granulocytes, 0.15+/-0.10; chronic myelocytic leukemia granulocytes, barely detectable activity. Properties of leukocytes enzymes resembled those of methyltransferases previously studied in bacteria and other animal cells. Granulocytes and chronic myelocytic leukemia cells contain a factor or factors that inhibits Escherichia coli enzyme. The data suggest that the prominence of this cobalamin-dependent enzyme in lymphocytes and other mononuclear cell types may be related to their potential for cell division.

DNA methyltransferase inhibitor CDA-II inhibits myogenic differentiation

International Nuclear Information System (INIS)

Chen, Zirong; Jin, Guorong; Lin, Shuibin; Lin, Xiumei; Gu, Yumei; Zhu, Yujuan; Hu, Chengbin; Zhang, Qingjiong; Wu, Lizi; Shen, Huangxuan

2012-01-01

Highlights: ► CDA-II inhibits myogenic differentiation in a dose-dependent manner. ► CDA-II repressed expression of muscle transcription factors and structural proteins. ► CDA-II inhibited proliferation and migration of C2C12 myoblasts. -- Abstract: CDA-II (cell differentiation agent II), isolated from healthy human urine, is a DNA methyltransferase inhibitor. Previous studies indicated that CDA-II played important roles in the regulation of cell growth and certain differentiation processes. However, it has not been determined whether CDA-II affects skeletal myogenesis. In this study, we investigated effects of CDA-II treatment on skeletal muscle progenitor cell differentiation, migration and proliferation. We found that CDA-II blocked differentiation of murine myoblasts C2C12 in a dose-dependent manner. CDA-II repressed expression of muscle transcription factors, such as Myogenin and Mef2c, and structural proteins, such as myosin heavy chain (Myh3), light chain (Mylpf) and MCK. Moreover, CDA-II inhibited C1C12 cell migration and proliferation. Thus, our data provide the first evidence that CDA-II inhibits growth and differentiation of muscle progenitor cells, suggesting that the use of CDA-II might affect skeletal muscle functions.

Sequence specificity of DNA cleavage by Micrococcus luteus γ endonuclease

International Nuclear Information System (INIS)

Hentosh, P.; Henner, W.D.; Reynolds, R.J.

1985-01-01

DNA fragments of defined sequence have been used to determine the sites of cleavage by γ-endonuclease activity in extracts prepared from Micrococcus luteus. End-labeled DNA restriction fragments of pBR322 DNA that had been irradiated under nitrogen in the presence of potassium iodide or t-butanol were treated with M. luteus γ endonuclease and analyzed on irradiated DNA preferentially at the positions of cytosines and thymines. DNA cleavage occurred immediately to the 3' side of pyrimidines in irradiated DNA and resulted in fragments that terminate in a 5'-phosphoryl group. These studies indicate that both altered cytosines and thymines may be important DNA lesions requiring repair after exposure to γ radiation

Transcriptional changes in epigenetic modifiers associated with gene silencing in the intestine of the sea cucumber, Apostichopus japonicus (Selenka), during aestivation

Science.gov (United States)

Wang, Tianming; Yang, Hongsheng; Zhao, Huan; Chen, Muyan; Wang, Bing

2011-11-01

The sea cucumber, Apostichopus japonicus, undergoes aestivation to improve survival during periods of high-temperature. During aestivation, the metabolic rate is depressed to reduce the consumption of reserved energy. We evaluated the role of epigenetic modification on global gene silencing during metabolic rate depression in the sea cucumber. We compared the expression of epigenetic modifiers in active and aestivating sea cucumbers. The expression of three genes involved in DNA methylation and chromatin remodeling (DNA (cytosine-5)-methyltransferase 1, Methyl-CpG-binding domain protein 2), and Chromodomain-helicase-DNA-binding protein 5) was significantly higher during aestivation (Days 20 and 40). Similarly, we observed an increase in the expression of genes involved in histone acetylation (Histone deacetylase 3) and Histone-binding protein RBBP4) during the early (Days 5 and 10) and late phases (Days 20 and 40) of aestivation. There was no change in the expression of KAT2B, a histone acetyltransferase. However, the expression of histone methylation associated modifiers (Histone-arginine methyltransferase CARMER and Histone-lysine N-methyltransferase MLL5) was significantly higher after 5 d in the aestivating group. The results suggest that the expression of epigenetic modifiers involved in DNA methylation, chromatin remodeling, histone acetylation, and histone methylation is upregulated during aestivation. We hypothesize that these changes regulate global gene silencing during aestivation in A. japonicus.

Molecular Data for a Biochemical Model of DNA Radiation Damage: Electron Impact Ionization and Dissociative Ionization of DNA Bases and Sugar-Phosphate Backbone

Science.gov (United States)

Dateo, Christopher E.; Fletcher, Graham D.

2004-01-01

As part of the database for building up a biochemical model of DNA radiation damage, electron impact ionization cross sections of sugar-phosphate backbone and DNA bases have been calculated using the improved binary-encounter dipole (iBED) model. It is found that the total ionization cross sections of C3'- and C5'-deoxyribose-phospate, two conformers of the sugar-phosphate backbone, are close to each other. Furthermore, the sum of the ionization cross sections of the separate deoxyribose and phosphate fragments is in close agreement with the C3'- and C5'-deoxyribose-phospate cross sections, differing by less than 10%. Of the four DNA bases, the ionization cross section of guanine is the largest, then in decreasing order, adenine, thymine, and cytosine. The order is in accordance with the known propensity of oxidation of the bases by ionizing radiation. Dissociative ionization (DI), a process that both ionizes and dissociates a molecule, is investigated for cytosine. The DI cross section for the formation of H and (cytosine-Hl)(+), with the cytosine ion losing H at the 1 position, is also reported. The threshold of this process is calculated to be 17.1 eV. Detailed analysis of ionization products such as in DI is important to trace the sequential steps in the biochemical process of DNA damage.

O6-Methylguanine-DNA methyltransferase status in neuroendocrine tumours: prognostic relevance and association with response to alkylating agents.

Science.gov (United States)

Walter, T; van Brakel, B; Vercherat, C; Hervieu, V; Forestier, J; Chayvialle, J-A; Molin, Y; Lombard-Bohas, C; Joly, M-O; Scoazec, J-Y

2015-02-03

O(6)-Methylguanine-DNA methyltransferase (MGMT) loss of expression has been suggested to be predictive of response to temozolomide in neuroendocrine tumours (NETs), but so far, only limited data are available. We evaluated the prognostic and predictive value of MGMT status, assessed by two molecular methods and immunohistochemistry, in a large series of NETs of different origins. A total of 107 patients, including 53 treated by alkylants (temozolomide, dacarbazine or streptozotocin), were retrospectively studied. In each case, we used methyl-specific PCR (MS-PCR) and pyrosequencing for evaluation of promoter methylation and immunohistochemistry for evaluation of protein status. MGMT promoter methylation was detected in 12 out of 99 (12%) interpretable cases by MS-PCR and in 24 out of 99 (24%) by pyrosequencing. O(6)-Methylguanine-DNA methyltransferase loss of expression was observed in 29 out of 89 (33%) interpretable cases. Status of MGMT was not correlated with overall survival (OS) from diagnosis. Progression-free survival and OS from first alkylant use (temozolomide, dacarbazine and streptozotocin) were higher in patients with MGMT protein loss (respectively, 20.2 vs 7.6 months, Palkylant-based chemotherapy in NETs.

Evidence Suggesting Absence of Mitochondrial DNA Methylation

DEFF Research Database (Denmark)

Mechta, Mie; Ingerslev, Lars R; Fabre, Odile

2017-01-01

, 16S, ND5 and CYTB, suggesting that mtDNA supercoiled structure blocks the access to bisulfite conversion. Here, we identified an artifact of mtDNA bisulfite sequencing that can lead to an overestimation of mtDNA methylation levels. Our study supports that cytosine methylation is virtually absent...

The expression of spinal methyl-CpG-binding protein 2, DNA methyltransferases and histone deacetylases is modulated in persistent pain states

Directory of Open Access Journals (Sweden)

Tochiki Keri K

2012-02-01

Full Text Available Abstract Background DNA CpG methylation is carried out by DNA methyltransferases and induces chromatin remodeling and gene silencing through a transcription repressor complex comprising the methyl-CpG-binding protein 2 (MeCP2 and a subset of histone deacetylases. Recently, we have found that MeCP2 activity had a crucial role in the pattern of gene expression seen in the superficial dorsal horn rapidly after injection of Complete Freund's Adjuvant (CFA in the rat ankle joint. The aim of the present study was to analyse the changes in expression of MeCP2, DNA methyltransferases and a subset of histone deacetylases in the superficial dorsal horn during the maintenance phase of persistent pain states. In this process, the cell specific expression of MeCP2 was also investigated. Results Using immunohistochemistry, we found that neurones, oligodendrocytes and astrocytes expressed MeCP2. Microglia, oligodendrocyte precursor cells and Schwann cells never showed any positive stain for MeCP2. Quantitative analyses showed that MeCP2 expression was increased in the superficial dorsal horn 7 days following CFA injection in the ankle joint but decreased 7 days following spared nerve injury. Overall, the expression of DNA methyltransferases and a subset of histone deacetylases followed the same pattern of expression. However, there were no significant changes in the expression of the MeCP2 targets that we had previously shown are regulated in the early time points following CFA injection in the ankle joint. Finally, the expression of MeCP2 was also down regulated in damaged dorsal root ganglion neurones following spared nerve injury. Conclusion Our results strongly suggest that changes in chromatin compaction, regulated by the binding of MeCP2 complexes to methylated DNA, are involved in the modulation of gene expression in the superficial dorsal horn and dorsal root ganglia during the maintenance of persistent pain states.

Molecular data for a biochemical model of DNA damage: Electron impact ionization and dissociative ionization cross sections of DNA bases and sugar-phosphate backbone

International Nuclear Information System (INIS)

Huo, Winifred M.; Dateo, Christopher E.; Fletcher, Graham D.

2006-01-01

As part of the database for building up a biochemical model of DNA radiation damage, electron impact ionization cross sections of sugar-phosphate backbone and DNA bases have been calculated using the improved binary-encounter dipole (iBED) model. It is found that the total ionization cross sections of C 3 ' - and C 5 ' -deoxyribose-phosphate, two conformers of the sugar-phosphate backbone, are close to each other. Furthermore, the sum of the ionization cross sections of the separate deoxyribose and phosphate fragments is in close agreement with the C 3 ' - and C 5 ' -deoxyribose-phosphate cross sections, differing by less than 10%, an indication that a building-up principle may be applicable. Of the four DNA bases, the ionization cross section of guanine is the largest, then in decreasing order, adenine, thymine, and cytosine. The order is in accordance with the known propensity of oxidation of the bases by ionizing radiation. Dissociative ionization (DI), a process that both ionizes and dissociates a molecule, is investigated for cytosine. The DI cross section for the formation of H and (cytosine-H1) + , with the cytosine ion losing H at the 1 position, is also reported. The threshold of this process is calculated to be 16.9eV. Detailed analysis of ionization products such as in DI is important to trace the sequential steps in the biochemical process of DNA damage

DNA Methylation Alterations at 5'-CCGG Sites in the Interspecific and Intraspecific Hybridizations Derived from Brassica rapa and B. napus.

Directory of Open Access Journals (Sweden)

Wanshan Xiong

Full Text Available DNA methylation is an important regulatory mechanism for gene expression that involved in the biological processes of development and differentiation in plants. To investigate the association of DNA methylation with heterosis in Brassica, a set of intraspecific hybrids in Brassica rapa and B. napus and interspecific hybrids between B. rapa and B. napus, together with parental lines, were used to monitor alterations in cytosine methylation at 5'-CCGG sites in seedlings and buds by methylation-sensitive amplification polymorphism analysis. The methylation status of approximately a quarter of the methylation sites changed between seedlings and buds. These alterations were related closely to the genomic structure and heterozygous status among accessions. The methylation status in the majority of DNA methylation sites detected in hybrids was the same as that in at least one of the parental lines in both seedlings and buds. However, the association between patterns of cytosine methylation and heterosis varied among different traits and between tissues in hybrids of Brassica, although a few methylation loci were associated with heterosis. Our data suggest that changes in DNA methylation at 5'-CCGG sites are not associated simply with heterosis in the interspecific and intraspecific hybridizations derived from B. rapa and B. napus.

Sensitive electrochemical assaying of DNA methyltransferase activity based on mimic-hybridization chain reaction amplified strategy.

Science.gov (United States)

Zhang, Linqun; Liu, Yuanjian; Li, Ying; Zhao, Yuewu; Wei, Wei; Liu, Songqin

2016-08-24

A mimic-hybridization chain reaction (mimic-HCR) amplified strategy was proposed for sensitive electrochemically detection of DNA methylation and methyltransferase (MTase) activity In the presence of methylated DNA, DNA-gold nanoparticles (DNA-AuNPs) were captured on the electrode by sandwich-type assembly. It then triggered mimic-HCR of two hairpin probes to produce many long double-helix chains for numerous hexaammineruthenium (III) chloride ([Ru(NH3)6](3+), RuHex) inserting. As a result, the signal for electrochemically detection of DNA MTase activity could be amplified. If DNA was non-methylated, however, the sandwich-type assembly would not form because the short double-stranded DNAs (dsDNA) on the Au electrode could be cleaved and digested by restriction endonuclease HpaII (HapII) and exonuclease III (Exo III), resulting in the signal decrement. Based on this, an electrochemical approach for detection of M.SssI MTase activity with high sensitivity was developed. The linear range for M.SssI MTase activity was from 0.05 U mL(-1) to 10 U mL(-1), with a detection limit down to 0.03 U mL(-1). Moreover, this detecting strategy held great promise as an easy-to-use and highly sensitive method for other MTase activity and inhibition detection by exchanging the corresponding DNA sequence. Copyright © 2016 Elsevier B.V. All rights reserved.

Differences in substrate specificity of C(5)-substituted or C(5)-unsubstituted pyrimidine nucleotides by DNA polymerases from thermophilic bacteria, archaea, and phages.

Science.gov (United States)

Sawai, Hiroaki; Nagashima, Junichi; Kuwahara, Msayasu; Kitagata, Rina; Tamura, Takehiro; Matsui, Ikuo

2007-09-01

The pyrimidine bases of RNA are uracil (U) and cytosine (C), while thymine (T) and C are used for DNA. The C(5) position of C and U is unsubstituted, whereas the C(5) of T is substituted with a Me group. Miller et al. hypothesized that various C(5)-substituted uracil derivatives were formed during chemical evolution, and that C(5)-substituted U derivatives may have played important roles in the transition from an 'RNA world' to a 'DNA-RNA-protein world'. Hyperthermophilic bacteria and archaea are considered to be primitive organisms that are evolutionarily close to the universal ancestor of all life on earth. Thus, we examined the substrate specificity of several C(5)-substituted or C(5)-unsubstituted dUTP and dCTP analogs for several DNA polymerases from hyperthermophilic bacteria, hyperthermophilic archaea, and viruses during PCR or primer extension reaction. The substrate specificity of the C(5)-substituted or C(5)-unsubstituted pyrimidine nucleotides varied greatly depending on the type of DNA polymerase. The significance of this difference in substrate specificity in terms of the origin and evolution of the DNA replication system is discussed briefly.

[Association of etheno-DNA adduct and DNA methylation level among workers exposed to diesel engine exhaust].

Science.gov (United States)

Shen, M L; He, Z N; Zhang, X; Duan, H W; Niu, Y; Bin, P; Ye, M; Meng, T; Dai, Y F; Yu, S F; Chen, W; Zheng, Y X

2017-06-06

Objective: To investigate the association between etheno-DNA adduct and the promoter of DNA methylation levels of cyclin dependent kinase inhibitor 2A (P16), Ras association domain family 1 (RASSF1A) and O-6-methylguanine-DNA methyltransferase (MGMT) in workers with occupational exposure to diesel engine exhaust (DEE). Methods: We recruited 124 diesel engine testing workers as DEE exposure group and 112 water pump operator in the same area as control group in Henan province in 2012 using cluster sampling. The demographic data were obtained by questionnaire survey; urine after work and venous blood samples were collected from each subject. The urinary etheno-DNA adducts were detected using UPLC-MS/MS, including 1,N6-etheno-2'-deoxyadenosine (εdA) and 3,N4-etheno-2'-deoxycytidine(εdC). The DNA methylation levels of P16, RASSF1A, and MGMT were evaluated using bisulfite-pyrosequencing assay. The percentage of methylation was expressed as the 5-methylcytosine (5mC) over the sum of cytosines (%5mC). Spearman correlation and multiple linear regression were applied to analyze the association between etheno-DNA adducts and DNA methylation of P16, RASSF1A, and MGMT. Results: The median ( P (25)- P (75)) of urinary εdA level was 230.00 (98.04-470.91) pmol/g creatinine in DEE exposure group, and 102.10 (49.95-194.48) creatinine in control group. The level of εdA was higher in DEE exposure group than control group ( P 0.05) . Multiple linear regression confirmed the negative correlation between εdA and DNA methylation levels of P16, RASSF1A, and MGMT in non-smoking group (β (95 %CI ) was -0.068 (-0.132--0.003), -0.082 (-0.159--0.004) and -0.048 (-0.090--0.007), P values were 0.039, 0.039 and 0.024, respectively). Moreover, εdC was negative associated with DNA methylation level of MGMT in non-smoking group (β (95 %CI ) was -0.094 (-0.179--0.008), P= 0.032). Conclusion: DEE exposure could induce the increased of εdA and decreased of DNA methylation levels of P16, RASSF1A

A probabilistic generative model for quantification of DNA modifications enables analysis of demethylation pathways.

Science.gov (United States)

Äijö, Tarmo; Huang, Yun; Mannerström, Henrik; Chavez, Lukas; Tsagaratou, Ageliki; Rao, Anjana; Lähdesmäki, Harri

2016-03-14

We present a generative model, Lux, to quantify DNA methylation modifications from any combination of bisulfite sequencing approaches, including reduced, oxidative, TET-assisted, chemical-modification assisted, and methylase-assisted bisulfite sequencing data. Lux models all cytosine modifications (C, 5mC, 5hmC, 5fC, and 5caC) simultaneously together with experimental parameters, including bisulfite conversion and oxidation efficiencies, as well as various chemical labeling and protection steps. We show that Lux improves the quantification and comparison of cytosine modification levels and that Lux can process any oxidized methylcytosine sequencing data sets to quantify all cytosine modifications. Analysis of targeted data from Tet2-knockdown embryonic stem cells and T cells during development demonstrates DNA modification quantification at unprecedented detail, quantifies active demethylation pathways and reveals 5hmC localization in putative regulatory regions.

Elastic electron scattering from the DNA bases cytosine and thymine

International Nuclear Information System (INIS)

Colyer, C. J.; Bellm, S. M.; Lohmann, B.; Blanco, F.; Garcia, G.

2011-01-01

Cross-section data for electron scattering from biologically relevant molecules are important for the modeling of energy deposition in living tissue. Relative elastic differential cross sections have been measured for cytosine and thymine using the crossed-beam method. These measurements have been performed for six discrete electron energies between 60 and 500 eV and for detection angles between 15 deg. and 130 deg. Calculations have been performed via the screen-corrected additivity rule method and are in good agreement with the present experiment.

An optimized rapid bisulfite conversion method with high recovery of cell-free DNA.

Science.gov (United States)

Yi, Shaohua; Long, Fei; Cheng, Juanbo; Huang, Daixin

2017-12-19

Methylation analysis of cell-free DNA is a encouraging tool for tumor diagnosis, monitoring and prognosis. Sensitivity of methylation analysis is a very important matter due to the tiny amounts of cell-free DNA available in plasma. Most current methods of DNA methylation analysis are based on the difference of bisulfite-mediated deamination of cytosine between cytosine and 5-methylcytosine. However, the recovery of bisulfite-converted DNA based on current methods is very poor for the methylation analysis of cell-free DNA. We optimized a rapid method for the crucial steps of bisulfite conversion with high recovery of cell-free DNA. A rapid deamination step and alkaline desulfonation was combined with the purification of DNA on a silica column. The conversion efficiency and recovery of bisulfite-treated DNA was investigated by the droplet digital PCR. The optimization of the reaction results in complete cytosine conversion in 30 min at 70 °C and about 65% of recovery of bisulfite-treated cell-free DNA, which is higher than current methods. The method allows high recovery from low levels of bisulfite-treated cell-free DNA, enhancing the analysis sensitivity of methylation detection from cell-free DNA.

Planarizing cytosine: The S1 state structure, vibrations, and nonradiative dynamics of jet-cooled 5,6-trimethylenecytosine

Science.gov (United States)

Trachsel, Maria A.; Lobsiger, Simon; Schär, Tobias; Blancafort, Lluís; Leutwyler, Samuel

2017-06-01

We measure the S0 → S1 spectrum and time-resolved S1 state nonradiative dynamics of the "clamped" cytosine derivative 5,6-trimethylenecytosine (TMCyt) in a supersonic jet, using two-color resonant two-photon ionization (R2PI), UV/UV holeburning, and ns time-resolved pump/delayed ionization. The experiments are complemented with spin-component scaled second-order approximate coupled cluster (SCS-CC2), time-dependent density functional theory, and multi-state second-order perturbation-theory (MS-CASPT2) ab initio calculations. While the R2PI spectrum of cytosine breaks off ˜500 cm-1 above its 000 band, that of TMCyt extends up to +4400 cm-1 higher, with over a hundred resolved vibronic bands. Thus, clamping the cytosine C5-C6 bond allows us to explore the S1 state vibrations and S0 → S1 geometry changes in detail. The TMCyt S1 state out-of-plane vibrations ν1', ν3', and ν5' lie below 420 cm-1, and the in-plane ν11', ν12', and ν23' vibrational fundamentals appear at 450, 470, and 944 cm-1. S0 → S1 vibronic simulations based on SCS-CC2 calculations agree well with experiment if the calculated ν1', ν3', and ν5' frequencies are reduced by a factor of 2-3. MS-CASPT2 calculations predict that the ethylene-type S1 ⇝ S0 conical intersection (CI) increases from +366 cm-1 in cytosine to >6000 cm-1 in TMCyt, explaining the long lifetime and extended S0 → S1 spectrum. The lowest-energy S1 ⇝ S0 CI of TMCyt is the "amino out-of-plane" (OPX) intersection, calculated at +4190 cm-1. The experimental S1 ⇝ S0 internal conversion rate constant at the S1(v'=0 ) level is kI C=0.98 -2.2 ṡ1 08 s-1, which is ˜10 times smaller than in 1-methylcytosine and cytosine. The S1(v'=0 ) level relaxes into the T1(3π π *) state by intersystem crossing with kI S C=0.41 -1.6 ṡ1 08 s-1. The T1 state energy is measured to lie 24 580 ±560 cm-1 above the S0 state. The S1(v'=0 ) lifetime is τ =2.9 ns, resulting in an estimated fluorescence quantum yield of Φf l=24 %. Intense

Micrococcus luteus correndonucleases. II. Mechanism of action of two endonucleases specific for DNA containing pyrimidine dimers

International Nuclear Information System (INIS)

Riazuddin, S.; Grossman, L.

1977-01-01

Py--Py correndonucleases I and II from Micrococcus luteus act exclusively on thymine-thymine, cytosine-cytosine, and thymine-cytosine cyclobutyl dimers in DNA, catalyzing incision 5' to the damage and generating 3'-hydroxyl and 5'-phosphoryl termini. Both enzymes initiate excision of pyrimidine dimers in vitro by correxonucleases and DNA polymerase I. The respective incised DNAs, however, differ in their ability to act as substrate for phage T4 polynucleotide ligase or bacterial alkaline phosphatase, suggesting that each endonuclease is specific for a conformationally unique site. The possibility that their respective action generates termini which represent different degrees of single strandedness is suggested by the unequal protection by Escherichia coli binding protein from the hydrolytic action of exonuclease VII

Molecular cloning and functional expression of a stress-induced multifunctional O-methyltransferase with pinosylvin methyltransferase activity from Scots pine (Pinus sylvestris L.).

Science.gov (United States)

Chiron, H; Drouet, A; Claudot, A C; Eckerskorn, C; Trost, M; Heller, W; Ernst, D; Sandermann, H

2000-12-01

Formation of pinosylvin (PS) and pinosylvin 3-O-monomethyl ether (PSM), as well as the activities of stilbene synthase (STS) and S-adenosyl-1-methionine (SAM):pinosylvin O-methyltransferase (PMT), were induced strongly in needles of Scots pine seedlings upon ozone treatment, as well as in cell suspension cultures of Scots pine upon fungal elicitation. A SAM-dependent PMT protein was purified and partially characterised. A cDNA encoding PMT was isolated from an ozone-induced Scots pine cDNA library. Southern blot analysis of the genomic DNA suggested the presence of a gene family. The deduced protein sequence showed the typical highly conserved regions of O-methyltransferases (OMTs), and average identities of 20-56% to known OMTs. PMT expressed in Escherichia coli corresponded to that of purified PMT (40 kDa) from pine cell cultures. The recombinant enzyme catalysed the methylation of PS, caffeic acid, caffeoyl-CoA and quercetin. Several other substances, such as astringenin, resveratrol, 5-OH-ferulic acid, catechol and luteolin, were also methylated. Recombinant PMT thus had a relatively broad substrate specificity. Treatment of 7-year old Scots pine trees with ozone markedly increased the PMT mRNA level. Our results show that PMT represents a new SAM-dependent OMT for the methylation of stress-induced pinosylvin in Scots pine needles.

Reconsolidation of a cocaine associated memory requires DNA methyltransferase activity in the basolateral amygdala

Science.gov (United States)

Shi, Hai-Shui; Luo, Yi-Xiao; Yin, Xi; Wu, Hong-Hai; Xue, Gai; Geng, Xu-Hong; Hou, Yan-Ning

2015-01-01

Drug addiction is considered an aberrant form of learning, and drug-associated memories evoked by the presence of associated stimuli (drug context or drug-related cues) contribute to recurrent craving and reinstatement. Epigenetic changes mediated by DNA methyltransferase (DNMT) have been implicated in the reconsolidation of fear memory. Here, we investigated the role of DNMT activity in the reconsolidation of cocaine-associated memories. Rats were trained over 10 days to intravenously self-administer cocaine by nosepokes. Each injection was paired with a light/tone conditioned stimulus (CS). After acquisition of stable self-administration behaviour, rats underwent nosepoke extinction (10 d) followed by cue-induced reactivation and subsequent cue-induced and cocaine-priming + cue-induced reinstatement tests or subsequently tested to assess the strength of the cocaine-associated cue as a conditioned reinforcer to drive cocaine seeking behaviour. Bilateral intra-basolateral amygdala (BLA) infusion of the DNMT inhibitor5-azacytidine (5-AZA, 1 μg per side) immediately following reactivation decreased subsequent reinstatement induced by cues or cocaine priming as well as cue-maintained cocaine-seeking behaviour. In contrast, delayed intra-BLA infusion of 5-AZA 6 h after reactivation or 5-AZA infusion without reactivation had no effect on subsequent cue-induced reinstatement. These findings indicate that memory reconsolidation for a cocaine-paired stimulus depends critically on DNMT activity in the BLA. PMID:26289919

The Helicobacter pylori HpyAXII restriction–modification system limits exogenous DNA uptake by targeting GTAC sites but shows asymmetric conservation of the DNA methyltransferase and restriction endonuclease components

Science.gov (United States)

Humbert, Olivier; Salama, Nina R.

2008-01-01

The naturally competent organism Helicobacter pylori encodes a large number of restriction–modification (R–M) systems that consist of a restriction endonuclease and a DNA methyltransferase. R–M systems are not only believed to limit DNA exchange among bacteria but may also have other cellular functions. We report a previously uncharacterized H. pylori type II R–M system, M.HpyAXII/R.HpyAXII. We show that this system targets GTAC sites, which are rare in the H. pylori chromosome but numerous in ribosomal RNA genes. As predicted, this type II R–M system showed attributes of a selfish element. Deletion of the methyltransferase M.HpyAXII is lethal when associated with an active endonuclease R.HpyAXII unless compensated by adaptive mutation or gene amplification. R.HpyAXII effectively restricted both unmethylated plasmid and chromosomal DNA during natural transformation and was predicted to belong to the novel ‘half pipe’ structural family of endonucleases. Analysis of a panel of clinical isolates revealed that R.HpyAXII was functional in a small number of H. pylori strains (18.9%, n = 37), whereas the activity of M.HpyAXII was highly conserved (92%, n = 50), suggesting that GTAC methylation confers a selective advantage to H. pylori. However, M.HpyAXII activity did not enhance H. pylori fitness during stomach colonization of a mouse infection model. PMID:18978016

Rapid, large-scale purification and characterization of Ada protein (O sup 6 methylguanine-DNA methyltransferase) of E. coli

Energy Technology Data Exchange (ETDEWEB)

Bhattacharyya, D.; Tano, K.; Bunick, G.J.; Uberbacher, E.C.; Mitra, S. (Oak Ridge National Laboratory, TN (USA)); Behnke, W.D. (Univ. of Cincinnati College of Medicine, OH (USA))

1988-07-25

The E. coli Ada protein (O{sup 6}-methylguanine-DNA methyltransferase) has been purified using a high-level expression vector with a yield of about 3 mg per liter of E. coli culture. The 39-kDa protein has an extinction coefficient (E{sup 280nm}{sub 1%}) of 5.3. Its isoelectric point of 7.1 is lower than that predicted from the amino acid content. The homogeneous Ada protein is fully active as a methyl acceptor from O{sup 6}-methylguanine in DNA. Its reaction with O{sup 6}-methylguanine in a synthetic DNA has a second-order rate constant of 1.1 {times} 10{sup 9} M{sup {minus}1} min{sup {minus}1} at 0{degree}C. Both the native form and the protein methylated at Cys-69 are monomeric. The CD spectrum suggests a low {alpha}-helical content and the radius of gyration of 23 {angstrom} indicates a compact, globular shape. The middle region of the protein is sensitive to a variety of proteases, including an endogenous activity in E. coli, suggesting that the protein is composed of N-terminal and C-terminal domains connected by a hinge region. E. coli B has a higher level of this protease than does K12.

Human C6orf211 Encodes Armt1, a Protein Carboxyl Methyltransferase that Targets PCNA and Is Linked to the DNA Damage Response

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J. Jefferson P. Perry

2015-03-01

Full Text Available Recent evidence supports the presence of an L-glutamyl methyltransferase(s in eukaryotic cells, but this enzyme class has been defined only in certain prokaryotic species. Here, we characterize the human C6orf211 gene product as “acidic residue methyltransferase-1” (Armt1, an enzyme that specifically targets proliferating cell nuclear antigen (PCNA in breast cancer cells, predominately methylating glutamate side chains. Armt1 homologs share structural similarities with the SAM-dependent methyltransferases, and negative regulation of activity by automethylation indicates a means for cellular control. Notably, shRNA-based knockdown of Armt1 expression in two breast cancer cell lines altered survival in response to genotoxic stress. Increased sensitivity to UV, adriamycin, and MMS was observed in SK-Br-3 cells, while in contrast, increased resistance to these agents was observed in MCF7 cells. Together, these results lay the foundation for defining the mechanism by which this post-translational modification operates in the DNA damage response (DDR.

DNA methyltransferase 3A gene polymorphism contributes to daily life stress susceptibility

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Barliana MI

2017-12-01

Full Text Available Melisa I Barliana,1,2 Shintya N Amalya,1 Ivan S Pradipta,3 Sofa D Alfian,3 Arif SW Kusuma,1,2 Tiana Milanda,1,4 Rizky Abdulah3,4 1Department of Biological Pharmacy, Biotechnology Pharmacy Laboratory, 2Pharmacy Services Development Research Center, 3Department of Pharmacology and Clinical Pharmacy, Clinical Pharmacy Laboratory, 4Center for Drug Discovery and Product Development, Faculty of Pharmacy, Universitas Padjadjaran, Jatinangor, West Java, Indonesia Abstract: Daily life stress markedly affects the response toward stressful stimuli. DNA methy­lation is one of the factors that regulate this response, and is a normal mechanism of somatic cell growth, but its regulatory gene variations may cause alterations in the stress response. The aim of the present study was to investigate genotypic variants of the DNA methyltransferase 3A (DNMT3A gene in 129 healthy subjects and evaluate its association with daily life stress. Blood samples were collected, and genomic DNA was isolated. DNA was amplified using specific tetra primers for DNMT3A (C/T rs11683424 and visualized following 2% agarose gel electrophoresis. The association of DNMT3A genetic variants with daily life stress was analyzed using the Kessler Psychological Distress Scale (K10. We observed that the distribution of subjects with genotype CC (wild type, CT (heteromutant, and TT (homomutant was 13.95%, 81.4%, and 4.65%, respectively. Genetic variations significantly affected the daily life stress condition (p=0.04 in Indonesian healthy subjects, but most of the subjects with the CT phenotype were classified in a stress condition. Keywords: daily life stressor, DNA methylation, epigenetic, Kessler Psychological Distress Scale (K10, rs11683424, DNMT3A

Methylation effect on the ohmic resistance of a poly-GC DNA-like chain

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Moura, F.A.B.F. de, E-mail: fidelis@fis.ufal.br [Instituto de Física, Universidade Federal de Alagoas, Maceió AL 57072-970 (Brazil); Lyra, M.L. [Instituto de Física, Universidade Federal de Alagoas, Maceió AL 57072-970 (Brazil); Almeida, M.L. de; Ourique, G.S.; Fulco, U.L.; Albuquerque, E.L. [Departamento de Biofísica e Farmacologia, Universidade Federal do Rio Grande do Norte, 59072-970, Natal-RN (Brazil)

2016-10-14

We determine, by using a tight-binding model Hamiltonian, the characteristic current–voltage (IxV) curves of a 5-methylated cytosine single strand poly-GC DNA-like finite segment, considering the methyl groups attached laterally to a random fraction of the cytosine basis. Striking, we found that the methylation significantly impacts the ohmic resistance (R) of the DNA-like segments, indicating that measurements of R can be used as a biosensor tool to probe the presence of anomalous methylation. - Highlights: • Ohmic resistance of finite segments of poly-CG DNA-like segments. • Possibility for the development of biosensor devices. • Methylation effect and electronic transport in DNA-like segments.

Contrasting roles for DNA methyltransferases and histone deacetylases in single-item and associative recognition memory.

Science.gov (United States)

Scott, Hannah; Smith, Anna E; Barker, Gareth R; Uney, James B; Warburton, E Clea

2017-03-01

Recognition memory enables us to judge whether we have encountered a stimulus before and to recall associated information, including where the stimulus was encountered. The perirhinal cortex (PRh) is required for judgment of stimulus familiarity, while hippocampus (HPC) and medial prefrontal cortex (mPFC) are additionally involved when spatial information associated with a stimulus needs to be remembered. While gene expression is known to be essential for the consolidation of long-term recognition memory, the underlying regulatory mechanisms are not fully understood. Here we investigated the roles of two epigenetic mechanisms, DNA methylation and histone deacetylation, in recognition memory. Infusion of DNA methyltransferase inhibitors into PRh impaired performance in novel object recognition and object-in-place tasks while infusions into HPC or mPFC impaired object-in-place performance only. In contrast, inhibition of histone deacetylases in PRh, but not mPFC, enhanced recognition memory. These results support the emerging role of epigenetic processes in learning and memory.

Contrasting roles for DNA methyltransferases and histone deacetylases in single-item and associative recognition memory

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Hannah Scott

2017-03-01

Full Text Available Recognition memory enables us to judge whether we have encountered a stimulus before and to recall associated information, including where the stimulus was encountered. The perirhinal cortex (PRh is required for judgment of stimulus familiarity, while hippocampus (HPC and medial prefrontal cortex (mPFC are additionally involved when spatial information associated with a stimulus needs to be remembered. While gene expression is known to be essential for the consolidation of long-term recognition memory, the underlying regulatory mechanisms are not fully understood. Here we investigated the roles of two epigenetic mechanisms, DNA methylation and histone deacetylation, in recognition memory. Infusion of DNA methyltransferase inhibitors into PRh impaired performance in novel object recognition and object-in-place tasks while infusions into HPC or mPFC impaired object-in-place performance only. In contrast, inhibition of histone deacetylases in PRh, but not mPFC, enhanced recognition memory. These results support the emerging role of epigenetic processes in learning and memory.

Assessing the fidelity of ancient DNA sequences amplified from nuclear genes

DEFF Research Database (Denmark)

Binladen, Jonas; Wiuf, Carsten Henrik; Gilbert, M. Thomas P.

2006-01-01

To date, the field of ancient DNA has relied almost exclusively on mitochondrial DNA (mtDNA) sequences. However, a number of recent studies have reported the successful recovery of ancient nuclear DNA (nuDNA) sequences, thereby allowing the characterization of genetic loci directly involved...... in phenotypic traits of extinct taxa. It is well documented that postmortem damage in ancient mtDNA can lead to the generation of artifactual sequences. However, as yet no one has thoroughly investigated the damage spectrum in ancient nuDNA. By comparing clone sequences from 23 fossil specimens, recovered from...... adenine), respectively. Type 2 transitions are by far the most dominant and increase relative to those of type 1 with damage load. The results suggest that the deamination of cytosine (and 5-methyl cytosine) to uracil (and thymine) is the main cause of miscoding lesions in both ancient mtDNA and nu...

Caught in the act: visualization of an intermediate in the DNA base-flipping pathway induced by HhaI methyltransferase | Center for Cancer Research

Science.gov (United States)

HHAI methyltransferase (blue ribbon) bound to oligonucleotide (strands with bonds colored yellow and green) containing a pseudorotationally constrained sugar analogue at the target position (orange bonds with cyan atoms). The south-constrained pseudosugar is rotated about its flanking phosphodiester bonds, 90° from its initial position in B-form DNA, but short of a completely

2′-O Methylation of Internal Adenosine by Flavivirus NS5 Methyltransferase

Science.gov (United States)

Dong, Hongping; Chang, David C.; Hua, Maggie Ho Chia; Lim, Siew Pheng; Chionh, Yok Hian; Hia, Fabian; Lee, Yie Hou; Kukkaro, Petra; Lok, Shee-Mei; Dedon, Peter C.; Shi, Pei-Yong

2012-01-01

RNA modification plays an important role in modulating host-pathogen interaction. Flavivirus NS5 protein encodes N-7 and 2′-O methyltransferase activities that are required for the formation of 5′ type I cap (m7GpppAm) of viral RNA genome. Here we reported, for the first time, that flavivirus NS5 has a novel internal RNA methylation activity. Recombinant NS5 proteins of West Nile virus and Dengue virus (serotype 4; DENV-4) specifically methylates polyA, but not polyG, polyC, or polyU, indicating that the methylation occurs at adenosine residue. RNAs with internal adenosines substituted with 2′-O-methyladenosines are not active substrates for internal methylation, whereas RNAs with adenosines substituted with N6-methyladenosines can be efficiently methylated, suggesting that the internal methylation occurs at the 2′-OH position of adenosine. Mass spectroscopic analysis further demonstrated that the internal methylation product is 2′-O-methyladenosine. Importantly, genomic RNA purified from DENV virion contains 2′-O-methyladenosine. The 2′-O methylation of internal adenosine does not require specific RNA sequence since recombinant methyltransferase of DENV-4 can efficiently methylate RNAs spanning different regions of viral genome, host ribosomal RNAs, and polyA. Structure-based mutagenesis results indicate that K61-D146-K181-E217 tetrad of DENV-4 methyltransferase forms the active site of internal methylation activity; in addition, distinct residues within the methyl donor (S-adenosyl-L-methionine) pocket, GTP pocket, and RNA-binding site are critical for the internal methylation activity. Functional analysis using flavivirus replicon and genome-length RNAs showed that internal methylation attenuated viral RNA translation and replication. Polymerase assay revealed that internal 2′-O-methyladenosine reduces the efficiency of RNA elongation. Collectively, our results demonstrate that flavivirus NS5 performs 2′-O methylation of internal adenosine of

Determination of DNA methylation associated with Acer rubrum (red maple) adaptation to metals: analysis of global DNA modifications and methylation-sensitive amplified polymorphism.

Science.gov (United States)

Kim, Nam-Soo; Im, Min-Ji; Nkongolo, Kabwe

2016-08-01

Red maple (Acer rubum), a common deciduous tree species in Northern Ontario, has shown resistance to soil metal contamination. Previous reports have indicated that this plant does not accumulate metals in its tissue. However, low level of nickel and copper corresponding to the bioavailable levels in contaminated soils in Northern Ontario causes severe physiological damages. No differentiation between metal-contaminated and uncontaminated populations has been reported based on genetic analyses. The main objective of this study was to assess whether DNA methylation is involved in A. rubrum adaptation to soil metal contamination. Global cytosine and methylation-sensitive amplified polymorphism (MSAP) analyses were carried out in A. rubrum populations from metal-contaminated and uncontaminated sites. The global modified cytosine ratios in genomic DNA revealed a significant decrease in cytosine methylation in genotypes from a metal-contaminated site compared to uncontaminated populations. Other genotypes from a different metal-contaminated site within the same region appear to be recalcitrant to metal-induced DNA alterations even ≥30 years of tree life exposure to nickel and copper. MSAP analysis showed a high level of polymorphisms in both uncontaminated (77%) and metal-contaminated (72%) populations. Overall, 205 CCGG loci were identified in which 127 were methylated in either outer or inner cytosine. No differentiation among populations was established based on several genetic parameters tested. The variations for nonmethylated and methylated loci were compared by analysis of molecular variance (AMOVA). For methylated loci, molecular variance among and within populations was 1.5% and 13.2%, respectively. These values were low (0.6% for among populations and 5.8% for within populations) for unmethylated loci. Metal contamination is seen to affect methylation of cytosine residues in CCGG motifs in the A. rubrum populations that were analyzed.

DNA Methyltransferases Modulate Hepatogenic Lineage Plasticity of Mesenchymal Stromal Cells

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Chien-Wei Lee

2017-07-01

Full Text Available The irreversibility of developmental processes in mammalian cells has been challenged by rising evidence that de-differentiation of hepatocytes occurs in adult liver. However, whether reversibility exists in mesenchymal stromal cell (MSC-derived hepatocytes (dHeps remains elusive. In this study, we find that hepatogenic differentiation (HD of MSCs is a reversible process and is modulated by DNA methyltransferases (DNMTs. DNMTs are regulated by transforming growth factor β1 (TGFβ1, which in turn controls hepatogenic differentiation and de-differentiation. In addition, a stepwise reduction in TGFβ1 concentrations in culture media increases DNMT1 and decreases DNMT3 in primary hepatocytes (Heps and confers Heps with multi-differentiation potentials similarly to MSCs. Hepatic lineage reversibility of MSCs and lineage conversion of Heps are regulated by DNMTs in response to TGFβ1. This previously unrecognized TGFβ1-DNMTs-MSC-HD axis may further increase the understanding the normal and pathological processes in the liver, as well as functions of MSCs after transplantation to treat liver diseases.

Replication protein A (RPA hampers the processive action of APOBEC3G cytosine deaminase on single-stranded DNA.

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Artem G Lada

Full Text Available Editing deaminases have a pivotal role in cellular physiology. A notable member of this superfamily, APOBEC3G (A3G, restricts retroviruses, and Activation Induced Deaminase (AID generates antibody diversity by localized deamination of cytosines in DNA. Unconstrained deaminase activity can cause genome-wide mutagenesis and cancer. The mechanisms that protect the genomic DNA from the undesired action of deaminases are unknown. Using the in vitro deamination assays and expression of A3G in yeast, we show that replication protein A (RPA, the eukaryotic single-stranded DNA (ssDNA binding protein, severely inhibits the deamination activity and processivity of A3G.We found that mutations induced by A3G in the yeast genomic reporter are changes of a single nucleotide. This is unexpected because of the known property of A3G to catalyze multiple deaminations upon one substrate encounter event in vitro. The addition of recombinant RPA to the oligonucleotide deamination assay severely inhibited A3G activity. Additionally, we reveal the inverse correlation between RPA concentration and the number of deaminations induced by A3G in vitro on long ssDNA regions. This resembles the "hit and run" single base substitution events observed in yeast.Our data suggest that RPA is a plausible antimutator factor limiting the activity and processivity of editing deaminases in the model yeast system. Because of the similar antagonism of yeast RPA and human RPA with A3G in vitro, we propose that RPA plays a role in the protection of the human genome cell from A3G and other deaminases when they are inadvertently diverged from their natural targets. We propose a model where RPA serves as one of the guardians of the genome that protects ssDNA from the destructive processive activity of deaminases by non-specific steric hindrance.

Replication protein A (RPA) hampers the processive action of APOBEC3G cytosine deaminase on single-stranded DNA.

Science.gov (United States)

Lada, Artem G; Waisertreiger, Irina S-R; Grabow, Corinn E; Prakash, Aishwarya; Borgstahl, Gloria E O; Rogozin, Igor B; Pavlov, Youri I

2011-01-01

Editing deaminases have a pivotal role in cellular physiology. A notable member of this superfamily, APOBEC3G (A3G), restricts retroviruses, and Activation Induced Deaminase (AID) generates antibody diversity by localized deamination of cytosines in DNA. Unconstrained deaminase activity can cause genome-wide mutagenesis and cancer. The mechanisms that protect the genomic DNA from the undesired action of deaminases are unknown. Using the in vitro deamination assays and expression of A3G in yeast, we show that replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA) binding protein, severely inhibits the deamination activity and processivity of A3G. We found that mutations induced by A3G in the yeast genomic reporter are changes of a single nucleotide. This is unexpected because of the known property of A3G to catalyze multiple deaminations upon one substrate encounter event in vitro. The addition of recombinant RPA to the oligonucleotide deamination assay severely inhibited A3G activity. Additionally, we reveal the inverse correlation between RPA concentration and the number of deaminations induced by A3G in vitro on long ssDNA regions. This resembles the "hit and run" single base substitution events observed in yeast. Our data suggest that RPA is a plausible antimutator factor limiting the activity and processivity of editing deaminases in the model yeast system. Because of the similar antagonism of yeast RPA and human RPA with A3G in vitro, we propose that RPA plays a role in the protection of the human genome cell from A3G and other deaminases when they are inadvertently diverged from their natural targets. We propose a model where RPA serves as one of the guardians of the genome that protects ssDNA from the destructive processive activity of deaminases by non-specific steric hindrance.

YebU is a m5C methyltransferase specific for 16 S rRNA nucleotide 1407

DEFF Research Database (Denmark)

Andersen, Niels Møller; Douthwaite, Stephen

2006-01-01

generally require specific enzymes, and only one m5C rRNA methyltransferase, RsmB (formerly Fmu) that methylates nucleotide C967, has previously been identified. BLAST searches of the E.coli genome revealed a single gene, yebU, with sufficient similarity to rsmB to encode a putative m5C RNA...... methyltransferase. This suggested that the yebU gene product modifies C1407 and/or C1962. Here, we analysed the E.coli rRNAs by matrix assisted laser desorption/ionization mass spectrometry and show that inactivation of the yebU gene leads to loss of methylation at C1407 in 16 S rRNA, but does not interfere...

CDKL5 is a brain MeCP2 target gene regulated by DNA methylation.

Science.gov (United States)

Carouge, Delphine; Host, Lionel; Aunis, Dominique; Zwiller, Jean; Anglard, Patrick

2010-06-01

Rett syndrome and its "early-onset seizure" variant are severe neurodevelopmental disorders associated with mutations within the MECP2 and the CDKL5 genes. Antidepressants and drugs of abuse induce the expression of the epigenetic factor MeCP2, thereby influencing chromatin remodeling. We show that increased MeCP2 levels resulted in the repression of Cdkl5 in rat brain structures in response to cocaine, as well as in cells exposed to serotonin, or overexpressing MeCP2. In contrast, Cdkl5 was induced by siRNA-mediated knockdown of Mecp2 and by DNA-methyltransferase inhibitors, demonstrating its regulation by MeCP2 and by DNA methylation. Cdkl5 gene methylation and its methylation-dependent binding to MeCP2 were increased in the striatum of cocaine-treated rats. Our data demonstrate that Cdkl5 is a MeCP2-repressed target gene providing a link between genes the mutation of which generates overlapping symptoms. They highlight DNA methylation changes as a potential mechanism participating in the long-term plasticity triggered by pharmacological agents.

Cyclopentenyl cytosine induces apoptosis and increases cytarabine-induced apoptosis in a T-lymphoblastic leukemic cell-line

NARCIS (Netherlands)

Verschuur, A. C.; Brinkman, J.; van Gennip, A. H.; Leen, R.; Vet, R. J.; Evers, L. M.; Voûte, P. A.; van Kuilenburg, A. B.

2001-01-01

Cyclopentenyl cytosine (CPEC) is a nucleoside-analogue that decreases the concentrations of cytidine triphosphate (CTP) and deoxycytidine triphosphate (dCTP) in leukemic cells by inhibiting the enzyme CTP synthetase, resulting in a decreased synthesis of RNA and DNA. Low concentrations of dCTP

Mutagenic effect of radionuclides incorporated into DNA of Drosophila melanogaster. Progress report, 1975--1976

International Nuclear Information System (INIS)

Lee, W.R.

1976-01-01

Progress is reported on research in the following areas: determination of mutagenic effects of tritium incorporated into DNA using 32 P, 33 P, and 14 C; dosimetry studies using number of disintegrations per minute per sperm cell; effect of the 5-position of the label of the cytosine moiety on mutation frequency; distribution of labeled thymine and comparison with labeled cytosine and a non-DNA labeled arginine-rich protein; detection of temperature sensitive mutants; induction of mosaics by x radiation; and methods for determining quantitatively the location of tritium in the deoxycytidine moiety

Mutations in the DNA methyltransferase gene, DNMT3A, cause an overgrowth syndrome with intellectual disability

Science.gov (United States)

Tatton-Brown, Katrina; Seal, Sheila; Ruark, Elise; Harmer, Jenny; Ramsay, Emma; del Vecchio Duarte, Silvana; Zachariou, Anna; Hanks, Sandra; O’Brien, Eleanor; Aksglaede, Lise; Baralle, Diana; Dabir, Tabib; Gener, Blanca; Goudie, David; Homfray, Tessa; Kumar, Ajith; Pilz, Daniela T; Selicorni, Angelo; Temple, I Karen; Van Maldergem, Lionel; Yachelevich, Naomi; van Montfort, Robert; Rahman, Nazneen

2014-01-01

Overgrowth disorders are a heterogeneous group of conditions characterised by increased growth parameters and variable other clinical features, such as intellectual disability and facial dysmorphism1. To identify novel causes of human overgrowth we performed exome sequencing in 10 proband-parent trios and detected two de novo DNMT3A mutations. We identified 11 additional de novo mutations through DNMT3A sequencing of a further 142 individuals with overgrowth. The mutations were all located in functional DNMT3A domains and protein modelling suggests they interfere with domain-domain interactions and histone binding. No similar mutations were present in 1000 UK population controls (13/152 vs 0/1000; P<0.0001). Mutation carriers had a distinctive facial appearance, intellectual disability and increased height. DNMT3A encodes a key methyltransferase essential for establishing the methylation imprint in embryogenesis and is commonly somatically mutated in acute myeloid leukaemia2-4. Thus DNMT3A joins an emerging group of epigenetic DNA and histone modifying genes associated with both developmental growth disorders and haematological malignancies5. PMID:24614070

Epigenetic editing of DNA Methylation and DeRewriting DNA Methylation Signatures at Will : The Curable Genome Within Reach?

NARCIS (Netherlands)

Stolzenburg, Sabine; Goubert, Désirée; Rots, Marianne

2016-01-01

DNA methyltransferases are important enzymes in a broad range of organisms. Dysfunction of DNA methyltransferases in humans leads to many severe diseases, including cancer. This book focuses on the biochemical properties of these enzymes, describing their structures and mechanisms in bacteria,

Identification and characterization of DNAzymes targeting DNA methyltransferase I for suppressing bladder cancer proliferation

Energy Technology Data Exchange (ETDEWEB)

Wang, Xiangbo; Zhang, Lu; Ding, Nianhua; Yang, Xinghui; Zhang, Jin; He, Jiang; Li, Zhi; Sun, Lun-Quan, E-mail: lunquansun@csu.edu.cn

2015-05-29

Epigenetic inactivation of genes plays a critical role in many important human diseases, especially in cancer. A core mechanism for epigenetic inactivation of the genes is methylation of CpG islands in genome DNA, which is catalyzed by DNA methyltransferases (DNMTs). The inhibition of DNMTs may lead to demethylation and expression of the silenced tumor suppressor genes. Although DNMT inhibitors are currently being developed as potential anticancer agents, only limited success is achieved due to substantial toxicity. Here, we utilized a multiplex selection system to generate efficient RNA-cleaving DNAzymes targeting DNMT1. The lead molecule from the selection was shown to possess efficient kinetic profiles and high efficiency in inhibiting the enzyme activity. Transfection of the DNAzyme caused significant down-regulation of DNMT1 expression and reactivation of p16 gene, resulting in reduced cell proliferation of bladder cancers. This study provides an alternative for targeting DNMTs for potential cancer therapy. - Highlights: • Identified DNMT1-targeted DNAzymes by multiplex selection system. • Biochemically characterized a lead DNAzyme with high kinetic efficiency. • Validated DNMT1-targeted DNAzyme in its enzymatic and cellular activities.

Investigating the potential role of genetic and epigenetic variation of DNA methyltransferase genes in hyperplastic polyposis syndrome.

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Musa Drini

2011-02-01

Full Text Available Hyperplastic Polyposis Syndrome (HPS is a condition associated with multiple serrated polyps, and an increased risk of colorectal cancer (CRC. At least half of CRCs arising in HPS show a CpG island methylator phenotype (CIMP, potentially linked to aberrant DNA methyltransferase (DNMT activity. CIMP is associated with methylation of tumor suppressor genes including regulators of DNA mismatch repair (such as MLH1, MGMT, and negative regulators of Wnt signaling (such as WIF1. In this study, we investigated the potential for interaction of genetic and epigenetic variation in DNMT genes, in the aetiology of HPS.We utilized high resolution melting (HRM analysis to screen 45 cases with HPS for novel sequence variants in DNMT1, DNMT3A, DNMT3B, and DNMT3L. 21 polyps from 13 patients were screened for BRAF and KRAS mutations, with assessment of promoter methylation in the DNMT1, DNMT3A, DNMT3B, DNMT3L MLH1, MGMT, and WIF1 gene promoters.No pathologic germline mutations were observed in any DNA-methyltransferase gene. However, the T allele of rs62106244 (intron 10 of DNMT1 gene was over-represented in cases with HPS (p<0.01 compared with population controls. The DNMT1, DNMT3A and DNMT3B promoters were unmethylated in all instances. Interestingly, the DNMT3L promoter showed low levels of methylation in polyps and normal colonic mucosa relative to matched disease free cells with methylation level negatively correlated to expression level in normal colonic tissue. DNMT3L promoter hypomethylation was more often found in polyps harbouring KRAS mutations (p = 0.0053. BRAF mutations were common (11 out of 21 polyps, whilst KRAS mutations were identified in 4 of 21 polyps.Genetic or epigenetic alterations in DNMT genes do not appear to be associated with HPS, but further investigation of genetic variation at rs62106244 is justified given the high frequency of the minor allele in this case series.

Atomic Insight into the Altered O6-Methylguanine-DNA Methyltransferase Protein Architecture in Gastric Cancer.

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Naveed Anjum Chikan

Full Text Available O6-methylguanine-DNA methyltransferase (MGMT is one of the major DNA repair protein that counteracts the alkalyting agent-induced DNA damage by replacing O6-methylguanine (mutagenic lesion back to guanine, eventually suppressing the mismatch errors and double strand crosslinks. Exonic alterations in the form of nucleotide polymorphism may result in altered protein structure that in turn can lead to the loss of function. In the present study, we focused on the population feared for high exposure to alkylating agents owing to their typical and specialized dietary habits. To this end, gastric cancer patients pooled out from the population were selected for the mutational screening of a specific error prone region of MGMT gene. We found that nearly 40% of the studied neoplastic samples harbored missense mutation at codon151 resulting into Serine to Isoleucine variation. This variation resulted in bringing about the structural disorder, subsequently ensuing into a major stoichiometric variance in recognition domain, substrate binding and selectivity loop of the active site of the MGMT protein, as observed under virtual microscope of molecular dynamics simulation (MDS. The atomic insight into MGMT protein by computational approach showed a significant change in the intra molecular hydrogen bond pattern, thus leading to the observed structural anomalies. To further examine the mutational implications on regulatory plugs of MGMT that holds the protein in a DNA-Binding position, a MDS based analysis was carried out on, all known physically interacting amino acids essentially clustered into groups based on their position and function. The results generated by physical-functional clustering of protein indicated that the identified mutation in the vicinity of the active site of MGMT protein causes the local and global destabilization of a protein by either eliminating the stabilizing salt bridges in cluster C3, C4, and C5 or by locally destabilizing the

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

International Nuclear Information System (INIS)

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

2015-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2015-04-15

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

Human catechol-O-methyltransferase: Cloning and expression of the membrane-associated form

International Nuclear Information System (INIS)

Bertocci, B.; Miggiano, V.; Da Prada, M.; Dembic, Z.; Lahm, H.W.; Malherbe, P.

1991-01-01

A cDNA clone for human catechol-O-methyltransferase was isolated from a human hepatoma cell line (Hep G2) cDNA library by hybridization screening with a porcine cDNA probe. The cDNA clone was sequenced and found to have an insert of 1226 nucleotides. The deduced primary structure of hCOMT is composed of 271 amino acid residues with the predicted molecular mass of 30 kDa. At its N terminus it has a hydrophobic segment of 21 amino acid residues that may be responsible for insertion of hCOMT into the endoplasmic reticulum membrane. The primary structure of hCOMT exhibits high homology to the porcine partial cDNA sequence (93%). The deduced amino acid sequence contains two tryptic peptide sequences (T-22, T-33) found in porcine liver catechol-O-methyltransferase (CEMT). The coding region of hCOMT cDNA was placed under the control of the cytomegalovirus promoter to transfect human kidney 293 cells. The recombinant hCOMT was shown by immunoblot analysis to be mainly associated with the membrane fraction. RNA blot analysis revealed one COMT mRNA transcript of 1.4 kilobases in Hep G2 poly(A) + RNA

Effect of Rap1 binding on DNA distortion and potassium permanganate hypersensitivity.

Science.gov (United States)

Le Bihan, Yann-Vaï; Matot, Béatrice; Pietrement, Olivier; Giraud-Panis, Marie-Josèphe; Gasparini, Sylvaine; Le Cam, Eric; Gilson, Eric; Sclavi, Bianca; Miron, Simona; Le Du, Marie-Hélène

2013-03-01

Repressor activator protein 1 (Rap1) is an essential factor involved in transcription and telomere stability in the budding yeast Saccharomyces cerevisiae. Its interaction with DNA causes hypersensitivity to potassium permanganate, suggesting local DNA melting and/or distortion. In this study, various Rap1-DNA crystal forms were obtained using specifically designed crystal screens. Analysis of the DNA conformation showed that its distortion was not sufficient to explain the permanganate reactivity. However, anomalous data collected at the Mn edge using a Rap1-DNA crystal soaked in potassium permanganate solution indicated that the DNA conformation in the crystal was compatible with interaction with permanganate ions. Sequence-conservation analysis revealed that double-Myb-containing Rap1 proteins all carry a fully conserved Arg580 at a position that may favour interaction with permanganate ions, although it is not involved in the hypersensitive cytosine distortion. Permanganate reactivity assays with wild-type Rap1 and the Rap1[R580A] mutant demonstrated that Arg580 is essential for hypersensitivity. AFM experiments showed that wild-type Rap1 and the Rap1[R580A] mutant interact with DNA over 16 successive binding sites, leading to local DNA stiffening but not to accumulation of the observed local distortion. Therefore, Rap1 may cause permanganate hypersensitivity of DNA by forming a pocket between the reactive cytosine and Arg580, driving the permanganate ion towards the C5-C6 bond of the cytosine.

Nanoswitches based on DNA base pairs: why adenine-thymine is less suitable than guanine-cytosine

NARCIS (Netherlands)

Fonseca Guerra, C.; van der Wijst, T.; Bickelhaupt, F.M.

2006-01-01

Substituted Watson-Crick guanine-cytosine (GC) base pairs were recently shown to yield robust three-state nanoswitches. Here, we address the question: Can such supramolecular switches also be based on Watson-Crick adenine-thymine (AT) base pairs? We have theoretically analyzed AT pairs in which

O6-methylguanine DNA-methyltransferase (MGMT) overexpression in melanoma cells induces resistance to nitrosoureas and temozolomide but sensitizes to mitomycin C

International Nuclear Information System (INIS)

Passagne, Isabelle; Evrard, Alexandre; Depeille, Philippe; Cuq, Pierre; Cupissol, Didier; Vian, Laurence

2006-01-01

Alkylating agents play an important role in the chemotherapy of malignant melanomas. The activity of alkylating agents depends on their capacity to form alkyl adducts with DNA, in some cases causing cross-linking of DNA strands. However, the use of these agents is limited by cellular resistance induced by the DNA repair enzyme O 6 -methylguanine DNA-methyltransferase (MGMT) which removes alkyl groups from alkylated DNA strands. To determine to what extent the expression of MGMT in melanoma cells induces resistance to alkylating agents, the human cell line CAL77 Mer- (i.e., MGMT deficient) were transfected with pcMGMT vector containing human MGMT cDNA. Several clones expressing MGMT at a high level were selected to determine their sensitivity to chemotherapeutic drugs. Melanoma-transfected cells were found to be significantly less sensitive to nitrosoureas (carmustine, fotemustine, streptozotocin) and temozolomide with an increase of IC 5 values between 3 and 14 when compared to parent cells. No difference in cell survival rates between MGMT-proficient and -deficient cells was observed for melphalan, chlorambucil, busulphan, thiotepa and cisplatin which preferentially induce N 7 guanine lesions. Surprisingly, MGMT overexpression increased the sensitivity of CAL77 cells to mitomycin C by approximately 10-fold. Treatment of clonal cell lines with buthionine-[S,R]-sulfoximine (BSO), an inhibitor of γ-glutamylcysteine synthetase which depletes cellular glutathione, completely reversed this unexpected increase in sensitivity to mitomycin C. This observation suggests that glutathione is involved in the sensitivity of MGMT-transfected cells to mitomycin C and may act synergistically with MGMT via an unknown mechanism

Electrochemical DNA probe for Hg(2+) detection based on a triple-helix DNA and Multistage Signal Amplification Strategy.

Science.gov (United States)

Wang, Huan; Zhang, Yihe; Ma, Hongmin; Ren, Xiang; Wang, Yaoguang; Zhang, Yong; Wei, Qin

2016-12-15

In this work, an ultrasensitive electrochemical sensor was developed for detection of Hg(2+). Gold nanoparticles decorated bovine serum albumin reduction of graphene oxide (AuNP-BSA-rGO) were used as subsurface material for the immobilization of triple-helix DNA. The triple-helix DNA containing a thiol labelled single-stranded DNA (sDNA) and a thymine-rich DNA (T-rich DNA), which could be unwinded in the present of Hg(2+) to form more stable thymine-Hg(2+)-thymine (T-Hg(2+)-T) complex. T-Hg(2+)-T complex was then removed and the sDNA was left on the electrode. At this time, gold nanoparticle carrying thiol labelled cytosine-rich complementary DNA (cDNA-AuNP) could bind with the free sDNA. Meanwhile, the other free cDNA on AuNP could bind with each other in the present of Ag(+) to form the stable cytosine-Ag(+)-cytosine (C-Ag(+)-C) complex and circle amplification. Plenty of C-Ag(+)-C could form silver nanoclusters by electrochemical reduction and the striping signal of Ag could be measured for purpose of the final electrochemical detection of Hg(2+). This sensor could detect Hg(2+) over a wide concentration range from 0.1 to 130nM with a detection limit of 0.03nM. Copyright © 2016 Elsevier B.V. All rights reserved.

Fluorescent quenching-based quantitative detection of specific DNA/RNA using a BODIPY® FL-labeled probe or primer

Science.gov (United States)

Kurata, Shinya; Kanagawa, Takahiro; Yamada, Kazutaka; Torimura, Masaki; Yokomaku, Toyokazu; Kamagata, Yoichi; Kurane, Ryuichiro

2001-01-01

We have developed a simple method for the quantitative detection of specific DNA or RNA molecules based on the finding that BODIPY® FL fluorescence was quenched by its interaction with a uniquely positioned guanine. This approach makes use of an oligonucleotide probe or primer containing a BODIPY® FL-modified cytosine at its 5′-end. When such a probe was hybridized with a target DNA, its fluorescence was quenched by the guanine in the target, complementary to the modified cytosine, and the quench rate was proportional to the amount of target DNA. This widely applicable technique will be used directly with larger samples or in conjunction with the polymerase chain reaction to quantify small DNA samples. PMID:11239011

Detection of DNA damage based on metal-mediated molecular beacon and DNA strands displacement reaction

Science.gov (United States)

Xiong, Yanxiang; Wei, Min; Wei, Wei; Yin, Lihong; Pu, Yuepu; Liu, Songqin

2014-01-01

DNA hairpin structure probes are usually designed by forming intra-molecular duplex based on Watson-Crick hydrogen bonds. In this paper, a molecular beacon based on silver ions-mediated cytosine-Ag+-cytosine base pairs was used to detect DNA. The inherent characteristic of the metal ligation facilitated the design of functional probe and the adjustment of its binding strength compared to traditional DNA hairpin structure probes, which make it be used to detect DNA in a simple, rapid and easy way with the help of DNA strands displacement reaction. The method was sensitive and also possesses the good specificity to differentiate the single base mismatched DNA from the complementary DNA. It was also successfully applied to study the damage effect of classic genotoxicity chemicals such as styrene oxide and sodium arsenite on DNA, which was significant in food science, environmental science and pharmaceutical science.

AID/APOBEC cytosine deaminase induces genome-wide kataegis

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Lada Artem G

2012-12-01

Full Text Available Abstract Clusters of localized hypermutation in human breast cancer genomes, named “kataegis” (from the Greek for thunderstorm, are hypothesized to result from multiple cytosine deaminations catalyzed by AID/APOBEC proteins. However, a direct link between APOBECs and kataegis is still lacking. We have sequenced the genomes of yeast mutants induced in diploids by expression of the gene for PmCDA1, a hypermutagenic deaminase from sea lamprey. Analysis of the distribution of 5,138 induced mutations revealed localized clusters very similar to those found in tumors. Our data provide evidence that unleashed cytosine deaminase activity is an evolutionary conserved, prominent source of genome-wide kataegis events. Reviewers This article was reviewed by: Professor Sandor Pongor, Professor Shamil R. Sunyaev, and Dr Vladimir Kuznetsov.

Mutations in the DNA methyltransferase gene DNMT3A cause an overgrowth syndrome with intellectual disability.

Science.gov (United States)

Tatton-Brown, Katrina; Seal, Sheila; Ruark, Elise; Harmer, Jenny; Ramsay, Emma; Del Vecchio Duarte, Silvana; Zachariou, Anna; Hanks, Sandra; O'Brien, Eleanor; Aksglaede, Lise; Baralle, Diana; Dabir, Tabib; Gener, Blanca; Goudie, David; Homfray, Tessa; Kumar, Ajith; Pilz, Daniela T; Selicorni, Angelo; Temple, I Karen; Van Maldergem, Lionel; Yachelevich, Naomi; van Montfort, Robert; Rahman, Nazneen

2014-04-01

Overgrowth disorders are a heterogeneous group of conditions characterized by increased growth parameters and other variable clinical features such as intellectual disability and facial dysmorphism. To identify new causes of human overgrowth, we performed exome sequencing in ten proband-parent trios and detected two de novo DNMT3A mutations. We identified 11 additional de novo mutations by sequencing DNMT3A in a further 142 individuals with overgrowth. The mutations alter residues in functional DNMT3A domains, and protein modeling suggests that they interfere with domain-domain interactions and histone binding. Similar mutations were not present in 1,000 UK population controls (13/152 cases versus 0/1,000 controls; P < 0.0001). Mutation carriers had a distinctive facial appearance, intellectual disability and greater height. DNMT3A encodes a DNA methyltransferase essential for establishing methylation during embryogenesis and is commonly somatically mutated in acute myeloid leukemia. Thus, DNMT3A joins an emerging group of epigenetic DNA- and histone-modifying genes associated with both developmental growth disorders and hematological malignancies.

A novel fluorescent probe (dtpa-bis(cytosine)) for detection of Eu(III) in rare earth metal ions

Science.gov (United States)

Yang, Fan; Ren, Peipei; Liu, Guanhong; Song, Youtao; Bu, Naishun; Wang, Jun

2018-03-01

In this paper, a novel fluorescent probe, dtpa-bis(cytosine), was designed and synthesized for detecting europium (Eu3 +) ion. Upon addition of Eu3 + ions into the dtpa-bis(cytosine) solution, the fluorescence intensity can strongly be enhanced. Conversely, adding other rare earth metal ions, such as Y3 +, Ce3 +, Pr3 +, Nd3 +, Sm3 +, Gd3 +, Tb3 +, Dy3 +, Ho3 +, Er3 +, Yb3 + and Lu3 +, into dtpa-bis(cytosine) solution, the fluorescence intensity is decreased slightly. Some parameters affecting the fluorescence intensity of dtpa-bis(cytosine) solution in the presence of Eu3 + ions were investigated, including solution pH value, Eu3 + ion concentration and interfering substances. The detection mechanism of Eu3 + ion using dtpa-bis(cytosine) as fluorescent probe was proposed. Under optimum conditions, the fluorescence emission intensities of EuIII-dtpa-bis(cytosine) at 375 nm in the concentration range of 0.50 × 10- 5 mol • L- 1-5.00 × 10- 5 mol • L- 1 of Eu3 + ion display a better linear relationship. The limit of detection (LOD) was determined as 8.65 × 10- 7 mol • L- 1 and the corresponding correlation coefficient (R2) of the linear equation is 0.9807. It is wished that the proposed method could be applied for sensitively and selectively detecting Eu3 + ion.

DNA-functionalized gold nanoparticle-based fluorescence polarization for the sensitive detection of silver ions.

Science.gov (United States)

Wang, Gongke; Wang, Shuangli; Yan, Changling; Bai, Guangyue; Liu, Yufang

2018-04-05

Despite their practical applications, Ag + ions are environmental pollutants and affect human health. So the effective detection methods of Ag + ions are imperative. Herein, we developed a simple, sensitive, selective, and cost-effective fluorescence polarization sensor for Ag + detection in aqueous solution using thiol-DNA-functionalized gold nanoparticles (AuNPs). In this sensing strategy, Ag + ions can specifically interact with a cytosine-cytosine (CC) mismatch in DNA duplexes and form stable metal-mediated cytosine-Ag + -cytosine (C-Ag + -C) base pairs. The formation of the C-Ag + -C complex results in evident changes in the molecular volume and fluorescence polarization signal. To achieve our aims, we prepared two complementary DNA strands containing C-base mismatches (probe A: 5'-SH-A 10 -TACCACTCCTCAC-3' and probe B: 5'-TCCTCACCAGTCCTA-FAM-3'). The stable hybridization between probe A and probe B occurs with the formation of the C-Ag + -C complex in the presence of Ag + ions, leading to obvious fluorescence quenching in comparison to the system without AuNP enhancement. The assay can be used to identify nanomolar levels of Ag + within 6 min at room temperature, and has extremely high specificity for Ag + , even in the presence of higher concentrations of interfering metal ions. Furthermore, the sensor was successfully applied to the detection of Ag + ions in environmental water samples and showed excellent selectivity and high sensitivity, implying its promising application in the future. Copyright © 2018 Elsevier B.V. All rights reserved.

Stress-induced gene expression and behavior are controlled by DNA methylation and methyl donor availability in the dentate gyrus

Science.gov (United States)

Saunderson, Emily A.; Spiers, Helen; Gutierrez-Mecinas, Maria; Trollope, Alexandra F.; Shaikh, Abeera; Mill, Jonathan; Reul, Johannes M. H. M.

2016-01-01

Stressful events evoke long-term changes in behavioral responses; however, the underlying mechanisms in the brain are not well understood. Previous work has shown that epigenetic changes and immediate-early gene (IEG) induction in stress-activated dentate gyrus (DG) granule neurons play a crucial role in these behavioral responses. Here, we show that an acute stressful challenge [i.e., forced swimming (FS)] results in DNA demethylation at specific CpG (5′-cytosine–phosphate–guanine-3′) sites close to the c-Fos (FBJ murine osteosarcoma viral oncogene homolog) transcriptional start site and within the gene promoter region of Egr-1 (early growth response protein 1) specifically in the DG. Administration of the (endogenous) methyl donor S-adenosyl methionine (SAM) did not affect CpG methylation and IEG gene expression at baseline. However, administration of SAM before the FS challenge resulted in an enhanced CpG methylation at the IEG loci and suppression of IEG induction specifically in the DG and an impaired behavioral immobility response 24 h later. The stressor also specifically increased the expression of the de novo DNA methyltransferase Dnmt3a [DNA (cytosine-5-)-methyltransferase 3 alpha] in this hippocampus region. Moreover, stress resulted in an increased association of Dnmt3a enzyme with the affected CpG loci within the IEG genes. No effects of SAM were observed on stress-evoked histone modifications, including H3S10p-K14ac (histone H3, phosphorylated serine 10 and acetylated lysine-14), H3K4me3 (histone H3, trimethylated lysine-4), H3K9me3 (histone H3, trimethylated lysine-9), and H3K27me3 (histone H3, trimethylated lysine-27). We conclude that the DNA methylation status of IEGs plays a crucial role in FS-induced IEG induction in DG granule neurons and associated behavioral responses. In addition, the concentration of available methyl donor, possibly in conjunction with Dnmt3a, is critical for the responsiveness of dentate neurons to environmental

Advances in DNA methylation: 5-hydroxymethylcytosine revisited

DEFF Research Database (Denmark)

Dahl, Christina; Grønbæk, Kirsten; Guldberg, Per

2011-01-01

modification involved in gene regulation, X-chromosome inactivation, genomic imprinting, long-term silencing of transposons and cancer development is well described. 5hmC, on the other hand, has only recently entered center stage when it was shown that the Ten-Eleven-Translocation (TET) family of oxygenases...... in cancer development, and developing sequencing methodologies that can accurately distinguish among cytosine, 5mC and 5hmC at single-base-pair resolution....

Mitochondrial targeting of human O6-methylguanine DNA methyltransferase protects against cell killing by chemotherapeutic alkylating agents.

Science.gov (United States)

Cai, Shanbao; Xu, Yi; Cooper, Ryan J; Ferkowicz, Michael J; Hartwell, Jennifer R; Pollok, Karen E; Kelley, Mark R

2005-04-15

DNA repair capacity of eukaryotic cells has been studied extensively in recent years. Mammalian cells have been engineered to overexpress recombinant nuclear DNA repair proteins from ectopic genes to assess the impact of increased DNA repair capacity on genome stability. This approach has been used in this study to specifically target O(6)-methylguanine DNA methyltransferase (MGMT) to the mitochondria and examine its impact on cell survival after exposure to DNA alkylating agents. Survival of human hematopoietic cell lines and primary hematopoietic CD34(+) committed progenitor cells was monitored because the baseline repair capacity for alkylation-induced DNA damage is typically low due to insufficient expression of MGMT. Increased DNA repair capacity was observed when K562 cells were transfected with nuclear-targeted MGMT (nucl-MGMT) or mitochondrial-targeted MGMT (mito-MGMT). Furthermore, overexpression of mito-MGMT provided greater resistance to cell killing by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) than overexpression of nucl-MGMT. Simultaneous overexpression of mito-MGMT and nucl-MGMT did not enhance the resistance provided by mito-MGMT alone. Overexpression of either mito-MGMT or nucl-MGMT also conferred a similar level of resistance to methyl methanesulfonate (MMS) and temozolomide (TMZ) but simultaneous overexpression in both cellular compartments was neither additive nor synergistic. When human CD34(+) cells were infected with oncoretroviral vectors that targeted O(6)-benzylguanine (6BG)-resistant MGMT (MGMT(P140K)) to the nucleus or the mitochondria, committed progenitors derived from infected cells were resistant to 6BG/BCNU or 6BG/TMZ. These studies indicate that mitochondrial or nuclear targeting of MGMT protects hematopoietic cells against cell killing by BCNU, TMZ, and MMS, which is consistent with the possibility that mitochondrial DNA damage and nuclear DNA damage contribute equally to alkylating agent-induced cell killing during chemotherapy.

Patterns of cytosine methylation in an elite rice hybrid and its parental lines, detected by a methylation-sensitive amplification polymorphism technique.

Science.gov (United States)

Xiong, L Z; Xu, C G; Saghai Maroof, M A; Zhang, Q

1999-04-01

DNA methylation is known to play an important role in the regulation of gene expression in eukaryotes. In this study, we assessed the extent and pattern of cytosine methylation in the rice genome, using the technique of methylation-sensitive amplified polymorphism (MSAP), which is a modification of the amplified fragment length polymorphism (AFLP) method that makes use of the differential sensitivity of a pair of isoschizomers to cytosine methylation. The tissues assayed included seedlings and flag leaves of an elite rice hybrid, Shanyou 63, and the parental lines Zhenshan 97 and Minghui 63. In all, 1076 fragments, each representing a recognition site cleaved by either or both of the isoschizomers, were amplified using 16 pairs of selective primers. A total of 195 sites were found to be methylated at cytosines in one or both parents, and the two parents showed approximately the same overall degree of methylation (16.3%), as revealed by the incidence of differential digestion by the isoschizomers. Four classes of patterns were identified in a comparative assay of cytosine methylation in the parents and hybrid; increased methylation was detected in the hybrid compared to the parents at some of the recognition sites, while decreased methylation in the hybrid was detected at other sites. A small proportion of the sites was found to be differentially methylated in seedlings and flag leaves; DNA from young seedlings was methylated to a greater extent than that from flag leaves. Almost all of the methylation patterns detected by MSAP could be confirmed by Southern analysis using the isolated amplified fragments as probes. The results clearly demonstrate that the MSAP technique is highly efficient for large-scale detection of cytosine methylation in the rice genome. We believe that the technique can be adapted for use in other plant species.

An RNA polymerase II-and AGO4-associated protein acts in RNA-directed DNA methylation

KAUST Repository

Gao, Zhihuan

2010-04-21

DNA methylation is an important epigenetic mark in many eukaryotes. In plants, 24-nucleotide small interfering RNAs (siRNAs) bound to the effector protein, Argonaute 4 (AGO4), can direct de novo DNA methylation by the methyltransferase DRM2 (refs 2, 4-6). Here we report a new regulator of RNA-directed DNA methylation (RdDM) in Arabidopsis: RDM1. Loss-of-function mutations in the RDM1 gene impair the accumulation of 24-nucleotide siRNAs, reduce DNA methylation, and release transcriptional gene silencing at RdDM target loci. RDM1 encodes a small protein that seems to bind single-stranded methyl DNA, and associates and co-localizes with RNA polymerase II (Pol II, also known as NRPB), AGO4 and DRM2 in the nucleus. Our results indicate that RDM1 is a component of the RdDM effector complex and may have a role in linking siRNA production with pre-existing or de novo cytosine methylation. Our results also indicate that, although RDM1 and Pol V (also known as NRPE) may function together at some RdDM target sites in the peri-nucleolar siRNA processing centre, Pol II rather than Pol V is associated with the RdDM effector complex at target sites in the nucleoplasm. © 2010 Macmillan Publishers Limited. All rights reserved.

Analysis of DNA Hydroxymethylation Using Colorimetric Assay.

Science.gov (United States)

Golubov, Andrey; Kovalchuk, Igor

2017-01-01

Hydroxymethylcytosine (hmC or 5-hmC) is a nitrogen base occurring as a result of cytosine methylation followed by replacing a methyl group with a hydroxyl group through active oxidation. 5-hmC is considered to be one of the forms of epigenetic modification and is suggested as an intermediate step in a semi-active loss of DNA methylation mark. 5-hmC plays an important role in the epigenetic regulation of gene expression in animals, although its role in plants remains controversial. Here, we present a colorimetric method of quantification of 5-hmC using Brassica rapa DNA.

Achaete-scute complex homolog-1 promotes DNA repair in the lung carcinogenesis through matrix metalloproteinase-7 and O(6-methylguanine-DNA methyltransferase.

Directory of Open Access Journals (Sweden)

Xiao-Yang Wang

Full Text Available Lung cancer is the leading cause of cancer-related deaths in the world. Achaete-scute complex homolog-1 (Ascl1 is a member of the basic helix-loop-helix (bHLH transcription factor family that has multiple functions in the normal and neoplastic lung such as the regulation of neuroendocrine differentiation, prevention of apoptosis and promotion of tumor-initiating cells. We now show that Ascl1 directly regulates matrix metalloproteinase-7 (MMP-7 and O(6-methylguanine-DNA methyltransferase (MGMT. Loss- and gain-of-function experiments in human bronchial epithelial and lung carcinoma cell lines revealed that Ascl1, MMP-7 and MGMT are able to protect cells from the tobacco-specific nitrosamine NNK-induced DNA damage and the alkylating agent cisplatin-induced apoptosis. We also examined the role of Ascl1 in NNK-induced lung tumorigenesis in vivo. Using transgenic mice which constitutively expressed human Ascl1 in airway lining cells, we found that there was a delay in lung tumorigenesis. We conclude that Ascl1 potentially enhances DNA repair through activation of MMP-7 and MGMT which may impact lung carcinogenesis and chemoresistance. The study has uncovered a novel and unexpected function of Ascl1 which will contribute to better understanding of lung carcinogenesis and the broad implications of transcription factors in tobacco-related carcinogenesis.

Structure of the human gene encoding the protein repair L-isoaspartyl (D-aspartyl) O-methyltransferase.

Science.gov (United States)

DeVry, C G; Tsai, W; Clarke, S

1996-11-15

The protein L-isoaspartyl/D-aspartyl O-methyltransferase (EC 2.1.1.77) catalyzes the first step in the repair of proteins damaged in the aging process by isomerization or racemization reactions at aspartyl and asparaginyl residues. A single gene has been localized to human chromosome 6 and multiple transcripts arising through alternative splicing have been identified. Restriction enzyme mapping, subcloning, and DNA sequence analysis of three overlapping clones from a human genomic library in bacteriophage P1 indicate that the gene spans approximately 60 kb and is composed of 8 exons interrupted by 7 introns. Analysis of intron/exon splice junctions reveals that all of the donor and acceptor splice sites are in agreement with the mammalian consensus splicing sequence. Determination of transcription initiation sites by primer extension analysis of poly(A)+ mRNA from human brain identifies multiple start sites, with a major site 159 nucleotides upstream from the ATG start codon. Sequence analysis of the 5'-untranslated region demonstrates several potential cis-acting DNA elements including SP1, ETF, AP1, AP2, ARE, XRE, CREB, MED-1, and half-palindromic ERE motifs. The promoter of this methyltransferase gene lacks an identifiable TATA box but is characterized by a CpG island which begins approximately 723 nucleotides upstream of the major transcriptional start site and extends through exon 1 and into the first intron. These features are characteristic of housekeeping genes and are consistent with the wide tissue distribution observed for this methyltransferase activity.

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

Science.gov (United States)

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

2010-08-23

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

Fingerprints of Both Watson-Crick and Hoogsteen Isomers of the Isolated (Cytosine-Guanine)H+ Pair.

Science.gov (United States)

Cruz-Ortiz, Andrés F; Rossa, Maximiliano; Berthias, Francis; Berdakin, Matías; Maitre, Philippe; Pino, Gustavo A

2017-11-16

 Gas phase protonated guanine-cytosine (CGH + ) pair was generated using an electrospray ionization source from solutions at two different pH (5.8 and 3.2). Consistent evidence from MS/MS fragmentation patterns and differential ion mobility spectra (DIMS) point toward the presence of two isomers of the CGH + pair, whose relative populations depend strongly on the pH of the solution. Gas phase infrared multiphoton dissociation (IRMPD) spectroscopy in the 900-1900 cm -1 spectral range further confirms that the Watson-Crick isomer is preferentially produced (91%) at pH = 5.8, while the Hoogsteen isomer predominates (66%) at pH = 3.2). These fingerprint signatures are expected to be useful for the development of new analytical methodologies and to trigger isomer selective photochemical studies of protonated DNA base pairs.

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

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Kimberly D Siegmund

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

Rationalization of activity cliffs of a sulfonamide inhibitor of DNA methyltransferases with induced-fit docking.

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Medina-Franco, José L; Méndez-Lucio, Oscar; Yoo, Jakyung

2014-02-21

Inhibitors of human DNA methyltransferases (DNMT) are of increasing interest to develop novel epi-drugs for the treatment of cancer and other diseases. As the number of compounds with reported DNMT inhibition is increasing, molecular docking is shedding light to elucidate their mechanism of action and further interpret structure-activity relationships. Herein, we present a structure-based rationalization of the activity of SW155246, a distinct sulfonamide compound recently reported as an inhibitor of human DNMT1 obtained from high-throughput screening. We used flexible and induce-fit docking to develop a binding model of SW155246 with a crystallographic structure of human DNMT1. Results were in excellent agreement with experimental information providing a three-dimensional structural interpretation of 'activity cliffs', e.g., analogues of SW155246 with a high structural similarity to the sulfonamide compound, but with no activity in the enzymatic assay.

Variation in the loss of O6-methylguanine-DNA methyltransferase during immortalization of human fibroblasts.

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Green, M H; Karran, P; Lowe, J E; Priestley, A; Arlett, C F; Mayne, L

1990-01-01

We have examined O6-methylguanine-DNA methyltransferase (MT) activity in four human fibroblast cell lines during immortalization. Transfection of primary fibroblasts with the plasmid pSV3gpt or pSV3neo, which encode the SV40 large T antigen, confers a transformed phenotype but not immediate immortality. After a period of growth (pre-crisis) the cells enter a quiescent phase (crisis) from which an immortal clone of cells eventually grows out. From measurements of MT activity in extracts of cells taken at different defined stages of the immortalization process, we conclude that the establishment of a Mex- (MT-deficient) cell population is not specifically associated with cellular transformation or with any particular stage of immortalization. It appears that in different cell populations the change from Mex+ to Mex- may occur at different times during the immortalization process and that the change may be very abrupt.

Rationalization of Activity Cliffs of a Sulfonamide Inhibitor of DNA Methyltransferases with Induced-Fit Docking

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José L. Medina-Franco

2014-02-01

Full Text Available Inhibitors of human DNA methyltransferases (DNMT are of increasing interest to develop novel epi-drugs for the treatment of cancer and other diseases. As the number of compounds with reported DNMT inhibition is increasing, molecular docking is shedding light to elucidate their mechanism of action and further interpret structure–activity relationships. Herein, we present a structure-based rationalization of the activity of SW155246, a distinct sulfonamide compound recently reported as an inhibitor of human DNMT1 obtained from high-throughput screening. We used flexible and induce-fit docking to develop a binding model of SW155246 with a crystallographic structure of human DNMT1. Results were in excellent agreement with experimental information providing a three-dimensional structural interpretation of ‘activity cliffs’, e.g., analogues of SW155246 with a high structural similarity to the sulfonamide compound, but with no activity in the enzymatic assay.

N3 and O2 Protonated Conformers of the Cytosine Mononucleotides Coexist in the Gas Phase

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Wu, R. R.; Hamlow, L. A.; He, C. C.; Nei, Y.-w.; Berden, G.; Oomens, J.; Rodgers, M. T.

2017-08-01

The gas-phase conformations of the protonated forms of the DNA and RNA cytosine mononucleotides, [pdCyd+H]+ and [pCyd+H]+, are examined by infrared multiple photon dissociation (IRMPD) action spectroscopy over the IR fingerprint and hydrogen-stretching regions complemented by electronic structure calculations. The low-energy conformations of [pdCyd+H]+ and [pCyd+H]+ and their relative stabilities are computed at the B3LYP/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) and MP2(full)/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) levels of theory. Comparisons of the measured IRMPD action spectra and B3LYP/6-311+G(d,p) linear IR spectra computed for the low-energy conformers allow the conformers present in the experiments to be determined. Similar to that found in previous IRMPD action spectroscopy studies of the protonated forms of the cytosine nucleosides, [dCyd+H]+ and [Cyd+H]+, both N3 and O2 protonated cytosine mononucleotides exhibiting an anti orientation of cytosine are found to coexist in the experimental population. The 2'-hydroxyl substituent does not significantly influence the most stable conformations of [pCyd+H]+ versus those of [pdCyd+H]+, as the IRMPD spectral profiles of [pdCyd+H]+ and [pCyd+H]+ are similar. However, the presence of the 2'-hydroxyl substituent does influence the relative intensities of the measured IRMPD bands. Comparisons to IRMPD spectroscopy studies of the deprotonated forms of the cytosine mononucleotides, [pdCyd-H]- and [pCyd-H]-, provide insight into the effects of protonation versus deprotonation on the conformational features of the nucleobase and sugar moieties. Likewise, comparisons to results of IRMPD spectroscopy studies of the protonated cytosine nucleosides provide insight into the influence of the phosphate moiety on structure. Comparison with previous ion mobility results shows the superiority of IRMPD spectroscopy for distinguishing various protonation sites.

Investigation of the effect of radiation of DNA methylation patterns

International Nuclear Information System (INIS)

Kalinich, J.F.

1986-01-01

Four lines of cultured mammalian cells were used in this project: V79A03, a Chinese hamster lung fibroblast; HeLa S-3, an epithelial cell line from a human cervix carcinoma; CHO K-1, an epithelial cell line from a Chinese hamster ovary; and C-1300 N1E-115, a mouse neuroblastoma line. The 5-methylcytosine levels in DNA following exposure to cobalt-60 gamma radiation were measured. Induction of metallothionein in V79A03 cells and acetylcholinesterase in C-1300 N1E-115 cells after irradiation was determined and the effect of radiation on cytoplasmic and nuclear levels of DNA methyltransferase was studied and nuclear demethylase activity assayed. This study showed gamma radiation resulted in a decrease of 5-methylcytosine levels in the DNA of cultured mammalian cells. This radiation-induced hypomethylation resulted in the induction of acetylcholinesterase in mouse neuroblastoma cells and metallothionein in Chinese hamster lung fibroblasts and was caused by a decrease in DNA methyltransferase activity in the nucleus after irradiation and not by the presence of DNA demethylase

Superimposed Code Theorectic Analysis of DNA Codes and DNA Computing

Science.gov (United States)

2010-03-01

that the hybridization that occurs between a DNA strand and its Watson - Crick complement can be used to perform mathematical computation. This research...ssDNA single stranded DNA WC Watson – Crick A Adenine C Cytosine G Guanine T Thymine ... Watson - Crick (WC) duplex, e.g., TCGCA TCGCA . Note that non-WC duplexes can form and such a formation is called a cross-hybridization. Cross

Evidence that steroid 5alpha-reductase isozyme genes are differentially methylated in human lymphocytes.

Science.gov (United States)

Rodríguez-Dorantes, M; Lizano-Soberón, M; Camacho-Arroyo, I; Calzada-León, R; Morimoto, S; Téllez-Ascencio, N; Cerbón, M A

2002-03-01

The synthesis of dihydrotestosterone (DHT) is catalyzed by steroid 5alpha-reductase isozymes 1 and 2, and this function determines the development of the male phenotype during embriogenesis and the growth of androgen sensitive tissues during puberty. The aim of this study was to determine the cytosine methylation status of 5alpha-reductase isozymes types 1 and 2 genes in normal and in 5alpha-reductase deficient men. Genomic DNA was obtained from lymphocytes of both normal subjects and patients with primary 5alpha-reductase deficiency due to point mutations in 5alpha-reductase 2 gene. Southern blot analysis of 5alpha-reductase types 1 and 2 genes from DNA samples digested with HpaII presented a different cytosine methylation pattern compared to that observed with its isoschizomer MspI, indicating that both genes are methylated in CCGG sequences. The analysis of 5alpha-reductase 1 gene from DNA samples digested with Sau3AI and its isoschizomer MboI which recognize methylation in GATC sequences showed an identical methylation pattern. In contrast, 5alpha-reductase 2 gene digested with Sau3AI presented a different methylation pattern to that of the samples digested with MboI, indicating that steroid 5alpha-reductase 2 gene possess methylated cytosines in GATC sequences. Analysis of exon 4 of 5alpha-reductase 2 gene after metabisulfite PCR showed that normal and deficient subjects present a different methylation pattern, being more methylated in patients with 5alpha-reductase 2 mutated gene. The overall results suggest that 5alpha-reductase genes 1 and 2 are differentially methylated in lymphocytes from normal and 5alpha-reductase deficient patients. Moreover, the extensive cytosine methylation pattern observed in exon 4 of 5alpha-reductase 2 gene in deficient patients, points out to an increased rate of mutations in this gene.

Epigenetic contribution to successful polyploidizations: variation in global cytosine methylation along an extensive ploidy series in Dianthus broteri (Caryophyllaceae).

Science.gov (United States)

Alonso, Conchita; Balao, Francisco; Bazaga, Pilar; Pérez, Ricardo

2016-11-01

Polyploidization is a significant evolutionary force in plants which involves major genomic and genetic changes, frequently regulated by epigenetic factors. We explored whether natural polyploidization in Dianthus broteri complex resulted in substantial changes in global DNA cytosine methylation associated to ploidy. Global cytosine methylation was estimated by high-performance liquid chromatography (HPLC) in 12 monocytotypic populations with different ploidies (2×, 4×, 6×, 12×) broadly distributed within D. broteri distribution range. The effects of ploidy level and local variation on methylation were assessed by generalized linear mixed models (GLMMs). Dianthus broteri exhibited a higher methylation percent (˜33%) than expected by its monoploid genome size and a large variation among study populations (range: 29.3-35.3%). Global methylation tended to increase with ploidy but did not significantly differ across levels due to increased variation within the highest-order polyploidy categories. Methylation varied more among hexaploid and dodecaploid populations, despite such cytotypes showing more restricted geographic location and increased genetic relatedness than diploids and tetraploids. In this study, we demonstrate the usefulness of an HPLC method in providing precise and genome reference-free global measure of DNA cytosine methylation, suitable to advance current knowledge of the roles of this epigenetic mechanism in polyploidization processes. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Protein arginine methyltransferase 5 regulates multiple signaling pathways to promote lung cancer cell proliferation

International Nuclear Information System (INIS)

Sheng, Xiumei; Wang, Zhengxin

2016-01-01

Protein arginine methyltransferase 5 (PRMT5) catalyzes the formation of symmetrical dimethylation of arginine residues in proteins. WD repeat domain 77 (WDR77), also known as p44, MEP50, or WD45, forms a stoichiometric complex with PRMT5. The PRMT5/p44 complex is required for cellular proliferation of lung and prostate epithelial cells during earlier stages of development and is re-activated during prostate and lung tumorigenesis. The molecular mechanisms by which PRMT5 and p44 promote cellular proliferation are unknown. Expression of PRMT5 and p44 in lung and prostate cancer cells was silenced and their target genes were identified. The regulation of target genes was validated in various cancer cells during lung development and tumorigenesis. Altered expression of target genes was achieved by ectopic cDNA expression and shRNA-mediated silencing. PRMT5 and p44 regulate expression of a specific set of genes encoding growth and anti-growth factors, including receptor tyrosine kinases and antiproliferative proteins. Genes whose expression was suppressed by PRMT5 and p44 encoded anti-growth factors and inhibited cell growth when ectopically expressed. In contrast, genes whose expression was enhanced by PRMT5 and p44 encoded growth factors and increased cell growth when expressed. Altered expression of target genes is associated with re-activation of PRMT5 and p44 during lung tumorigenesis. Our data provide the molecular basis by which PRMT5 and p44 regulate cell growth and lay a foundation for further investigation of their role in lung tumor initiation. The online version of this article (doi:10.1186/s12885-016-2632-3) contains supplementary material, which is available to authorized users

Radiation effects on DNA methylation in mice

International Nuclear Information System (INIS)

Komura, J.; Kurishita, A.; Miyamura, Y.; Ono, T.; Tawa, R.; Sakurai, H.

1992-01-01

Effects of ionizing radiation on DNA methylation in liver, brain and spleen were examined by high performance liquid chromatography (HPLC). The total methylated cytosine level in the genome was reduced within 8 hours after 3.8 Gy of irradiation in liver of adult mice. But no appreciable effect was observed in brain and spleen. When mice were irradiated at newborn, liver DNA revealed no change in methylated cytosine level. Even though slight effects of radiation were detected in he methylation of the c-myc and c-fos genes, they were only temporary and no long-term effects were observed. These data suggest that the effect of radiation on DNA methylation in vivo is not prevailing a DNA damage, but rather influenced much through biological parameters. (author)

DNA methyltransferase 3b is dispensable for mouse neural crest development.

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Bridget T Jacques-Fricke

Full Text Available The neural crest is a population of multipotent cells that migrates extensively throughout vertebrate embryos to form diverse structures. Mice mutant for the de novo DNA methyltransferase DNMT3b exhibit defects in two neural crest derivatives, the craniofacial skeleton and cardiac ventricular septum, suggesting that DNMT3b activity is necessary for neural crest development. Nevertheless, the requirement for DNMT3b specifically in neural crest cells, as opposed to interacting cell types, has not been determined. Using a conditional DNMT3b allele crossed to the neural crest cre drivers Wnt1-cre and Sox10-cre, neural crest DNMT3b mutants were generated. In both neural crest-specific and fully DNMT3b-mutant embryos, cranial neural crest cells exhibited only subtle migration defects, with increased numbers of dispersed cells trailing organized streams in the head. In spite of this, the resulting cranial ganglia, craniofacial skeleton, and heart developed normally when neural crest cells lacked DNMT3b. This indicates that DNTM3b is not necessary in cranial neural crest cells for their development. We conclude that defects in neural crest derivatives in DNMT3b mutant mice reflect a requirement for DNMT3b in lineages such as the branchial arch mesendoderm or the cardiac mesoderm that interact with neural crest cells during formation of these structures.

Detecting single-abasic residues within a DNA strand immobilized in a biological nanopore using an integrated CMOS sensor.

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Kim, Jungsuk; Maitra, Raj D; Pedrotti, Ken; Dunbar, William B

2013-02-01

In this paper, we demonstrate the application of a novel current-measuring sensor (CMS) customized for nanopore applications. The low-noise CMS is fabricated in a 0.35μm CMOS process and is implemented in experiments involving DNA captured in an α-hemolysin (α-HL) nanopore. Specifically, the CMS is used to build a current amplitude map as a function of varying positions of a single-abasic residue within a homopolymer cytosine single-stranded DNA (ssDNA) that is captured and held in the pore. Each ssDNA is immobilized using a biotin-streptavidin linkage. Five different DNA templates are measured and compared: one all-cytosine ssDNA, and four with a single-abasic residue substitution that resides in or near the ~1.5nm aperture of the α-HL channel when the strand is immobilized. The CMOS CMS is shown to resolves the ~5Å displacements of the abasic residue within the varying templates. The demonstration represents an advance in application-specific circuitry that is optimized for small-footprint nanopore applications, including genomic sequencing.

Ligation bias in Illumina next-generation DNA libraries

DEFF Research Database (Denmark)

Seguin-Orlando, Andaine; Schubert, Mikkel; Clary, Joel

2013-01-01

Ancient DNA extracts consist of a mixture of endogenous molecules and contaminant DNA templates, often originating from environmental microbes. These two populations of templates exhibit different chemical characteristics, with the former showing depurination and cytosine deamination by-products,...... for authenticating ancient sequence data. Consequently, we show that models adequate for estimating post-mortem DNA damage levels must be robust to the molecular tools used for building ancient DNA libraries.......Ancient DNA extracts consist of a mixture of endogenous molecules and contaminant DNA templates, often originating from environmental microbes. These two populations of templates exhibit different chemical characteristics, with the former showing depurination and cytosine deamination by......-products, resulting from post-mortem DNA damage. Such chemical modifications can interfere with the molecular tools used for building second-generation DNA libraries, and limit our ability to fully characterize the true complexity of ancient DNA extracts. In this study, we first use fresh DNA extracts to demonstrate...

Aberrant regulation of DNA methylation in amyotrophic lateral sclerosis: a new target of disease mechanisms.

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Martin, Lee J; Wong, Margaret

2013-10-01

Amyotrophic lateral sclerosis (ALS) is the third most common adult-onset neurodegenerative disease. A diagnosis is fatal owing to degeneration of motor neurons in brain and spinal cord that control swallowing, breathing, and movement. ALS can be inherited, but most cases are not associated with a family history of the disease. The mechanisms causing motor neuron death in ALS are still unknown. Given the suspected complex interplay between multiple genes, the environment, metabolism, and lifestyle in the pathogenesis of ALS, we have hypothesized that the mechanisms of disease in ALS involve epigenetic contributions that can drive motor neuron degeneration. DNA methylation is an epigenetic mechanism for gene regulation engaged by DNA methyltransferase (Dnmt)-catalyzed methyl group transfer to carbon-5 in cytosine residues in gene regulatory promoter and nonpromoter regions. Recent genome-wide analyses have found differential gene methylation in human ALS. Neuropathologic assessments have revealed that motor neurons in human ALS show significant abnormalities in Dnmt1, Dnmt3a, and 5-methylcytosine. Similar changes are seen in mice with motor neuron degeneration, and Dnmt3a was found abundantly at synapses and in mitochondria. During apoptosis of cultured motor neuron-like cells, Dnmt1 and Dnmt3a protein levels increase, and 5-methylcytosine accumulates. Enforced expression of Dnmt3a, but not Dnmt1, induces degeneration of cultured neurons. Truncation mutation of the Dnmt3a catalytic domain and Dnmt3a RNAi blocks apoptosis of cultured neurons. Inhibition of Dnmt catalytic activity with small molecules RG108 and procainamide protects motor neurons from excessive DNA methylation and apoptosis in cell culture and in a mouse model of ALS. Thus, motor neurons can engage epigenetic mechanisms to cause their degeneration, involving Dnmts and increased DNA methylation. Aberrant DNA methylation in vulnerable cells is a new direction for discovering mechanisms of ALS

Methyl transfer in glucosinolate biosynthesis mediated by indole glucosinolate O-Methyltransferase 5

DEFF Research Database (Denmark)

Pfalz, Marina; Mukhaimar, Maisara; Perreau, François

2016-01-01

in position 1 (1-IG modification) or 4 (4-IG modification). Products of the 4-IG modification pathway mediate plant-enemy interactions and are particularly important for Arabidopsis innate immunity. While CYP81Fs encoding cytochrome P450 monooxygenases and IGMTs encoding indole glucosinolate O...... with moderate similarity to previously characterized IGMTs, encodes the methyltransferase that is responsible for the conversion of 1OHI3M to 1MOI3M. Disruption of IGMT5 function increases resistance against the root-knot nematode Meloidogyne javanica and suggests a potential role for the 1-IG modification...

Targeted inhibition of osteosarcoma tumor growth by bone marrow-derived mesenchymal stem cells expressing cytosine deaminase/5-fluorocytosine in tumor-bearing mice.

Science.gov (United States)

NguyenThai, Quynh-Anh; Sharma, Neelesh; Luong, Do Huynh; Sodhi, Simrinder Singh; Kim, Jeong-Hyun; Kim, Nameun; Oh, Sung-Jong; Jeong, Dong Kee

2015-01-01

Mesenchymal stem cells (MSCs) are considered as an attractive approach for gene or drug delivery in cancer therapy. In the present study, the ability of human bone marrow-derived MSCs expressing the cytosine deaminase/5-fluorocytosine prodrug (CD/5-FC MSCs) to target the human osteosarcoma cell line Cal72 was evaluated. The stable CD/5-FC MSC cell line was established by transfection of pEGFP containing the cytosine deaminase gene into MSCs with G418 selection. The anti-tumor effect was verified by a bystander effect assay in vitro and co-injection of Cal72 and CD/5-FC MSCs in cancer-bearing mice. The therapeutic CD/5-FC MSCs retained the characteristics of multipotent cells, such as differentiation into adipocytes/osteocytes and expression of mesenchymal markers (CD90 and CD44), and showed migration toward Cal72 cells to a greater extent than the native MSCs. The bystander effect assay showed that the CD/5-FC MSCs significantly augmented Cal72 cytotoxicity in direct co-culture and in the presence of 5-FC through the application of conditioned medium. In osteosarcoma-bearing mice, the CD/5-FC MSCs inhibited tumor growth compared to control mice subcutaneously injected with only Cal72 cells. Taken together, these findings suggest that CD/5-FC MSCs may be suitable for targeting human osteosarcoma. Copyright © 2015 John Wiley & Sons, Ltd.

DNA methyltransferases contribute to the fungal development, stress tolerance and virulence of the entomopathogenic fungus Metarhizium robertsii.

Science.gov (United States)

Wang, Yulong; Wang, Tiantian; Qiao, Lintao; Zhu, Jianyu; Fan, Jinrui; Zhang, Tingting; Wang, Zhang-Xun; Li, Wanzhen; Chen, Anhui; Huang, Bo

2017-05-01

DNA methylation is an important epigenetic mark in mammals, plants, and fungi and depends on multiple genetic pathways involving de novo and maintenance DNA methyltransferases (DNMTases). Metarhizium robertsii, a model system for investigating insect-fungus interactions, has been used as an environmentally friendly alternative to chemical insecticides. However, little is known concerning the molecular basis for DNA methylation. Here, we report on the roles of two DNMTases (MrRID and MrDIM-2) by characterizing ΔMrRID, ΔMrDIM-2, and ΔRID/ΔDIM-2 mutants. The results showed that approximately 71, 10, and 8% of m C sites remained in the ΔMrRID, ΔMrDIM-2, and ΔRID/ΔDIM-2 strains, respectively, compared with the wild-type (WT) strain. Further analysis showed that MrRID regulates the specificity of DNA methylation and MrDIM-2 is responsible for most DNA methylation, implying an interaction or cooperation between MrRID and MrDIM-2 for DNA methylation. Moreover, the ΔMrDIM-2 and ΔRID/ΔDIM-2 strains showed more defects in radial growth and conidial production compared to the WT. Under ultraviolet (UV) irradiation or heat stress, an obvious reduction in spore viability was observed for all the mutant strains compared to the WT. The spore median lethal times (LT 50 s) for the ΔMrDIM-2 and ΔRID/ΔDIM-2 strains in the greater wax moth, Galleria mellonella, were decreased by 47.7 and 65.9%, respectively, which showed that MrDIM-2 is required for full fungal virulence. Our data advances the understanding of the function of DNMTase in entomopathogenic fungi, which should contribute to future epigenetic investigations in fungi.

The Aorta-Gonad-Mesonephros Organ Culture Recapitulates 5hmC Reorganization and Replication-Dependent and Independent Loss of DNA Methylation in the Germline.

Science.gov (United States)

Calvopina, Joseph Hargan; Cook, Helene; Vincent, John J; Nee, Kevin; Clark, Amander T

2015-07-01

Removal of cytosine methylation from the genome is critical for reprogramming and transdifferentiation and plays a central role in our understanding of the fundamental principles of embryo lineage development. One of the major models for studying cytosine demethylation is the mammalian germ line during the primordial germ cell (PGC) stage of embryo development. It is now understood that oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) is required to remove cytosine methylation in a locus-specific manner in PGCs; however, the mechanisms downstream of 5hmC are controversial and hypothesized to involve either active demethylation or replication-coupled loss. In the current study, we used the aorta-gonad-mesonephros (AGM) organ culture model to show that this model recapitulates germ line reprogramming, including 5hmC reorganization and loss of cytosine methylation from Snrpn and H19 imprinting control centers (ICCs). To directly address the hypothesis that cell proliferation is required for cytosine demethylation, we blocked PI3-kinase-dependent PGC proliferation and show that this leads to a G1 and G2/M cell cycle arrest in PGCs, together with retained levels of cytosine methylation at the Snrpn ICC, but not at the H19 ICC. Taken together, the AGM organ culture model is an important tool to evaluate mechanisms of locus-specific demethylation and the role of PI3-kinase-dependent PGC proliferation in the locus-specific removal of cytosine methylation from the genome.

Interaction of Cu+ with cytosine and formation of i-motif-like C-M+-C complexes: alkali versus coinage metals

NARCIS (Netherlands)

Gao, J.; Berden, G.; Rodgers, M.T.; Oomens, J.

2016-01-01

The Watson-Crick structure of DNA is among the most well-known molecular structures of our time. However, alternative base-pairing motifs are also known to occur, often depending on base sequence, pH, or the presence of cations. Pairing of cytosine (C) bases induced by the sharing of a single proton

DNA binding of the p21 repressor ZBTB2 is inhibited by cytosine hydroxymethylation

Energy Technology Data Exchange (ETDEWEB)

Lafaye, Céline; Barbier, Ewa; Miscioscia, Audrey; Saint-Pierre, Christine [Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E_3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France); Kraut, Alexandra; Couté, Yohann [Etude de la Dynamique des Protéomes, Biologie à Grande Echelle, UMR S_1038 CEA/INSERM/UJF-Grenoble 1, iRTSV, 17 rue des Martyrs, Grenoble F-38054 (France); Plo, Isabelle [INSERM, U1009, Institut Gustave Roussy, Université Paris 11, 114 rue Edouard Vaillant, Villejuif F-94805 (France); Gasparutto, Didier; Ravanat, Jean-Luc [Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E_3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France); Breton, Jean, E-mail: jean.breton@cea.fr [Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E_3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France)

2014-03-28

Highlights: • 5-hmC epigenetic modification is measurable in HeLa, SH-SY5Y and UT7-MPL cell lines. • ZBTB2 binds to DNA probes containing 5-mC but not to sequences containing 5-hmC. • This differential binding is verified with DNA sequences involved in p21 regulation. - Abstract: Recent studies have demonstrated that the modified base 5-hydroxymethylcytosine (5-hmC) is detectable at various rates in DNA extracted from human tissues. This oxidative product of 5-methylcytosine (5-mC) constitutes a new and important actor of epigenetic mechanisms. We designed a DNA pull down assay to trap and identify nuclear proteins bound to 5-hmC and/or 5-mC. We applied this strategy to three cancerous cell lines (HeLa, SH-SY5Y and UT7-MPL) in which we also measured 5-mC and 5-hmC levels by HPLC-MS/MS. We found that the putative oncoprotein Zinc finger and BTB domain-containing protein 2 (ZBTB2) is associated with methylated DNA sequences and that this interaction is inhibited by the presence of 5-hmC replacing 5-mC. As published data mention ZBTB2 recognition of p21 regulating sequences, we verified that this sequence specific binding was also alleviated by 5-hmC. ZBTB2 being considered as a multifunctional cell proliferation activator, notably through p21 repression, this work points out new epigenetic processes potentially involved in carcinogenesis.

DNA binding of the p21 repressor ZBTB2 is inhibited by cytosine hydroxymethylation

International Nuclear Information System (INIS)

3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" data-affiliation=" (Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" >Lafaye, Céline; 3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" data-affiliation=" (Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" >Barbier, Ewa; 3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" data-affiliation=" (Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" >Miscioscia, Audrey; 3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" data-affiliation=" (Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" >Saint-Pierre, Christine; 1038 CEA/INSERM/UJF-Grenoble 1, iRTSV, 17 rue des Martyrs, Grenoble F-38054 (France))" data-affiliation=" (Etude de la Dynamique des Protéomes, Biologie à Grande Echelle, UMR S1038 CEA/INSERM/UJF-Grenoble 1, iRTSV, 17 rue des Martyrs, Grenoble F-38054 (France))" >Kraut, Alexandra; 1038 CEA/INSERM/UJF-Grenoble 1, iRTSV, 17 rue des Martyrs, Grenoble F-38054 (France))" data-affiliation=" (Etude de la Dynamique des Protéomes, Biologie à Grande Echelle, UMR S1038 CEA/INSERM/UJF-Grenoble 1, iRTSV, 17 rue des Martyrs, Grenoble F-38054 (France))" >Couté, Yohann; Plo, Isabelle; 3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" data-affiliation=" (Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" >Gasparutto, Didier; 3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" data-affiliation=" (Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" >Ravanat, Jean-Luc; 3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" data-affiliation=" (Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" >Breton, Jean

2014-01-01

Highlights: • 5-hmC epigenetic modification is measurable in HeLa, SH-SY5Y and UT7-MPL cell lines. • ZBTB2 binds to DNA probes containing 5-mC but not to sequences containing 5-hmC. • This differential binding is verified with DNA sequences involved in p21 regulation. - Abstract: Recent studies have demonstrated that the modified base 5-hydroxymethylcytosine (5-hmC) is detectable at various rates in DNA extracted from human tissues. This oxidative product of 5-methylcytosine (5-mC) constitutes a new and important actor of epigenetic mechanisms. We designed a DNA pull down assay to trap and identify nuclear proteins bound to 5-hmC and/or 5-mC. We applied this strategy to three cancerous cell lines (HeLa, SH-SY5Y and UT7-MPL) in which we also measured 5-mC and 5-hmC levels by HPLC-MS/MS. We found that the putative oncoprotein Zinc finger and BTB domain-containing protein 2 (ZBTB2) is associated with methylated DNA sequences and that this interaction is inhibited by the presence of 5-hmC replacing 5-mC. As published data mention ZBTB2 recognition of p21 regulating sequences, we verified that this sequence specific binding was also alleviated by 5-hmC. ZBTB2 being considered as a multifunctional cell proliferation activator, notably through p21 repression, this work points out new epigenetic processes potentially involved in carcinogenesis

Minimal traumatic brain injury causes persistent changes in DNA methylation at BDNF gene promoters in rat amygdala: A possible role in anxiety-like behaviors.

Science.gov (United States)

Sagarkar, Sneha; Bhamburkar, Tanmayi; Shelkar, Gajanan; Choudhary, Amit; Kokare, Dadasaheb M; Sakharkar, Amul J

2017-10-01

Minimal traumatic brain injury (MTBI) often transforms into chronic neuropsychiatric conditions including anxiety, the underlying mechanisms of which are largely unknown. In the present study, we employed the closed-head injury paradigm to induce MTBI in rats and examined whether DNA methylation can explain long-term changes in the expression of the brain-derived neurotrophic factor (BDNF) in the amygdala as well as trauma-induced anxiety-like behaviors. The MTBI caused anxiety-like behaviors and altered the expression of DNA methyltransferase (DNMT) isoforms (DNMT1, DNMT3a, and DNMT3b) and factors involved in DNA demethylation such as the growth arrest and DNA damage 45 (GADD45a and GADD45b). After 30days of MTBI, the over-expression of DNMT3a and DNMT3b corresponded to heightened DNMT activity, whereas the mRNA levels of GADD45a and GADD45b were declined. The methylated cytosine levels at the BDNF promoters (Ip, IVp and IXp) were increased in the amygdala of the trauma-induced animals; these coincided negatively with the mRNA levels of exon IV and IXa, but not of exon I. Interestingly, treatment with 5-azacytidine, a pan DNMT inhibitor, normalized the MTBI-induced DNMT activity and DNA hypermethylation at exon IVp and IXp. Furthermore, 5-azacytidine also corrected the deficits in the expression of exons IV and IXa and reduced the anxiety-like behaviors. These results suggest that the DNMT-mediated DNA methylation at the BDNF IVp and IXp might be involved in the regulation of BDNF gene expression in the amygdala. Further, it could also be related to MTBI-induced anxiety-like behaviors via the regulation of synaptic plasticity. Copyright © 2017 Elsevier Inc. All rights reserved.

miRNAting control of DNA methylation

Indian Academy of Sciences (India)

DNA methylation is a type of epigenetic modification where a methyl group is added to the cytosine or adenine residue of a given DNA sequence. It has been observed that DNA methylation is achieved by some collaborative agglomeration of certain proteins and non-coding RNAs. The assembly of IDN2 and its ...

Three-Dimensional Structure and Catalytic Mechanism of Cytosine Deaminase

Energy Technology Data Exchange (ETDEWEB)

R Hall; A Fedorov; C Xu; E Fedorov; S Almo; F Raushel

2011-12-31

Cytosine deaminase (CDA) from E. coli is a member of the amidohydrolase superfamily. The structure of the zinc-activated enzyme was determined in the presence of phosphonocytosine, a mimic of the tetrahedral reaction intermediate. This compound inhibits the deamination of cytosine with a K{sub i} of 52 nM. The zinc- and iron-containing enzymes were characterized to determine the effect of the divalent cations on activation of the hydrolytic water. Fe-CDA loses activity at low pH with a kinetic pKa of 6.0, and Zn-CDA has a kinetic pKa of 7.3. Mutation of Gln-156 decreased the catalytic activity by more than 5 orders of magnitude, supporting its role in substrate binding. Mutation of Glu-217, Asp-313, and His-246 significantly decreased catalytic activity supporting the role of these three residues in activation of the hydrolytic water molecule and facilitation of proton transfer reactions. A library of potential substrates was used to probe the structural determinants responsible for catalytic activity. CDA was able to catalyze the deamination of isocytosine and the hydrolysis of 3-oxauracil. Large inverse solvent isotope effects were obtained on k{sub cat} and k{sub cat}/K{sub m}, consistent with the formation of a low-barrier hydrogen bond during the conversion of cytosine to uracil. A chemical mechanism for substrate deamination by CDA was proposed.

[Novel Approaches in DNA Methylation Studies - MS-HRM Analysis and Electrochemistry].

Science.gov (United States)

Bartošík, M; Ondroušková, E

Cytosine methylation in DNA is an epigenetic mechanism regulating gene expression and plays a vital role in cell differentiation or proliferation. Tumor cells often exhibit aberrant DNA methylation, e.g. hypermethylation of tumor suppressor gene promoters. New methods, capable of determining methylation status of specific DNA sequences, are thus being developed. Among them, MS-HRM (methylation-specific high resolution melting) and electrochemistry offer relatively inexpensive instrumentation, fast assay times and possibility of screening multiple samples/DNA regions simultaneously. MS-HRM is due to its sensitivity and simplicity an interesting alternative to already established techniques, including methylation-specific PCR or bisulfite sequencing. Electrochemistry, when combined with suitable electroactive labels and electrode surfaces, has been applied in several unique strategies for discrimination of cytosines and methylcytosines. Both techniques were successfully tested in analysis of DNA methylation within promoters of important tumor suppressor genes and could thus help in achieving more precise diagnostics and prognostics of cancer. Aberrant methylation of promoters has already been described in hundreds of genes associated with tumorigenesis and could serve as important biomarker if new methods applicable into clinical practice are sufficiently advanced.Key words: DNA methylation - 5-methylcytosine - HRM analysis - melting temperature - DNA duplex - electrochemistry - nucleic acid hybridizationThis work was supported by MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 6. 5. 2016Accepted: 16. 5. 2016.

Hydrogen bond disruption in DNA base pairs from (14)C transmutation.

Science.gov (United States)

Sassi, Michel; Carter, Damien J; Uberuaga, Blas P; Stanek, Christopher R; Mancera, Ricardo L; Marks, Nigel A

2014-09-04

Recent ab initio molecular dynamics simulations have shown that radioactive carbon does not normally fragment DNA bases when it decays. Motivated by this finding, density functional theory and Bader analysis have been used to quantify the effect of C → N transmutation on hydrogen bonding in DNA base pairs. We find that (14)C decay has the potential to significantly alter hydrogen bonds in a variety of ways including direct proton shuttling (thymine and cytosine), thermally activated proton shuttling (guanine), and hydrogen bond breaking (cytosine). Transmutation substantially modifies both the absolute and relative strengths of the hydrogen bonding pattern, and in two instances (adenine and cytosine), the density at the critical point indicates development of mild covalent character. Since hydrogen bonding is an important component of Watson-Crick pairing, these (14)C-induced modifications, while infrequent, may trigger errors in DNA transcription and replication.

Genome-wide nucleosome map and cytosine methylation levels of an ancient human genome

DEFF Research Database (Denmark)

Pedersen, Jakob Skou; Valen, Eivind; Velazquez, Amhed Missael Vargas

2014-01-01

Epigenetic information is available from contemporary organisms, but is difficult to track back in evolutionary time. Here, we show that genome-wide epigenetic information can be gathered directly from next-generation sequence reads of DNA isolated from ancient remains. Using the genome sequence...... data generated from hair shafts of a 4000-yr-old Paleo-Eskimo belonging to the Saqqaq culture, we generate the first ancient nucleosome map coupled with a genome-wide survey of cytosine methylation levels. The validity of both nucleosome map and methylation levels were confirmed by the recovery...

Binding of the antitumor drug nogalamycin and its derivatives to DNA: Structural comparison

International Nuclear Information System (INIS)

Gao, Yi-Gui; Liaw, Yen-Chywan; Robinson, H.; Wang, A. H.-J.

1990-01-01

The three-dimensional molecular structures of the complexes between a novel antitumor drug nogalamycin and its derivative U-58872 with a modified DNA hexamer d[m 5 CGT(pS)Am 5 CG] have been determined at 1.7- and 1.8-angstrom resolution, respectively, by X-ray diffraction analyses. Both structures (in space group P6 1 ) have been refined with constrained refinement procedure to final R factors of 0.208 (3386 reflections) and 0.196 (2143 reflections). In both complexes, two nogalamycins bind to the DNA hexamer double helix in a 2:1 ratio with the elongated aglycon chromophore intercalated between the CpG steps at both ends of the helix. The aglycon chromophore spans across the GC Watson-Crick base pairs with its nogalose lying in the minor groove and the aminoglucose lying in the major groove of the distorted B-DNA double helix. Most of the sugars remain in the C2'-endo pucker family, except three deoxycytidine residues (terminal C1, C7, and internal C5). All nucleotides are in the anti conformation. Specific hydrogen bonds are found in the complex between the drug and guanine-cytosine bases in both grooves of the helix. One hydroxyl group of the aminoglucose donates a hydrogen bond to the N7 of guanine, while the other receives a hydrogen bond from the N4 amino group of cytosine. The orientation of these two hydrogen bonds suggests that nogalamycin prefers a GC base pair with its aglycon chromophore intercalating at the 5'-side of a guanine (between NpG), or at the 3'-side of a cytosine (between CpN) with the sugars pointing toward the GC base pair. The binding of nogalamycin to DNA requires that the base pairs in DNA open up transiently to allow the bulky sugars to go through, suggesting that nogalamycin prefers GC sequences embedded in a stretch of AT sequences

Methylator phenotype of malignant germ cell tumours in children identifies strong candidates for chemotherapy resistance.

Science.gov (United States)

Jeyapalan, J N; Noor, D A Mohamed; Lee, S-H; Tan, C L; Appleby, V A; Kilday, J P; Palmer, R D; Schwalbe, E C; Clifford, S C; Walker, D A; Murray, M J; Coleman, N; Nicholson, J C; Scotting, P J

2011-08-09

Yolk sac tumours (YSTs) and germinomas are the two major pure histological subtypes of germ cell tumours. To date, the role of DNA methylation in the aetiology of this class of tumour has only been analysed in adult testicular forms and with respect to only a few genes. A bank of paediatric tumours was analysed for global methylation of LINE-1 repeat elements and global methylation of regulatory elements using GoldenGate methylation arrays. Both germinomas and YSTs exhibited significant global hypomethylation of LINE-1 elements. However, in germinomas, methylation of gene regulatory regions differed little from control samples, whereas YSTs exhibited increased methylation at a large proportion of the loci tested, showing a 'methylator' phenotype, including silencing of genes associated with Caspase-8-dependent apoptosis. Furthermore, we found that the methylator phenotype of YSTs was coincident with higher levels of expression of the DNA methyltransferase, DNA (cytosine-5)-methyltransferase 3B, suggesting a mechanism underlying the phenotype. Epigenetic silencing of a large number of potential tumour suppressor genes in YSTs might explain why they exhibit a more aggressive natural history than germinomas and silencing of genes associated with Caspase-8-dependent cell death might explain the relative resistance of YSTs to conventional therapy.

Catalytic mechanism and inhibition of tRNA (Uracil-5-)methyltransferase: evidence for covalent catalysis

International Nuclear Information System (INIS)

Santi, D.V.; Hardy, L.W.

1987-01-01

tRNA (Ura-5-) methyltransferase catalyzes the transfer of a methyl group from S-adenosylmethionine (AdoMet) to the 5-carbon of a specific Urd residue in tRNA. This results in stoichiometric release of tritium from [5- 3 H] Urd-labeled substrate tRNA isolated from methyltransferase-deficient Escherichia coli. The enzyme also catalyzes an AdoMet-independent exchange reaction between [5- 3 H]-Urd-labeled substrate tRNA and protons of water at a rate that is about 1% that of the normal methylation reaction, but with identical stoichiometry. S-Adenosylhomocysteine inhibits the rate of the exchange reaction by 2-3-fold, whereas an analog having the sulfur of AdoMet replaced by nitrogen accelerates the exchange reaction 9-fold. In the presence (but not absence) of AdoMet, 5-fluorouracil-substituted tRNA (FUra-tRNA) leads to the first-order inactivation of the enzyme. This is accompanied by the formation of a stable covalent complex containing the enzyme, FUra-tRNA, and the methyl group AdoMet. A mechanism for catalysis is proposed that explains both the 5-H exchange reaction and the inhibition by FUra-tRNA: the enzyme forms a covalent Michael adduct with substrate or inhibitor tRNA by attack of a nucleophilic group of the enzyme at carbon 6 of the pyrimidine residue to be modified. As a result, an anion equivalent is generated at carbon 5 that is sufficiently reactive to be methylated by AdoMet. Preliminary experiments and precedents suggest that the nucleophilic catalyst of the enzyme is a thiol group of cysteine. The potent irreversible inhibition by FUra-tRNA suggest that a mechanism for the RNA effects of FUra may also involve irreversible inhibition of RNA-modifying enzymes

Surface study of gallium- and aluminum- doped graphenes upon adsorption of cytosine: DFT calculations

International Nuclear Information System (INIS)

Shokuhi Rad, Ali; Zareyee, Daryoush; Peyravi, Majid; Jahanshahi, Mohsen

2016-01-01

Highlights: • P1 and P4 are the most stable adsorption configurations for cytosine. • NBO analysis show n-type semiconductor property for both Al- and Ga-doped graphenes. • Important changes in the HOMO and LUMO of doped graphene upon adsorption of cytosine. • Increase in the conductivity of system when cytosine is adsorbed on doped graphenes. - Abstract: The adsorption of cytosine molecule on Al- and Ga- doped graphenes is studied using first-principles density functional theory (DFT) calculations. The energetically most stable geometries of cytosine on both Al- and Ga- doped graphenes are determined and the adsorption energies are calculated. The net charge of transfer as well as local charge of doped atoms upon adsorption of cytosine are studied by natural bond orbitals (NBO) analysis. Orbital hybridizing of complexes was searched by frontier molecular orbital theory (FMO), and density of states (DOS). Depending on the side of cytosine, there are four possible sites for its adsorption on doped graphene; denoted as P1, P2, P3, and P4, respectively. The order of binding energy in the case of Al-doped graphene is found as P1 > P4 > P3 > P2. Interestingly, the order in the case of Ga-doped graphene changes to: P4 ∼ P1 > P3 > P2. Both surfaces show superior adsorbent property, resulting chemisorption of cytosine, especially at P1 and P4 position configurations. The NBO charge analysis reveals that the charge transfers from Al- and Ga- doped graphene sheets to cytosine. The electronic properties of both surfaces undertake important changes after cytosine adsorption, which indicates notable change in its electrical conductivity.

Surface study of gallium- and aluminum- doped graphenes upon adsorption of cytosine: DFT calculations

Energy Technology Data Exchange (ETDEWEB)

Shokuhi Rad, Ali, E-mail: a.shokuhi@gmail.com [Department of Chemical Engineering, Qaemshahr Branch, Islamic Azad University, Qaemshahr (Iran, Islamic Republic of); Zareyee, Daryoush [Department of Chemistry, Qaemshahr Branch, Islamic Azad University, Qaemshahr (Iran, Islamic Republic of); Peyravi, Majid; Jahanshahi, Mohsen [Faculty of Chemical Engineering, Babol University of Technology, Babol (Iran, Islamic Republic of)

2016-12-30

Highlights: • P1 and P4 are the most stable adsorption configurations for cytosine. • NBO analysis show n-type semiconductor property for both Al- and Ga-doped graphenes. • Important changes in the HOMO and LUMO of doped graphene upon adsorption of cytosine. • Increase in the conductivity of system when cytosine is adsorbed on doped graphenes. - Abstract: The adsorption of cytosine molecule on Al- and Ga- doped graphenes is studied using first-principles density functional theory (DFT) calculations. The energetically most stable geometries of cytosine on both Al- and Ga- doped graphenes are determined and the adsorption energies are calculated. The net charge of transfer as well as local charge of doped atoms upon adsorption of cytosine are studied by natural bond orbitals (NBO) analysis. Orbital hybridizing of complexes was searched by frontier molecular orbital theory (FMO), and density of states (DOS). Depending on the side of cytosine, there are four possible sites for its adsorption on doped graphene; denoted as P1, P2, P3, and P4, respectively. The order of binding energy in the case of Al-doped graphene is found as P1 > P4 > P3 > P2. Interestingly, the order in the case of Ga-doped graphene changes to: P4 ∼ P1 > P3 > P2. Both surfaces show superior adsorbent property, resulting chemisorption of cytosine, especially at P1 and P4 position configurations. The NBO charge analysis reveals that the charge transfers from Al- and Ga- doped graphene sheets to cytosine. The electronic properties of both surfaces undertake important changes after cytosine adsorption, which indicates notable change in its electrical conductivity.

Zinc-fingers and homeoboxes 1 (ZHX1) binds DNA methyltransferase (DNMT) 3B to enhance DNMT3B-mediated transcriptional repression

International Nuclear Information System (INIS)

Kim, Sung-Hak; Park, Jinah; Choi, Moon-Chang; Kim, Hwang-Phill; Park, Jung-Hyun; Jung, Yeonjoo; Lee, Ju-Hee; Oh, Do-Youn; Im, Seock-Ah; Bang, Yung-Jue; Kim, Tae-You

2007-01-01

DNA methyltransferases (DNMT) 3B is a de novo DNMT that represses transcription independent of DNMT activity. In order to gain a better insight into DNMT3B-mediated transcriptional repression, we performed a yeast two-hybrid analysis using DNMT3B as a bait. Of the various binding candidates, ZHX1, a member of zinc-finger and homeobox protein, was found to interact with DNMT3B in vivo and in vitro. N-terminal PWWP domain of DNMT3B was required for its interaction with homeobox motifs of ZHX1. ZHX1 contains nuclear localization signal at C-terminal homeobox motif, and both ZHX1 and DNMT3B were co-localized in nucleus. Furthermore, we found that ZHX1 enhanced the transcriptional repression mediated by DNMT3B when DNMT3B is directly targeted to DNA. These results showed for First the direct linkage between DNMT and zinc-fingers homeoboxes protein, leading to enhanced gene silencing by DNMT3B

DNMT1 is associated with cell cycle and DNA replication gene sets in diffuse large B-cell lymphoma.

Science.gov (United States)

Loo, Suet Kee; Ab Hamid, Suzina Sheikh; Musa, Mustaffa; Wong, Kah Keng

2018-01-01

Dysregulation of DNA (cytosine-5)-methyltransferase 1 (DNMT1) is associated with the pathogenesis of various types of cancer. It has been previously shown that DNMT1 is frequently expressed in diffuse large B-cell lymphoma (DLBCL), however its functions remain to be elucidated in the disease. In this study, we gene expression profiled (GEP) shRNA targeting DNMT1(shDNMT1)-treated germinal center B-cell-like DLBCL (GCB-DLBCL)-derived cell line (i.e. HT) compared with non-silencing shRNA (control shRNA)-treated HT cells. Independent gene set enrichment analysis (GSEA) performed using GEPs of shRNA-treated HT cells and primary GCB-DLBCL cases derived from two publicly-available datasets (i.e. GSE10846 and GSE31312) produced three separate lists of enriched gene sets for each gene sets collection from Molecular Signatures Database (MSigDB). Subsequent Venn analysis identified 268, 145 and six consensus gene sets from analyzing gene sets in C2 collection (curated gene sets), C5 sub-collection [gene sets from gene ontology (GO) biological process ontology] and Hallmark collection, respectively to be enriched in positive correlation with DNMT1 expression profiles in shRNA-treated HT cells, GSE10846 and GSE31312 datasets [false discovery rate (FDR) 0.8) with DNMT1 expression and significantly downregulated (log fold-change <-1.35; p<0.05) following DNMT1 silencing in HT cells. These results suggest the involvement of DNMT1 in the activation of cell cycle and DNA replication in DLBCL cells. Copyright © 2017 Elsevier GmbH. All rights reserved.

Quantitation of O6-methylguanine-DNA methyltransferase gene messenger RNA in gliomas by means of real-time RT-PCR and clinical response to nitrosoureas.

Science.gov (United States)

Tanaka, Satoshi; Oka, Hidehiro; Fujii, Kiyotaka; Watanabe, Kaoru; Nagao, Kumi; Kakimoto, Atsushi

2005-09-01

1. O6-methylguanine-DNA methyltransferase (MGMT) mRNA was measured in 50 malignant gliomas that had received 1-(4-amino-2-methyl-5-pyrimidynyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) after the resection of the tumor by real-time reverse transcription-polymerase chain reaction (RT-PCR) using TaqMan probe. 2. The mean absolute value of MGMTmRNA normalized to the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for 50 tumors was 1.29 x 10(4)+/- 1.28 x 10(4) copy/microg RNA (mean +/- SD). The amount of MGMTmRNA less than 6 x 10(3) copy/microg RNA was the most significant factor in predicting the initial effect of treatment with ACNU by multi-variant regression analysis (p = 0.0157). 3. These results suggest that quantitation of MGMTmRNA is the excellent method for predicting for the effect of ACNU in glioma therapy.

Inhibition of Y-box binding protein-1 slows the growth of glioblastoma multiforme and sensitizes to temozolomide independent O6-methylguanine-DNA methyltransferase.

Science.gov (United States)

Gao, Yuanyuan; Fotovati, Abbas; Lee, Cathy; Wang, Michelle; Cote, Gilbert; Guns, Emma; Toyota, Brian; Faury, Damien; Jabado, Nada; Dunn, Sandra E

2009-12-01

Glioblastoma multiforme (GBM) is an aggressive type of brain tumor where 5 years. In adults, GBM is the most common type of brain tumor. It is rarer in children, where it constitutes approximately 15% of all brain tumors diagnosed. These tumors are often invasive, making surgical resection difficult. Further, they can be refractory to current therapies such as temozolomide. The current dogma is that temozolomide resistance rests on the expression of O6-methylguanine-DNA methyltransferase (MGMT) because it cleaves methylated DNA adducts formed by the drug. Our laboratory recently reported that another drug resistance gene known as the Y-box binding protein-1 (YB-1) is highly expressed in primary GBM but not in normal brain tissues based on the evaluation of primary tumors. We therefore questioned whether GBM depend on YB-1 for growth and/or response to temozolomide. Herein, we report that YB-1 inhibition reduced tumor cell invasion and growth in monolayer as well as in soft agar. Moreover, blocking this protein ultimately delayed tumor onset in mice. Importantly, inhibiting YB-1 enhanced temozolomide sensitivity in a manner that was independent of MGMT in models of adult and pediatric GBM. In conclusion, inhibiting YB-1 may be a novel way to improve the treatment of GBM.

Intrinsic resistance to aminoglycosides in Enterococcus faecium is conferred by the 16S rRNA m5C1404-specific methyltransferase EfmM

DEFF Research Database (Denmark)

Galimand, Marc; Schmitt, Emmanuelle; Panvert, Michel

2011-01-01

methyltransferase, as well as by the previously characterized aac(6')-Ii that encodes a 6'-N-aminoglycoside acetyltransferase. Inactivation of efmM in E. faecium increases susceptibility to the aminoglycosides kanamycin and tobramycin, and, conversely, expression of a recombinant version of efmM in Escherichia coli...... confers resistance to these drugs. The EfmM protein shows significant sequence similarity to E. coli RsmF (previously called YebU), which is a 5-methylcytidine (m(5)C) methyltransferase modifying 16S rRNA nucleotide C1407. The target for EfmM is shown by mass spectrometry to be a neighboring 16S r...

Exceptionally High Protection of Photocarcinogenesis by Topical Application of (--Epi gal locatechin-3-Gal late in Hydrophilic Cream in SKH-1 Hairless Mouse Model: Relationship to Inhibition of UVB-Induced Global DNA Hypomethylation

Directory of Open Access Journals (Sweden)

Anshu Mittal

2003-11-01

Full Text Available (--Epigallocatechin-3-gal late (EGCG has been shown to have potent antiphotocarcinogenic activity, but it was required to develop a cream-based formulation for topical application. For topical application, we tested hydrophilic cream as a vehicle for EGCG. Treatment with EGCG (≈ 1 mg/cm2 skin area in hydrophilic cream resulted in exceptionally high protection against photocarcinogenesis when determined in terms of tumor incidence, tumor multiplicity, and tumor size in a SKI-11-11 hairless mouse model. EGCG also inhibited malignant transformation of ultraviolet B (UVB-induced papillomas to carcinomas. In order to determine the mechanism of prevention of photocarcinogenesis, we determined the effect of EGCG on global DNA methylation pattern using monoclonal antibodies against 5-methyl cytosine and DNA methyltransferase in the long-term UV-irradiated skin because altered DNA methylation silencing is recognized as a molecular hallmark of human cancer. We found that treatment with EGCG resulted in significant inhibition of UVBinduced global DNA hypomethylation pattern. Longterm application of EGCG did not show any apparent sign of toxicity in mice when determined in terms of skin appearance, lean mass, total bone mineral content, and total bone mineral density but showed reduction in fat mass when analyzed using dual-energy X-ray absorptiometry. These data suggest that hydrophilic cream could be a suitable vehicle for topical application of EGCG, and that EGCG is a promising candidate for future cancer therapies based on its influence on the epigenetic pathway.

Structural Chemistry of Human RNA Methyltransferases.

Science.gov (United States)

Schapira, Matthieu

2016-03-18

RNA methyltransferases (RNMTs) play important roles in RNA stability, splicing, and epigenetic mechanisms. They constitute a promising target class that is underexplored by the medicinal chemistry community. Information of relevance to drug design can be extracted from the rich structural coverage of human RNMTs. In this work, the structural chemistry of this protein family is analyzed in depth. Unlike most methyltransferases, RNMTs generally feature a substrate-binding site that is largely open on the cofactor-binding pocket, favoring the design of bisubstrate inhibitors. Substrate purine or pyrimidines are often sandwiched between hydrophobic walls that can accommodate planar ring systems. When the substrate base is laying on a shallow surface, a 5' flanking base is sometimes anchored in a druggable cavity. The cofactor-binding site is structurally more diverse than in protein methyltransferases and more druggable in SPOUT than in Rossman-fold enzymes. Finally, conformational plasticity observed both at the substrate and cofactor binding sites may be a challenge for structure-based drug design. The landscape drawn here may inform ongoing efforts toward the discovery of the first human RNMT inhibitors.

Elucidaton of DNA methylation changes in response to ionizng radiation induced double strand breaks

Energy Technology Data Exchange (ETDEWEB)

Herrlitz, Maren Linda

2014-07-04

Recent studies indicate that epigenetic modifications like DNA methylation are involved in the DNA damage response to ionizing radiation (IR). In this doctoral thesis DNA methylation changes after treatment with IR within one replication cycle time frame (≤ 24 hours) were investigated and a decrease in DNA methylation at short times after IR was observed. This fast decrease cannot be explained by a passive, replication-dependent mechanism due to reduced DNMT (DNA methyltransferase) expression. Rather it is conceivable that changing the enzymatic activity of enzymes may lead to changes in DNA methylation as a response to IR. In this context especially TET (ten eleven translocation) enzymes might play a role. These oxidize Methylcytosine (5mC) to Hydroxymethylcytosine (5hmC) and further to formylcytosine (5fC) and carboxylcytosine (5caC), being eventually replaced by cytosine via base excision repair (BER) mechanism. For the analysis of a TET-dependent DNA demethylation an appropriate experimental cellular system was established. Therefore, global 5mC levels, TET2 expression levels and 5hmC levels in different cell lines were investigated. An anti-correlation between 5mC levels and TET2 expression was shown for all cell lines, while 5hmC and 5mC levels were correlated only in a part of the investigated cell lines and no correlation between 5hmC and TET2 expression was observed. NIH/3T3 cells, showing no TET2 expression, were chosen for experiments in which the catalytic domain of TET2 (TET2CD-GFP) was overexpressed. Using two different techniques (immunofluorescence and ELISA-based colorimetric analysis), an increase in 5hmC abundance was demonstrated as a response to TET2CD-GFP overexpression, indicating that ectopically expressed TET2 is active. Similar results were obtained when TET2CD-GFP was overexpressed in U-2 OS cells, which have a high expression level of TET2. TET2CD-GFP overexpression also led to its accumulation in nucleoli. Whether this observation

Elucidaton of DNA methylation changes in response to ionizng radiation induced double strand breaks

International Nuclear Information System (INIS)

Herrlitz, Maren Linda

2014-01-01

Recent studies indicate that epigenetic modifications like DNA methylation are involved in the DNA damage response to ionizing radiation (IR). In this doctoral thesis DNA methylation changes after treatment with IR within one replication cycle time frame (≤ 24 hours) were investigated and a decrease in DNA methylation at short times after IR was observed. This fast decrease cannot be explained by a passive, replication-dependent mechanism due to reduced DNMT (DNA methyltransferase) expression. Rather it is conceivable that changing the enzymatic activity of enzymes may lead to changes in DNA methylation as a response to IR. In this context especially TET (ten eleven translocation) enzymes might play a role. These oxidize Methylcytosine (5mC) to Hydroxymethylcytosine (5hmC) and further to formylcytosine (5fC) and carboxylcytosine (5caC), being eventually replaced by cytosine via base excision repair (BER) mechanism. For the analysis of a TET-dependent DNA demethylation an appropriate experimental cellular system was established. Therefore, global 5mC levels, TET2 expression levels and 5hmC levels in different cell lines were investigated. An anti-correlation between 5mC levels and TET2 expression was shown for all cell lines, while 5hmC and 5mC levels were correlated only in a part of the investigated cell lines and no correlation between 5hmC and TET2 expression was observed. NIH/3T3 cells, showing no TET2 expression, were chosen for experiments in which the catalytic domain of TET2 (TET2CD-GFP) was overexpressed. Using two different techniques (immunofluorescence and ELISA-based colorimetric analysis), an increase in 5hmC abundance was demonstrated as a response to TET2CD-GFP overexpression, indicating that ectopically expressed TET2 is active. Similar results were obtained when TET2CD-GFP was overexpressed in U-2 OS cells, which have a high expression level of TET2. TET2CD-GFP overexpression also led to its accumulation in nucleoli. Whether this observation

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

Promoter DNA hypermethylation and gene repression in undifferentiated Arabidopsis cells.

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María Berdasco

Full Text Available Maintaining and acquiring the pluripotent cell state in plants is critical to tissue regeneration and vegetative multiplication. Histone-based epigenetic mechanisms are important for regulating this undifferentiated state. Here we report the use of genetic and pharmacological experimental approaches to show that Arabidopsis cell suspensions and calluses specifically repress some genes as a result of promoter DNA hypermethylation. We found that promoters of the MAPK12, GSTU10 and BXL1 genes become hypermethylated in callus cells and that hypermethylation also affects the TTG1, GSTF5, SUVH8, fimbrin and CCD7 genes in cell suspensions. Promoter hypermethylation in undifferentiated cells was associated with histone hypoacetylation and primarily occurred at CpG sites. Accordingly, we found that the process specifically depends on MET1 and DRM2 methyltransferases, as demonstrated with DNA methyltransferase mutants. Our results suggest that promoter DNA methylation may be another important epigenetic mechanism for the establishment and/or maintenance of the undifferentiated state in plant cells.

A unique regulatory phase of DNA methylation in the early mammalian embryo

OpenAIRE

Smith, Zachary D.; Chan, Michelle M.; Mikkelsen, Tarjei S.; Gu, Hongcang; Gnirke, Andreas; Regev, Aviv; Meissner, Alexander

2012-01-01

Summary DNA methylation is highly dynamic during mammalian embryogenesis. It is broadly accepted that the paternal genome is actively depleted of 5-methyl cytosine at fertilization, followed by passive loss that reaches a minimum at the blastocyst stage. However, this model is based on limited data, and to date no base-resolution maps exist to support and refine it. Here, we generated genome-scale DNA methylation maps in mouse gametes and through post-implantation embryogenesis. We find that ...

Crystallization and preliminary X-ray crystallographic characterization of TrmFO, a folate-dependent tRNA methyltransferase from Thermotoga maritima

International Nuclear Information System (INIS)

Cicmil, Nenad

2008-01-01

T. maritima TrmFO was overexpressed, purified and crystallized. A diffraction data set was collected to a resolution of 2.6 Å. TrmFO, previously classified as GID, is a methyltransferase that catalyzes the formation of 5-methyluridine or ribothymidine (T) at position 54 in tRNA in some Gram-positive bacteria. To date, TrmFO is the only characterized tRNA methyltransferase that does not use S-adenosylmethionine as the methyl-group donor. Instead, the donor of the methyl group is N 5 ,N 10 -methylenetetrahydrofolate. The crystallization and preliminary X-ray crystallographic studies of TrmFO are reported here. The recombinant protein, cloned from Thermotoga maritima genomic DNA, was overproduced in Esherichia coli and crystallized in 25%(v/v) PEG 4000, 100 mM NaCl and sodium citrate buffer pH 5.0 at 291 K using the hanging-drop vapor-diffusion method. The plate-shaped crystals diffracted to 2.6 Å and belong to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 79.94, b = 92.46, c = 127.20 Å

Cross-Talk between Dnmt2-Dependent tRNA Methylation and Queuosine Modification

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Ann E. Ehrenhofer-Murray

2017-02-01

Full Text Available Enzymes of the Dnmt2 family of methyltransferases have yielded a number of unexpected discoveries. The first surprise came more than ten years ago when it was realized that, rather than being DNA methyltransferases, Dnmt2 enzymes actually are transfer RNA (tRNA methyltransferases for cytosine-5 methylation, foremost C38 (m5C38 of tRNAAsp. The second unanticipated finding was our recent discovery of a nutritional regulation of Dnmt2 in the fission yeast Schizosaccharomyces pombe. Significantly, the presence of the nucleotide queuosine in tRNAAsp strongly stimulates Dnmt2 activity both in vivo and in vitro in S. pombe. Queuine, the respective base, is a hypermodified guanine analog that is synthesized from guanosine-5’-triphosphate (GTP by bacteria. Interestingly, most eukaryotes have queuosine in their tRNA. However, they cannot synthesize it themselves, but rather salvage it from food or from gut microbes. The queuine obtained from these sources comes from the breakdown of tRNAs, where the queuine ultimately was synthesized by bacteria. Queuine thus has been termed a micronutrient. This review summarizes the current knowledge of Dnmt2 methylation and queuosine modification with respect to translation as well as the organismal consequences of the absence of these modifications. Models for the functional cooperation between these modifications and its wider implications are discussed.

O(6)-methylguanine DNA-methyltransferase (MGMT) overexpression in melanoma cells induces resistance to nitrosoureas and temozolomide but sensitizes to mitomycin C.

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Passagne, Isabelle; Evrard, Alexandre; Depeille, Philippe; Cuq, Pierre; Cupissol, Didier; Vian, Laurence

2006-03-01

Alkylating agents play an important role in the chemotherapy of malignant melanomas. The activity of alkylating agents depends on their capacity to form alkyl adducts with DNA, in some cases causing cross-linking of DNA strands. However, the use of these agents is limited by cellular resistance induced by the DNA repair enzyme O(6)-methylguanine DNA-methyltransferase (MGMT) which removes alkyl groups from alkylated DNA strands. To determine to what extent the expression of MGMT in melanoma cells induces resistance to alkylating agents, the human cell line CAL77 Mer- (i.e., MGMT deficient) were transfected with pcMGMT vector containing human MGMT cDNA. Several clones expressing MGMT at a high level were selected to determine their sensitivity to chemotherapeutic drugs. Melanoma-transfected cells were found to be significantly less sensitive to nitrosoureas (carmustine, fotemustine, streptozotocin) and temozolomide with an increase of IC(50) values between 3 and 14 when compared to parent cells. No difference in cell survival rates between MGMT-proficient and -deficient cells was observed for melphalan, chlorambucil, busulphan, thiotepa and cisplatin which preferentially induce N(7) guanine lesions. Surprisingly, MGMT overexpression increased the sensitivity of CAL77 cells to mitomycin C by approximately 10-fold. Treatment of clonal cell lines with buthionine-[S,R]-sulfoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase which depletes cellular glutathione, completely reversed this unexpected increase in sensitivity to mitomycin C. This observation suggests that glutathione is involved in the sensitivity of MGMT-transfected cells to mitomycin C and may act synergistically with MGMT via an unknown mechanism.

Selective small-chemical inhibitors of protein arginine methyltransferase 5 with anti-lung cancer activity.

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Gui-Mei Kong

Full Text Available Protein arginine methyltransferase 5 (PRMT5 plays critical roles in a wide variety of biological processes, including tumorigenesis. By screening a library of small chemical compounds, we identified eight compounds that selectively inhibit the PRMT5 enzymatic activity, with IC50 values ranging from 0.1 to 6 μM. Molecular docking simulation and site-directed mutagenesis indicated that identified compounds target the substrate-binding site in PRMT5. Treatment of lung cancer cells with identified inhibitors led to inhibition of the symmetrical arginine methylation of SmD3 and histones and the cellular proliferation. Oral administration of the inhibitor demonstrated antitumor activity in a lung tumor xenograft model. Thus, identified PRMT5-specific small-molecule inhibitors would help elucidate the biological roles of PRMT5 and serve as lead compounds for future drug development.

Optoelectronic studies on heterocyclic bases of deoxyribonucleic acid for DNA photonics.

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El-Diasty, Fouad; Abdel-Wahab, Fathy

2015-10-01

The optoelectronics study of large molecules, particularly π-stacking molecules, such as DNA is really an extremely difficult task. We perform first electronic structure calculations on the heterocyclic bases of 2'-deoxyribonucleic acid based on Lorentz-Fresnel dispersion theory. In the UV-VIS range of spectrum, many of the optoelectronic parameters for DNA four bases namely adenine, guanine, cytosine and thymine are calculated and discussed. The results demonstrate that adenine has the highest hyperpolarizability, whereas thymine has the lowest hyperpolarizability. Cytosine has the lower average oscillator energy and the higher lattice energy. Thymine infers the most stable nucleic base with the lower phonon energy. Thymine also has the highest average oscillator energy and the lower lattice energy. Moreover, the four nucleic acid bases have large band gap energies less than 5 eV with a semiconducting behavior. Guanine shows the smallest band gap and the highest Fermi level energy, whereas adenine elucidates the highest band gap energy. Copyright © 2015. Published by Elsevier B.V.

Genome-wide tracking of unmethylated DNA Alu repeats in normal and cancer cells

DEFF Research Database (Denmark)

Rodriguez, Jairo; Vives, Laura; Jordà, Mireia

2008-01-01

Methylation of the cytosine is the most frequent epigenetic modification of DNA in mammalian cells. In humans, most of the methylated cytosines are found in CpG-rich sequences within tandem and interspersed repeats that make up to 45% of the human genome, being Alu repeats the most common family....

In planta assays involving epigenetically silenced genes reveal inhibition of cytosine methylation by genistein

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Arase Sachiko

2012-03-01

Full Text Available Abstract Background Cytosine methylation is involved in epigenetic control of gene expression in a wide range of organisms. An increasing number of examples indicate that changing the frequency of cytosine methylation in the genome is a feasible tool to engineer novel traits in plants. Although demethylating effects of compounds have been analyzed in human cultured cells in terms of suppressing cancer, their effect in plant cells has not been analyzed extensively. Here, we developed in planta assay systems to detect inhibition of cytosine methylation using plants that contain a transgene transcriptionally silenced by an epigenetic mechanism. Results Seeds of two transgenic plants were used: a petunia line that has been identified as a revertant of the co-suppression of the chalcone synthase-A (CHS-A gene and contains CHS-A transgenes whose transcription is repressed; Nicotiana benthamiana plants that contain the green fluorescent protein (GFP reporter gene whose transcription is repressed through virus-induced transcriptional gene silencing. Seeds of these plants were sown on a medium that contained a demethylating agent, either 5-azacytidine or trichostatin A, and the restoration of the transcriptionally active state of the transgene was detected in seedlings. Using these systems, we found that genistein, a major isoflavonoid compound, inhibits cytosine methylation, thus restoring transgene transcription. Genistein also restored the transcription of an epigenetically silenced endogenous gene in Arabidopsis plants. Conclusions Our assay systems allowed us to assess the inhibition of cytosine methylation, in particular of maintenance of methylation, by compounds in plant cells. These results suggest a novel role of flavonoids in plant cells and that genistein is useful for modifying the epigenetic state of plant genomes.

Chloroplast overexpression of rice caffeic acid O-methyltransferase increases melatonin production in chloroplasts via the 5-methoxytryptamine pathway in transgenic rice plants.

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Choi, Geun-Hee; Lee, Hyoung Yool; Back, Kyoungwhan

2017-08-01

Recent analyses of the enzymatic features of various melatonin biosynthetic genes from bacteria, animals, and plants have led to the hypothesis that melatonin could be synthesized via the 5-methoxytryptamine (5-MT) pathway. 5-MT is known to be synthesized in vitro from serotonin by the enzymatic action of O-methyltransferases, including N-acetylserotonin methyltransferase (ASMT) and caffeic acid O-methyltransferase (COMT), leading to melatonin synthesis by the subsequent enzymatic reaction with serotonin N-acetyltransferase (SNAT). Here, we show that 5-MT was produced and served as a precursor for melatonin synthesis in plants. When rice seedlings were challenged with senescence treatment, 5-MT levels and melatonin production were increased in transgenic rice seedlings overexpressing the rice COMT in chloroplasts, while no such increases were observed in wild-type or transgenic seedlings overexpressing the rice COMT in the cytosol, suggesting a 5-MT transport limitation from the cytosol to chloroplasts. In contrast, cadmium treatment led to results different from those in senescence. The enhanced melatonin production was not observed in the chloroplast COMT lines relative over the cytosol COMT lines although 5-MT levels were equally induced in all genotypes upon cadmium treatment. The transgenic seedlings with enhanced melatonin in their chloroplasts exhibited improved seedling growth vs the wild type under continuous light conditions. This is the first report describing enhanced melatonin production in chloroplasts via the 5-MT pathway with the ectopic overexpression of COMT in chloroplasts in plants. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Nanomechanical DNA origami pH sensors.

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Kuzuya, Akinori; Watanabe, Ryosuke; Yamanaka, Yusei; Tamaki, Takuya; Kaino, Masafumi; Ohya, Yuichi

2014-10-16

Single-molecule pH sensors have been developed by utilizing molecular imaging of pH-responsive shape transition of nanomechanical DNA origami devices with atomic force microscopy (AFM). Short DNA fragments that can form i-motifs were introduced to nanomechanical DNA origami devices with pliers-like shape (DNA Origami Pliers), which consist of two levers of 170-nm long and 20-nm wide connected at a Holliday-junction fulcrum. DNA Origami Pliers can be observed as in three distinct forms; cross, antiparallel and parallel forms, and cross form is the dominant species when no additional interaction is introduced to DNA Origami Pliers. Introduction of nine pairs of 12-mer sequence (5'-AACCCCAACCCC-3'), which dimerize into i-motif quadruplexes upon protonation of cytosine, drives transition of DNA Origami Pliers from open cross form into closed parallel form under acidic conditions. Such pH-dependent transition was clearly imaged on mica in molecular resolution by AFM, showing potential application of the system to single-molecular pH sensors.

Sites of termination of in vitro DNA synthesis on psoralen phototreated single-stranded templates

International Nuclear Information System (INIS)

Piette, J.; Hearst, J.

1985-01-01

Single-stranded DNA has been photochemically induced to react with 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and used as substrate for DNA replication with E. coli DNA polymerase I large fragment. By using the dideoxy sequencing procedure, it is possible to map the termination sites on the template photoreacted with HMT. These sites occur at the nucleotides preceding each thymine residue (and a few cytosine residues), emphasizing the fact that in a single-stranded stretch of DNA, HMT reacts with each thymine residue without any specificity regarding the flanking base sequence of the thymine residues. In addition, termination of DNA synthesis due to psoralen-adducted thymine is not influenced by the efficiency of the 3'-5' exonuclease proof-reading activity of the DNA polymerase. (author)

Electron attachment to the guanine-cytosine nucleic acid base pair and the effects of monohydration and proton transfer.

Science.gov (United States)

Gupta, Ashutosh; Jaeger, Heather M; Compaan, Katherine R; Schaefer, Henry F

2012-05-17

The guanine-cytosine (GC) radical anion and its interaction with a single water molecule is studied using ab initio and density functional methods. Z-averaged second-order perturbation theory (ZAPT2) was applied to GC radical anion for the first time. Predicted spin densities show that the radical character is localized on cytosine. The Watson-Crick monohydrated GC anion is compared to neutral GC·H2O, as well as to the proton-transferred analogue on the basis of structural and energetic properties. In all three systems, local minima are identified that correspond to water positioned in the major and minor grooves of macromolecular DNA. On the anionic surface, two novel structures have water positioned above or below the GC plane. On the neutral and anionic surfaces, the global minimum can be described as water interacting with the minor groove. These structures are predicted to have hydration energies of 9.7 and 11.8 kcal mol(-1), respectively. Upon interbase proton-transfer (PT), the anionic global minimum has water positioned in the major groove, and the hydration energy increases to 13.4 kcal mol(-1). PT GC·H2O(•-) has distonic character; the radical character resides on cytosine, while the negative charge is localized on guanine. The effects of proton transfer are further investigated through the computed adiabatic electron affinities (AEA) of GC and monohydrated GC, and the vertical detachment energies (VDE) of the corresponding anions. Monohydration increases the AEAs and VDEs by only 0.1 eV, while proton-transfer increases the VDEs substantially (0.8 eV). The molecular charge distribution of monohydrated guanine-cytosine radical anion depends heavily on interbase proton transfer.

The dynamics of DNA methylation and hydroxymethylation during amelogenesis.

Science.gov (United States)

Yoshioka, Hirotaka; Minamizaki, Tomoko; Yoshiko, Yuji

2015-11-01

Amelogenesis is a multistep process that relies on specific temporal and spatial signaling networks between the dental epithelium and mesenchymal tissues. Epigenetic modifications of key developmental genes in this process may be closely linked to a network of molecular events. However, the role of epigenetic regulation in amelogenesis remains unclear. Here, we have uncovered the spatial distributions of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) to determine epigenetic events in the mandibular incisors of mice. Immunohistochemistry and dot blotting showed that 5-hmC in ameloblasts increased from the secretory stage to the later maturation stage. We also demonstrated the distribution of 5-mC-positive ameloblasts with punctate nuclear labeling from sometime after the initiation of the secretory stage to the later maturation stage; however, dot blotting failed to detect this change. No obvious alteration of 5-mC/5-hmC staining in odontoblasts and dental pulp cells was observed. Concomitant with quantitative expression data, immunohistochemistry showed that maintenance DNA methyltransferase DNMT1 was highly expressed in immature dental epithelial cells and subsequently decreased at later stages of development. Meanwhile, de novo DNA methyltransferase Dnmt3a and Dnmt3b and DNA demethylase Tet family genes were universally expressed, except Tet1 that was highly expressed in immature dental epithelial cells. Thus, DNMT1 may sustain the undifferentiated status of dental epithelial cells through the maintenance of DNA methylation, while the hydroxylation of 5-mC may occur through the whole differentiation process by TET activity. Taken together, these data indicate that the dynamic changes of 5-mC and 5-hmC may be critical for the regulation of amelogenesis.

4'-Methyl derivatives of 5-MOP and 5-MOA: synthesis, photoreactivity, and photobiological activity.

Science.gov (United States)

Gia, O; Anselmo, A; Conconi, M T; Antonello, C; Uriarte, E; Caffieri, S

1996-10-25

The synthesis and photobiological activity of four new 4'-methyl derivatives of 5-MOP (5-methoxypsoralen) and 5-MOA (5-methoxyangelicin), i.e., 4,4'-dimethyl-5-methoxypsoralen, 3,4'-dimethyl-5-methoxypsoralen, 4,4'-dimethyl-5-methoxyangelicin, and 3,4'-dimethyl-5-methoxyangelicin, are described. All these compounds photobind efficiently to DNA. The DNA-photobinding process was investigated using various nucleic acid structures such as double-helix DNA, bacterial DNA, and synthetic polydeoxyribonucleotides. Photoreaction experiments showed that, unlike 8-MOP (8-methoxypsoralen) and 5-MOP, both angular derivatives bind thymine and cytosine with the same efficiency. The principal nucleoside-psoralen monoadducts were isolated and characterized after enzymatic digestion or acid hydrolysis. Biological activity studies revealed a good correlation with the extent of covalent photoaddition. Moreover, the two angular derivatives and the 4,4'-dimethyl-5-methoxypsoralen were unable to induce skin erythema, in striking contrast with the reference drugs, 8-MOP and 5-MOP; only the 3,4'-dimethyl-5-methoxypsoralen caused erythema, although to a substantially lower extent than that induced by the two parent compounds.

pH-Modulated Watson-Crick duplex-quadruplex equilibria of guanine-rich and cytosine-rich DNA sequences 140 base pairs upstream of the c-kit transcription initiation site.

Science.gov (United States)

Bucek, Pavel; Jaumot, Joaquim; Aviñó, Anna; Eritja, Ramon; Gargallo, Raimundo

2009-11-23

Guanine-rich regions of DNA are sequences capable of forming G-quadruplex structures. The formation of a G-quadruplex structure in a region 140 base pairs (bp) upstream of the c-kit transcription initiation site was recently proposed (Fernando et al., Biochemistry, 2006, 45, 7854). In the present study, the acid-base equilibria and the thermally induced unfolding of the structures formed by a guanine-rich region and by its complementary cytosine-rich strand in c-kit were studied by means of circular dichroism and molecular absorption spectroscopies. In addition, competition between the Watson-Crick duplex and the isolated structures was studied as a function of pH value and temperature. Multivariate data analysis methods based on both hard and soft modeling were used to allow accurate quantification of the various acid-base species present in the mixtures. Results showed that the G-quadruplex and i-motif coexist with the Watson-Crick duplex over the pH range from 3.0 to 6.5, approximately, under the experimental conditions tested in this study. At pH 7.0, the duplex is practically the only species present.

Das DNA-Puzzle

Science.gov (United States)

Kirchner, Stefan

Im Jahre 1953 wurde von James Watson und Francis Crick erstmalig der strukturelle Aufbau der sogenannten DNA (Desoxyribonukleinsäure) beschrieben, welche das Erbgut jedes Lebewesens enthält. Der wesentliche Teil des Erbguts wird dabei durch eine sehr lange Folge der vier Basen Adenin (A), Cytosin (C), Guanin (G) und Thymin (T) codiert. Seit einigen Jahren ist es möglich, die Folge der vier Basen zu einer gegebenen DNA zu bestimmen. Biologen bezeichnen diesen Vorgang als Sequenzierung.

Characterization of the Relationship of CDKL5 with MeCP2 and Dnmt1 in PrimaryRat Cortical Neurons

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Zhi Yi

Full Text Available ABSTRACT Cyclin-dependent kinase-like 5 (CDKL5 is a protein kinase that is homologous to mitogen-activated protein kinases (MAPKs and cyclin-dependent kinases (CDKs. Mutations in the CDKL5 gene cause X-linked infantile spasms and phenotypes that overlap with that of Rett syndrome, which is a neurodevelopmental disorder caused primarily by mutations in the methyl CpG binding protein 2 gene (MECP2. Previous studies in transfected cell lines showed that CDKL5 interacts with MeCP2 and DNA (cytosine-5-methyltransferase 1 (Dnmt1. However, little is known about the relationships of CDKL5 with interacting proteins in primary neuronal cultures. In this study, we investigated the expression patterns of CDKL5, MeCP2 and Dnmt1, and their interaction in cultured rat cortical neurons. Using real-time PCR analysis, we found that CDKL5, MeCP2 and Dnmt1 have similar expression patterns at the mRNA level. In contrast, the expression patterns of those proteins at the protein level are different and could be inversely correlated, as shown by western blotting. Using co-immunoprecipitation, we further demonstrated that CDKL5 interacts with MeCP2 and Dnmt1 in primary rat cortical neurons. These data suggest that a functional link exists among CDKL5, MeCP2 and Dnmt1 during neuronal development and may provide further insight into the pathogenesis of Rett syndrome.

Interaction of Cu(+) with cytosine and formation of i-motif-like C-M(+)-C complexes: alkali versus coinage metals.

Science.gov (United States)

Gao, Juehan; Berden, Giel; Rodgers, M T; Oomens, Jos

2016-03-14

The Watson-Crick structure of DNA is among the most well-known molecular structures of our time. However, alternative base-pairing motifs are also known to occur, often depending on base sequence, pH, or the presence of cations. Pairing of cytosine (C) bases induced by the sharing of a single proton (C-H(+)-C) may give rise to the so-called i-motif, which occurs primarily in expanded trinucleotide repeats and the telomeric region of DNA, particularly at low pH. At physiological pH, silver cations were recently found to stabilize C dimers in a C-Ag(+)-C structure analogous to the hemiprotonated C-dimer. Here we use infrared ion spectroscopy in combination with density functional theory calculations at the B3LYP/6-311G+(2df,2p) level to show that copper in the 1+ oxidation state induces an analogous formation of C-Cu(+)-C structures. In contrast to protons and these transition metal ions, alkali metal ions induce a different dimer structure, where each ligand coordinates the alkali metal ion in a bidentate fashion in which the N3 and O2 atoms of both cytosine ligands coordinate to the metal ion, sacrificing hydrogen-bonding interactions between the ligands for improved chelation of the metal cation.

Cytosine methylation dysregulation in neonates following intrauterine growth restriction.

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Francine Einstein

2010-01-01

Full Text Available Perturbations of the intrauterine environment can affect fetal development during critical periods of plasticity, and can increase susceptibility to a number of age-related diseases (e.g., type 2 diabetes mellitus; T2DM, manifesting as late as decades later. We hypothesized that this biological memory is mediated by permanent alterations of the epigenome in stem cell populations, and focused our studies specifically on DNA methylation in CD34+ hematopoietic stem and progenitor cells from cord blood from neonates with intrauterine growth restriction (IUGR and control subjects.Our epigenomic assays utilized a two-stage design involving genome-wide discovery followed by quantitative, single-locus validation. We found that changes in cytosine methylation occur in response to IUGR of moderate degree and involving a restricted number of loci. We also identify specific loci that are targeted for dysregulation of DNA methylation, in particular the hepatocyte nuclear factor 4alpha (HNF4A gene, a well-known diabetes candidate gene not previously associated with growth restriction in utero, and other loci encoding HNF4A-interacting proteins.Our results give insights into the potential contribution of epigenomic dysregulation in mediating the long-term consequences of IUGR, and demonstrate the value of this approach to studies of the fetal origin of adult disease.

ORF Sequence: NC_002162 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available NC_002162 gi|13358092 >gi|13358092|ref|NP_078366.1| cytosine-specific methyltransferase [Ureap...RILFDLQKLNQLPQFLLLENVNNMLSKQHKLDYDMWTKSLKQLGYSTCTFQLNALDYGSAQRRKRVYAISILNYDGLIDSNGNILDLEAPIFDGKQKQLKDVLKTNYK

Crystallization and preliminary X-ray crystallographic characterization of TrmFO, a folate-dependent tRNA methyltransferase from Thermotoga maritima

Energy Technology Data Exchange (ETDEWEB)

Cicmil, Nenad, E-mail: cicmil@uiuc.edu [Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801 (United States)

2008-03-01

T. maritima TrmFO was overexpressed, purified and crystallized. A diffraction data set was collected to a resolution of 2.6 Å. TrmFO, previously classified as GID, is a methyltransferase that catalyzes the formation of 5-methyluridine or ribothymidine (T) at position 54 in tRNA in some Gram-positive bacteria. To date, TrmFO is the only characterized tRNA methyltransferase that does not use S-adenosylmethionine as the methyl-group donor. Instead, the donor of the methyl group is N{sup 5},N{sup 10}-methylenetetrahydrofolate. The crystallization and preliminary X-ray crystallographic studies of TrmFO are reported here. The recombinant protein, cloned from Thermotoga maritima genomic DNA, was overproduced in Esherichia coli and crystallized in 25%(v/v) PEG 4000, 100 mM NaCl and sodium citrate buffer pH 5.0 at 291 K using the hanging-drop vapor-diffusion method. The plate-shaped crystals diffracted to 2.6 Å and belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 79.94, b = 92.46, c = 127.20 Å.

MicroRNA-29a Alleviates Bile Duct Ligation Exacerbation of Hepatic Fibrosis in Mice through Epigenetic Control of Methyltransferases

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Ya-Ling Yang

2017-01-01

Full Text Available MicroRNA-29 (miR-29 is found to modulate hepatic stellate cells’ (HSCs activation and, thereby, reduces liver fibrosis pathogenesis. Histone methyltransferase regulation of epigenetic reactions reportedly participates in hepatic fibrosis. This study is undertaken to investigate the miR-29a regulation of the methyltransferase signaling and epigenetic program in hepatic fibrosis progression. miR-29a transgenic mice (miR-29aTg mice and wild-type littermates were subjected to bile duct-ligation (BDL to develop cholestatic liver fibrosis. Primary HSCs were transfected with a miR-29a mimic and antisense inhibitor. Profibrogenic gene expression, histone methyltransferases and global genetic methylation were probed with real-time quantitative RT-PCR, immunohistochemical stain, Western blot and ELISA. Hepatic tissue in miR-29aTg mice displayed weak fibrotic matrix as evidenced by Sirius Red staining concomitant with low fibrotic matrix collagen 1α1 expression within affected tissues compared to the wild-type mice. miR-29a overexpression reduced the BDL exaggeration of methyltransferases, DNMT1, DNMT3b and SET domain containing 1A (SET1A expression. It also elevated phosphatase and tensin homolog deleted on chromosome 10 (PTEN signaling within liver tissue. In vitro, miR-29a mimic transfection lowered collagen 1α1, DNMT1, DNMT3b and SET1A expression in HSCs. Gain of miR-29a signaling resulted in DNA hypomethylation and high PTEN expression. This study shines a new light on miR-29a inhibition of methyltransferase, a protective effect to maintain the DNA hypomethylation state that decreases fibrogenic activities in HSC. These robust analyses also highlight the miR-29a regulation of epigenetic actions to ameliorate excessive fibrosis during cholestatic liver fibrosis development.

Molecular cloning, characterization and expression of the caffeic acid O-methyltransferase (COMT) ortholog from kenaf (Hibiscus cannabinus)

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We cloned the full-length of the gene putatively encoding caffeic acid O-methyltransferase (COMT) from kenaf (Hibiscus cannabinus L.) using degenerate primers and the RACE (rapid amplification of cDNA ends) method. Kenaf is an herbaceous and rapidly growing dicotyledonous plant with great potential ...

Alterations of 5-Hydroxymethylcytosine in Human Cancers

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Ali Yesilkanal

2013-06-01

Full Text Available Prior to 2009, 5-methylcytosine (5-mC was thought to be the only biologically significant cytosine modification in mammalian DNA. With the discovery of the TET enzymes, which convert 5-methylcytosine (5-mC to 5-hydroxymethylcytosine (5-hmC, however, intense interest has emerged in determining the biological function of 5-hmC. Here, we review the techniques used to study 5-hmC and evidence that alterations to 5-hmC physiology play a functional role in the molecular pathogenesis of human cancers.

MTAP deletion confers enhanced dependency on the PRMT5 arginine methyltransferase in cancer cells | Office of Cancer Genomics

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The discovery of cancer dependencies has the potential to inform therapeutic strategies and to identify putative drug targets. Integrating data from comprehensive genomic profiling of cancer cell lines and from functional characterization of cancer cell dependencies, we discovered that loss of the enzyme methylthioadenosine phosphorylase (MTAP) confers a selective dependence on protein arginine methyltransferase 5 (PRMT5) and its binding partner WDR77. MTAP is frequently lost due to its proximity to the commonly deleted tumor suppressor gene, CDKN2A.

RNA-directed DNA methylation: Mechanisms and functions

KAUST Repository

Mahfouz, Magdy M.

2010-07-01

Epigenetic RNA based gene silencing mechanisms play a major role in genome stability and control of gene expression. Transcriptional gene silencing via RNA-directed DNA methylation (RdDM) guides the epigenetic regulation of the genome in response to disease states, growth, developmental and stress signals. RdDM machinery is composed of proteins that produce and modify 24-nt- long siRNAs, recruit the RdDM complex to genomic targets, methylate DNA and remodel chromatin. The final DNA methylation pattern is determined by either DNA methyltransferase alone or by the combined action of DNA methyltransferases and demethylases. The dynamic interaction between RdDM and demethylases may render the plant epigenome plastic to growth, developmental, and environmental cues. The epigenome plasticity may allow the plant genome to assume many epigenomes and to have the right epigenome at the right time in response to intracellular or extracellular stimuli. This review discusses recent advances in RdDM research and considers future perspectives.

Nanomechanical DNA Origami pH Sensors

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Akinori Kuzuya

2014-10-01

Full Text Available Single-molecule pH sensors have been developed by utilizing molecular imaging of pH-responsive shape transition of nanomechanical DNA origami devices with atomic force microscopy (AFM. Short DNA fragments that can form i-motifs were introduced to nanomechanical DNA origami devices with pliers-like shape (DNA Origami Pliers, which consist of two levers of 170-nm long and 20-nm wide connected at a Holliday-junction fulcrum. DNA Origami Pliers can be observed as in three distinct forms; cross, antiparallel and parallel forms, and cross form is the dominant species when no additional interaction is introduced to DNA Origami Pliers. Introduction of nine pairs of 12-mer sequence (5'-AACCCCAACCCC-3', which dimerize into i-motif quadruplexes upon protonation of cytosine, drives transition of DNA Origami Pliers from open cross form into closed parallel form under acidic conditions. Such pH-dependent transition was clearly imaged on mica in molecular resolution by AFM, showing potential application of the system to single-molecular pH sensors.

A Contracted DNA Repeat in LHX3 Intron 5 Is Associated with Aberrant Splicing and Pituitary Dwarfism in German Shepherd Dogs

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Voorbij, Annemarie M. W. Y.; van Steenbeek, Frank G.; Vos-Loohuis, Manon; Martens, Ellen E. C. P.; Hanson-Nilsson, Jeanette M.; van Oost, Bernard A.; Kooistra, Hans S.; Leegwater, Peter A.

2011-01-01

Dwarfism in German shepherd dogs is due to combined pituitary hormone deficiency of unknown genetic cause. We localized the recessively inherited defect by a genome wide approach to a region on chromosome 9 with a lod score of 9.8. The region contains LHX3, which codes for a transcription factor essential for pituitary development. Dwarfs have a deletion of one of six 7 bp repeats in intron 5 of LHX3, reducing the intron size to 68 bp. One dwarf was compound heterozygous for the deletion and an insertion of an asparagine residue in the DNA-binding homeodomain of LHX3, suggesting involvement of the gene in the disorder. An exon trapping assay indicated that the shortened intron is not spliced efficiently, probably because it is too small. We applied bisulfite conversion of cytosine to uracil in RNA followed by RT-PCR to analyze the splicing products. The aberrantly spliced RNA molecules resulted from either skipping of exon 5 or retention of intron 5. The same splicing defects were observed in cDNA derived from the pituitary of dwarfs. A survey of similarly mutated introns suggests that there is a minimal distance requirement between the splice donor and branch site of 50 nucleotides. In conclusion, a contraction of a DNA repeat in intron 5 of canine LHX3 leads to deficient splicing and is associated with pituitary dwarfism. PMID:22132174

Recognition of 5-Hydroxymethylcytosine by the Uhrf1 SRA Domain

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Bultmann, Sebastian; Casa, Valentina; Cardoso, M. Cristina; Antes, Iris; Leonhardt, Heinrich

2011-01-01

Recent discovery of 5-hydroxymethylcytosine (5hmC) in genomic DNA raises the question how this sixth base is recognized by cellular proteins. In contrast to the methyl-CpG binding domain (MBD) of MeCP2, we found that the SRA domain of Uhrf1, an essential factor in DNA maintenance methylation, binds 5hmC and 5-methylcytosine containing substrates with similar affinity. Based on the co-crystal structure, we performed molecular dynamics simulations of the SRA:DNA complex with the flipped cytosine base carrying either of these epigenetic modifications. Our data indicate that the SRA binding pocket can accommodate 5hmC and stabilizes the flipped base by hydrogen bond formation with the hydroxyl group. PMID:21731699

A common mutation in the 5,10-methylenetetrahydrofolate reductase gene affects genomic DNA methylation through an interaction with folate status

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Friso, Simonetta; Choi, Sang-Woon; Girelli, Domenico; Mason, Joel B.; Dolnikowski, Gregory G.; Bagley, Pamela J.; Olivieri, Oliviero; Jacques, Paul F.; Rosenberg, Irwin H.; Corrocher, Roberto; Selhub, Jacob

2002-01-01

DNA methylation, an essential epigenetic feature of DNA that modulates gene expression and genomic integrity, is catalyzed by methyltransferases that use the universal methyl donor S-adenosyl-l-methionine. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the synthesis of 5-methyltetrahydrofolate (5-methylTHF), the methyl donor for synthesis of methionine from homocysteine and precursor of S-adenosyl-l-methionine. In the present study we sought to determine the effect of folate status on genomic DNA methylation with an emphasis on the interaction with the common C677T mutation in the MTHFR gene. A liquid chromatography/MS method for the analysis of nucleotide bases was used to assess genomic DNA methylation in peripheral blood mononuclear cell DNA from 105 subjects homozygous for this mutation (T/T) and 187 homozygous for the wild-type (C/C) MTHFR genotype. The results show that genomic DNA methylation directly correlates with folate status and inversely with plasma homocysteine (tHcy) levels (P < 0.01). T/T genotypes had a diminished level of DNA methylation compared with those with the C/C wild-type (32.23 vs.62.24 ng 5-methylcytosine/μg DNA, P < 0.0001). When analyzed according to folate status, however, only the T/T subjects with low levels of folate accounted for the diminished DNA methylation (P < 0.0001). Moreover, in T/T subjects DNA methylation status correlated with the methylated proportion of red blood cell folate and was inversely related to the formylated proportion of red blood cell folates (P < 0.03) that is known to be solely represented in those individuals. These results indicate that the MTHFR C677T polymorphism influences DNA methylation status through an interaction with folate status. PMID:11929966

Designing cyclopentapeptide inhibitor as potential antiviral drug for dengue virus ns5 methyltransferase.

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Idrus, Syarifuddin; Tambunan, Usman Sumo Friend; Zubaidi, Ahmad Ardilla

2012-01-01

NS5 methyltransferase (Mtase) has a crucial role in the replication of dengue virus. There are two active sites on NS5 Mtase i.e., SAM and RNA-cap binding sites. Inhibition of the NS5 Mtase activity is expected to prevent the propagation of dengue virus. This study was conducted to design cyclic peptide ligands as enzyme inhibitors of dengue virus NS5 Mtase through computational approach. Cyclopentapeptides were designed as ligand of SAM binding site as much as 1635 and 736 cyclopentpeptides were designed as ligand of RNA-cap binding site. Interaction between ligand and NS5 Mtase has been conducted on the Docking simulation. The result shows that cyclopentapeptide CTWYC was the best peptide candidate on SAM binding site, with estimated free binding energy -30.72 kca/mol. Cyclopentapeptide CYEFC was the best peptide on RNA-cap binding site with estimated free binding energy -22.89 kcal/mol. Both peptides did not have tendency toward toxicity properties. So it is expected that both CTWYC and CYEFC ligands could be used as a potential antiviral drug candidates, which can inhibit the SAM and RNA-cap binding sites of dengue virus NS5 Mtase.

Histone H3 lysine 36 methyltransferase mobilizes NER factors to regulate tolerance against alkylation damage in fission yeast.

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Lim, Kim Kiat; Nguyen, Thi Thuy Trang; Li, Adelicia Yongling; Yeo, Yee Phan; Chen, Ee Sin

2018-04-09

The Set2 methyltransferase and its target, histone H3 lysine 36 (H3K36), affect chromatin architecture during the transcription and repair of DNA double-stranded breaks. Set2 also confers resistance against the alkylating agent, methyl methanesulfonate (MMS), through an unknown mechanism. Here, we show that Schizosaccharomyces pombe (S. pombe) exhibit MMS hypersensitivity when expressing a set2 mutant lacking the catalytic histone methyltransferase domain or a H3K36R mutant (reminiscent of a set2-null mutant). Set2 acts synergistically with base excision repair factors but epistatically with nucleotide excision repair (NER) factors, and determines the timely nuclear accumulation of the NER initiator, Rhp23, in response to MMS. Set2 facilitates Rhp23 recruitment to chromatin at the brc1+ locus, presumably to repair alkylating damage and regulate the expression of brc1+ in response to MMS. Set2 also show epistasis with DNA damage checkpoint proteins; regulates the activation of Chk1, a DNA damage response effector kinase; and acts in a similar functional group as proteins involved in homologous recombination. Consistently, Set2 and H3K36 ensure the dynamicity of Rhp54 in DNA repair foci formation after MMS treatment. Overall, our results indicate a novel role for Set2/H3K36me in coordinating the recruitment of DNA repair machineries to timely manage alkylating damage.

miR-125b targets DNMT3b and mediates p53 DNA methylation involving in the vascular smooth muscle cells proliferation induced by homocysteine

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Cao, ChengJian [Key Laboratory of Basic Research in Cardio-Cerebral Vascular Diseases, Ningxia Medical University, Yinchuan (China); Zhang, HuiPing [Department of Prenatal Diagnosis Center, General Hospital of Ningxia Medical University, Yinchuan (China); Zhao, Li [Department of Medical Laboratory, Ningxia Medical University, Yinchuan (China); Zhou, Longxia [Department of Basic Medicine, Ningxia Medical University, Yinchuan (China); Zhang, Minghao; Xu, Hua [Key Laboratory of Basic Research in Cardio-Cerebral Vascular Diseases, Ningxia Medical University, Yinchuan (China); Department of Basic Medicine, Ningxia Medical University, Yinchuan (China); Han, Xuebo [Department of Medical Laboratory, Ningxia Medical University, Yinchuan (China); Li, Guizhong; Yang, Xiaoling [Key Laboratory of Basic Research in Cardio-Cerebral Vascular Diseases, Ningxia Medical University, Yinchuan (China); Department of Basic Medicine, Ningxia Medical University, Yinchuan (China); Jiang, YiDeng, E-mail: jyjcyxy@yeah.net [Key Laboratory of Basic Research in Cardio-Cerebral Vascular Diseases, Ningxia Medical University, Yinchuan (China); Department of Basic Medicine, Ningxia Medical University, Yinchuan (China)

2016-09-10

MicroRNAs (miRNAs) are short non-coding RNA and play crucial roles in a wide array of biological processes, including cell proliferation, differentiation and apoptosis. Our previous studies found that homocysteine(Hcy) can stimulate the proliferation of vascular smooth muscle cells (VSMCs), however, the underlying mechanisms were not fully elucidated. Here, we found proliferation of VSMCs induced by Hcy was of correspondence to the miR-125b expression reduced both in vitro and in the ApoE knockout mice, the hypermethylation of p53, its decreased expression, and DNA (cytosine-5)-methyltransferase 3b (DNMT3b) up-regulated. And, we found DNMT3b is a target of miR-125b, which was verified by the Dual-Luciferase reporter assay and western blotting. Besides, the siRNA interference for DNMT3b significantly decreased the methylation level of p53, which unveiled the causative role of DNMT3b in p53 hypermethylation. miR-125b transfection further confirmed its regulative roles on p53 gene methylation status and the VSMCs proliferation. Our data suggested that a miR-125b-DNMT3b-p53 signal pathway may exist in the VSMCs proliferation induced by Hcy.

miR-125b targets DNMT3b and mediates p53 DNA methylation involving in the vascular smooth muscle cells proliferation induced by homocysteine

International Nuclear Information System (INIS)

Cao, ChengJian; Zhang, HuiPing; Zhao, Li; Zhou, Longxia; Zhang, Minghao; Xu, Hua; Han, Xuebo; Li, Guizhong; Yang, Xiaoling; Jiang, YiDeng

2016-01-01

MicroRNAs (miRNAs) are short non-coding RNA and play crucial roles in a wide array of biological processes, including cell proliferation, differentiation and apoptosis. Our previous studies found that homocysteine(Hcy) can stimulate the proliferation of vascular smooth muscle cells (VSMCs), however, the underlying mechanisms were not fully elucidated. Here, we found proliferation of VSMCs induced by Hcy was of correspondence to the miR-125b expression reduced both in vitro and in the ApoE knockout mice, the hypermethylation of p53, its decreased expression, and DNA (cytosine-5)-methyltransferase 3b (DNMT3b) up-regulated. And, we found DNMT3b is a target of miR-125b, which was verified by the Dual-Luciferase reporter assay and western blotting. Besides, the siRNA interference for DNMT3b significantly decreased the methylation level of p53, which unveiled the causative role of DNMT3b in p53 hypermethylation. miR-125b transfection further confirmed its regulative roles on p53 gene methylation status and the VSMCs proliferation. Our data suggested that a miR-125b-DNMT3b-p53 signal pathway may exist in the VSMCs proliferation induced by Hcy.

Molecular mechanisms of adaptive response to alkylating agents in Escherichia coli and some remarks on O(6)-methylguanine DNA-methyltransferase in other organisms.

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Kleibl, Karol

2002-09-01

Alkylating agents are environmental genotoxic agents with mutagenic and carcinogenic potential, however, their properties are also exploited in the treatment of malignant diseases. O(6)-Methylguanine is an important adduct formed by methylating agents that, if not repaired, can lead to mutations and death. Its repair is carried out by O(6)-methylguanine DNA-methyltransferase (MTase) in an unique reaction in which methyl groups are transferred to the cysteine acceptor site of the protein itself. Exposure of Escherichia coli cells to sublethal concentrations of methylating agents triggers the expression of a set of genes, which allows the cells to tolerate DNA lesions, and this kind of inducible repair is called the adaptive response. The MTase of E. coli, encoded by the ada gene was the first MTase to be discovered and one of best characterised. Its repair and regulatory mechanisms are understood in considerable detail and this bacterial protein played a key role in identification of its counterparts in other living organisms. This review summarises the nature of alkylation damage in DNA and our current knowledge about the adaptive response in E. coli. I also include a brief mention of MTases from other organisms with the emphasis on the human MTase, which could play a crucial role in both cancer prevention and cancer treatment.

Chromosomal replication incompatibility in Dam methyltransferase deficient Escherichia coli cells

DEFF Research Database (Denmark)

Freiesleben, Ulrik Von

1996-01-01

Dam methyltransferase deficient Escherichia coli cells containing minichromosomes were constructed. Free plasmid DNA could not be detected in these cells and the minichromosomes were found to be integrated in multiple copies in the origin of replication (oriC) region of the host chromosome....... The absence of the initiation cascade in Dam- cells is proposed to account for this observation of apparent incompatibility between plasmid and chromosomal copies of oriC. Studies using oriC-pBR322 chimeric plasmids and their deletion derivatives indicated that the incompatibility determinant is an intact...

Cytosine Methylation Dysregulation in Neonates Following Intrauterine Growth Restriction

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Bhagat, Tushar D.; Fazzari, Melissa J.; Verma, Amit; Barzilai, Nir; Greally, John M.

2010-01-01

Background Perturbations of the intrauterine environment can affect fetal development during critical periods of plasticity, and can increase susceptibility to a number of age-related diseases (e.g., type 2 diabetes mellitus; T2DM), manifesting as late as decades later. We hypothesized that this biological memory is mediated by permanent alterations of the epigenome in stem cell populations, and focused our studies specifically on DNA methylation in CD34+ hematopoietic stem and progenitor cells from cord blood from neonates with intrauterine growth restriction (IUGR) and control subjects. Methods and Findings Our epigenomic assays utilized a two-stage design involving genome-wide discovery followed by quantitative, single-locus validation. We found that changes in cytosine methylation occur in response to IUGR of moderate degree and involving a restricted number of loci. We also identify specific loci that are targeted for dysregulation of DNA methylation, in particular the hepatocyte nuclear factor 4α (HNF4A) gene, a well-known diabetes candidate gene not previously associated with growth restriction in utero, and other loci encoding HNF4A-interacting proteins. Conclusions Our results give insights into the potential contribution of epigenomic dysregulation in mediating the long-term consequences of IUGR, and demonstrate the value of this approach to studies of the fetal origin of adult disease. PMID:20126273

5-Aza-2'-deoxycytidine in the medial prefrontal cortex regulates alcohol-related behavior and Ntf3-TrkC expression in rats.

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Xiaomeng Qiao

Full Text Available Recent studies have indicated that DNA methylation plays an important role in the development of alcohol abuse. 5-Aza-2'-deoxycytidine (5-Aza-dc, an inhibitor of DNA methyltransferases, was FDA approved for myelodysplastic syndrome treatment. However, it is unclear whether 5-Aza-dc is involved in alcohol abuse. In this study, using a chronic alcohol exposure model in rats, 5-Aza-dc was injected into the medial prefrontal cortex (mPFC. Alcohol-drinking behavior and the anxiety related behavior were evaluated by two-bottle choice and open field test. We found that 5-Aza-dc injection into the mPFC significantly decreased alcohol consumption and alcohol preference in alcohol-exposure rats, corresponding to the reduced blood alcohol levels. Although 5-Aza-dc potentiated the anxiety-like behavior of alcohol-exposure rats, it had no effect on the locomotor activity. Moreover, both of the mRNA and protein levels of DNA Methyltransferase 3A (DNMT3A and DNMT3B in the mPFC were upregulated after 35 days of alcohol exposure and this upregulation could be reversed by 5-Aza-dc treatment. Additionally, 5-Aza-dc reversed the alcohol-induced downregulation of neurotrophin-3 (Ntf3, correspondingly the expression of its receptor-TrkC was reduced. These findings identified a functional role of 5-Aza-dc in alcohol-related behavioral phenotypes and one of the potential target genes, Ntf3. We also provide novel evidence for DNA methyltransferases as potential therapeutic targets in alcohol abuse.

Epigenetic modulation upon exposure of lung fibroblasts to TiO2 and ZnO nanoparticles: alterations in DNA methylation

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Patil NA

2016-09-01

Full Text Available Nayana A Patil,1,2 WN Gade,2 Deepti D Deobagkar1 1Department of Zoology, Molecular Biology Research Laboratory, Centre of Advanced Studies, 2Department of Biotechnology, Proteomic Research Laboratory, Savitribai Phule Pune University, Pune, India Abstract: Titanium dioxide (TiO2 and zinc oxide (ZnO nanoparticles (NPs are promising candidates for numerous applications in consumer products. This will lead to increased human exposure, thus posing a threat to human health. Both these types of NPs have been studied for their cell toxicity, immunotoxicity, and genotoxicity. However, effects of these NPs on epigenetic modulations have not been studied. Epigenetics is an important link in the genotype and phenotype modulation and misregulation can often lead to lifestyle diseases. In this study, we have evaluated the DNA methylation-based epigenetic changes upon exposure to various concentrations of NPs. The investigation was designed to evaluate global DNA methylation, estimating the corresponding methyltransferase activity and expression of Dnmt gene using lung fibroblast (MRC5 cell line as lungs are the primary route of entry and target of occupational exposure to TiO2 and ZnO NPs. Enzyme-linked immunosorbent assay-based immunochemical assay revealed dose-related decrease in global DNA methylation and DNA methyltransferase activity. We also found direct correlation between the concentration of NPs, global methylation levels, and expression levels of Dnmt1, 3A, and 3B genes upon exposure. This is the first study to investigate effect of exposure to TiO2 and ZnO on DNA methylation levels in MRC5 cells. Epigenetic processes are known to play an important role in reprogramming and adaptation ability of an organism and can have long-term consequences. We suggest that changes in DNA methylation can serve as good biomarkers for early exposure to NPs since they occur at concentrations well below the sublethal levels. Our results demonstrate a clear

Anhydrous crystals of DNA bases are wide gap semiconductors.

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Maia, F F; Freire, V N; Caetano, E W S; Azevedo, D L; Sales, F A M; Albuquerque, E L

2011-05-07

We present the structural, electronic, and optical properties of anhydrous crystals of DNA nucleobases (guanine, adenine, cytosine, and thymine) found after DFT (Density Functional Theory) calculations within the local density approximation, as well as experimental measurements of optical absorption for powders of these crystals. Guanine and cytosine (adenine and thymine) anhydrous crystals are predicted from the DFT simulations to be direct (indirect) band gap semiconductors, with values 2.68 eV and 3.30 eV (2.83 eV and 3.22 eV), respectively, while the experimentally estimated band gaps we have measured are 3.83 eV and 3.84 eV (3.89 eV and 4.07 eV), in the same order. The electronic effective masses we have obtained at band extremes show that, at low temperatures, these crystals behave like wide gap semiconductors for electrons moving along the nucleobases stacking direction, while the hole transport are somewhat limited. Lastly, the calculated electronic dielectric functions of DNA nucleobases crystals in the parallel and perpendicular directions to the stacking planes exhibit a high degree of anisotropy (except cytosine), in agreement with published experimental results.

Ribonucleotides Linked to DNA of Herpes Simplex Virus Type 1

Science.gov (United States)

Hirsch, Ivan; Vonka, Vladimír

1974-01-01

Cells of a continuous cell line derived from rabbit embryo fibroblasts were infected with herpes simplex type 1 virus (HSV-1) and maintained in the presence of either [5-3H]uridine or [methyl-3H]thymidine or 32PO43−. Nucleocapsids were isolated from the cytoplasmic fraction, partially purified, and treated with DNase and RNase. From the pelleted nucleocapsids, DNA was extracted and purified by centrifugation in sucrose and cesium sulfate gradients. The acid-precipitable radioactivity of [5-3H]uridine-labeled DNA was partially susceptible to pancreatic RNase and alkaline treatment; the susceptibility to the enzyme decreased with increasing salt concentration. No drop of activity of DNA labeled with [3H]thymidine was observed either after RNase or alkali treatment. Base composition analysis of [5-3H]uridine-labeled DNA showed that the radioactivity was recovered as uracil and cytosine. In the cesium sulfate gradient, the purified [5-3H]uridine-labeled DNA banded at the same position as the 32P-labeled DNA. The present data tend to suggest that ribonucleotide sequences are present in HSV DNA, that they are covalently attached to the viral DNA, and that they can form double-stranded structures. PMID:4364894

High-resolution analysis of the 5'-end transcriptome using a next generation DNA sequencer.

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Shin-ichi Hashimoto

Full Text Available Massively parallel, tag-based sequencing systems, such as the SOLiD system, hold the promise of revolutionizing the study of whole genome gene expression due to the number of data points that can be generated in a simple and cost-effective manner. We describe the development of a 5'-end transcriptome workflow for the SOLiD system and demonstrate the advantages in sensitivity and dynamic range offered by this tag-based application over traditional approaches for the study of whole genome gene expression. 5'-end transcriptome analysis was used to study whole genome gene expression within a colon cancer cell line, HT-29, treated with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5Aza. More than 20 million 25-base 5'-end tags were obtained from untreated and 5Aza-treated cells and matched to sequences within the human genome. Seventy three percent of the mapped unique tags were associated with RefSeq cDNA sequences, corresponding to approximately 14,000 different protein-coding genes in this single cell type. The level of expression of these genes ranged from 0.02 to 4,704 transcripts per cell. The sensitivity of a single sequence run of the SOLiD platform was 100-1,000 fold greater than that observed from 5'end SAGE data generated from the analysis of 70,000 tags obtained by Sanger sequencing. The high-resolution 5'end gene expression profiling presented in this study will not only provide novel insight into the transcriptional machinery but should also serve as a basis for a better understanding of cell biology.

O6-alkylguanine-DNA alkyltransferase activity correlates with the therapeutic response of human rhabdomyosarcoma xenografts to 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea

International Nuclear Information System (INIS)

Brent, T.P.; Houghton, P.J.; Houghton, J.A.

1985-01-01

Immune-deprived female CBA/CaJ mice bearing xenografts of six different human rhabdomyosarcoma lines were treated with 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (MeCCNU). Tumor responses were compared with levels of O 6 -methylguanine-DNA methyltransferase activity because of evidence indicating that repair of DNA interstrand cross-link precursors, mediated by the transferase, may be an important determinant of MeCCNU cytotoxicity. Levels of methyltransferase in tumor extracts were measured by determining the loss of O 6 -methylguanine from 3 H-labeled methylated DNA. Five of the six tumor lines examined showed either no response to MeCCNU or regrowth after an incomplete response. In each instance, the extent of tumor regression correlated with the level of O 6 -methylguanine-DNA methyltransferase activity in tumor extracts. These results suggest that O 6 -methylguanine-DNA methyltransferase levels in human tumor cells may be a clinically useful predictor of sensitivity to the chloroethylnitrosoureas

Lysine methyltransferase G9a is not required for DNMT3A/3B anchoring to methylated nucleosomes and maintenance of DNA methylation in somatic cells

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Sharma Shikhar

2012-01-01

Full Text Available Abstract Background DNA methylation, histone modifications and nucleosome occupancy act in concert for regulation of gene expression patterns in mammalian cells. Recently, G9a, a H3K9 methyltransferase, has been shown to play a role in establishment of DNA methylation at embryonic gene targets in ES cells through recruitment of de novo DNMT3A/3B enzymes. However, whether G9a plays a similar role in maintenance of DNA methylation in somatic cells is still unclear. Results Here we show that G9a is not essential for maintenance of DNA methylation in somatic cells. Knockdown of G9a has no measurable effect on DNA methylation levels at G9a-target loci. DNMT3A/3B remain stably anchored to nucleosomes containing methylated DNA even in the absence of G9a, ensuring faithful propagation of methylated states in cooperation with DNMT1 through somatic divisions. Moreover, G9a also associates with nucleosomes in a DNMT3A/3B and DNA methylation-independent manner. However, G9a knockdown synergizes with pharmacologic inhibition of DNMTs resulting in increased hypomethylation and inhibition of cell proliferation. Conclusions Taken together, these data suggest that G9a is not involved in maintenance of DNA methylation in somatic cells but might play a role in re-initiation of de novo methylation after treatment with hypomethylating drugs, thus serving as a potential target for combinatorial treatments strategies involving DNMTs inhibitors.

The DNA methyltransferase inhibitor zebularine exerts antitumor effects and reveals BATF2 as a poor prognostic marker for childhood medulloblastoma.

Science.gov (United States)

Andrade, Augusto Faria; Borges, Kleiton Silva; Suazo, Veridiana Kiill; Geron, Lenisa; Corrêa, Carolina Alves Pereira; Castro-Gamero, Angel Mauricio; de Vasconcelos, Elton José Rosas; de Oliveira, Ricardo Santos; Neder, Luciano; Yunes, José Andres; Dos Santos Aguiar, Simone; Scrideli, Carlos Alberto; Tone, Luiz Gonzaga

2017-02-01

Medulloblastoma (MB) is the most common solid tumor among pediatric patients and corresponds to 20 % of all pediatric intracranial tumors in this age group. Its treatment currently involves significant side effects. Epigenetic changes such as DNA methylation may contribute to its development and progression. DNA methyltransferase (DNMT) inhibitors have shown promising anticancer effects. The agent Zebularine acts as an inhibitor of DNA methylation and shows low toxicity and high efficacy, being a promising adjuvant agent for anti-cancer chemotherapy. Several studies have reported its effects on different types of tumors; however, there are no studies reporting its effects on MB. We analyzed its potential anticancer effects in four pediatric MB cell lines. The treatment inhibited proliferation and clonogenicity, increased the apoptosis rate and the number of cells in the S phase (p < 0.05), as well as the expression of p53, p21, and Bax, and decreased cyclin A, Survivin and Bcl-2 proteins. In addition, the combination of zebularine with the chemotherapeutic agents vincristine and cisplatin resulted in synergism and antagonism, respectively. Zebularine also modulated the activation of the SHH pathway, reducing SMO and GLI1 levels and one of its targets, PTCH1, without changing SUFU levels. A microarray analysis revealed different pathways modulated by the drug, including the Toll-Like Receptor pathway and high levels of the BATF2 gene. The low expression of this gene was associated with a worse prognosis in MB. Taken together, these data suggest that Zebularine may be a potential drug for further in vivo studies of MB treatment.

Pleiotropic phenotypes of the salt-tolerant and cytosine ...

Indian Academy of Sciences (India)

2SKA Institution for Research, Education and Development (SKAIRED), 4/11 Sarv Priya Vihar, ... wild-type parent cv Nirmal were characterized for overall cytosine methylation at .... (ii) altered in the expression of genes involved in the perfor-.

Mitochondrial DNA: A Blind Spot in Neuroepigenetics.

Science.gov (United States)

Manev, Hari; Dzitoyeva, Svetlana; Chen, Hu

2012-04-01

Neuroepigenetics, which includes nuclear DNA modifications such as 5-methylcytosine and 5-hydoxymethylcytosine and modifications of nuclear proteins such as histones, is emerging as the leading field in molecular neuroscience. Historically, a functional role for epigenetic mechanisms, including in neuroepigenetics, has been sought in the area of the regulation of nuclear transcription. However, one important compartment of mammalian cell DNA, different from nuclear but equally important for physiological and pathological processes (including in the brain), mitochondrial DNA has for the most part not had a systematic epigenetic characterization. The importance of mitochondria and mitochondrial DNA (particularly its mutations) in central nervous system physiology and pathology has long been recognized. Only recently have mechanisms of mitochondrial DNA methylation and hydroxymethylation, including the discovery of mitochondrial DNA-methyltransferases and the presence and the functionality of 5-methylcytosine and 5-hydroxymethylcytosine in mitochondrial DNA (e.g., in modifying the transcription of mitochondrial genome), been unequivocally recognized as a part of mammalian mitochondrial physiology. Here we summarize for the first time evidence supporting the existence of these mechanisms and we propose the term "mitochondrial epigenetics" to be used when referring to them. Currently, neuroepigenetics does not include mitochondrial epigenetics - a gap that we expect to close in the near future.

Silver (I) as DNA glue: Ag+-mediated guanine pairing revealed by removing Watson-Crick constraints

Science.gov (United States)

Swasey, Steven M.; Leal, Leonardo Espinosa; Lopez-Acevedo, Olga; Pavlovich, James; Gwinn, Elisabeth G.

2015-01-01

Metal ion interactions with DNA have far-reaching implications in biochemistry and DNA nanotechnology. Ag+ is uniquely interesting because it binds exclusively to the bases rather than the backbone of DNA, without the toxicity of Hg2+. In contrast to prior studies of Ag+ incorporation into double-stranded DNA, we remove the constraints of Watson-Crick pairing by focusing on homo-base DNA oligomers of the canonical bases. High resolution electro-spray ionization mass spectrometry reveals an unanticipated Ag+-mediated pairing of guanine homo-base strands, with higher stability than canonical guanine-cytosine pairing. By exploring unrestricted binding geometries, quantum chemical calculations find that Ag+ bridges between non-canonical sites on guanine bases. Circular dichroism spectroscopy shows that the Ag+-mediated structuring of guanine homobase strands persists to at least 90 °C under conditions for which canonical guanine-cytosine duplexes melt below 20 °C. These findings are promising for DNA nanotechnology and metal-ion based biomedical science. PMID:25973536

Silver (I) as DNA glue: Ag(+)-mediated guanine pairing revealed by removing Watson-Crick constraints.

Science.gov (United States)

Swasey, Steven M; Leal, Leonardo Espinosa; Lopez-Acevedo, Olga; Pavlovich, James; Gwinn, Elisabeth G

2015-05-14

Metal ion interactions with DNA have far-reaching implications in biochemistry and DNA nanotechnology. Ag(+) is uniquely interesting because it binds exclusively to the bases rather than the backbone of DNA, without the toxicity of Hg(2+). In contrast to prior studies of Ag(+) incorporation into double-stranded DNA, we remove the constraints of Watson-Crick pairing by focusing on homo-base DNA oligomers of the canonical bases. High resolution electro-spray ionization mass spectrometry reveals an unanticipated Ag(+)-mediated pairing of guanine homo-base strands, with higher stability than canonical guanine-cytosine pairing. By exploring unrestricted binding geometries, quantum chemical calculations find that Ag(+) bridges between non-canonical sites on guanine bases. Circular dichroism spectroscopy shows that the Ag(+)-mediated structuring of guanine homobase strands persists to at least 90 °C under conditions for which canonical guanine-cytosine duplexes melt below 20 °C. These findings are promising for DNA nanotechnology and metal-ion based biomedical science.

Silver (I) as DNA glue: Ag+-mediated guanine pairing revealed by removing Watson-Crick constraints

Science.gov (United States)

Swasey, Steven M.; Leal, Leonardo Espinosa; Lopez-Acevedo, Olga; Pavlovich, James; Gwinn, Elisabeth G.

2015-05-01

Metal ion interactions with DNA have far-reaching implications in biochemistry and DNA nanotechnology. Ag+ is uniquely interesting because it binds exclusively to the bases rather than the backbone of DNA, without the toxicity of Hg2+. In contrast to prior studies of Ag+ incorporation into double-stranded DNA, we remove the constraints of Watson-Crick pairing by focusing on homo-base DNA oligomers of the canonical bases. High resolution electro-spray ionization mass spectrometry reveals an unanticipated Ag+-mediated pairing of guanine homo-base strands, with higher stability than canonical guanine-cytosine pairing. By exploring unrestricted binding geometries, quantum chemical calculations find that Ag+ bridges between non-canonical sites on guanine bases. Circular dichroism spectroscopy shows that the Ag+-mediated structuring of guanine homobase strands persists to at least 90 °C under conditions for which canonical guanine-cytosine duplexes melt below 20 °C. These findings are promising for DNA nanotechnology and metal-ion based biomedical science.

A contracted DNA repeat in LHX3 intron 5 is associated with aberrant splicing and pituitary dwarfism in German shepherd dogs.

Directory of Open Access Journals (Sweden)

Annemarie M W Y Voorbij

Full Text Available Dwarfism in German shepherd dogs is due to combined pituitary hormone deficiency of unknown genetic cause. We localized the recessively inherited defect by a genome wide approach to a region on chromosome 9 with a lod score of 9.8. The region contains LHX3, which codes for a transcription factor essential for pituitary development. Dwarfs have a deletion of one of six 7 bp repeats in intron 5 of LHX3, reducing the intron size to 68 bp. One dwarf was compound heterozygous for the deletion and an insertion of an asparagine residue in the DNA-binding homeodomain of LHX3, suggesting involvement of the gene in the disorder. An exon trapping assay indicated that the shortened intron is not spliced efficiently, probably because it is too small. We applied bisulfite conversion of cytosine to uracil in RNA followed by RT-PCR to analyze the splicing products. The aberrantly spliced RNA molecules resulted from either skipping of exon 5 or retention of intron 5. The same splicing defects were observed in cDNA derived from the pituitary of dwarfs. A survey of similarly mutated introns suggests that there is a minimal distance requirement between the splice donor and branch site of 50 nucleotides. In conclusion, a contraction of a DNA repeat in intron 5 of canine LHX3 leads to deficient splicing and is associated with pituitary dwarfism.

ORF Alignment: NC_002162 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available NC_002162 gi|13358092 >6mhtA 61 323 7 298 3e-31 ... ref|NP_078366.1| cytosine-specifi...ic ... methyltransferase [Ureaplasma parvum serovar 3 str. ATCC ... 700970] pir||D82880 cytosi...ne-specific methyltransferase ... UU528 [imported] - Ureaplasma urealyticu...FGMKKGSGTRSGLLWEIERILFDLQKLNQ 60 ... Query: 127 LNYDGLIDSNGNILDLEAPIFDGKQKQLKDVLKTNY...KIEKYYQEAYLAQPNRTFSRIKYV 186 ... LNYDGLIDSNGNILDLEAPIFDGKQKQLKDVLKTNYKIEKYYQEAYLAQPNRTFSRIKYV Sbjct: 121 LNYDGLIDSNGNILDLEAP

Distribution of the type III DNA methyltransferases modA, modB and modD among Neisseria meningitidis genotypes: implications for gene regulation and virulence.

Science.gov (United States)

Tan, Aimee; Hill, Dorothea M C; Harrison, Odile B; Srikhanta, Yogitha N; Jennings, Michael P; Maiden, Martin C J; Seib, Kate L

2016-02-12

Neisseria meningitidis is a human-specific bacterium that varies in invasive potential. All meningococci are carried in the nasopharynx, and most genotypes are very infrequently associated with invasive meningococcal disease; however, those belonging to the 'hyperinvasive lineages' are more frequently associated with sepsis or meningitis. Genome content is highly conserved between carriage and disease isolates, and differential gene expression has been proposed as a major determinant of the hyperinvasive phenotype. Three phase variable DNA methyltransferases (ModA, ModB and ModD), which mediate epigenetic regulation of distinct phase variable regulons (phasevarions), have been identified in N. meningitidis. Each mod gene has distinct alleles, defined by their Mod DNA recognition domain, and these target and methylate different DNA sequences, thereby regulating distinct gene sets. Here 211 meningococcal carriage and >1,400 disease isolates were surveyed for the distribution of meningococcal mod alleles. While modA11-12 and modB1-2 were found in most isolates, rarer alleles (e.g., modA15, modB4, modD1-6) were specific to particular genotypes as defined by clonal complex. This suggests that phase variable Mod proteins may be associated with distinct phenotypes and hence invasive potential of N. meningitidis strains.

Environmentally sensitive molecular probes reveal mutations and epigenetic 5-methyl cytosine in human oncogenes

DEFF Research Database (Denmark)

Taskova, M.; Barducci, M. C.; Astakhova, K.

2017-01-01

There is currently an unmet need for reliable tools that allow for direct detection and quantification of modifications in genomic DNA. For example, in cancer research and clinical diagnostics, target DNA has to be amplified and sequenced in order to reveal mutations. For 5-methylcytosine detecti...

A novel multifunctional O-methyltransferase implicated in a dual methylation pathway associated with lignin biosynthesis in loblolly pine.

Science.gov (United States)

Li, L; Popko, J L; Zhang, X H; Osakabe, K; Tsai, C J; Joshi, C P; Chiang, V L

1997-05-13

S-adenosyl-L-methionine (SAM)-dependent O-methyltransferases (OMTs) catalyze the methylation of hydroxycinnamic acid derivatives for the synthesis of methylated plant polyphenolics, including lignin. The distinction in the extent of methylation of lignins in angiosperms and gymnosperms, mediated by substrate-specific OMTs, represents one of the fundamental differences in lignin biosynthesis between these two classes of plants. In angiosperms, two types of structurally and functionally distinct lignin pathway OMTs, caffeic acid 3-O-methyltransferases (CAOMTs) and caffeoyl CoA 3-O-methyltransferases (CCoAOMTs), have been reported and extensively studied. However, little is known about lignin pathway OMTs in gymnosperms. We report here the first cloning of a loblolly pine (Pinus taeda) xylem cDNA encoding a multifunctional enzyme, SAM:hydroxycinnamic Acids/hydroxycinnamoyl CoA Esters OMT (AEOMT). The deduced protein sequence of AEOMT is partially similar to, but clearly distinguishable from, that of CAOMTs and does not exhibit any significant similarity with CCoAOMT protein sequences. However, functionally, yeast-expressed AEOMT enzyme catalyzed the methylation of CAOMT substrates, caffeic and 5-hydroxyferulic acids, as well as CCoAOMT substrates, caffeoyl CoA and 5-hydroxyferuloyl CoA esters, with similar specific activities and was completely inactive with substrates associated with flavonoid synthesis. The lignin-related substrates were also efficiently methylated in crude extracts of loblolly pine secondary xylem. Our results support the notion that, in the context of amino acid sequence and biochemical function, AEOMT represents a novel SAM-dependent OMT, with both CAOMT and CCoAOMT activities and thus the potential to mediate a dual methylation pathway in lignin biosynthesis in loblolly pine xylem.

A novel multifunctional O-methyltransferase implicated in a dual methylation pathway associated with lignin biosynthesis in loblolly pine

Science.gov (United States)

Li, Laigeng; Popko, Jacqueline L.; Zhang, Xing-Hai; Osakabe, Keishi; Tsai, Chung-Jui; Joshi, Chandrashekhar P.; Chiang, Vincent L.

1997-01-01

S-adenosyl-l-methionine (SAM)-dependent O-methyltransferases (OMTs) catalyze the methylation of hydroxycinnamic acid derivatives for the synthesis of methylated plant polyphenolics, including lignin. The distinction in the extent of methylation of lignins in angiosperms and gymnosperms, mediated by substrate-specific OMTs, represents one of the fundamental differences in lignin biosynthesis between these two classes of plants. In angiosperms, two types of structurally and functionally distinct lignin pathway OMTs, caffeic acid 3-O-methyltransferases (CAOMTs) and caffeoyl CoA 3-O-methyltransferases (CCoAOMTs), have been reported and extensively studied. However, little is known about lignin pathway OMTs in gymnosperms. We report here the first cloning of a loblolly pine (Pinus taeda) xylem cDNA encoding a multifunctional enzyme, SAM:hydroxycinnamic Acids/hydroxycinnamoyl CoA Esters OMT (AEOMT). The deduced protein sequence of AEOMT is partially similar to, but clearly distinguishable from, that of CAOMTs and does not exhibit any significant similarity with CCoAOMT protein sequences. However, functionally, yeast-expressed AEOMT enzyme catalyzed the methylation of CAOMT substrates, caffeic and 5-hydroxyferulic acids, as well as CCoAOMT substrates, caffeoyl CoA and 5-hydroxyferuloyl CoA esters, with similar specific activities and was completely inactive with substrates associated with flavonoid synthesis. The lignin-related substrates were also efficiently methylated in crude extracts of loblolly pine secondary xylem. Our results support the notion that, in the context of amino acid sequence and biochemical function, AEOMT represents a novel SAM-dependent OMT, with both CAOMT and CCoAOMT activities and thus the potential to mediate a dual methylation pathway in lignin biosynthesis in loblolly pine xylem. PMID:9144260

Functions that Protect Escherichia coli from Tightly Bound DNA-Protein Complexes Created by Mutant EcoRII Methyltransferase.

Science.gov (United States)

Henderson, Morgan L; Kreuzer, Kenneth N

2015-01-01

Expression of mutant EcoRII methyltransferase protein (M.EcoRII-C186A) in Escherichia coli leads to tightly bound DNA-protein complexes (TBCs), located sporadically on the chromosome rather than in tandem arrays. The mechanisms behind the lethality induced by such sporadic TBCs are not well studied, nor is it clear whether very tight binding but non-covalent complexes are processed in the same way as covalent DNA-protein crosslinks (DPCs). Using 2D gel electrophoresis, we found that TBCs induced by M.EcoRII-C186A block replication forks in vivo. Specific bubble molecules were detected as spots on the 2D gel, only when M.EcoRII-C186A was induced, and a mutation that eliminates a specific EcoRII methylation site led to disappearance of the corresponding spot. We also performed a candidate gene screen for mutants that are hypersensitive to TBCs induced by M.EcoRII-C186A. We found several gene products necessary for protection against these TBCs that are known to also protect against DPCs induced with wild-type M.EcoRII (after 5-azacytidine incorporation): RecA, RecBC, RecG, RuvABC, UvrD, FtsK, XerCD and SsrA (tmRNA). In contrast, the RecFOR pathway and Rep helicase are needed for protection against TBCs but not DPCs induced by M.EcoRII. We propose that stalled fork processing by RecFOR and RecA promotes release of tightly bound (but non-covalent) blocking proteins, perhaps by licensing Rep helicase-driven dissociation of the blocking M.EcoRII-C186A. Our studies also argued against the involvement of several proteins that might be expected to protect against TBCs. We took the opportunity to directly compare the sensitivity of all tested mutants to two quinolone antibiotics, which target bacterial type II topoisomerases and induce a unique form of DPC. We uncovered rep, ftsK and xerCD as novel quinolone hypersensitive mutants, and also obtained evidence against the involvement of a number of functions that might be expected to protect against quinolones.

Functions that Protect Escherichia coli from Tightly Bound DNA-Protein Complexes Created by Mutant EcoRII Methyltransferase.

Directory of Open Access Journals (Sweden)

Morgan L Henderson

Full Text Available Expression of mutant EcoRII methyltransferase protein (M.EcoRII-C186A in Escherichia coli leads to tightly bound DNA-protein complexes (TBCs, located sporadically on the chromosome rather than in tandem arrays. The mechanisms behind the lethality induced by such sporadic TBCs are not well studied, nor is it clear whether very tight binding but non-covalent complexes are processed in the same way as covalent DNA-protein crosslinks (DPCs. Using 2D gel electrophoresis, we found that TBCs induced by M.EcoRII-C186A block replication forks in vivo. Specific bubble molecules were detected as spots on the 2D gel, only when M.EcoRII-C186A was induced, and a mutation that eliminates a specific EcoRII methylation site led to disappearance of the corresponding spot. We also performed a candidate gene screen for mutants that are hypersensitive to TBCs induced by M.EcoRII-C186A. We found several gene products necessary for protection against these TBCs that are known to also protect against DPCs induced with wild-type M.EcoRII (after 5-azacytidine incorporation: RecA, RecBC, RecG, RuvABC, UvrD, FtsK, XerCD and SsrA (tmRNA. In contrast, the RecFOR pathway and Rep helicase are needed for protection against TBCs but not DPCs induced by M.EcoRII. We propose that stalled fork processing by RecFOR and RecA promotes release of tightly bound (but non-covalent blocking proteins, perhaps by licensing Rep helicase-driven dissociation of the blocking M.EcoRII-C186A. Our studies also argued against the involvement of several proteins that might be expected to protect against TBCs. We took the opportunity to directly compare the sensitivity of all tested mutants to two quinolone antibiotics, which target bacterial type II topoisomerases and induce a unique form of DPC. We uncovered rep, ftsK and xerCD as novel quinolone hypersensitive mutants, and also obtained evidence against the involvement of a number of functions that might be expected to protect against quinolones.

Protection of hematopoietic cells from O(6)-alkylation damage by O(6)-methylguanine DNA methyltransferase gene transfer: studies with different O(6)-alkylating agents and retroviral backbones.

Science.gov (United States)

Jansen, M; Bardenheuer, W; Sorg, U R; Seeber, S; Flasshove, M; Moritz, T

2001-07-01

Overexpression of O(6)-methylguanine DNA methyltransferase (MGMT) can protect hematopoietic cells from O(6)-alkylation damage. To identify possible clinical applications of this technology we compared the effect of MGMT gene transfer on the hematotoxicity induced by different O(6)-alkylating agents in clinical use: the chloroethylnitrosoureas ACNU, BCNU, CCNU and the tetrazine derivative temozolomide. In addition, various retroviral vectors expressing the MGMT-cDNA were investigated to identify optimal viral backbones for hematoprotection by MGMT expression. Protection from ACNU, BCNU, CCNU or temozolomide toxicity was evaluated utilizing a Moloney murine leukemia virus-based retroviral vector (N2/Zip-PGK-MGMT) to transduce primary murine bone marrow cells. Increased resistance in murine colony-forming units (CFU) was demonstrated for all four drugs. In comparison to mock-transduced controls, after transduction with N2/Zip-PGK-MGMT the IC50 for CFU increased on average 4.7-fold for ACNU, 2.5-fold for BCNU, 6.3-fold for CCNU and 1.5-fold for temozolomide. To study the effect of the retroviral backbone on hematoprotection various vectors expressing the human MGMT-cDNA from a murine embryonic sarcoma virus LTR (MSCV-MGMT) or a hybrid spleen focus-forming/murine embryonic sarcoma virus LTR (SF1-MGMT) were compared with the N2/Zip-PGK-MGMT vector. While all vectors increased resistance of transduced human CFU to ACNU, the SF1-MGMT construct was most efficient especially at high ACNU concentrations (8-12 microg/ml). Similar results were obtained for protection of murine high-proliferative-potential colony-forming cells. These data may help to optimize treatment design and retroviral constructs in future clinical studies aiming at hematoprotection by MGMT gene transfer.

Erasure of Tet-Oxidized 5-Methylcytosine by a SRAP Nuclease

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Soo-Mi Kweon

2017-10-01

Full Text Available Enzymatic oxidation of 5-methylcytosine (5mC in DNA by the Tet dioxygenases reprograms genome function in embryogenesis and postnatal development. Tet-oxidized derivatives of 5mC such as 5-hydroxymethylcytosine (5hmC act as transient intermediates in DNA demethylation or persist as durable marks, yet how these alternative fates are specified at individual CpGs is not understood. Here, we report that the SOS response-associated peptidase (SRAP domain protein Srap1, the mammalian ortholog of an ancient protein superfamily associated with DNA damage response operons in bacteria, binds to Tet-oxidized forms of 5mC in DNA and catalyzes turnover of these bases to unmodified cytosine by an autopeptidase-coupled nuclease. Biallelic inactivation of murine Srap1 causes embryonic sublethality associated with widespread accumulation of ectopic 5hmC. These findings establish a function for a class of DNA base modification-selective nucleases and position Srap1 as a determinant of 5mC demethylation trajectories during mammalian embryonic development.

Different Binding Properties and Function of CXXC Zinc Finger Domains in Dnmt1 and Tet1

Science.gov (United States)

Meilinger, Daniela; Bultmann, Sebastian; Fellinger, Karin; Hasenöder, Stefan; Wang, Mengxi; Qin, Weihua; Söding, Johannes; Spada, Fabio; Leonhardt, Heinrich

2011-01-01

Several mammalian proteins involved in chromatin and DNA modification contain CXXC zinc finger domains. We compared the structure and function of the CXXC domains in the DNA methyltransferase Dnmt1 and the methylcytosine dioxygenase Tet1. Sequence alignment showed that both CXXC domains have a very similar framework but differ in the central tip region. Based on the known structure of a similar MLL1 domain we developed homology models and designed expression constructs for the isolated CXXC domains of Dnmt1 and Tet1 accordingly. We show that the CXXC domain of Tet1 has no DNA binding activity and is dispensable for catalytic activity in vivo. In contrast, the CXXC domain of Dnmt1 selectively binds DNA substrates containing unmethylated CpG sites. Surprisingly, a Dnmt1 mutant construct lacking the CXXC domain formed covalent complexes with cytosine bases both in vitro and in vivo and rescued DNA methylation patterns in dnmt1−/− embryonic stem cells (ESCs) just as efficiently as wild type Dnmt1. Interestingly, neither wild type nor ΔCXXC Dnmt1 re-methylated imprinted CpG sites of the H19a promoter in dnmt1−/− ESCs, arguing against a role of the CXXC domain in restraining Dnmt1 methyltransferase activity on unmethylated CpG sites. PMID:21311766

Different binding properties and function of CXXC zinc finger domains in Dnmt1 and Tet1.

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Carina Frauer

2011-02-01

Full Text Available Several mammalian proteins involved in chromatin and DNA modification contain CXXC zinc finger domains. We compared the structure and function of the CXXC domains in the DNA methyltransferase Dnmt1 and the methylcytosine dioxygenase Tet1. Sequence alignment showed that both CXXC domains have a very similar framework but differ in the central tip region. Based on the known structure of a similar MLL1 domain we developed homology models and designed expression constructs for the isolated CXXC domains of Dnmt1 and Tet1 accordingly. We show that the CXXC domain of Tet1 has no DNA binding activity and is dispensable for catalytic activity in vivo. In contrast, the CXXC domain of Dnmt1 selectively binds DNA substrates containing unmethylated CpG sites. Surprisingly, a Dnmt1 mutant construct lacking the CXXC domain formed covalent complexes with cytosine bases both in vitro and in vivo and rescued DNA methylation patterns in dnmt1⁻/⁻ embryonic stem cells (ESCs just as efficiently as wild type Dnmt1. Interestingly, neither wild type nor ΔCXXC Dnmt1 re-methylated imprinted CpG sites of the H19a promoter in dnmt1⁻/⁻ ESCs, arguing against a role of the CXXC domain in restraining Dnmt1 methyltransferase activity on unmethylated CpG sites.

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.

Evaluation of oxidative DNA damage promoted by storage in sperm from sex-reversed rainbow trout.

Science.gov (United States)

Pérez-Cerezales, S; Martínez-Páramo, S; Cabrita, E; Martínez-Pastor, F; de Paz, P; Herráez, M P

2009-03-01

Short-term storage and cryopreservation of sperm are two common procedures in aquaculture, used for routine practices in artificial insemination reproduction and gene banking, respectively. Nevertheless, both procedures cause injuries affecting sperm motility, viability, cell structure and DNA stability, which diminish reproductive success. DNA modification is considered extremely important, especially when sperm storage is carried out with gene banking purposes. DNA damage caused by sperm storage is not well characterized and previous studies have reported simple and double strand breaks that have been attributed to oxidative events promoted by the generation of free radicals during storage. The objective of this study was to reveal DNA fragmentation and to explore the presence of oxidized bases that could be produced by oxidative events during short-term storage and cryopreservation in sex-reversed rainbow trout (Oncorhynchus mykiss) spermatozoa. Sperm from six males was analyzed separately. Different aliquots of the samples were stored 2h (fresh) or 5 days at 4 degrees C or were cryopreserved. Then spermatozoa were analyzed using the Comet assay, as well as combining this method with digestion with two endonucleases from Escherichia coli (Endonuclease III, that cut in oxidized cytosines, and FPG, cutting in oxidized guanosines). Both storage procedures yielded DNA fragmentation, but only short-term storage oxidative events were clearly detected, showing that oxidative processes affect guanosines rather than cytosines. Cryopreservation increases DNA fragmentation but the presence of oxidized bases was not noticed, suggesting that mechanisms other than oxidative stress could be involved in DNA fragmentation promoted by freezing.

Determination of oxidation products of 5-methylcytosine in plants by chemical derivatization coupled with liquid chromatography/tandem mass spectrometry analysis.

Science.gov (United States)

Tang, Yang; Xiong, Jun; Jiang, Han-Peng; Zheng, Shu-Jian; Feng, Yu-Qi; Yuan, Bi-Feng

2014-08-05

Cytosine methylation (5-methylcytosine, 5-mC) in DNA is an important epigenetic mark that has regulatory roles in various biological processes. In plants, active DNA demethylation can be achieved through direct cleavage by DNA glycosylases, followed by replacement of 5-mC with cytosine by base excision repair (BER) machinery. Recent studies in mammals have demonstrated 5-mC can be sequentially oxidized to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-foC), and 5-carboxylcytosine (5-caC) by Ten-eleven translocation (TET) proteins. The consecutive oxidations of 5-mC constitute the active DNA demethylation pathway in mammals, which raised the possible presence of oxidation products of 5-mC (5-hmC, 5-foC, and 5-caC) in plant genomes. However, there is no definitive evidence supporting the presence of these modified bases in plant genomic DNA, especially for 5-foC and 5-caC. Here we developed a chemical derivatization strategy combined with liquid chromatography-electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method to determine 5-formyl-2'-deoxycytidine (5-fodC) and 5-carboxyl-2'-deoxycytidine (5-cadC). Derivatization of 5-fodC and 5-cadC by Girard's reagents (GirD, GirT, and GirP) significantly increased the detection sensitivities of 5-fodC and 5-cadC by 52-260-fold. Using this method, we demonstrated the widespread existence of 5-fodC and 5-cadC in genomic DNA of various plant tissues, indicating that active DNA demethylation in plants may go through an alternative pathway similar to mammals besides the pathway of direct DNA glycosylases cleavage combined with BER. Moreover, we found that environmental stresses of drought and salinity can change the contents of 5-fodC and 5-cadC in plant genomes, suggesting the functional roles of 5-fodC and 5-cadC in response to environmental stresses.

DNA Methylation: A Frontier in Tooth Organogenesis and Developmental Dental Defects.

Science.gov (United States)

Wan, Mian; Li, Hongyu; Zhou, Yachuan; Du, Wei; Xu, Xin; Ye, Ling; Zhou, Xuedong; Zheng, Liwei

2018-01-01

Tooth development relies on interactions between epithelial and mesenchymal tissues, which are controlled by sophisticated networks of conserved signaling. The signaling networks regulating odontogenesis have been well characterized, but the epigenetic mechanisms underlying remain to be elucidated. In this review, we describe current researches regarding the control of various genes expression by DNA methylation during odontogenesis, summarize genomic mapping of DNA methylation in various stages of tooth formation and diverse dental tissues by high-throughput approaches, and highlight the roles of DNA methylation in odontogenesis. Researches on mammals have revealed that the genomic methylation, which occurs on cytosine residues, regulates certain genes transcription. Consequently, DNA methylation plays a crucial role in spatiotemporal organization of signaling pathways, and is essential for organogenesis. Recently, mounting evidence proves that methylation of genomes contributes to the spatiotemporal gene dynamics during odontogenesis. With emerging new technologies of mapping cytosine modifications in global genome, investigators are seeking an overall view of DNA methylome dynamics that characterize genetic information to manifest across incredibly varied tooth development stages, dental tissues, and developmental dental defects. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Towards an understanding of CG methylation in DNA transcription

International Nuclear Information System (INIS)

Chela-Flores, J.; Migoni, R.L.

1989-09-01

A simple model of DNA is considered in which the nucleotides cytosine (C) and guanine (G) are not assumed to be identical, and in which macroscopic thermodynamic quantities may be calculated exactly. The H bonds between the C and G nucleotides are assumed to be Morse potentials. We discuss the statistical mechanics of the DNA molecule in the configuration (5'...GGG...3'; 3'...CCC...5'), which may be copied by RNA polymerase into a messenger RNA (mRNA) strand (5'...CCC...3'). This model suggests that replacements of C by 5-methylcytosine (5mC) may be a secondary effect in the inhibition of genetic expression, not interfering directly with the formation of an open state. An experimental test is suggested. The implications of this result are discussed for a related system, in which the enzyme methylase is known to methylate almost exclusively those Cs that are followed by Gs as a regulatory strategy employed by some eukaryotes. (author). 14 refs, 2 figs

Identification of a Novel Protein Arginine Methyltransferase 5 Inhibitor in Non-small Cell Lung Cancer by Structure-Based Virtual Screening

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Qianqian Wang

2018-03-01

Full Text Available Protein arginine methyltransferase 5 (PRMT5 is able to regulate gene transcription by catalyzing the symmetrical dimethylation of arginine residue of histone, which plays a key role in tumorigenesis. Many efforts have been taken in discovering small-molecular inhibitors against PRMT5, but very few were reported and most of them were SAM-competitive. EPZ015666 is a recently reported PRMT5 inhibitor with a new binding site, which is different from S-adenosylmethionine (SAM-binding pocket. This new binding site provides a new clue for the design and discovery of potent and specific PRMT5 inhibitors. In this study, the structure-based virtual screening targeting this site was firstly performed to identify potential PRMT5 inhibitors. Then, the bioactivity of the candidate compound was studied. MTT results showed that compound T1551 decreased cell viability of A549 and H460 non-small cell lung cancer cell lines. By inhibiting the methyltransferase activity of PRMT5, T1551 reduced the global level of H4R3 symmetric dimethylation (H4R3me2s. T1551 also downregulated the expression of oncogene FGFR3 and eIF4E, and disturbed the activation of related PI3K/AKT/mTOR and ERK signaling in A549 cell. Finally, we investigated the conformational spaces and identified collective motions important for description of T1551/PRMT5 complex by using molecular dynamics simulation and normal mode analysis methods. This study provides a novel non-SAM-competitive hit compound for developing small molecules targeting PRMT5 in non-small cell lung cancer.

Analysis of DNA Cytosine Methylation Patterns Using Methylation-Sensitive Amplification Polymorphism (MSAP).

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Guevara, María Ángeles; de María, Nuria; Sáez-Laguna, Enrique; Vélez, María Dolores; Cervera, María Teresa; Cabezas, José Antonio

2017-01-01

Different molecular techniques have been developed to study either the global level of methylated cytosines or methylation at specific gene sequences. One of them is the methylation-sensitive amplified polymorphism technique (MSAP) which is a modification of amplified fragment length polymorphism (AFLP). It has been used to study methylation of anonymous CCGG sequences in different fungi, plants, and animal species. The main variation of this technique resides on the use of isoschizomers with different methylation sensitivity (such as HpaII and MspI) as a frequent-cutter restriction enzyme. For each sample, MSAP analysis is performed using both EcoRI/HpaII- and EcoRI/MspI-digested samples. A comparative analysis between EcoRI/HpaII and EcoRI/MspI fragment patterns allows the identification of two types of polymorphisms: (1) methylation-insensitive polymorphisms that show common EcoRI/HpaII and EcoRI/MspI patterns but are detected as polymorphic amplified fragments among samples and (2) methylation-sensitive polymorphisms which are associated with the amplified fragments that differ in their presence or absence or in their intensity between EcoRI/HpaII and EcoRI/MspI patterns. This chapter describes a detailed protocol of this technique and discusses the modifications that can be applied to adjust the technology to different species of interest.

[Characteristic and clinical significance of DNA methyltransferase 3B overexpression in endometrial carcinoma].

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Dong, Y; Zhou, M; Ba, X J; Si, J W; Li, W T; Wang, Y; Li, D; Li, T

2016-10-18

To determine the clinicopathological significance of the DNA methyltransferase 3B (DNMT3B) overexpression in endometrial carcinomas and to evaluate its correlation with hormone receptor status. Immunohistochemistry was performed to assess the expression of DNMT3B and hormone receptors in 104 endometrial carcinomas. DNMT3B overexpression occurred frequently in endometrioid carcinoma (EC, 54.8%) more than in nonendometrioid carcinoma (NEC, 30.0%) with statistical significance (P=0.028). Furthermore, there was a trend that EC with worse clinico-pathological variables and shorter survival had a higher DNMT3B expression, and the correlation between DNMT3B and tumor grade reached statistical significance (P=0.019).A negative correlation between DNMT3B and estrogen receptor (ER) or progesterone receptor (PR) expression was found in EC. NMT3B overexpression occurred frequently in the ER or PR negative subgroups (78.9%, 86.7%) more than in the positive subgroups (47.7%, 47.8%) with statistical significance (P=0.016, P=0.006). In addition, the DNMT3B overexpression increased in tumors with both ER and PR negative expression (92.9%, P=0.002). However, no such correlation was found in NEC (P>0.05). Sequence analyses demonstrated multiple ER and PR binding sites in the promoter regions of DNMT3B gene. This study showed that the expression of DNMT3B in EC and NEC was different. DNMT3B overexpression in EC was associated with the worse clinicopathological variables and might have predictive value. The methylation status of EC and NEC maybe different. In addition, in EC, DNMT3B overexpression negatively correlated with ER or PR expression. In NEC, the correlation between DNMT3B and ER or PR status was not present.

Effect of DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine on radiosensitivity of the human lung cancer cells in three-dimensional culture

International Nuclear Information System (INIS)

Pan Dong; Xue Gang; Li Xiaoman; Chen Yaxiong; Ren Zhenxin; Du Yarong; Hu Burong

2014-01-01

5-Aza-CdR is a specific inhibitor of DNMTs which could suppress tumor growth by demethylation of genomic DNA. There have only few studies thus far concerning it as radiosensitizers in three-dimensional (3D) cells. The principal aim of this study is to evaluate the effects of 5-Aza-CdR on the radiosensitivity of A549 cells in monolayer (2D) and 3D cultures in an attempt to find out a new combination treatments with radiotherapy. The cell proliferation was detected by MTT assay after pretreated with different doses of 5-Aza-CdR for 72 h. A549 cells were treated with or without 5-Aza-CdR (2, 5 μmol/L) for 72 h before be exposed to X-rays of 1, 2, 4, 6 Gy, respectively. The DNA damage was evaluated by micronucleus assay and clonogenic assays. Pretreatment with 5-Aza-CdR inhibited the A549 cell proliferation significantly. More micronucleus were observed after irradiation in 3D cells pretreated with 2 and 5 μmol/L concentration of drug than those without treatment. The survival fractions of cells pretreated by both 2 and 5 μmol/L drug reduced significantly in 3D cultures after irradiation. These significances, however, were found in 2D cells pretreated by only 5 μmol/L drug. Our results suggest that 5-Aza-CdR can inhibit the A549 cells proliferation and apparently enhance the radiosensitivity of cells in 3D cultures. Using of the low dose 5-Aza-CdR in clinical radiotherapy may reduce side effects and enhance effectively the cancer target therapy. (authors)

Optical Characterization of Oligonucleotide DNA Influenced by Magnetic Fields

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Seyedeh Maryam Banihashemian

2013-09-01

Full Text Available UV-VIS spectroscopic analysis of oligonucleotide DNA exposed to different magnetic fields was performed in order to investigate the relationship between DNA extinction coefficients and optical parameters according to magnetic-field strength. The results with the oligonucleotides adenine-thymine 100 mer (AT-100 DNA and cytosine-guanine 100 mer (CG-100 DNA indicate that the magnetic field influences DNA molar extinction coefficients and refractive indexes. The imaginary parts of the refractive index and molar extinction coefficients of the AT-100 and CG-100 DNA decreased after exposure to a magnetic field of 750 mT due to cleavage of the DNA oligonucleotides into smaller segments.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1).

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Nwaobi, Sinifunanya E; Olsen, Michelle L

2015-09-26

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Small Molecule Inhibitors That Selectively Block Dengue Virus Methyltransferase*

Science.gov (United States)

Lim, Siew Pheng; Sonntag, Louis Sebastian; Noble, Christian; Nilar, Shahul H.; Ng, Ru Hui; Zou, Gang; Monaghan, Paul; Chung, Ka Yan; Dong, Hongping; Liu, Boping; Bodenreider, Christophe; Lee, Gladys; Ding, Mei; Chan, Wai Ling; Wang, Gang; Jian, Yap Li; Chao, Alexander Theodore; Lescar, Julien; Yin, Zheng; Vedananda, T. R.; Keller, Thomas H.; Shi, Pei-Yong

2011-01-01

Crystal structure analysis of Flavivirus methyltransferases uncovered a flavivirus-conserved cavity located next to the binding site for its cofactor, S-adenosyl-methionine (SAM). Chemical derivatization of S-adenosyl-homocysteine (SAH), the product inhibitor of the methylation reaction, with substituents that extend into the identified cavity, generated inhibitors that showed improved and selective activity against dengue virus methyltransferase (MTase), but not related human enzymes. Crystal structure of dengue virus MTase with a bound SAH derivative revealed that its N6-substituent bound in this cavity and induced conformation changes in residues lining the pocket. These findings demonstrate that one of the major hurdles for the development of methyltransferase-based therapeutics, namely selectivity for disease-related methyltransferases, can be overcome. PMID:21147775

A SAM-dependent methyltransferase cotranscribed with arsenate reductase alters resistance to peptidyl transferase center-binding antibiotics in Azospirillum brasilense Sp7.

Science.gov (United States)

Singh, Sudhir; Singh, Chhaya; Tripathi, Anil Kumar

2014-05-01

The genome of Azospirillum brasilense harbors a gene encoding S-adenosylmethionine-dependent methyltransferase, which is located downstream of an arsenate reductase gene. Both genes are cotranscribed and translationally coupled. When they were cloned and expressed individually in an arsenate-sensitive strain of Escherichia coli, arsenate reductase conferred tolerance to arsenate; however, methyltransferase failed to do so. Sequence analysis revealed that methyltransferase was more closely related to a PrmB-type N5-glutamine methyltransferase than to the arsenate detoxifying methyltransferase ArsM. Insertional inactivation of prmB gene in A. brasilense resulted in an increased sensitivity to chloramphenicol and resistance to tiamulin and clindamycin, which are known to bind at the peptidyl transferase center (PTC) in the ribosome. These observations suggested that the inability of prmB:km mutant to methylate L3 protein might alter hydrophobicity in the antibiotic-binding pocket of the PTC, which might affect the binding of chloramphenicol, clindamycin, and tiamulin differentially. This is the first report showing the role of PrmB-type N5-glutamine methyltransferases in conferring resistance to tiamulin and clindamycin in any bacterium.

Adenovirus-mediated interleukin-12 gene transfer combined with cytosine deaminase followed by 5-fluorocytosine treatment exerts potent antitumor activity in Renca tumor-bearing mice

International Nuclear Information System (INIS)

Hwang, Kyung-Sun; Cho, Won-Kyung; Yoo, Jinsang; Yun, Hwan-Jung; Kim, Samyong; Im, Dong-Soo

2005-01-01

Therapeutic gene transfer affords a clinically feasible and safe approach to cancer treatment but a more effective modality is needed to improve clinical outcomes. Combined transfer of therapeutic genes with different modes of actions may be a means to this end. Interleukin-12 (IL-12), a heterodimeric immunoregulatory cytokine composed of covalently linked p35 and p40 subunits, has antitumor activity in animal models. The enzyme/prodrug strategy using cytosine deaminase (CD) and 5-fluorocytosine (5-FC) has been used for cancer gene therapy. We have evaluated the antitumor effect of combining IL-12 with CD gene transfer in mice bearing renal cell carcinoma (Renca) tumors. Adenoviral vectors were constructed encoding one or both subunits of murine IL-12 (Ad.p35, Ad.p40 and Ad.IL-12) or cytosine deaminase (Ad.CD). The functionality of the IL-12 or CD gene products expressed from these vectors was validated by splenic interferon (IFN)-γ production or viability assays in cultured cells. Ad.p35 plus Ad.p40, or Ad.IL-12, with or without Ad.CD, were administered (single-dose) intratumorally to Renca tumor-bearing mice. The animals injected with Ad.CD also received 5-FC intraperitoneally. The antitumor effects were then evaluated by measuring tumor regression, mean animal survival time, splenic natural killer (NK) cell activity and IFN-γ production. The inhibition of tumor growth in mice treated with Ad.p35 plus Ad.p40 and Ad.CD, followed by injection of 5-FC, was significantly greater than that in mice treated with Ad.CD/5-FC, a mixture of Ad.p35 plus Ad.p40, or Ad.GFP (control). The combined gene transfer increased splenic NK cell activity and IFN-γ production by splenocytes. Ad.CD/5-FC treatment significantly increased the antitumor effect of Ad.IL-12 in terms of tumor growth inhibition and mean animal survival time. The results suggest that adenovirus-mediated IL-12 gene transfer combined with Ad.CD followed by 5-FC treatment may be useful for treating cancers

The anti-CMS technique for genome-wide mapping of 5-hydroxymethylcytosine.

Science.gov (United States)

Huang, Yun; Pastor, William A; Zepeda-Martínez, Jorge A; Rao, Anjana

2012-10-01

5-Hydroxymethylcytosine (5hmC) is a recently discovered base in the mammalian genome, produced upon oxidation of 5-methylcytosine (5mC) in a process catalyzed by TET proteins. The biological functions of 5hmC and further oxidation products of 5mC are under intense investigation, as they are likely intermediates in DNA demethylation pathways. Here we describe a novel protocol to profile 5hmC at a genome-wide scale. This approach is based on sodium bisulfite-mediated conversion of 5hmC to cytosine-5-methylenesulfonate (CMS); CMS-containing DNA fragments are then immunoprecipitated using a CMS-specific antiserum. The anti-CMS technique is highly specific with a low background, and is much less dependent on 5hmC density than anti-5hmC immunoprecipitation (IP). Moreover, it does not enrich for CA and CT repeats, as noted for 5hmC DNA IP using antibodies to 5hmC. The anti-CMS protocol takes 3 d to complete.

Raman microspectroscopic study of effects of Na(I) and Mg(II) ions on low pH induced DNA structural changes.

Science.gov (United States)

Muntean, C M; Segers-Nolten, G M J

2003-01-01

In this work a confocal Raman microspectrometer is used to investigate the influence of Na(+) and Mg(2+) ions on the DNA structural changes induced by low pH. Measurements are carried out on calf thymus DNA at neutral pH (7) and pH 3 in the presence of low and high concentrations of Na(+) and Mg(2+) ions, respectively. It is found that low concentrations of Na(+) ions do not protect DNA against binding of H(+). High concentrations of monovalent ions can prevent protonation of the DNA double helix. Our Raman spectra show that low concentrations of Mg(2+) ions partly protect DNA against protonation of cytosine (line at 1262 cm(-1)) but do not protect adenine and guanine N(7) against binding of H(+) (characteristic lines at 1304 and 1488 cm(-1), respectively). High concentrations of Mg(2+) can prevent protonation of cytosine and protonation of adenine (disruption of AT pairs). By analyzing the line at 1488 cm(-1), which obtains most of its intensity from a guanine vibration, high magnesium salt protect the N(7) of guanine against protonation. A high salt concentration can prevent protonation of guanine, cytosine, and adenine in DNA. Higher salt concentrations cause less DNA protonation than lower salt concentrations. Magnesium ions are found to be more effective in protecting DNA against binding of H(+) as compared with calcium ions presented in a previous study. Divalent metal cations (Mg(2+), Ca(2+)) are more effective in protecting DNA against protonation than monovalent ions (Na(+)). Copyright 2003 Wiley Periodicals, Inc. Biopolymers (Biospectroscopy) 72: 000-000, 2003

LINE1 CpG-DNA Hypomethylation in Granulosa Cells and Blood Leukocytes Is Associated With PCOS and Related Traits.

Science.gov (United States)

Sagvekar, Pooja; Mangoli, Vijay; Desai, Sadhana; Patil, Anushree; Mukherjee, Srabani

2017-04-01

Altered global DNA methylation is indicative of epigenomic instability concerning chronic diseases. Investigating its incidence and association with polycystic ovary syndrome (PCOS) is essential to understand the etiopathogenesis of this disorder. We assessed global DNA methylation differences in peripheral blood leukocytes (PBLs) and cumulus granulosa cells (CGCs) of controls and women with PCOS; and their association with PCOS and its traits. This study included a total of 102 controls and women with PCOS. Forty-one women undergoing controlled ovarian hyperstimulation (COH) and 61 women not undergoing COH were recruited from in vitro fertilization (IVF) and infertility clinics. DNA methylation was measured by ELISA for 5'-methyl-cytosine content and bisulfite sequencing of 5'-untranslated region (5'-UTR) of long interspersed nucleotide element-1 (LINE1/L1). Total 5'-methyl-cytosine and L1 methylation levels in PBLs and CGCs were similar between controls and women with PCOS. Methylation assessed at CpG sites of L1 5'-UTR revealed a single CpG-site (CpG-4) to be consistently hypomethylated in PBLs of both PCOS groups and CGCs of stimulated PCOS group. In unstimulated women, hypomethylation at CpG-4 was strongly associated with PCOS susceptibility, whereas in stimulated group it showed strong associations with PCOS and its hormonal traits. Furthermore, CGCs demonstrated consistent global and CpG-DNA hypomethylation relative to PBLs, irrespective of normal or disease states. Our study revealed strong association of single hypomethylated CpG-site with PCOS. Identification and characterization of more such methyl-CpG signatures in repetitive elements in larger study populations would provide valuable epigenetic insights into PCOS. Copyright © 2017 by the Endocrine Society

Label free detection of 5′ hydroxymethylcytosine within CpG Islands using optical sensors

Science.gov (United States)

Hawk, Rasheeda M.; Armani, Andrea M.

2014-01-01

Significant research has been invested in correlating genetic variations with different disease probabilities. Recently, it has become apparent that other DNA modifications, such as the addition of a methyl or hydroxymethyl group to cytosine, can also play a role. While these modifications do not change the sequence, they can negatively impact the function. Therefore, it is critical to be able to both read the genetic code and identify these modifications. Currently, the detection of hydroxymethylated cytosine (5′hmC) and the two closely related variants, cytosine (C) and 5′methylcytosine (5′mC), relies on a combination of nucleotide modification steps, followed by PCR and gene sequencing. However, this approach is not ideal because transcription errors which are inherent to the PCR process can be misinterpreted as fluctuations in the relative C:5′mC:5′hmC concentrations. As such, an alternative method which does not rely on PCR or nucleotide modification is desirable. One approach is based on label-free optical resonant cavity sensors. In the present work, toroidal resonant cavity sensors are functionalized with antibodies to enable label-free detection and discrimination between C, 5′mC, and 5′hmC in real-time without PCR. Specifically, epoxide chemistry is used to covalently attach the 5′hmC antibody to the surface of the cavity. Subsequently, to thoroughly characterize the sensor platform, detection of C, 5′mC, and 5′hmC is performed over a concentration range from pM to nM. At low (pM) concentrations, the hydroxymethylated cytosine produces a significantly larger signal than the structurally similar epigenetic markers; thus demonstrating the applicability of this platform. PMID:25461158

DNA-methyltransferase 3B 39179 G > T polymorphism and risk of sporadic colorectal cancer in a subset of Iranian population

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Abdolreza Daraei

2011-01-01

Full Text Available Background: Epigenetic event is a biological regulation that influences the expression of various genes involved in cancer. DNA methylation is established by DNA methyltransferases, particularly DNAmethyltransferase 3B (DNMT3B. It seems to play an oncogenic role in the creation of abnormal methylation during tumorigenesis. The polymorphisms of the DNMT3B gene may influence DNMT3B activity in DNA methylation and increase the susceptibility to several cancers. These genetic polymorphisms have been studied in several cancers in different populations. Methods: In this study, we performed a case-control study with 125 colorectal cancer patients and 135 cancer-free controls to evaluate the association between DNMT3B G39179T polymorphism (rs1569686 in the promoter region and the risk of sporadic colorectal cancer. Up to now, few studies have investigated the role of this gene variant in sporadic colorectal cancer with no familial history. The genotypes of DNMT3B G39179T polymorphism was analyzed by PCR-RFLP. Results: We found that compared with G allele carriers, statistically the DNMT3B TT genotype (%34 was significantly associated with increased risk of colorectal cancer (adjusted OR, 3.993, 95% CI, 1.726-9.238, P = 0.001. Compared with DNMT3B TT genotype, the GT and GG genotypes had lower risk of developing sporadic colorectal cancer (OR = 0.848, 95% CI = 0.436-1.650. Conclusions: Our findings were consistent with that of previously reported case-control studies with colorectal cancer. These results suggest that the DNMT3B G39179T polymorphism influences DNMT3B expression, thus contributing to the genetic susceptibility to colorectal cancer. Further mechanistic studies are needed to unravel the causal molecular mechanisms.

Synthesis of aldehyde-linked nucleotides and DNA and their bioconjugations with lysine and peptides through reductive amination.

Science.gov (United States)

Raindlová, Veronika; Pohl, Radek; Hocek, Michal

2012-03-26

5-(5-Formylthienyl)-, 5-(4-formylphenyl)- and 5-(2-fluoro-5-formylphenyl)cytosine 2'-deoxyribonucleoside mono- (dC(R)MP) and triphosphates (dC(R)TP) were prepared by aqueous Suzuki-Miyaura cross-coupling of 5-iodocytosine nucleotides with the corresponding formylarylboronic acids. The dC(R)TPs were excellent substrates for DNA polymerases and were incorporated into DNA by primer extension or PCR. Reductive aminations of the model dC(R)MPs with lysine or lysine-containing tripeptide were studied and optimized. In aqueous phosphate buffer (pH 6.7) the yields of the reductive aminations with tripeptide III were up to 25 %. Bioconjugation of an aldehyde-containing DNA with a lysine-containing tripeptide was achieved through reductive amination in yields of up to 90 % in aqueous phosphate buffer. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of Cytosine Arabinoside, 3-Aminobenzamide and Hydroxyurea on the frequencies of radiation-induced micronuclei and aneuploidy in human lymphocytes

International Nuclear Information System (INIS)

Cho, Yoon Hee; Kim, Yang Jee; Ha, Sung Whan; Chung, Hai Won; Kang, Chang Mo

2005-01-01

This study was carried out to examine the effect of the DNA repair inhibitors, Cytosine Arabinoside(Ara C), 3-Aminobenzamide(3AB) and Hydroxyurea(HU) on the frequencies of radiation-induced MicroNuclei(MNi) and aneuploidy. Irradiated lymphocytes(1-3Gy) were treated with DNA repair inhibitors, Ara C, 3AB and HU for 3 hours and CBMN assay - FISH technique with DNA probe for chromosome 1 and 4 was performed. The frequencies of x-ray induced MNi and aneuploidy of chromosome 1 and 4 were increased in a dose-dependent manner. Ara C, 3AB and HU enhanced the frequencies of radiation-induced MNi and the frequencies of radiation-induced aneuploidy of chromosome 1 and 4 were enhanced by HU and Ara C while no effect was observed by 3AB. The frequency of radiation-induced aneuploidy of chromosome 1 was higher than that of chromosome 4. These results suggest that there are different mechanisms involved in the formation of MNi and aneuploidy by radiation

Effect of Cytosine Arabinoside, 3-Aminobenzamide and Hydroxyurea on the frequencies of radiation-induced micronuclei and aneuploidy in human lymphocytes

Energy Technology Data Exchange (ETDEWEB)

Cho, Yoon Hee; Kim, Yang Jee; Ha, Sung Whan; Chung, Hai Won [Seoul National Univ., Seoul (Korea, Republic of); Kang, Chang Mo [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

2005-12-15

This study was carried out to examine the effect of the DNA repair inhibitors, Cytosine Arabinoside(Ara C), 3-Aminobenzamide(3AB) and Hydroxyurea(HU) on the frequencies of radiation-induced MicroNuclei(MNi) and aneuploidy. Irradiated lymphocytes(1-3Gy) were treated with DNA repair inhibitors, Ara C, 3AB and HU for 3 hours and CBMN assay - FISH technique with DNA probe for chromosome 1 and 4 was performed. The frequencies of x-ray induced MNi and aneuploidy of chromosome 1 and 4 were increased in a dose-dependent manner. Ara C, 3AB and HU enhanced the frequencies of radiation-induced MNi and the frequencies of radiation-induced aneuploidy of chromosome 1 and 4 were enhanced by HU and Ara C while no effect was observed by 3AB. The frequency of radiation-induced aneuploidy of chromosome 1 was higher than that of chromosome 4. These results suggest that there are different mechanisms involved in the formation of MNi and aneuploidy by radiation.

Simultaneous determination of cytosine arabinoside, daunorubicin and etoposide in human plasma

DEFF Research Database (Denmark)

Krogh-Madsen, Mikkel; Hansen, Steen Honoré; Honoré, Per Hartvig

2010-01-01

A method for simultaneous bioanalysis of the three cytotoxic drugs cytosine arabinoside, daunorubicin and etoposide in human plasma was developed and validated. A HPLC method with ultra-violet and fluorescence detection, preceded by mixed-mode cation-exchange solid phase extraction sample....... The overall precision (% relative standard deviation) was within 0.2-13.5% and the recovery ranged between 86.1% and 110.1% for the three drugs at all concentrations tested. Plasma samples were stable for at least two months when stored at -20 degrees C. The method was successfully applied to quantification...

Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations

OpenAIRE

van Aelst, Kara; Saikrishnan, Kayarat; Szczelkun, Mark D

2015-01-01

The prokaryotic Type ISP restriction-modification enzymes are single-chain proteins comprising an Mrr-family nuclease, a superfamily 2 helicase-like ATPase, a coupler domain, a methyltransferase, and a DNA-recognition domain. Upon recognising an unmodified DNA target site, the helicase-like domain hydrolyzes ATP to cause site release (remodeling activity) and to then drive downstream translocation consuming 1-2 ATP per base pair (motor activity). On an invading foreign DNA, double-strand brea...

DNA methyltransferase 1-targeting miRNA-148aof dairymilk: apotential bioactive modifier of thehumanepigenome

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Bodo C. Melnik

2017-09-01

Full Text Available Background: The perception of milk has changed from a “simple food” to a more sophisticated bioactive functional signaling system that promotes mTORC1-driven postnatal anabolism, growth, and development of the newborn infant. Accumulating evidence supports the view that milk´s miRNAs significantly contribute to these processes. The most abundant miRNA of milk found in milk fat and milk exosomes is miRNA-148a, which targets DNA methyltransferase 1 (DNMT1, a pivotal epigenetic regulator that suppresses transcription. Furthermore, milk-derived miRNA-125b, miRNA-30d, and miRNA-25 target TP53, the guardian of the genome that interacts with DNMT1 and regulates metabolism, cell kinetics, and apoptosis. Thus, the question arose whether cow´s milk-derived miRNAs may modify epigenetic regulation of the human milk consumer. Methods: To understand the potential impact of dairy milk consumption on human epigenetics, we have analyzed all relevant research-based bioinformatics data related to milk, milk miRNAs, epigenetic regulation, and lactation performance with special attention to bovine miRNAs that modify gene expression of DNA methyltransferase 1 (DNMT1 and p53 (TP53, the two guardians of the mammalian genome. By means of translational research and comparative functional genomics, we investigated the potential impact of cow´s milk miRNAs on epigenetic regulation of human DNMT1, TP53, FOXP3, and FTO, which are critically involved in immunologic and metabolic programming respectively. miRNA sequences have been obtained from mirbase.org. miRNA-target site prediction has been performed using TargetScan release 7.0. Results: The most abundant miRNA of cow´s milk is miRNA-148a, which represents more than 10% of all miRNAs of cow´s milk, survives pasteurization and refrigerated storage. The seed sequence of human and bovine miRNA-148a-3p is identical. Furthermore, human and bovine DNMT1 mRNA share 88% identity. The miRNA-148a 7mer seed is conserved in

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

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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.

Characterization of Dnmt1 Binding and DNA Methylation on Nucleosomes and Nucleosomal Arrays.

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Anna Schrader

Full Text Available The packaging of DNA into nucleosomes and the organisation into higher order structures of chromatin limits the access of sequence specific DNA binding factors to DNA. In cells, DNA methylation is preferentially occuring in the linker region of nucleosomes, suggesting a structural impact of chromatin on DNA methylation. These observations raise the question whether DNA methyltransferases are capable to recognize the nucleosomal substrates and to modify the packaged DNA. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the maintenance DNA methyltransferase Dnmt1. Our binding studies show that Dnmt1 has a DNA length sensing activity, binding cooperatively to DNA, and requiring a minimal DNA length of 20 bp. Dnmt1 needs linker DNA to bind to nucleosomes and most efficiently recognizes nucleosomes with symmetric DNA linkers. Footprinting experiments reveal that Dnmt1 binds to both DNA linkers exiting the nucleosome core. The binding pattern correlates with the efficient methylation of DNA linkers. However, the enzyme lacks the ability to methylate nucleosomal CpG sites on mononucleosomes and nucleosomal arrays, unless chromatin remodeling enzymes create a dynamic chromatin state. In addition, our results show that Dnmt1 functionally interacts with specific chromatin remodeling enzymes to enable complete methylation of hemi-methylated DNA in chromatin.

The yield of fission neutron-induced chromatid aberrations in G[sub 2]-stage human lymphocytes: effect of caffeine, hydroxyurea and cytosine arabinoside post-irradiation

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Antoccia, A.; Tanzarella, C. (La Sapienza Univ., Rome (Italy)); Palitti, F. (Tuscia Univ., Viterbo (Italy) La Sapienza Univ., Rome (Italy)); Raggi, T. (Tuscia Univ., Viterbo (Italy)); Catena, C. (ENEA, Casaccia (Italy). Centro Ricerche Energia)

1992-11-01

To evaluate the influence of inhibitors of DNA synthesis/repair on the yield of chromosomal aberrations in the G[sub 2] phase of the cell cycle, whole-blood cultures of human lymphocytes were exposed to various doses of fission neutrons or X-rays and treated post-irradiation during the last 2.45 h before harvesting, with 5mM hydroxyurea (HU) and 0.05 mM cytosine arabinoside (ara-C). The presence of caffeine and HU strongly potentiated the yield of chromatid-type aberrations induced by both neutrons and X-rays. No potentiating effect, except at the highest dose of neutrons, was observed when irradiated cells were subsequently treated with ara-C. In addition, neutron-induced mitotic delay was shortened by treatment with caffeine, mainly within the first 2 h after irradiation. (Author).

Global DNA hypermethylation-associated cancer chemotherapy resistance and its reversion with the demethylating agent hydralazine

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Benitez-Bribiesca Luis

2006-08-01

Full Text Available Abstract Background The development of resistance to cytotoxic chemotherapy continues to be a major obstacle for successful anticancer therapy. It has been shown that cells exposed to toxic concentrations of commonly used cancer chemotherapy agents develop DNA hypermetylation. Hence, demethylating agents could play a role in overcoming drug resistance. Methods MCF-7 cells were rendered adriamycin-resistant by weekly treatment with adriamycin. Wild-type and the resulting MCF-7/Adr cells were analyzed for global DNA methylation. DNA methyltransferase activity and DNA methyltransferase (dnmt gene expression were also determined. MCF-7/Adr cells were then subjected to antisense targeting of dnmt1, -3a, and -b genes and to treatment with the DNA methylation inhibitor hydralazine to investigate whether DNA demethylation restores sensitivity to adriamycin. Results MCF-7/Adr cells exhibited the multi-drug resistant phenotype as demonstrated by adriamycin resistance, mdr1 gene over-expression, decreased intracellular accumulation of adriamycin, and cross-resistance to paclitaxel. The mdr phenotype was accompanied by global DNA hypermetylation, over-expression of dnmt genes, and increased DNA methyltransferase activity as compared with wild-type MCF-7 cells. DNA demethylation through antisense targeting of dnmts or hydralazine restored adriamycin sensitivity of MCF-7/Adr cells to a greater extent than verapamil, a known inhibitor of mdr protein, suggesting that DNA demethylation interferes with the epigenetic reprogramming that participates in the drug-resistant phenotype. Conclusion We provide evidence that DNA hypermethylation is at least partly responsible for development of the multidrug-resistant phenotype in the MCF-7/Adr model and that hydralazine, a known DNA demethylating agent, can revert the resistant phenotype.

DNA nanotechnology: a future perspective

Science.gov (United States)

2013-01-01

In addition to its genetic function, DNA is one of the most distinct and smart self-assembling nanomaterials. DNA nanotechnology exploits the predictable self-assembly of DNA oligonucleotides to design and assemble innovative and highly discrete nanostructures. Highly ordered DNA motifs are capable of providing an ultra-fine framework for the next generation of nanofabrications. The majority of these applications are based upon the complementarity of DNA base pairing: adenine with thymine, and guanine with cytosine. DNA provides an intelligent route for the creation of nanoarchitectures with programmable and predictable patterns. DNA strands twist along one helix for a number of bases before switching to the other helix by passing through a crossover junction. The association of two crossovers keeps the helices parallel and holds them tightly together, allowing the assembly of bigger structures. Because of the DNA molecule's unique and novel characteristics, it can easily be applied in a vast variety of multidisciplinary research areas like biomedicine, computer science, nano/optoelectronics, and bionanotechnology. PMID:23497147

Biophysical and molecular docking insight into the interaction of cytosine β-D arabinofuranoside with human serum albumin

International Nuclear Information System (INIS)

Alam, Parvez; Chaturvedi, Sumit Kumar; Anwar, Tamanna; Siddiqi, Mohammad Khursheed; Ajmal, Mohd Rehan; Badr, Gamal; Mahmoud, Mohamed H.; Hasan Khan, Rizwan

2015-01-01

Interaction of pharmacologically important anticancer drug cytosine β-D arabinofuranoside with human serum albumin (HSA) at physiological pH 7.4 has been studied by utilizing various spectroscopic and molecular docking strategies. Fluorescence results revealed that cytosine β-D arabinofuranoside interacts with HSA through static quenching mechanism with binding affinity of 2.4×10 3 M −1 . The average binding distance between drug and Trp 214 of HSA was found to be 2.23 nm on the basis of the theory of Förster's energy transfer. Synchronous fluorescence data indicated that interaction of drug with HSA changed the microenvironment around the tryptophan residue. UV–visible spectroscopy and circular dichroism results deciphered the complex formation and conformational alterations in the HSA respectively. Dynamic light scattering was utilized to understand the topology of protein in absence and presence of drug. Thermodynamic parameters obtained from isothermal titration calorimetry (ΔH=−26.01 kJ mol −1 and TΔS=6.5 kJ mol −1 ) suggested the involvement of van der Waal interaction and hydrogen bonding. Molecular docking and displacement study with site specific markers suggested that cytosine β-D arabinofuranoside binds to subdomain IB of HSA which is also known as the hemin binding site. This study will be helpful to understand the binding mechanism of cytosine β-D arabinofuranoside with HSA and associated alterations. - Highlights: • Comprehensive insight into the interaction of CBDA with HSA. • The interaction process is spontaneous and exothermic. • The main governing forces for stabilizing HSA–CBDA complex are van der Waal interaction and hydrogen bonding. • CBDA binds at subdomain IB on HSA

Regulation of DNA Methylation Patterns by CK2-Mediated Phosphorylation of Dnmt3a

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Rachel Deplus

2014-08-01

Full Text Available DNA methylation is a central epigenetic modification that is established by de novo DNA methyltransferases. The mechanisms underlying the generation of genomic methylation patterns are still poorly understood. Using mass spectrometry and a phosphospecific Dnmt3a antibody, we demonstrate that CK2 phosphorylates endogenous Dnmt3a at two key residues located near its PWWP domain, thereby downregulating the ability of Dnmt3a to methylate DNA. Genome-wide DNA methylation analysis shows that CK2 primarily modulates CpG methylation of several repeats, most notably of Alu SINEs. This modulation can be directly attributed to CK2-mediated phosphorylation of Dnmt3a. We also find that CK2-mediated phosphorylation is required for localization of Dnmt3a to heterochromatin. By revealing phosphorylation as a mode of regulation of de novo DNA methyltransferase function and by uncovering a mechanism for the regulation of methylation at repetitive elements, our results shed light on the origin of DNA methylation patterns.

Modulation of Epstein–Barr Virus Nuclear Antigen 2-dependent transcription by protein arginine methyltransferase 5

International Nuclear Information System (INIS)

Liu, Cheng-Der; Cheng, Chi-Ping; Fang, Jia-Shih; Chen, Ling-Chih; Zhao, Bo; Kieff, Elliott; Peng, Chih-Wen

2013-01-01

Highlights: ► Catalytic active PRMT5 substantially binds to the EBNA2 RG domain. ► PRMT5 augments the EBNA2-dependent transcription. ► PRMT5 triggers the symmetric dimethylation of the EBNA2 RG domain. ► PRMT5 enhances the promoter occupancy of EBNA2 on its target promoters. -- Abstract: Epstein–Barr Virus Nuclear Antigen (EBNA) 2 features an Arginine–Glycine repeat (RG) domain at amino acid positions 335–360, which is a known target for protein arginine methyltransferaser 5 (PRMT5). In this study, we performed protein affinity pull-down assays to demonstrate that endogenous PRMT5 derived from lymphoblastoid cells specifically associated with the protein bait GST-E2 RG. Transfection of a plasmid expressing PRMT5 induced a 2.5- to 3-fold increase in EBNA2-dependent transcription of both the LMP1 promoter in AKATA cells, which contain the EBV genome endogenously, and a Cp-Luc reporter plasmid in BJAB cells, which are EBV negative. Furthermore, we showed that there was a 2-fold enrichment of EBNA2 occupancy in target promoters in the presence of exogenous PRMT5. Taken together, we show that PRMT5 triggers the symmetric dimethylation of EBNA2 RG domain to coordinate with EBNA2-mediated transcription. This modulation suggests that PRMT5 may play a role in latent EBV infection

Modulation of Epstein–Barr Virus Nuclear Antigen 2-dependent transcription by protein arginine methyltransferase 5

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Liu, Cheng-Der; Cheng, Chi-Ping; Fang, Jia-Shih; Chen, Ling-Chih [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China); Zhao, Bo; Kieff, Elliott [Department of Medicine and Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women’s Hospital and Harvard Medical School, 181 Longwood Ave., Boston 02115, MA (United States); Peng, Chih-Wen, E-mail: pengcw@mail.tcu.edu.tw [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China)

2013-01-18

Highlights: ► Catalytic active PRMT5 substantially binds to the EBNA2 RG domain. ► PRMT5 augments the EBNA2-dependent transcription. ► PRMT5 triggers the symmetric dimethylation of the EBNA2 RG domain. ► PRMT5 enhances the promoter occupancy of EBNA2 on its target promoters. -- Abstract: Epstein–Barr Virus Nuclear Antigen (EBNA) 2 features an Arginine–Glycine repeat (RG) domain at amino acid positions 335–360, which is a known target for protein arginine methyltransferaser 5 (PRMT5). In this study, we performed protein affinity pull-down assays to demonstrate that endogenous PRMT5 derived from lymphoblastoid cells specifically associated with the protein bait GST-E2 RG. Transfection of a plasmid expressing PRMT5 induced a 2.5- to 3-fold increase in EBNA2-dependent transcription of both the LMP1 promoter in AKATA cells, which contain the EBV genome endogenously, and a Cp-Luc reporter plasmid in BJAB cells, which are EBV negative. Furthermore, we showed that there was a 2-fold enrichment of EBNA2 occupancy in target promoters in the presence of exogenous PRMT5. Taken together, we show that PRMT5 triggers the symmetric dimethylation of EBNA2 RG domain to coordinate with EBNA2-mediated transcription. This modulation suggests that PRMT5 may play a role in latent EBV infection.

Metal-mediated deamination of cytosine: experiment and DFT calculations

Czech Academy of Sciences Publication Activity Database

Šponer, Judit E.; Sanz Miguel, P. J.; Rodríguez-Santiago, L.; Erxleben, A.; Krumm, M.; Sodupe, M.; Šponer, Jiří; Lippert, B.

2004-01-01

Roč. 43, č. 40 (2004), s. 5396-5399 ISSN 1433-7851 R&D Projects: GA MŠk LN00A016 Institutional research plan: CEZ:AV0Z5004920 Keywords : cytosine * deamination * density functional calculations Subject RIV: BO - Biophysics Impact factor: 9.161, year: 2004

Experimental mitochondria-targeted DNA methylation identifies GpC methylation, not CpG methylation, as potential regulator of mitochondrial gene expression

NARCIS (Netherlands)

van der Wijst, Monique G. P.; van Tilburg, Amanda Y.; Ruiters, Marcel H. J.; Rots, Marianne G.

2017-01-01

Like the nucleus, mitochondria contain their own DNA and recent reports provide accumulating evidence that also the mitochondrial DNA (mtDNA) is subjective to DNA methylation. This evidence includes the demonstration of mitochondria-localised DNA methyltransferases and demethylases, and the

Global DNA Methylation in the Chestnut Blight Fungus Cryphonectria parasitica and Genome-Wide Changes in DNA Methylation Accompanied with Sectorization

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Kum-Kang So

2018-02-01

Full Text Available Mutation in CpBck1, an ortholog of the cell wall integrity mitogen-activated protein kinase kinase kinase (MAPKKK of Saccharomyces cerevisiae, in the chestnut blight fungus Cryphonectria parasitica resulted in a sporadic sectorization as culture proceeded. The progeny from the sectored area maintained the characteristics of the sector, showing a massive morphogenetic change, including robust mycelial growth without differentiation. Epigenetic changes were investigated as the genetic mechanism underlying this sectorization. Quantification of DNA methylation and whole-genome bisulfite sequencing revealed genome-wide DNA methylation of the wild-type at each nucleotide level and changes in DNA methylation of the sectored progeny. Compared to the wild-type, the sectored progeny exhibited marked genome-wide DNA hypomethylation but increased methylation sites. Expression analysis of two DNA methyltransferases, including two representative types of DNA methyltransferase (DNMTase, demonstrated that both were significantly down-regulated in the sectored progeny. However, functional analysis using mutant phenotypes of corresponding DNMTases demonstrated that a mutant of CpDmt1, an ortholog of RID of Neurospora crassa, resulted in the sectored phenotype but the CpDmt2 mutant did not, suggesting that the genetic basis of fungal sectorization is more complex. The present study revealed that a mutation in a signaling pathway component resulted in sectorization accompanied with changes in genome-wide DNA methylation, which suggests that this signal transduction pathway is important for epigenetic control of sectorization via regulation of genes involved in DNA methylation.

Cytosine methylation alteration in natural populations of Leymus chinensis induced by multiple abiotic stresses.

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Yingjie Yu

Full Text Available BACKGROUND: Human activity has a profound effect on the global environment and caused frequent occurrence of climatic fluctuations. To survive, plants need to adapt to the changing environmental conditions through altering their morphological and physiological traits. One known mechanism for phenotypic innovation to be achieved is environment-induced rapid yet inheritable epigenetic changes. Therefore, the use of molecular techniques to address the epigenetic mechanisms underpinning stress adaptation in plants is an important and challenging topic in biological research. In this study, we investigated the impact of warming, nitrogen (N addition, and warming+nitrogen (N addition stresses on the cytosine methylation status of Leymus chinensis Tzvel. at the population level by using the amplified fragment length polymorphism (AFLP, methylation-sensitive amplified polymorphism (MSAP and retrotransposon based sequence-specific amplification polymorphism (SSAP techniques. METHODOLOGY/PRINCIPAL FINDINGS: Our results showed that, although the percentages of cytosine methylation changes in SSAP are significantly higher than those in MSAP, all the treatment groups showed similar alteration patterns of hypermethylation and hypomethylation. It meant that the abiotic stresses have induced the alterations in cytosine methylation patterns, and the levels of cytosine methylation changes around the transposable element are higher than the other genomic regions. In addition, the identification and analysis of differentially methylated loci (DML indicated that the abiotic stresses have also caused targeted methylation changes at specific loci and these DML might have contributed to the capability of plants in adaptation to the abiotic stresses. CONCLUSIONS/SIGNIFICANCE: Our results demonstrated that abiotic stresses related to global warming and nitrogen deposition readily evoke alterations of cytosine methylation, and which may provide a molecular basis for rapid

Cytosine Methylation Alteration in Natural Populations of Leymus chinensis Induced by Multiple Abiotic Stresses

Science.gov (United States)

Yu, Yingjie; Yang, Xuejiao; Wang, Huaying; Shi, Fengxue; Liu, Ying; Liu, Jushan; Li, Linfeng; Wang, Deli; Liu, Bao

2013-01-01

Background Human activity has a profound effect on the global environment and caused frequent occurrence of climatic fluctuations. To survive, plants need to adapt to the changing environmental conditions through altering their morphological and physiological traits. One known mechanism for phenotypic innovation to be achieved is environment-induced rapid yet inheritable epigenetic changes. Therefore, the use of molecular techniques to address the epigenetic mechanisms underpinning stress adaptation in plants is an important and challenging topic in biological research. In this study, we investigated the impact of warming, nitrogen (N) addition, and warming+nitrogen (N) addition stresses on the cytosine methylation status of Leymus chinensis Tzvel. at the population level by using the amplified fragment length polymorphism (AFLP), methylation-sensitive amplified polymorphism (MSAP) and retrotransposon based sequence-specific amplification polymorphism (SSAP) techniques. Methodology/Principal Findings Our results showed that, although the percentages of cytosine methylation changes in SSAP are significantly higher than those in MSAP, all the treatment groups showed similar alteration patterns of hypermethylation and hypomethylation. It meant that the abiotic stresses have induced the alterations in cytosine methylation patterns, and the levels of cytosine methylation changes around the transposable element are higher than the other genomic regions. In addition, the identification and analysis of differentially methylated loci (DML) indicated that the abiotic stresses have also caused targeted methylation changes at specific loci and these DML might have contributed to the capability of plants in adaptation to the abiotic stresses. Conclusions/Significance Our results demonstrated that abiotic stresses related to global warming and nitrogen deposition readily evoke alterations of cytosine methylation, and which may provide a molecular basis for rapid adaptation by

Gas chromatographic determination of purines and pyrimidines from DNA using ethyl chloroformate as derivatizing reagent

International Nuclear Information System (INIS)

Brohi, R.O.Z.; Khuhawar, M.Y.; Laghari, A.J.; Channa, A.

2016-01-01

An analytical method has been proposed for the separation and determination of guanine, adenine, cytosine, thymine and uracil by gas chromatography (GC) following precolumn derivatization using ethyl chloroformate. The GC separation was achieved from HP-5 (30 m x 0.32 rnrn id) column with layer thickness 0.25 microm. The linear calibrations were observed within 0.5-50.0 micro mole/L for each of the compound and limits of detection were within 0.1-0.17 micro mol/L. The derivatization, separation and quantitation was repeatable with intra (n=5) and inter (n=5) variation in terms of peak height/peak area and retention time with relative standard deviation (RSD) within 4.70-6.43%. The method was applied for the analysis of isolated DNA from human blood and plant leaves after acid hydrolysis. The concentration of thymine, adenine, cytosine and guanine in blood samples were observed within 0.602-2.135 micro mol/L of each compounds with RSD 2.60- 6.00%. The recovery of the nucleobases by standard addition was calculated within 98-108% with RSD 2.5-7.8%. (author)

Gas Chromatographic Determination of Purines and Pyrimidines from DNA Using Ethyl Chloroformate as Derivatizing Reagent

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Rafi O. Zaman Brohi

2016-06-01

Full Text Available An analytical method has been proposed for the separation and determination of guanine, adenine, cytosine, thymine and uracil by gas chromatography (GC following precolumn derivatization using ethyl chloroformate. The GC separation was achieved from HP-5 (30 m × 0.32 mm id column with layer thickness 0.25 µm. The linear calibrations were observed within 0.5-50.0 µmol/L for each of the compound and limits of detection were within 0.1-0.17 µmol/L. The derivatization, separation and quantitation was repeatable with intra (n=5 and inter (n=5 variation in terms of peak height/peak area and retention time with relative standard deviation (RSD within 4.70-6.43%. The method was applied for the analysis of isolated DNA from human blood and plant leaves after acid hydrolysis. The concentration of thymine, adenine, cytosine and guanine in blood samples were observed within 0.602-2.135 µmol/L of each compounds with RSD 2.60-6.00%. The recovery of the nucleobases by standard addition was calculated within 98-108% with RSD 2.5-7.8%.

Conformational change in human DNA repair enzyme O6-methylguanine-DNA methyltransferase upon alkylation of its active site by SN1 (indirect-acting) and SN2 (direct-acting) alkylating agents: breaking a "salt-link".

Science.gov (United States)

Oh, H K; Teo, A K; Ali, R B; Lim, A; Ayi, T C; Yarosh, D B; Li, B F

1996-09-24

Human O6-methylguanine-DNA methyltransferase (MGMT) repairs DNA by transferring alkyl (R-) adducts from O6-alkylguanine (6RG) in DNA to its own cysteine residue at codon 145 (formation of R-MGMT). We show here that R-MGMT in cell extracts, which is sensitive to protease V8 cleavage at the glutamic acid residues at codons 30 (E30) and 172 (E172), can be specifically immunoprecipitated with an MGMT monoclonal antibody, Mab.3C7. This Mab recognizes an epitope of human MGMT including the lysine 107 (K107) which is within the most basic region that is highly conserved among mammalian MGMTs. Surprisingly, the K107L mutant protein is repair-deficient and readily cleaved by protease V8 similar to R-MGMT. We propose that R-MGMT adopted an altered conformation which exposed the Mab.3C7 epitope and rendered that protein sensitive to protease V8 attack. This proposal could be explained by the disruption of a structural "salt-link" within the molecule based on the available structural and biochemical data. The specific binding of Mab.3C7 to R-MGMT has been compared with the protease V8 method in the detection of R-MGMT in extracts of cells treated with low dosages of methyliodide (SN2) and O6-benzylguanine. Their identical behaviors in producing protease V8 sensitive R-MGMT and Mab.3C7 immunoprecipitates suggest that probably methyl iodide (an ineffective agent in producing 6RG in DNA) can directly alkylate the active site of cellular MGMT similar to O6-benzylguanine. The effectiveness of MeI in producing R-MGMT, i.e., inactivation of cellular MGMT, indicates that this agent can increase the effectiveness of environmental and endogenously produced alkylating carcinogens in producing the mutagenic O6-alkylguanine residues in DNA in vivo.

No effect of folic acid supplementation on global DNA methylation in men and women with moderately elevated homocysteine.

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Audrey Y Jung

Full Text Available A global loss of cytosine methylation in DNA has been implicated in a wide range of diseases. There is growing evidence that modifications in DNA methylation can be brought about by altering the intake of methyl donors such as folate. We examined whether long-term daily supplementation with 0.8 mg of folic acid would increase global DNA methylation compared with placebo in individuals with elevated plasma homocysteine. We also investigated if these effects were modified by MTHFR C677T genotype. Two hundred sixteen participants out of 818 subjects who had participated in a randomized double-blind placebo-controlled trial were selected, pre-stratified on MTHFR C677T genotype and matched on age and smoking status. They were allocated to receive either folic acid (0.8 mg/d; n = 105 or placebo treatment (n = 111 for three years. Peripheral blood leukocyte DNA methylation and serum and erythrocyte folate were assessed. Global DNA methylation was measured using liquid chromatography-tandem mass spectrometry and expressed as a percentage of 5-methylcytosines versus the total number of cytosine. There was no difference in global DNA methylation between those randomized to folic acid and those in the placebo group (difference = 0.008, 95%CI = -0.05,0.07, P = 0.79. There was also no difference between treatment groups when we stratified for MTHFR C677T genotype (CC, n = 76; CT, n = 70; TT, n = 70, baseline erythrocyte folate status or baseline DNA methylation levels. In moderately hyperhomocysteinemic men and women, long-term folic acid supplementation does not increase global DNA methylation in peripheral blood leukocytes.ClinicalTrials.gov NCT00110604.

No effect of folic acid supplementation on global DNA methylation in men and women with moderately elevated homocysteine.

Science.gov (United States)

Jung, Audrey Y; Smulders, Yvo; Verhoef, Petra; Kok, Frans J; Blom, Henk; Kok, Robert M; Kampman, Ellen; Durga, Jane

2011-01-01

A global loss of cytosine methylation in DNA has been implicated in a wide range of diseases. There is growing evidence that modifications in DNA methylation can be brought about by altering the intake of methyl donors such as folate. We examined whether long-term daily supplementation with 0.8 mg of folic acid would increase global DNA methylation compared with placebo in individuals with elevated plasma homocysteine. We also investigated if these effects were modified by MTHFR C677T genotype. Two hundred sixteen participants out of 818 subjects who had participated in a randomized double-blind placebo-controlled trial were selected, pre-stratified on MTHFR C677T genotype and matched on age and smoking status. They were allocated to receive either folic acid (0.8 mg/d; n = 105) or placebo treatment (n = 111) for three years. Peripheral blood leukocyte DNA methylation and serum and erythrocyte folate were assessed. Global DNA methylation was measured using liquid chromatography-tandem mass spectrometry and expressed as a percentage of 5-methylcytosines versus the total number of cytosine. There was no difference in global DNA methylation between those randomized to folic acid and those in the placebo group (difference = 0.008, 95%CI = -0.05,0.07, P = 0.79). There was also no difference between treatment groups when we stratified for MTHFR C677T genotype (CC, n = 76; CT, n = 70; TT, n = 70), baseline erythrocyte folate status or baseline DNA methylation levels. In moderately hyperhomocysteinemic men and women, long-term folic acid supplementation does not increase global DNA methylation in peripheral blood leukocytes.ClinicalTrials.gov NCT00110604.

The effect of thiopurine drugs on DNA methylation in relation to TPMT expression.

Science.gov (United States)

Hogarth, L A; Redfern, C P F; Teodoridis, J M; Hall, A G; Anderson, H; Case, M C; Coulthard, S A

2008-10-15

The thiopurine drugs 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are well-established agents for the treatment of leukaemia but their main modes of action are controversial. Thiopurine methyltransferase (TPMT) metabolises thiopurine drugs and influences their cytotoxic activity. TPMT, like DNA methyltransferases (DNMTs), transfers methyl groups from S-adenosylmethionine (SAM) and generates S-adenosylhomocysteine (SAH). Since SAM levels are dependent on de novo purine synthesis (DNPS) and the metabolic products of 6-TG and 6-MP differ in their ability to inhibit DNPS, we postulated that 6-TG compared to 6-MP would have differential effects on changes in SAM and SAH levels and global DNA methylation, depending on TPMT status. To test this hypothesis, we used a human embryonic kidney cell line with inducible TPMT. Although changes in SAM and SAH levels occurred with each drug, decrease in global DNA methylation more closely reflected a decrease in DNMT activity. Inhibition was influenced by TPMT for 6-TG, but not 6-MP. The decrease in global methylation and DNMT activity with 6-MP, or with 6-TG when TPMT expression was low, were comparable to 5-aza-2'-deoxycytidine. However, this was not reflected in changes in methylation at the level of an individual marker gene (MAGE1A). The results suggest that a non-TPMT metabolised metabolite of 6-MP and 6-TG and the TPMT-metabolised 6-MP metabolite 6-methylthioguanosine 5'-monophosphate, contribute to a decrease in DNMT levels and global DNA methylation. As demethylating agents have shown promise in leukaemia treatment, inhibition of DNA methylation by the thiopurine drugs may contribute to their cytotoxic affects.

EGNAS: an exhaustive DNA sequence design algorithm

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Kick Alfred

2012-06-01

Full Text Available Abstract Background The molecular recognition based on the complementary base pairing of deoxyribonucleic acid (DNA is the fundamental principle in the fields of genetics, DNA nanotechnology and DNA computing. We present an exhaustive DNA sequence design algorithm that allows to generate sets containing a maximum number of sequences with defined properties. EGNAS (Exhaustive Generation of Nucleic Acid Sequences offers the possibility of controlling both interstrand and intrastrand properties. The guanine-cytosine content can be adjusted. Sequences can be forced to start and end with guanine or cytosine. This option reduces the risk of “fraying” of DNA strands. It is possible to limit cross hybridizations of a defined length, and to adjust the uniqueness of sequences. Self-complementarity and hairpin structures of certain length can be avoided. Sequences and subsequences can optionally be forbidden. Furthermore, sequences can be designed to have minimum interactions with predefined strands and neighboring sequences. Results The algorithm is realized in a C++ program. TAG sequences can be generated and combined with primers for single-base extension reactions, which were described for multiplexed genotyping of single nucleotide polymorphisms. Thereby, possible foldback through intrastrand interaction of TAG-primer pairs can be limited. The design of sequences for specific attachment of molecular constructs to DNA origami is presented. Conclusions We developed a new software tool called EGNAS for the design of unique nucleic acid sequences. The presented exhaustive algorithm allows to generate greater sets of sequences than with previous software and equal constraints. EGNAS is freely available for noncommercial use at http://www.chm.tu-dresden.de/pc6/EGNAS.

Intramolecular tautomerisation and the conformational variability of some classical mutagens – cytosine derivatives: quantum chemical study

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Hovorun D. M.

2011-04-01

Full Text Available Aim. To determine the lifetime of the mutagenic cytosine derivatives through the investigation of the physicochemical mechanisms of their intramolecular proton transfer. Methods. Non-empirical quantum chemistry, the analysis of the electron density by means of Bader’s atoms in molecules (AIM theory and physicochemical kinetics were used. Results. It is shown that the modification of all investigated compounds, except DCyt, prevents their pairing in both mutagenic and canonical tautomeric forms with a base which is an interacting partner. This effect can inhibit their mutagenic potential. It is also established that Watson-Crick tautomeric hypothesis can be formally expanded for the investigated molecules so far as a lifetime of the mutagenic tautomers much more exceeds characteristic time for the incorporation of one nucleotides pair by DNA biosynthesis machinery. It seems that just within the frame of this hypothesis it will be possible to give an adequate explanation of the mechanisms of mutagenic action of N4-aminocytosine, N4-methoxycytosine, N4-hydroxycytosine and N4dehydrocytosine, which have much more energy advantageous imino form in comparison with amino form. Conclusions. For the first time the comprehensive conformational analysis of a number of classical mutagens, namely cytosine derivatives, has been performed using the methods of non-empirical quantum chemistry at the MP2/6-311++G (2df,pd//B3LYP/6-311++G(d,p level of theory

Array-based assay detects genome-wide 5-mC and 5-hmC in the brains of humans, non-human primates, and mice.

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Chopra, Pankaj; Papale, Ligia A; White, Andrew T J; Hatch, Andrea; Brown, Ryan M; Garthwaite, Mark A; Roseboom, Patrick H; Golos, Thaddeus G; Warren, Stephen T; Alisch, Reid S

2014-02-13

Methylation on the fifth position of cytosine (5-mC) is an essential epigenetic mark that is linked to both normal neurodevelopment and neurological diseases. The recent identification of another modified form of cytosine, 5-hydroxymethylcytosine (5-hmC), in both stem cells and post-mitotic neurons, raises new questions as to the role of this base in mediating epigenetic effects. Genomic studies of these marks using model systems are limited, particularly with array-based tools, because the standard method of detecting DNA methylation cannot distinguish between 5-mC and 5-hmC and most methods have been developed to only survey the human genome. We show that non-human data generated using the optimization of a widely used human DNA methylation array, designed only to detect 5-mC, reproducibly distinguishes tissue types within and between chimpanzee, rhesus, and mouse, with correlations near the human DNA level (R(2) > 0.99). Genome-wide methylation analysis, using this approach, reveals 6,102 differentially methylated loci between rhesus placental and fetal tissues with pathways analysis significantly overrepresented for developmental processes. Restricting the analysis to oncogenes and tumor suppressor genes finds 76 differentially methylated loci, suggesting that rhesus placental tissue carries a cancer epigenetic signature. Similarly, adapting the assay to detect 5-hmC finds highly reproducible 5-hmC levels within human, rhesus, and mouse brain tissue that is species-specific with a hierarchical abundance among the three species (human > rhesus > mouse). Annotation of 5-hmC with respect to gene structure reveals a significant prevalence in the 3'UTR and an association with chromatin-related ontological terms, suggesting an epigenetic feedback loop mechanism for 5-hmC. Together, these data show that this array-based methylation assay is generalizable to all mammals for the detection of both 5-mC and 5-hmC, greatly improving the utility of mammalian model systems

Chemical Probes of Histone Lysine Methyltransferases

Science.gov (United States)

2015-01-01

Growing evidence suggests that histone methyltransferases (HMTs, also known as protein methyltransferases (PMTs)) play an important role in diverse biological processes and human diseases by regulating gene expression and the chromatin state. Therefore, HMTs have been increasingly recognized by the biomedical community as a class of potential therapeutic targets. High quality chemical probes of HMTs, as tools for deciphering their physiological functions and roles in human diseases and testing therapeutic hypotheses, are critical for advancing this promising field. In this review, we focus on the discovery, characterization, and biological applications of chemical probes for HMTs. PMID:25423077

Dynamics of self-assembled cytosine nucleobases on graphene

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Saikia, Nabanita; Johnson, Floyd; Waters, Kevin; Pandey, Ravindra

2018-05-01

Molecular self-assembly of cytosine (C n ) bases on graphene was investigated using molecular dynamics methods. For free-standing C n bases, simulation conditions (gas versus aqueous) determine the nature of self-assembly; the bases prefer to aggregate in the gas phase and are stabilized by intermolecular H-bonds, while in the aqueous phase, the water molecules disrupt base-base interactions, which facilitate the formation of π-stacked domains. The substrate-induced effects, on the other hand, find the polarity and donor-acceptor sites of the bases to govern the assembly process. For example, in the gas phase, the assembly of C n bases on graphene displays short-range ordered linear arrays stabilized by the intermolecular H-bonds. In the aqueous phase, however, there are two distinct configurations for the C n bases assembly on graphene. For the first case corresponding to low surface coverage, the bases are dispersed on graphene and are isolated. The second configuration archetype is disordered linear arrays assembled with medium and high surface coverage. The simulation results establish the role of H-bonding, vdW π-stacking, and the influence of graphene surface towards the self-assembly. The ability to regulate the assembly into well-defined patterns can aid in the design of self-assembled nanostructures for the next-generation DNA based biosensors and nanoelectronic devices.

Ligation bias in illumina next-generation DNA libraries: implications for sequencing ancient genomes.

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Andaine Seguin-Orlando

Full Text Available Ancient DNA extracts consist of a mixture of endogenous molecules and contaminant DNA templates, often originating from environmental microbes. These two populations of templates exhibit different chemical characteristics, with the former showing depurination and cytosine deamination by-products, resulting from post-mortem DNA damage. Such chemical modifications can interfere with the molecular tools used for building second-generation DNA libraries, and limit our ability to fully characterize the true complexity of ancient DNA extracts. In this study, we first use fresh DNA extracts to demonstrate that library preparation based on adapter ligation at AT-overhangs are biased against DNA templates starting with thymine residues, contrarily to blunt-end adapter ligation. We observe the same bias on fresh DNA extracts sheared on Bioruptor, Covaris and nebulizers. This contradicts previous reports suggesting that this bias could originate from the methods used for shearing DNA. This also suggests that AT-overhang adapter ligation efficiency is affected in a sequence-dependent manner and results in an uneven representation of different genomic contexts. We then show how this bias could affect the base composition of ancient DNA libraries prepared following AT-overhang ligation, mainly by limiting the ability to ligate DNA templates starting with thymines and therefore deaminated cytosines. This results in particular nucleotide misincorporation damage patterns, deviating from the signature generally expected for authenticating ancient sequence data. Consequently, we show that models adequate for estimating post-mortem DNA damage levels must be robust to the molecular tools used for building ancient DNA libraries.

Modification of -Adenosyl--Homocysteine as Inhibitor of Nonstructural Protein 5 Methyltransferase Dengue Virus Through Molecular Docking and Molecular Dynamics Simulation

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Usman Sumo Friend Tambunan

2017-04-01

Full Text Available Dengue fever is still a major threat worldwide, approximately threatening two-fifths of the world’s population in tropical and subtropical countries. Nonstructural protein 5 (NS5 methyltransferase enzyme plays a vital role in the process of messenger RNA capping of dengue by transferring methyl groups from S -adenosyl- l -methionine to N7 atom of the guanine bases of RNA and the RNA ribose group of 2′OH, resulting in S -adenosyl- l -homocysteine (SAH. The modification of SAH compound was screened using molecular docking and molecular dynamics simulation, along with computational ADME-Tox (absorption, distribution, metabolism, excretion, and toxicity test. The 2 simulations were performed using Molecular Operating Environment (MOE 2008.10 software, whereas the ADME-Tox test was performed using various software. The modification of SAH compound was done using several functional groups that possess different polarities and properties, resulting in 3460 ligands to be docked. After conducting docking simulation, we earned 3 best ligands (SAH-M331, SAH-M2696, and SAH-M1356 based on ΔG binding and molecular interactions, which show better results than the standard ligands. Moreover, the results of molecular dynamics simulation show that the best ligands are still able to maintain the active site residue interaction with the binding site until the end of the simulation. After a series of molecular docking and molecular dynamics simulation were performed, we concluded that SAH-M1356 ligand is the most potential SAH-based compound to inhibit NS5 methyltransferase enzyme for treating dengue fever.

Potential benefits of combining cytosine deaminase/5-fluorocytosine gene therapy and irradiation for prostate cancer. Experimental study

Energy Technology Data Exchange (ETDEWEB)

Kato, Hiroaki; Koshida, Kiyoshi; Yokoyama, Kunihiko; Mizokami, Atsushi; Namiki, Mikio [Kanazawa Univ. (Japan). School of Medicine

2002-10-01

The purpose of this study was to investigate the potential of combining cytosine deaminase/5-fluorocytosine (CD/5-FC) gene therapy and radiation therapy (either external beam radiation or radioimmunotherapy [RIT]), for the treatment of prostate cancer. Tumor xenografts of CD-transduced LNCaP cells grown in the testes of severe combined immunodeficiency (SCID) mice were used to evaluate antitumor effect. The mice were injected intraperitoneally with 500 mg/kg of 5-FC, or with 5, 15 or 30 mg/kg of 5-fluorouracil (5-FU), for 9 days. The tumors were treated with fractionated radiation at a dose of 1 or 3 Gy/day for 3 days, or I-131 labelled anti-prostate specific antigen (anti-PSA) monoclonal antibody (mAb) administration at a subtherapeutic dose of 20 or 80 {mu}Ci. Intratumoral and serum concentrations of 5-FU were measured using high performance liquid chromatography. Mice treated with CD/5-FC gene therapy presented a significant tumor growth inhibition comparable to that obtained with 15 mg/kg, 5-FU systemic administration without marked weight loss. Treatment with CD/5-FC gene therapy resulted in higher tumor but lower serum concentrations of 5-FU than treatment with systemic 5-FU chemotherapy. An additive antitumor effect was obtained when CD/5-FC therapy was combined with 1 Gy irradiation, which by itself did not produce a significant antitumor effect. However, the efficacy of CD/5-FC therapy was not enhanced when combined with RIT, probably due to poor accumulation of the mAb as the tumor/blood ratio never exceeded 1. These findings indicate that CD/5-FC gene therapy for prostate cancer may function with enhanced antitumor effect when combined with external beam radiation. However, combining CD/5-FC gene therapy and RIT using an anti-PSA mAb may not be effective because of insufficient accumulation of the mAb at the target tumors. (author)

Potential benefits of combining cytosine deaminase/5-fluorocytosine gene therapy and irradiation for prostate cancer. Experimental study

International Nuclear Information System (INIS)

Kato, Hiroaki; Koshida, Kiyoshi; Yokoyama, Kunihiko; Mizokami, Atsushi; Namiki, Mikio

2002-01-01

The purpose of this study was to investigate the potential of combining cytosine deaminase/5-fluorocytosine (CD/5-FC) gene therapy and radiation therapy (either external beam radiation or radioimmunotherapy [RIT]), for the treatment of prostate cancer. Tumor xenografts of CD-transduced LNCaP cells grown in the testes of severe combined immunodeficiency (SCID) mice were used to evaluate antitumor effect. The mice were injected intraperitoneally with 500 mg/kg of 5-FC, or with 5, 15 or 30 mg/kg of 5-fluorouracil (5-FU), for 9 days. The tumors were treated with fractionated radiation at a dose of 1 or 3 Gy/day for 3 days, or I-131 labelled anti-prostate specific antigen (anti-PSA) monoclonal antibody (mAb) administration at a subtherapeutic dose of 20 or 80 μCi. Intratumoral and serum concentrations of 5-FU were measured using high performance liquid chromatography. Mice treated with CD/5-FC gene therapy presented a significant tumor growth inhibition comparable to that obtained with 15 mg/kg, 5-FU systemic administration without marked weight loss. Treatment with CD/5-FC gene therapy resulted in higher tumor but lower serum concentrations of 5-FU than treatment with systemic 5-FU chemotherapy. An additive antitumor effect was obtained when CD/5-FC therapy was combined with 1 Gy irradiation, which by itself did not produce a significant antitumor effect. However, the efficacy of CD/5-FC therapy was not enhanced when combined with RIT, probably due to poor accumulation of the mAb as the tumor/blood ratio never exceeded 1. These findings indicate that CD/5-FC gene therapy for prostate cancer may function with enhanced antitumor effect when combined with external beam radiation. However, combining CD/5-FC gene therapy and RIT using an anti-PSA mAb may not be effective because of insufficient accumulation of the mAb at the target tumors. (author)

Epigenetic variation, inheritance, and parent-of-origin effects of cytosine methylation in maize (Zea mays).

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Lauria, Massimiliano; Piccinini, Sara; Pirona, Raul; Lund, Gertrud; Viotti, Angelo; Motto, Mario

2014-03-01

Pure epigenetic variation, or epigenetic variation that is independent of genetic context, may provide a mechanism for phenotypic variation in the absence of DNA mutations. To estimate the extent of pure epigenetic variation within and across generations and to identify the DNA regions targeted, a group of eight plants derived from a highly inbred line of maize (Zea mays) was analyzed by the methylation-sensitive amplified polymorphism (MSAP) technique. We found that cytosine methylation (mC) differences among individuals accounted for up to 7.4% of CCGG sites investigated by MSAP. Of the differentially methylated fragments (DMFs) identified in the S0 generation, ∼12% were meiotically inherited for at least six generations. We show that meiotically heritable mC variation was consistently generated for an average of 0.5% CCGG sites per generation and that it largely occurred somatically. We provide evidence that mC variation can be established and inherited in a parent-of-origin manner, given that the paternal lineage is more prone to both forward and reverse mC changes. The molecular characterization of selected DMFs revealed that the variation was largely determined by CG methylation changes that map within gene regions. The expression analysis of genes overlapping with DMFs did not reveal an obvious correlation between mC variation and transcription, reinforcing the idea that the primary function of gene-body methylation is not to control gene expression. Because this study focuses on epigenetic variation in field-grown plants, the data presented herein pertain to spontaneous epigenetic changes of the maize genome in a natural context.

Biophysical and molecular docking insight into the interaction of cytosine β-D arabinofuranoside with human serum albumin

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Alam, Parvez; Chaturvedi, Sumit Kumar [Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, UP (India); Anwar, Tamanna [Center of Bioinformatics Research and Technology, Aligarh 202002 (India); Siddiqi, Mohammad Khursheed; Ajmal, Mohd Rehan [Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, UP (India); Badr, Gamal [Laboratory of Immunology & Molecular Physiology, Zoology Department, Faculty of Science, Assiut University, 71516 Assiut (Egypt); Mahmoud, Mohamed H. [Food Science and Nutrition Department, National Research Center, Dokki, Cairo (Egypt); Deanship of Scientific Research, King Saud University, Riyadh (Saudi Arabia); Hasan Khan, Rizwan, E-mail: rizwanhkhan@hotmail.com [Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, UP (India)

2015-08-15

Interaction of pharmacologically important anticancer drug cytosine β-D arabinofuranoside with human serum albumin (HSA) at physiological pH 7.4 has been studied by utilizing various spectroscopic and molecular docking strategies. Fluorescence results revealed that cytosine β-D arabinofuranoside interacts with HSA through static quenching mechanism with binding affinity of 2.4×10{sup 3} M{sup −1}. The average binding distance between drug and Trp{sup 214} of HSA was found to be 2.23 nm on the basis of the theory of Förster's energy transfer. Synchronous fluorescence data indicated that interaction of drug with HSA changed the microenvironment around the tryptophan residue. UV–visible spectroscopy and circular dichroism results deciphered the complex formation and conformational alterations in the HSA respectively. Dynamic light scattering was utilized to understand the topology of protein in absence and presence of drug. Thermodynamic parameters obtained from isothermal titration calorimetry (ΔH=−26.01 kJ mol{sup −1} and TΔS=6.5 kJ mol{sup −1}) suggested the involvement of van der Waal interaction and hydrogen bonding. Molecular docking and displacement study with site specific markers suggested that cytosine β-D arabinofuranoside binds to subdomain IB of HSA which is also known as the hemin binding site. This study will be helpful to understand the binding mechanism of cytosine β-D arabinofuranoside with HSA and associated alterations. - Highlights: • Comprehensive insight into the interaction of CBDA with HSA. • The interaction process is spontaneous and exothermic. • The main governing forces for stabilizing HSA–CBDA complex are van der Waal interaction and hydrogen bonding. • CBDA binds at subdomain IB on HSA.

Differential stabilities and sequence-dependent base pair opening dynamics of Watson-Crick base pairs with 5-hydroxymethylcytosine, 5-formylcytosine, or 5-carboxylcytosine.

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Szulik, Marta W; Pallan, Pradeep S; Nocek, Boguslaw; Voehler, Markus; Banerjee, Surajit; Brooks, Sonja; Joachimiak, Andrzej; Egli, Martin; Eichman, Brandt F; Stone, Michael P

2015-02-10

5-Hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) form during active demethylation of 5-methylcytosine (5mC) and are implicated in epigenetic regulation of the genome. They are differentially processed by thymine DNA glycosylase (TDG), an enzyme involved in active demethylation of 5mC. Three modified Dickerson-Drew dodecamer (DDD) sequences, amenable to crystallographic and spectroscopic analyses and containing the 5'-CG-3' sequence associated with genomic cytosine methylation, containing 5hmC, 5fC, or 5caC placed site-specifically into the 5'-T(8)X(9)G(10)-3' sequence of the DDD, were compared. The presence of 5caC at the X(9) base increased the stability of the DDD, whereas 5hmC or 5fC did not. Both 5hmC and 5fC increased imino proton exchange rates and calculated rate constants for base pair opening at the neighboring base pair A(5):T(8), whereas 5caC did not. At the oxidized base pair G(4):X(9), 5fC exhibited an increase in the imino proton exchange rate and the calculated kop. In all cases, minimal effects to imino proton exchange rates occurred at the neighboring base pair C(3):G(10). No evidence was observed for imino tautomerization, accompanied by wobble base pairing, for 5hmC, 5fC, or 5caC when positioned at base pair G(4):X(9); each favored Watson-Crick base pairing. However, both 5fC and 5caC exhibited intranucleobase hydrogen bonding between their formyl or carboxyl oxygens, respectively, and the adjacent cytosine N(4) exocyclic amines. The lesion-specific differences observed in the DDD may be implicated in recognition of 5hmC, 5fC, or 5caC in DNA by TDG. However, they do not correlate with differential excision of 5hmC, 5fC, or 5caC by TDG, which may be mediated by differences in transition states of the enzyme-bound complexes.

Differential Stabilities and Sequence-Dependent Base Pair Opening Dynamics of Watson–Crick Base Pairs with 5-Hydroxymethylcytosine, 5-Formylcytosine, or 5-Carboxylcytosine

Science.gov (United States)

2016-01-01

5-Hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) form during active demethylation of 5-methylcytosine (5mC) and are implicated in epigenetic regulation of the genome. They are differentially processed by thymine DNA glycosylase (TDG), an enzyme involved in active demethylation of 5mC. Three modified Dickerson–Drew dodecamer (DDD) sequences, amenable to crystallographic and spectroscopic analyses and containing the 5′-CG-3′ sequence associated with genomic cytosine methylation, containing 5hmC, 5fC, or 5caC placed site-specifically into the 5′-T8X9G10-3′ sequence of the DDD, were compared. The presence of 5caC at the X9 base increased the stability of the DDD, whereas 5hmC or 5fC did not. Both 5hmC and 5fC increased imino proton exchange rates and calculated rate constants for base pair opening at the neighboring base pair A5:T8, whereas 5caC did not. At the oxidized base pair G4:X9, 5fC exhibited an increase in the imino proton exchange rate and the calculated kop. In all cases, minimal effects to imino proton exchange rates occurred at the neighboring base pair C3:G10. No evidence was observed for imino tautomerization, accompanied by wobble base pairing, for 5hmC, 5fC, or 5caC when positioned at base pair G4:X9; each favored Watson–Crick base pairing. However, both 5fC and 5caC exhibited intranucleobase hydrogen bonding between their formyl or carboxyl oxygens, respectively, and the adjacent cytosine N4 exocyclic amines. The lesion-specific differences observed in the DDD may be implicated in recognition of 5hmC, 5fC, or 5caC in DNA by TDG. However, they do not correlate with differential excision of 5hmC, 5fC, or 5caC by TDG, which may be mediated by differences in transition states of the enzyme-bound complexes. PMID:25632825

Defining Driver DNA Methylation Changes in Human Cancer

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Gerd P. Pfeifer

2018-04-01

Full Text Available Human malignant tumors are characterized by pervasive changes in the patterns of DNA methylation. These changes include a globally hypomethylated tumor cell genome and the focal hypermethylation of numerous 5′-cytosine-phosphate-guanine-3′ (CpG islands, many of them associated with gene promoters. It has been challenging to link specific DNA methylation changes with tumorigenesis in a cause-and-effect relationship. Some evidence suggests that cancer-associated DNA hypomethylation may increase genomic instability. Promoter hypermethylation events can lead to silencing of genes functioning in pathways reflecting hallmarks of cancer, including DNA repair, cell cycle regulation, promotion of apoptosis or control of key tumor-relevant signaling networks. A convincing argument for a tumor-driving role of DNA methylation can be made when the same genes are also frequently mutated in cancer. Many of the most commonly hypermethylated genes encode developmental transcription factors, the methylation of which may lead to permanent gene silencing. Inactivation of such genes will deprive the cells in which the tumor may initiate from the option of undergoing or maintaining lineage differentiation and will lock them into a perpetuated stem cell-like state thus providing an additional window for cell transformation.

Non-Covalent Fluorescent Labeling of Hairpin DNA Probe Coupled with Hybridization Chain Reaction for Sensitive DNA Detection.

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Song, Luna; Zhang, Yonghua; Li, Junling; Gao, Qiang; Qi, Honglan; Zhang, Chengxiao

2016-04-01

An enzyme-free signal amplification-based assay for DNA detection was developed using fluorescent hairpin DNA probes coupled with hybridization chain reaction (HCR). The hairpin DNAs were designed to contain abasic sites in the stem moiety. Non-covalent labeling of the hairpin DNAs was achieved when a fluorescent ligand was bound to the abasic sites through hydrogen bonding with the orphan cytosine present on the complementary strand, accompanied by quench of ligand fluorescence. As a result, the resultant probes, the complex formed between the hairpin DNA and ligand, showed almost no fluorescence. Upon hybridization with target DNA, the probe underwent a dehybridization of the stem moiety containing an abasic site. The release of ligand from the abasic site to the solution resulted in an effective fluorescent enhancement, which can be used as a signal. Compared with a sensing system without HCR, a 20-fold increase in the sensitivity was achieved using the sensing system with HCR. The fluorescent intensity of the sensing system increased with the increase in target DNA concentration from 0.5 nM to 100 nM. A single mismatched target ss-DNA could be effectively discriminated from complementary target DNA. Genotyping of a G/C single-nucleotide polymorphism of polymerase chain reaction (PCR) products was successfully demonstrated with the sensing system. Therefore, integrating HCR strategy with non-covalent labeling of fluorescent hairpin DNA probes provides a sensitive and cost-effective DNA assay. © The Author(s) 2016.

DNA Methyltransferase 3B Gene Promoter and Interleukin-1 Receptor Antagonist Polymorphisms in Childhood Immune Thrombocytopenia

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Margarita Pesmatzoglou

2012-01-01

Full Text Available Primary immune thrombocytopenia (ITP is one of the most common blood diseases as well as the commonest acquired bleeding disorder in childhood. Although the etiology of ITP is unclear, in the pathogenesis of the disease, both environmental and genetic factors including polymorphisms of TNF-a, IL-10, and IL-4 genes have been suggested to be involved. In this study, we investigated the rs2424913 single-nucleotide polymorphism (SNP (C46359T in DNA methyltransferase 3B (DNMT3B gene promoter and the VNTR polymorphism of IL-1 receptor antagonist (IL-1 Ra intron-2 in 32 children (17 boys with the diagnosis of ITP and 64 healthy individuals. No significant differences were found in the genotype distribution of DNMT3B polymorphism between the children with ITP and the control group, whereas the frequency of allele T appeared significantly increased in children with ITP (P = 0.03, OR = 2, 95% CI: 1.06–3.94. In case of IL-1 Ra polymorphism, children with ITP had a significantly higher frequency of genotype I/II, compared to control group (P = 0.043, OR = 2.60, 95% CI: 1.02–6.50. Moreover, genotype I/I as well as allele I was overrepresented in the control group, suggesting that allele I may have a decreased risk for development of ITP. Our findings suggest that rs2424913 DNMT3B SNP as well as IL-1 Ra VNTR polymorphism may contribute to the susceptibility to ITP.

Structural basis of asymmetric DNA methylation and ATP-triggered long-range diffusion by EcoP15I

Science.gov (United States)

Gupta, Yogesh K.; Chan, Siu-Hong; Xu, Shuang-Yong; Aggarwal, Aneel K.

2015-06-01

Type III R-M enzymes were identified >40 years ago and yet there is no structural information on these multisubunit enzymes. Here we report the structure of a Type III R-M system, consisting of the entire EcoP15I complex (Mod2Res1) bound to DNA. The structure suggests how ATP hydrolysis is coupled to long-range diffusion of a helicase on DNA, and how a dimeric methyltransferase functions to methylate only one of the two DNA strands. We show that the EcoP15I motor domains are specifically adapted to bind double-stranded DNA and to facilitate DNA sliding via a novel `Pin' domain. We also uncover unexpected `division of labour', where one Mod subunit recognizes DNA, while the other Mod subunit methylates the target adenine--a mechanism that may extend to adenine N6 RNA methylation in mammalian cells. Together the structure sheds new light on the mechanisms of both helicases and methyltransferases in DNA and RNA metabolism.

Interrogating Key Positions of Size-Reduced TALE Repeats Reveals a Programmable Sensor of 5-Carboxylcytosine.

Science.gov (United States)

Maurer, Sara; Giess, Mario; Koch, Oliver; Summerer, Daniel

2016-12-16

Transcription-activator-like effector (TALE) proteins consist of concatenated repeats that recognize consecutive canonical nucleobases of DNA via the major groove in a programmable fashion. Since this groove displays unique chemical information for the four human epigenetic cytosine nucleobases, TALE repeats with epigenetic selectivity can be engineered, with potential to establish receptors for the programmable decoding of all human nucleobases. TALE repeats recognize nucleobases via key amino acids in a structurally conserved loop whose backbone is positioned very close to the cytosine 5-carbon. This complicates the engineering of selectivities for large 5-substituents. To interrogate a more promising structural space, we engineered size-reduced repeat loops, performed saturation mutagenesis of key positions, and screened a total of 200 repeat-nucleobase interactions for new selectivities. This provided insight into the structural requirements of TALE repeats for affinity and selectivity, revealed repeats with improved or relaxed selectivity, and resulted in the first selective sensor of 5-carboxylcytosine.

2′-deoxy-5,6-dihydro-5-azacytidine—a less toxic alternative of 2′-deoxy-5-azacytidine

Science.gov (United States)

Matoušová, Marika; Votruba, Ivan; Otmar, Miroslav; Tloušťová, Eva; Günterová, Jana

2011-01-01

Restoration of transcriptionally silenced genes by means of methyltransferases inhibitors plays a crucial role in the current therapy of myelodysplastic syndromes and certain types of leukemias. A comparative study of hypomethylating activities of a series of 5-azacytidine nucleosides: 5-azacytidine (AC), 2′-deoxy-5-azacytidine (DAC) and its α-anomer (α-DAC), 5,6-dihydro-5-azacytidine (DHAC), 2′-deoxy-5,6-dihydro-5-azacytidine (DHDAC, KP-1212) and its α-anomer (α-DHDAC), and of a 2-pyrimidone ribonucleoside (zebularine) was conducted. Methylation-specific PCR was employed to detect the efficiency of individual agents on cyclin-dependent kinase inhibitor 2B and thrombospondin-1 hypermethylated gene loci. Overall changes in DNA methylation level were quantified by direct estimation of 5-methyl-2′-deoxycytidine-5′-monophosphate by HPLC using digested genomic DNA. Flow cytometric analysis of cell cycle progression and apoptotic markers was used to determine cytotoxicity of the compounds. mRNA expression was measured using qRT-PCR. 2′-deoxy-5,6-dihydro-5-azacytidine was found to be less cytotoxic and more stable than 2′-deoxy-5-azacytidine at the doses that induce comparable DNA hypomethylation and gene reactivation. This makes it a valuable tool for epigenetic research and worth further investigations to elucidate its possible therapeutic potential. PMID:21566456

Role of type II protein arginine methyltransferase 5 in the regulation of Circadian Per1 gene.

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Jungtae Na

Full Text Available Circadian clocks are the endogenous oscillators that regulate rhythmic physiological and behavioral changes to correspond to daily light-dark cycles. Molecular dissections have revealed that transcriptional feedback loops of the circadian clock genes drive the molecular oscillation, in which PER/CRY complexes inhibit the transcriptional activity of the CLOCK/BMAL1 heterodimer to constitute a negative feedback loop. In this study, we identified the type II protein arginine methyltransferase 5 (PRMT5 as an interacting molecule of CRY1. Although the Prmt5 gene was constitutively expressed, increased interaction of PRMT5 with CRY1 was observed when the Per1 gene was repressed both in synchronized mouse liver and NIH3T3 cells. Moreover, rhythmic recruitment of PRMT5 and CRY1 to the Per1 gene promoter was found to be associated with an increased level of histone H4R3 dimethylation and Per1 gene repression. Consistently, decreased histone H4R3 dimethylation and altered rhythmic Per1 gene expression were observed in Prmt5-depleted cells. Taken together, these findings provide an insight into the link between histone arginine methylation by PRMT5 and transcriptional regulation of the circadian Per1 gene.

Effect of D-valine and cytosine arabinoside on [3H]thymidine incorporation in rat and rabbit epididymal epithelial cell cultures

International Nuclear Information System (INIS)

Orgebin-Crist, M.C.; Jonas-Davies, J.; Storey, P.; Olson, G.E.

1984-01-01

Epithelial cell enriched primary cultures were established from the rat and the rabbit epididymis. Epithelial cell aggregates, obtained after pronase digestion of minced epididymis, attached to the culture dish and after 72 h in vitro spread out to form discrete patches of cells. These cells have an epithelioid morphology and form a monolayer of closely apposed polygonal cells where DNA synthesis, as judged by [ 3 H]thymidine uptake, is very low. In L-valine medium the nonepithelial cell contamination was no more than 10% in rat and rabbit epididymal primary cultures. The labeling index of rat epididymal cells cultured in D-valine medium was significantly lower than that of cells cultured in L-valine medium. In contrast, the labeling index of rabbit epididymal cells cultured in D-valine medium was significantly higher than that of cells cultured in L-valine medium. Cytosine arabinoside decreased the number of labeled cells in both L-valine and D-valine cultures. From these results, it appears that D-valine is a selective agent for rat epididymal epithelial cells, but not for rabbit epithelial cells, and that cytosine arabinoside is a simple and effective means to control the proliferation of fibroblast-like cells in both rat and rabbit epididymal cell cultures

In-silico investigations into natural products as nonnucleoside DNA ...

African Journals Online (AJOL)

nucleoside DNA methyltransferase 1 (DNMT1) inhibitor of epimutation in gastric cancer. Methods: A dataset of reported non-nucleoside DNMT1 inhibitors was used to target the active site of crystallized DNMT1 protein. Molecular docking simulations ...

Epigenetic dysregulation underlies radiation-induced transgenerational genome instability in vivo

International Nuclear Information System (INIS)

Koturbash, Igor; Baker, Mike; Loree, Jonathan; Kutanzi, Kristy; Hudson, Darryl; Pogribny, Igor; Sedelnikova, Olga; Bonner, William; Kovalchuk, Olga

2006-01-01

Purpose: Although modern cancer radiation therapy has led to increased patient survival rates, the risk of radiation treatment-related complications is becoming a growing problem. Among various complications, radiation also poses a threat to the progeny of exposed parents. It causes transgenerational genome instability that is linked to transgenerational carcinogenesis. Although the occurrence of transgenerational genome instability, which manifests as elevated delayed and nontargeted mutation, has been well documented, the mechanisms by which it arises remain obscure. We hypothesized that epigenetic alterations may play a pivotal role in the molecular etiology of transgenerational genome instability. Methods and Materials: We studied the levels of cytosine DNA methylation in somatic tissues of unexposed offspring upon maternal, paternal, or combined parental exposure. Results: We observed a significant loss of global cytosine DNA methylation in the thymus tissue of the offspring upon combined parental exposure. The loss of DNA methylation was paralleled by a significant decrease in the levels of maintenance (DNMT1) and de novo methyltransferases DNMT3a and 3b and methyl-CpG-binding protein MeCP2. Along with profound changes in DNA methylation, we noted a significant accumulation of DNA strand breaks in thymus, which is a radiation carcinogenesis target organ. Conclusions: The observed changes were indicative of a profound epigenetic dysregulation in the offspring, which in turn could lead to genome destabilization and possibly could serve as precursor for transgenerational carcinogenesis. Future studies are clearly needed to address the cellular and carcinogenic repercussions of those changes

An RNA polymerase II-and AGO4-associated protein acts in RNA-directed DNA methylation

KAUST Repository

Gao, Zhihuan; Liu, Hai-Liang; Daxinger, Lucia; Pontes, Olga; He, Xinjian; Qian, Weiqiang; Lin, Huixin; Xie, Mingtang; Lorkovic, Zdravko J.; Zhang, ShouDong; Miki, Daisuke; Zhan, Xianqiang; Pontier, Dominique; Lagrange, Thierry; Jin, Hailing; Matzke, Antonius J.; Matzke, Marjori; Pikaard, Craig S.; Zhu, Jian-Kang

2010-01-01

DNA methylation is an important epigenetic mark in many eukaryotes. In plants, 24-nucleotide small interfering RNAs (siRNAs) bound to the effector protein, Argonaute 4 (AGO4), can direct de novo DNA methylation by the methyltransferase DRM2 (refs 2

Distinction between the Cfr Methyltransferase Conferring Antibiotic Resistance and the Housekeeping RlmN Methyltransferase

DEFF Research Database (Denmark)

Atkinson, Gemma C; Hansen, Lykke H; Tenson, Tanel

2013-01-01

The cfr gene encodes the Cfr methyltransferase that primarily methylates C-8 in A2503 of 23S rRNA in the peptidyl transferase region of bacterial ribosomes. The methylation provides resistance to six classes of antibiotics of clinical and veterinary importance. The rlmN gene encodes the Rlm......N methyltransferase that methylates C-2 in A2503 in 23S rRNA and A37 in tRNA, but RlmN does not significantly influence antibiotic resistance. The enzymes are homologous and use the same mechanism involving radical S-adenosyl methionine to methylate RNA via an intermediate involving a methylated cysteine....... The differentiation between the two classes is supported by previous and new experimental evidence from antibiotic resistance, primer extensions, and mass spectrometry. Finally, evolutionary aspects of the distribution of Cfr- and RlmN-like enzymes are discussed....

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

IRMPD Action Spectroscopy of Alkali Metal Cation-Cytosine Complexes: Effects of Alkali Metal Cation Size on Gas Phase Conformation

NARCIS (Netherlands)

Yang, B.; Wu, R.R.; Polfer, N.C.; Berden, G.; Oomens, J.; Rodgers, M.T.

2013-01-01

The gas-phase structures of alkali metal cation-cytosine complexes generated by electrospray ionization are probed via infrared multiple photon dissociation (IRMPD) action spectroscopy and theoretical calculations. IRMPD action spectra of five alkali metal cation-cytosine complexes exhibit both

Free radicals from irradiated lyophilized DNA: influence of water of hydration

International Nuclear Information System (INIS)

Huettermann, J.; Roehrig, M.; Koehnlein, W.

1992-01-01

Lyophilized DNA equilibrated with water vapour at various relative humidities (0-95% H 2 O or D 2 O) was X-irradiated at 77 K and analysed for free radicals by electron paramagnetic resonance (EPR) spectroscopy in the temperature range 77-280 K. Analysis of spectra according to variation in humidity, microwave power and temperature generally yielded a doublet and a triplet spectrum at 77 K. The doublet partially converted into the 5-thymyl radical (TH . ). DNA containing deuterated thymine (dTDNA) revealed that the doublet of ''normal'' DNA should be composed of two similar doublets, one of which should be assigned to the thymine anion, the other possibly the cytosine anion. The triplet signal was more stable and could be related to the guanine cation or its deprotonated successor. Several other patterns were detected among them an allyl radical in highly aquated DNA (95% humidity). Other features occurred either predominantly or exclusively in DNA equilibrated above 66% relative humidity and were ascribed to an influence of the secondary structure. (author)

No Effect of Folic Acid Supplementation on Global DNA Methylation in Men and Women with Moderately Elevated Homocysteine

NARCIS (Netherlands)