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Sample records for active dna demethylation

  1. Active DNA demethylation by DNA repair: Facts and uncertainties.

    PubMed

    Schuermann, David; Weber, Alain R; Schär, Primo

    2016-08-01

    Pathways that control and modulate DNA methylation patterning in mammalian cells were poorly understood for a long time, although their importance in establishing and maintaining cell type-specific gene expression was well recognized. The discovery of proteins capable of converting 5-methylcytosine (5mC) to putative substrates for DNA repair introduced a novel and exciting conceptual framework for the investigation and ultimate discovery of molecular mechanisms of DNA demethylation. Against the prevailing notion that DNA methylation is a static epigenetic mark, it turned out to be dynamic and distinct mechanisms appear to have evolved to effect global and locus-specific DNA demethylation. There is compelling evidence that DNA repair, in particular base excision repair, contributes significantly to the turnover of 5mC in cells. By actively demethylating DNA, DNA repair supports the developmental establishment as well as the maintenance of DNA methylation landscapes and gene expression patterns. Yet, while the biochemical pathways are relatively well-established and reviewed, the biological context, function and regulation of DNA repair-mediated active DNA demethylation remains uncertain. In this review, we will thus summarize and critically discuss the evidence that associates active DNA demethylation by DNA repair with specific functional contexts including the DNA methylation erasure in the early embryo, the control of pluripotency and cellular differentiation, the maintenance of cell identity, and the nuclear reprogramming. PMID:27247237

  2. Electrochemical DNA sensor-based strategy for sensitive detection of DNA demethylation and DNA demethylase activity.

    PubMed

    Shen, Qingming; Fan, Mengxing; Yang, Yin; Zhang, Hui

    2016-08-31

    DNA demethylation and demethylase activity play important roles in DNA self-repair, and their detection is key to early diagnosis of fatal diseases. Herein, a facile electrochemical DNA (E-DNA) sensor was developed for the sensitive detection of DNA demethylation and demethylase activity based on an enzyme cleavage strategy. The thiol modified hemi-methylated hairpin probe DNA (pDNA) was self-assembled on a Au electrode surface through the formation of AuS bonds. The hemi-methylated pDNA served as the substrate of DNA demethylase (using methyl-CpG-binding domain protein 2 (MBD2) as an example). Following demethylation, the hairpin stem was then recognized and cleaved by BstUI endonuclease. The ferrocene carboxylic acid (FcA)-tagged pDNA strands were released into the buffer solution from the electrode surface, resulting in a significant decrease of electrochemical signal and providing a means to observe DNA demethylation. The activity of DNA demethylase was analyzed in the concentration ranging from 0.5 to 500 ng mL(-1) with a limit of detection as low as 0.17 ng mL(-1). With high specificity and sensitivity, rapid response, and low cost, this simple E-DNA sensor provides a unique platform for the sensitive detection of DNA demethylation, DNA demethylase activity, and related molecular diagnostics and drug screening. PMID:27506345

  3. Active DNA demethylation at enhancers during the vertebrate phylotypic period.

    PubMed

    Bogdanović, Ozren; Smits, Arne H; de la Calle Mustienes, Elisa; Tena, Juan J; Ford, Ethan; Williams, Ruth; Senanayake, Upeka; Schultz, Matthew D; Hontelez, Saartje; van Kruijsbergen, Ila; Rayon, Teresa; Gnerlich, Felix; Carell, Thomas; Veenstra, Gert Jan C; Manzanares, Miguel; Sauka-Spengler, Tatjana; Ecker, Joseph R; Vermeulen, Michiel; Gómez-Skarmeta, José Luis; Lister, Ryan

    2016-04-01

    The vertebrate body plan and organs are shaped during a conserved embryonic phase called the phylotypic stage. However, the mechanisms that guide the epigenome through this transition and their evolutionary conservation remain elusive. Here we report widespread DNA demethylation of enhancers during the phylotypic period in zebrafish, Xenopus tropicalis and mouse. These enhancers are linked to developmental genes that display coordinated transcriptional and epigenomic changes in the diverse vertebrates during embryogenesis. Binding of Tet proteins to (hydroxy)methylated DNA and enrichment of 5-hydroxymethylcytosine in these regions implicated active DNA demethylation in this process. Furthermore, loss of function of Tet1, Tet2 and Tet3 in zebrafish reduced chromatin accessibility and increased methylation levels specifically at these enhancers, indicative of DNA methylation being an upstream regulator of phylotypic enhancer function. Overall, our study highlights a regulatory module associated with the most conserved phase of vertebrate embryogenesis and suggests an ancient developmental role for Tet dioxygenases. PMID:26928226

  4. The Emerging Nexus of Active DNA Demethylation and Mitochondrial Oxidative Metabolism in Post-Mitotic Neurons

    PubMed Central

    Meng, Huan; Chen, Guiquan; Gao, Hui-Ming; Song, Xiaoyu; Shi, Yun; Cao, Liu

    2014-01-01

    The variable patterns of DNA methylation in mammals have been linked to a number of physiological processes, including normal embryonic development and disease pathogenesis. Active removal of DNA methylation, which potentially regulates neuronal gene expression both globally and gene specifically, has been recently implicated in neuronal plasticity, learning and memory processes. Model pathways of active DNA demethylation involve ten-eleven translocation (TET) methylcytosine dioxygenases that are dependent on oxidative metabolites. In addition, reactive oxygen species (ROS) and oxidizing agents generate oxidative modifications of DNA bases that can be removed by base excision repair proteins. These potentially link the two processes of active DNA demethylation and mitochondrial oxidative metabolism in post-mitotic neurons. We review the current biochemical understanding of the DNA demethylation process and discuss its potential interaction with oxidative metabolism. We then summarise the emerging roles of both processes and their interaction in neural plasticity and memory formation and the pathophysiology of neurodegeneration. Finally, possible therapeutic approaches for neurodegenerative diseases are proposed, including reprogramming therapy by global DNA demethylation and mitohormesis therapy for locus-specific DNA demethylation in post-mitotic neurons. PMID:25490140

  5. An AP endonuclease functions in active DNA demethylation and gene imprinting in Arabidopsis [corrected].

    PubMed

    Li, Yan; Córdoba-Cañero, Dolores; Qian, Weiqiang; Zhu, Xiaohong; Tang, Kai; Zhang, Huiming; Ariza, Rafael R; Roldán-Arjona, Teresa; Zhu, Jian-Kang

    2015-01-01

    Active DNA demethylation in plants occurs through base excision repair, beginning with removal of methylated cytosine by the ROS1/DME subfamily of 5-methylcytosine DNA glycosylases. Active DNA demethylation in animals requires the DNA glycosylase TDG or MBD4, which functions after oxidation or deamination of 5-methylcytosine, respectively. However, little is known about the steps following DNA glycosylase action in the active DNA demethylation pathways in plants and animals. We show here that the Arabidopsis APE1L protein has apurinic/apyrimidinic endonuclease activities and functions downstream of ROS1 and DME. APE1L and ROS1 interact in vitro and co-localize in vivo. Whole genome bisulfite sequencing of ape1l mutant plants revealed widespread alterations in DNA methylation. We show that the ape1l/zdp double mutant displays embryonic lethality. Notably, the ape1l+/-zdp-/- mutant shows a maternal-effect lethality phenotype. APE1L and the DNA phosphatase ZDP are required for FWA and MEA gene imprinting in the endosperm and are important for seed development. Thus, APE1L is a new component of the active DNA demethylation pathway and, together with ZDP, regulates gene imprinting in Arabidopsis. PMID:25569774

  6. Overproduction of stomatal lineage cells in Arabidopsis mutants defective in active DNA demethylation

    PubMed Central

    Yamamuro, Chizuko; Miki, Daisuke; Zheng, Zhimin; Ma, Jun; Wang, Jing; Yang, Zhenbiao; Dong, Juan; Zhu, Jian-Kang

    2014-01-01

    DNA methylation is a reversible epigenetic mark regulating genome stability and function in many eukaryotes. In Arabidopsis, active DNA demethylation depends on the function of the ROS1 subfamily of genes that encode 5-methylcytosine DNA glycosylases/lyases. ROS1-mediated DNA demethylation plays a critical role in the regulation of transgenes, transposable elements and some endogenous genes, but there have been no reports of clear developmental phenotypes in ros1 mutant plants. Here we report that, in the ros1 mutant, the promoter region of the peptide ligand gene EPF2 is hypermethylated, which greatly reduces EPF2 expression and thereby leads to a phenotype of overproduction of stomatal lineage cells. EPF2 gene expression in ros1 is restored and the defective epidermal cell patterning is suppressed by mutations in genes in the RNA-directed DNA methylation pathway. Our results show that active DNA demethylation combats the activity of RNA-directed DNA methylation to influence the initiation of stomatal lineage cells. PMID:24898766

  7. The role of active DNA demethylation and Tet enzyme function in memory formation and cocaine action.

    PubMed

    Alaghband, Yasaman; Bredy, Timothy W; Wood, Marcelo A

    2016-06-20

    Active DNA modification is a major epigenetic mechanism that regulates gene expression in an experience-dependent manner, which is thought to establish stable changes in neuronal function and behavior. Recent discoveries regarding the Ten eleven translocation (Tet1-3) family of DNA hydroxylases have provided a new avenue for the study of active DNA demethylation, and may thus help to advance our understanding of how dynamic DNA modifications lead to long-lasting changes in brain regions underlying learning and memory, as well as drug-seeking and propensity for relapse following abstinence. Drug addiction is a complex, relapsing disorder in which compulsive drug-seeking behavior can persist despite aversive consequences. Therefore, understanding the molecular mechanisms that underlie the onset and persistence of drug addiction, as well as the pronounced propensity for relapse observed in addicts, is necessary for the development of selective treatments and therapies. In this mini-review, we provide an overview of the involvement of active DNA demethylation with an emphasis on the Tet family of enzymes and 5-hydroxymethylcytosine (5-hmC) in learning and memory, as well as in drug-seeking behavior. Memory and addiction share overlapping molecular, cellular, and circuit functions allowing research in one area to inform the other. Current discrepancies and directions for future studies focusing on the dynamic interplay between DNA methylation and demethylation, and how they orchestrate gene expression required for neuronal plasticity underlying memory formation, are discussed. PMID:26806038

  8. AP endonucleases process 5-methylcytosine excision intermediates during active DNA demethylation in Arabidopsis

    PubMed Central

    Lee, Jiyoon; Jang, Hosung; Shin, Hosub; Choi, Woo Lee; Mok, Young Geun; Huh, Jin Hoe

    2014-01-01

    DNA methylation is a primary epigenetic modification regulating gene expression and chromatin structure in many eukaryotes. Plants have a unique DNA demethylation system in that 5-methylcytosine (5mC) is directly removed by DNA demethylases, such as DME/ROS1 family proteins, but little is known about the downstream events. During 5mC excision, DME produces 3′-phosphor-α, β-unsaturated aldehyde and 3′-phosphate by successive β- and δ-eliminations, respectively. The kinetic studies revealed that these 3′-blocking lesions persist for a significant amount of time and at least two different enzyme activities are required to immediately process them. We demonstrate that Arabidopsis AP endonucleases APE1L, APE2 and ARP have distinct functions to process such harmful lesions to allow nucleotide extension. DME expression is toxic to E. coli due to excessive 5mC excision, but expression of APE1L or ARP significantly reduces DME-induced cytotoxicity. Finally, we propose a model of base excision repair and DNA demethylation pathway unique to plants. PMID:25228464

  9. Ligand-activated PPARα-dependent DNA demethylation regulates the fatty acid β-oxidation genes in the postnatal liver.

    PubMed

    Ehara, Tatsuya; Kamei, Yasutomi; Yuan, Xunmei; Takahashi, Mayumi; Kanai, Sayaka; Tamura, Erina; Tsujimoto, Kazutaka; Tamiya, Takashi; Nakagawa, Yoshimi; Shimano, Hitoshi; Takai-Igarashi, Takako; Hatada, Izuho; Suganami, Takayoshi; Hashimoto, Koshi; Ogawa, Yoshihiro

    2015-03-01

    The metabolic function of the liver changes sequentially during early life in mammals to adapt to the marked changes in nutritional environment. Accordingly, hepatic fatty acid β-oxidation is activated after birth to produce energy from breast milk lipids. However, how it is induced during the neonatal period is poorly understood. Here we show DNA demethylation and increased mRNA expression of the fatty acid β-oxidation genes in the postnatal mouse liver. The DNA demethylation does not occur in the fetal mouse liver under the physiologic condition, suggesting that it is specific to the neonatal period. Analysis of mice deficient in the nuclear receptor peroxisome proliferator-activated receptor α (PPARα) and maternal administration of a PPARα ligand during the gestation and lactation periods reveal that the DNA demethylation is PPARα dependent. We also find that DNA methylation of the fatty acid β-oxidation genes are reduced in the adult human liver relative to the fetal liver. This study represents the first demonstration that the ligand-activated PPARα-dependent DNA demethylation regulates the hepatic fatty acid β-oxidation genes during the neonatal period, thereby highlighting the role of a lipid-sensing nuclear receptor in the gene- and life-stage-specific DNA demethylation of a particular metabolic pathway. PMID:25311726

  10. SUMO-modification and elimination of the active DNA demethylation enzyme TDG in cultured human cells.

    PubMed

    Moriyama, Taishi; Fujimitsu, Yuka; Yoshikai, Yushi; Sasano, Takashi; Yamada, Koji; Murakami, Masataka; Urano, Takeshi; Sugasawa, Kaoru; Saitoh, Hisato

    2014-05-01

    Thymine DNA glycosylase (TDG) is a base excision repair enzyme that interacts with the small ubiquitin-related modifier (SUMO)-targeted ubiquitin E3 ligase RNF4 and functions in the active DNA demethylation pathway. Here we showed that both SUMOylated and non-modified forms of endogenous TDG fluctuated during the cell cycle and in response to drugs that perturbed cell cycle progression, including hydroxyurea and nocodazole. Additionally, we detected a SUMOylation-independent association between TDG and RNF4 in vitro as well as in vivo, and observed that both forms of TDG were efficiently degraded in RNF4-depleted cells when arrested at S phase. Our findings provide insights into the in vivo dynamics of TDG SUMOylation and further clarify the TDG-RNF4 interaction. PMID:24727457

  11. OGG1 is essential in oxidative stress induced DNA demethylation.

    PubMed

    Zhou, Xiaolong; Zhuang, Ziheng; Wang, Wentao; He, Lingfeng; Wu, Huan; Cao, Yan; Pan, Feiyan; Zhao, Jing; Hu, Zhigang; Sekhar, Chandra; Guo, Zhigang

    2016-09-01

    DNA demethylation is an essential cellular activity to regulate gene expression; however, the mechanism that triggers DNA demethylation remains unknown. Furthermore, DNA demethylation was recently demonstrated to be induced by oxidative stress without a clear molecular mechanism. In this manuscript, we demonstrated that 8-oxoguanine DNA glycosylase-1 (OGG1) is the essential protein involved in oxidative stress-induced DNA demethylation. Oxidative stress induced the formation of 8-oxoguanine (8-oxoG). We found that OGG1, the 8-oxoG binding protein, promotes DNA demethylation by interacting and recruiting TET1 to the 8-oxoG lesion. Downregulation of OGG1 makes cells resistant to oxidative stress-induced DNA demethylation, while over-expression of OGG1 renders cells susceptible to DNA demethylation by oxidative stress. These data not only illustrate the importance of base excision repair (BER) in DNA demethylation but also reveal how the DNA demethylation signal is transferred to downstream DNA demethylation enzymes. PMID:27251462

  12. An interplay of the base excision repair and mismatch repair pathways in active DNA demethylation.

    PubMed

    Grin, Inga; Ishchenko, Alexander A

    2016-05-01

    Active DNA demethylation (ADDM) in mammals occurs via hydroxylation of 5-methylcytosine (5mC) by TET and/or deamination by AID/APOBEC family enzymes. The resulting 5mC derivatives are removed through the base excision repair (BER) pathway. At present, it is unclear how the cell manages to eliminate closely spaced 5mC residues whilst avoiding generation of toxic BER intermediates and whether alternative DNA repair pathways participate in ADDM. It has been shown that non-canonical DNA mismatch repair (ncMMR) can remove both alkylated and oxidized nucleotides from DNA. Here, a phagemid DNA containing oxidative base lesions and methylated sites are used to examine the involvement of various DNA repair pathways in ADDM in murine and human cell-free extracts. We demonstrate that, in addition to short-patch BER, 5-hydroxymethyluracil and uracil mispaired with guanine can be processed by ncMMR and long-patch BER with concomitant removal of distant 5mC residues. Furthermore, the presence of multiple mispairs in the same MMR nick/mismatch recognition region together with BER-mediated nick formation promotes proficient ncMMR resulting in the reactivation of an epigenetically silenced reporter gene in murine cells. These findings suggest cooperation between BER and ncMMR in the removal of multiple mismatches that might occur in mammalian cells during ADDM. PMID:26843430

  13. An interplay of the base excision repair and mismatch repair pathways in active DNA demethylation

    PubMed Central

    Grin, Inga; Ishchenko, Alexander A.

    2016-01-01

    Active DNA demethylation (ADDM) in mammals occurs via hydroxylation of 5-methylcytosine (5mC) by TET and/or deamination by AID/APOBEC family enzymes. The resulting 5mC derivatives are removed through the base excision repair (BER) pathway. At present, it is unclear how the cell manages to eliminate closely spaced 5mC residues whilst avoiding generation of toxic BER intermediates and whether alternative DNA repair pathways participate in ADDM. It has been shown that non-canonical DNA mismatch repair (ncMMR) can remove both alkylated and oxidized nucleotides from DNA. Here, a phagemid DNA containing oxidative base lesions and methylated sites are used to examine the involvement of various DNA repair pathways in ADDM in murine and human cell-free extracts. We demonstrate that, in addition to short-patch BER, 5-hydroxymethyluracil and uracil mispaired with guanine can be processed by ncMMR and long-patch BER with concomitant removal of distant 5mC residues. Furthermore, the presence of multiple mispairs in the same MMR nick/mismatch recognition region together with BER-mediated nick formation promotes proficient ncMMR resulting in the reactivation of an epigenetically silenced reporter gene in murine cells. These findings suggest cooperation between BER and ncMMR in the removal of multiple mismatches that might occur in mammalian cells during ADDM. PMID:26843430

  14. Pharmacological DNA demethylation: Implications for cancer immunotherapy

    PubMed Central

    Roulois, David; Yau, Helen Loo; De Carvalho, Daniel D.

    2016-01-01

    ABSTRACT Recent studies have demonstrated that DNA demethylation agents can mimic a viral infection by induction of dsRNAs. This viral mimicry leads to an antiviral response mediated by the cytosolic pattern recognition receptor MDA5, followed by MAVS (IPS1) activation, IRF7 nuclear translocation and upregulation of type III Interferon and interferon-stimulated genes. PMID:27141349

  15. De novo DNA demethylation and noncoding transcription define active intergenic regulatory elements.

    PubMed

    Schlesinger, Felix; Smith, Andrew D; Gingeras, Thomas R; Hannon, Gregory J; Hodges, Emily

    2013-10-01

    Deep sequencing of mammalian DNA methylomes has uncovered a previously unpredicted number of discrete hypomethylated regions in intergenic space (iHMRs). Here, we combined whole-genome bisulfite sequencing data with extensive gene expression and chromatin-state data to define functional classes of iHMRs, and to reconstruct the dynamics of their establishment in a developmental setting. Comparing HMR profiles in embryonic stem and primary blood cells, we show that iHMRs mark an exclusive subset of active DNase hypersensitive sites (DHS), and that both developmentally constitutive and cell-type-specific iHMRs display chromatin states typical of distinct regulatory elements. We also observe that iHMR changes are more predictive of nearby gene activity than the promoter HMR itself, and that expression of noncoding RNAs within the iHMR accompanies full activation and complete demethylation of mature B cell enhancers. Conserved sequence features corresponding to iHMR transcript start sites, including a discernible TATA motif, suggest a conserved, functional role for transcription in these regions. Similarly, we explored both primate-specific and human population variation at iHMRs, finding that while enhancer iHMRs are more variable in sequence and methylation status than any other functional class, conservation of the TATA box is highly predictive of iHMR maintenance, reflecting the impact of sequence plasticity and transcriptional signals on iHMR establishment. Overall, our analysis allowed us to construct a three-step timeline in which (1) intergenic DHS are pre-established in the stem cell, (2) partial demethylation of blood-specific intergenic DHSs occurs in blood progenitors, and (3) complete iHMR formation and transcription coincide with enhancer activation in lymphoid-specified cells. PMID:23811145

  16. De novo DNA demethylation and noncoding transcription define active intergenic regulatory elements

    PubMed Central

    Schlesinger, Felix; Smith, Andrew D.; Gingeras, Thomas R.; Hannon, Gregory J.; Hodges, Emily

    2013-01-01

    Deep sequencing of mammalian DNA methylomes has uncovered a previously unpredicted number of discrete hypomethylated regions in intergenic space (iHMRs). Here, we combined whole-genome bisulfite sequencing data with extensive gene expression and chromatin-state data to define functional classes of iHMRs, and to reconstruct the dynamics of their establishment in a developmental setting. Comparing HMR profiles in embryonic stem and primary blood cells, we show that iHMRs mark an exclusive subset of active DNase hypersensitive sites (DHS), and that both developmentally constitutive and cell-type-specific iHMRs display chromatin states typical of distinct regulatory elements. We also observe that iHMR changes are more predictive of nearby gene activity than the promoter HMR itself, and that expression of noncoding RNAs within the iHMR accompanies full activation and complete demethylation of mature B cell enhancers. Conserved sequence features corresponding to iHMR transcript start sites, including a discernible TATA motif, suggest a conserved, functional role for transcription in these regions. Similarly, we explored both primate-specific and human population variation at iHMRs, finding that while enhancer iHMRs are more variable in sequence and methylation status than any other functional class, conservation of the TATA box is highly predictive of iHMR maintenance, reflecting the impact of sequence plasticity and transcriptional signals on iHMR establishment. Overall, our analysis allowed us to construct a three-step timeline in which (1) intergenic DHS are pre-established in the stem cell, (2) partial demethylation of blood-specific intergenic DHSs occurs in blood progenitors, and (3) complete iHMR formation and transcription coincide with enhancer activation in lymphoid-specified cells. PMID:23811145

  17. IL-2 and GM-CSF are regulated by DNA demethylation during activation of T cells, B cells and macrophages

    SciTech Connect

    Li, Yan; Ohms, Stephen J.; Shannon, Frances M.; Sun, Chao; Fan, Jun Y.

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer DNA methylation is dynamic and flexible and changes rapidly upon cell activation. Black-Right-Pointing-Pointer DNA methylation controls the inducible gene expression in a given cell type. Black-Right-Pointing-Pointer Some enzymes are involved in maintaining the methylation profile of immune cells. -- Abstract: DNA demethylation has been found to occur at the promoters of a number of actively expressed cytokines and is believed to play a critical role in transcriptional regulation. While many DNA demethylation studies have focused on T cell activation, proliferation and differentiation, changes in DNA methylation in other types of immune cells are less well studied. We found that the expression of two cytokines (IL-2 and GM-CSF) responded differently to activation in three types of immune cells: EL4, A20 and RAW264.7 cells. Using the McrBC and MeDIP approaches, we observed decreases in DNA methylation at a genome-wide level and at the promoters of the genes of these cytokines. The expression of several potential enzymes/co-enzymes involved in the DNA demethylation pathways seemed to be associated with immune cell activation.

  18. Methyl-CpG-binding domain protein MBD7 is required for active DNA demethylation in Arabidopsis.

    PubMed

    Wang, Chunlei; Dong, Xiaomei; Jin, Dan; Zhao, Yusheng; Xie, Shaojun; Li, Xiaojie; He, Xinjian; Lang, Zhaobo; Lai, Jinsheng; Zhu, Jian-Kang; Gong, Zhizhong

    2015-03-01

    Although researchers have established that DNA methylation and active demethylation are dynamically regulated in plant cells, the molecular mechanism for the regulation of active DNA demethylation is not well understood. By using an Arabidopsis (Arabidopsis thaliana) line expressing the Promoter RESPONSIVE TO DEHYDRATION 29A:LUCIFERASE (ProRD29A:LUC) and Promoter cauliflower mosaic virus 35S:NEOMYCIN PHOSPHOTRANSFERASE II (Pro35S:NPTII) transgenes, we isolated an mbd7 (for methyl-CpG-binding domain protein7) mutant. The mbd7 mutation causes an inactivation of the Pro35S:NPTII transgene but does not affect the expression of the ProRD29A:LUC transgene. The silencing of the Pro35S:NPTII reporter gene is associated with DNA hypermethylation of the reporter gene. MBD7 interacts physically with REPRESSOR OF SILENCING5/INCREASED DNA METHYLATION2, a protein in the small heat shock protein family. MBD7 prefers to target the genomic loci with high densities of DNA methylation around chromocenters. The Gypsy-type long terminal repeat retrotransposons mainly distributed around chromocenters are most affected by mbd7 in all transposons. Our results suggest that MBD7 is required for active DNA demethylation and antisilencing of the genomic loci with high densities of DNA methylation in Arabidopsis. PMID:25593350

  19. Biochemical reconstitution of TET1–TDG–BER-dependent active DNA demethylation reveals a highly coordinated mechanism

    PubMed Central

    Weber, Alain R.; Krawczyk, Claudia; Robertson, Adam B.; Kuśnierczyk, Anna; Vågbø, Cathrine B.; Schuermann, David; Klungland, Arne; Schär, Primo

    2016-01-01

    Cytosine methylation in CpG dinucleotides is an epigenetic DNA modification dynamically established and maintained by DNA methyltransferases and demethylases. Molecular mechanisms of active DNA demethylation began to surface only recently with the discovery of the 5-methylcytosine (5mC)-directed hydroxylase and base excision activities of ten–eleven translocation (TET) proteins and thymine DNA glycosylase (TDG). This implicated a pathway operating through oxidation of 5mC by TET proteins, which generates substrates for TDG-dependent base excision repair (BER) that then replaces 5mC with C. Yet, direct evidence for a productive coupling of TET with BER has never been presented. Here we show that TET1 and TDG physically interact to oxidize and excise 5mC, and proof by biochemical reconstitution that the TET–TDG–BER system is capable of productive DNA demethylation. We show that the mechanism assures a sequential demethylation of symmetrically methylated CpGs, thereby avoiding DNA double-strand break formation but contributing to the mutability of methylated CpGs. PMID:26932196

  20. Biochemical reconstitution of TET1-TDG-BER-dependent active DNA demethylation reveals a highly coordinated mechanism.

    PubMed

    Weber, Alain R; Krawczyk, Claudia; Robertson, Adam B; Kuśnierczyk, Anna; Vågbø, Cathrine B; Schuermann, David; Klungland, Arne; Schär, Primo

    2016-01-01

    Cytosine methylation in CpG dinucleotides is an epigenetic DNA modification dynamically established and maintained by DNA methyltransferases and demethylases. Molecular mechanisms of active DNA demethylation began to surface only recently with the discovery of the 5-methylcytosine (5mC)-directed hydroxylase and base excision activities of ten-eleven translocation (TET) proteins and thymine DNA glycosylase (TDG). This implicated a pathway operating through oxidation of 5mC by TET proteins, which generates substrates for TDG-dependent base excision repair (BER) that then replaces 5mC with C. Yet, direct evidence for a productive coupling of TET with BER has never been presented. Here we show that TET1 and TDG physically interact to oxidize and excise 5mC, and proof by biochemical reconstitution that the TET-TDG-BER system is capable of productive DNA demethylation. We show that the mechanism assures a sequential demethylation of symmetrically methylated CpGs, thereby avoiding DNA double-strand break formation but contributing to the mutability of methylated CpGs. PMID:26932196

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

    PubMed

    Rajput, Pallavi; Pandey, Vijaya; Kumar, Vijay

    2016-08-01

    The well-studied Pol II transcription factor Sp1 has not been investigated for its regulatory role in rDNA transcription. Here, we show that Sp1 bound to specific sites on rDNA and localized into the nucleoli during the G1 phase of cell cycle to activate rDNA transcription. It facilitated the recruitment of Pol I pre-initiation complex and impeded the binding of nucleolar remodeling complex (NoRC) to rDNA resulting in the formation of euchromatin active state. More importantly, Sp1 also orchestrated the site-specific binding of Gadd45a-nucleotide excision repair (NER) complex resulting in active demethylation and transcriptional activation of rDNA. Interestingly, knockdown of Sp1 impaired rDNA transcription due to reduced engagement of the Gadd45a-NER complex and hypermethylation of rDNA. Thus, the present study unveils a novel role of Sp1 in rDNA transcription involving promoter demethylation. PMID:27156884

  2. The cytosolic Fe-S cluster assembly component MET18 is required for the full enzymatic activity of ROS1 in active DNA demethylation

    PubMed Central

    Wang, Xiaokang; Li, Qi; Yuan, Wei; Cao, Zhendong; Qi, Bei; Kumar, Suresh; Li, Yan; Qian, Weiqiang

    2016-01-01

    DNA methylation patterns in plants are dynamically regulated by DNA methylation and active DNA demethylation in response to both environmental changes and development of plant. Beginning with the removal of methylated cytosine by ROS1/DME family of 5-methylcytosine DNA glycosylases, active DNA demethylation in plants occurs through base excision repair. So far, many components involved in active DNA demethylation remain undiscovered. Through a forward genetic screening of Arabidopsis mutants showing DNA hypermethylation at the EPF2 promoter region, we identified the conserved iron-sulfur cluster assembly protein MET18. MET18 dysfunction caused DNA hypermethylation at more than 1000 loci as well as the silencing of reporter genes and some endogenous genes. MET18 can directly interact with ROS1 in vitro and in vivo. ROS1 activity was reduced in the met18 mutant plants and point mutation in the conserved Fe-S cluster binding motif of ROS1 disrupted its biological function. Interestingly, a large number of DNA hypomethylated loci, especially in the CHH context, were identified from the met18 mutants and most of the hypo-DMRs were from TE regions. Our results suggest that MET18 can regulate both active DNA demethylation and DNA methylation pathways in Arabidopsis. PMID:27193999

  3. The cytosolic Fe-S cluster assembly component MET18 is required for the full enzymatic activity of ROS1 in active DNA demethylation.

    PubMed

    Wang, Xiaokang; Li, Qi; Yuan, Wei; Cao, Zhendong; Qi, Bei; Kumar, Suresh; Li, Yan; Qian, Weiqiang

    2016-01-01

    DNA methylation patterns in plants are dynamically regulated by DNA methylation and active DNA demethylation in response to both environmental changes and development of plant. Beginning with the removal of methylated cytosine by ROS1/DME family of 5-methylcytosine DNA glycosylases, active DNA demethylation in plants occurs through base excision repair. So far, many components involved in active DNA demethylation remain undiscovered. Through a forward genetic screening of Arabidopsis mutants showing DNA hypermethylation at the EPF2 promoter region, we identified the conserved iron-sulfur cluster assembly protein MET18. MET18 dysfunction caused DNA hypermethylation at more than 1000 loci as well as the silencing of reporter genes and some endogenous genes. MET18 can directly interact with ROS1 in vitro and in vivo. ROS1 activity was reduced in the met18 mutant plants and point mutation in the conserved Fe-S cluster binding motif of ROS1 disrupted its biological function. Interestingly, a large number of DNA hypomethylated loci, especially in the CHH context, were identified from the met18 mutants and most of the hypo-DMRs were from TE regions. Our results suggest that MET18 can regulate both active DNA demethylation and DNA methylation pathways in Arabidopsis. PMID:27193999

  4. Gadd45a promotes DNA demethylation through TDG.

    PubMed

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

    2015-04-30

    Growth arrest and DNA-damage-inducible protein 45 (Gadd45) family members have been implicated in DNA demethylation in vertebrates. However, it remained unclear how they contribute to the demethylation process. Here, we demonstrate that Gadd45a promotes active DNA demethylation through thymine DNA glycosylase (TDG) which has recently been shown to excise 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) generated in Ten-eleven-translocation (Tet)-initiated oxidative demethylation. The connection of Gadd45a with oxidative demethylation is evidenced by the enhanced activation of a methylated reporter gene in HEK293T cells expressing Gadd45a in combination with catalytically active TDG and Tet. Gadd45a interacts with TDG physically and increases the removal of 5fC and 5caC from genomic and transfected plasmid DNA by TDG. Knockout of both Gadd45a and Gadd45b from mouse ES cells leads to hypermethylation of specific genomic loci most of which are also targets of TDG and show 5fC enrichment in TDG-deficient cells. These observations indicate that the demethylation effect of Gadd45a is mediated by TDG activity. This finding thus unites Gadd45a with the recently defined Tet-initiated demethylation pathway. PMID:25845601

  5. Gadd45a promotes DNA demethylation through TDG

    PubMed Central

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

    2015-01-01

    Growth arrest and DNA-damage-inducible protein 45 (Gadd45) family members have been implicated in DNA demethylation in vertebrates. However, it remained unclear how they contribute to the demethylation process. Here, we demonstrate that Gadd45a promotes active DNA demethylation through thymine DNA glycosylase (TDG) which has recently been shown to excise 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) generated in Ten-eleven-translocation (Tet)—initiated oxidative demethylation. The connection of Gadd45a with oxidative demethylation is evidenced by the enhanced activation of a methylated reporter gene in HEK293T cells expressing Gadd45a in combination with catalytically active TDG and Tet. Gadd45a interacts with TDG physically and increases the removal of 5fC and 5caC from genomic and transfected plasmid DNA by TDG. Knockout of both Gadd45a and Gadd45b from mouse ES cells leads to hypermethylation of specific genomic loci most of which are also targets of TDG and show 5fC enrichment in TDG-deficient cells. These observations indicate that the demethylation effect of Gadd45a is mediated by TDG activity. This finding thus unites Gadd45a with the recently defined Tet-initiated demethylation pathway. PMID:25845601

  6. MET18 Connects the Cytosolic Iron-Sulfur Cluster Assembly Pathway to Active DNA Demethylation in Arabidopsis

    PubMed Central

    Tang, Kai; Zhang, Huiming; Mangrauthia, Satendra K.; Lei, Mingguang; Hsu, Chuan-Chih; Hou, Yueh-Ju; Wang, Chunguo; Li, Yan; Tao, W. Andy; Zhu, Jian-Kang

    2015-01-01

    DNA demethylation mediated by the DNA glycosylase ROS1 helps determine genomic DNA methylation patterns and protects active genes from being silenced. However, little is known about the mechanism of regulation of ROS1 enzymatic activity. Using a forward genetic screen, we identified an anti-silencing (ASI) factor, ASI3, the dysfunction of which causes transgene promoter hyper-methylation and silencing. Map-based cloning identified ASI3 as MET18, a component of the cytosolic iron-sulfur cluster assembly (CIA) pathway. Mutation in MET18 leads to hyper-methylation at thousands of genomic loci, the majority of which overlap with hypermethylated loci identified in ros1 and ros1dml2dml3 mutants. Affinity purification followed by mass spectrometry indicated that ROS1 physically associates with MET18 and other CIA components. Yeast two-hybrid and split luciferase assays showed that ROS1 can directly interact with MET18 and another CIA component, AE7. Site-directed mutagenesis of ROS1 indicated that the conserved iron-sulfur motif is indispensable for ROS1 enzymatic activity. Our results suggest that ROS1-mediated active DNA demethylation requires MET18-dependent transfer of the iron-sulfur cluster, highlighting an important role of the CIA pathway in epigenetic regulation. PMID:26492035

  7. MET18 Connects the Cytosolic Iron-Sulfur Cluster Assembly Pathway to Active DNA Demethylation in Arabidopsis.

    PubMed

    Duan, Cheng-Guo; Wang, Xingang; Tang, Kai; Zhang, Huiming; Mangrauthia, Satendra K; Lei, Mingguang; Hsu, Chuan-Chih; Hou, Yueh-Ju; Wang, Chunguo; Li, Yan; Tao, W Andy; Zhu, Jian-Kang

    2015-10-01

    DNA demethylation mediated by the DNA glycosylase ROS1 helps determine genomic DNA methylation patterns and protects active genes from being silenced. However, little is known about the mechanism of regulation of ROS1 enzymatic activity. Using a forward genetic screen, we identified an anti-silencing (ASI) factor, ASI3, the dysfunction of which causes transgene promoter hyper-methylation and silencing. Map-based cloning identified ASI3 as MET18, a component of the cytosolic iron-sulfur cluster assembly (CIA) pathway. Mutation in MET18 leads to hyper-methylation at thousands of genomic loci, the majority of which overlap with hypermethylated loci identified in ros1 and ros1dml2dml3 mutants. Affinity purification followed by mass spectrometry indicated that ROS1 physically associates with MET18 and other CIA components. Yeast two-hybrid and split luciferase assays showed that ROS1 can directly interact with MET18 and another CIA component, AE7. Site-directed mutagenesis of ROS1 indicated that the conserved iron-sulfur motif is indispensable for ROS1 enzymatic activity. Our results suggest that ROS1-mediated active DNA demethylation requires MET18-dependent transfer of the iron-sulfur cluster, highlighting an important role of the CIA pathway in epigenetic regulation. PMID:26492035

  8. DNA Methylation and Demethylation in Plant Immunity.

    PubMed

    Deleris, A; Halter, T; Navarro, L

    2016-08-01

    Detection of plant and animal pathogens triggers a massive transcriptional reprogramming, which is directed by chromatin-based processes, and ultimately results in antimicrobial immunity. Although the implication of histone modifications in orchestrating biotic stress-induced transcriptional reprogramming has been well characterized, very little was known, until recently, about the role of DNA methylation and demethylation in this process. In this review, we summarize recent findings on the dynamics and biological relevance of DNA methylation and demethylation in plant immunity against nonviral pathogens. In particular, we report the implications of these epigenetic regulatory processes in the transcriptional and co-transcriptional control of immune-responsive genes and discuss their relevance in fine-tuning antimicrobial immune responses. Finally, we discuss the possible yet elusive role of DNA methylation and demethylation in systemic immune responses, transgenerational immune priming, and de novo epiallelism, which could be adaptive. PMID:27491436

  9. Base-resolution profiling of active DNA demethylation using MAB-seq and caMAB-seq.

    PubMed

    Wu, Hao; Wu, Xiaoji; Zhang, Yi

    2016-06-01

    A complete understanding of the function of the ten-eleven translocation (TET) family of dioxygenase-mediated DNA demethylation requires new methods to quantitatively map oxidized 5-methylcytosine (5mC) bases at high resolution. We have recently developed a methylase-assisted bisulfite sequencing (MAB-seq) method that allows base-resolution mapping of 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), two oxidized 5mC bases indicative of active DNA demethylation events. In standard bisulfite sequencing (BS-seq), unmodified C, 5fC and 5caC are read as thymine; thus 5fC and 5caC cannot be distinguished from C. In MAB-seq, unmodified C is enzymatically converted to 5mC, allowing direct mapping of rare modifications such as 5fC and 5caC. By combining MAB-seq with chemical reduction of 5fC to 5hmC, we also developed caMAB-seq, a method for direct 5caC mapping. Compared with subtraction-based mapping methods, MAB-seq and caMAB-seq require less sequencing effort and enable robust statistical calling of 5fC and/or 5caC. MAB-seq and caMAB-seq can be adapted to map 5fC/5caC at the whole-genome scale (WG-MAB-seq), within specific genomic regions enriched for enhancer-marking histone modifications (chromatin immunoprecipitation (ChIP)-MAB-seq), or at CpG-rich sequences (reduced-representation (RR)-MAB-seq) such as gene promoters. The full protocol, including DNA preparation, enzymatic treatment, library preparation and sequencing, can be completed within 6-8 d. PMID:27172168

  10. Hepatitis B virus X protein induces EpCAM expression via active DNA demethylation directed by RelA in complex with EZH2 and TET2.

    PubMed

    Fan, H; Zhang, H; Pascuzzi, P E; Andrisani, O

    2016-02-11

    Chronic hepatitis B virus (HBV) infection is a major risk factor for developing hepatocellular carcinoma (HCC), and HBV X protein (HBx) acts as cofactor in hepatocarcinogenesis. In liver tumors from animals modeling HBx- and HBV-mediated hepatocarcinogenesis, downregulation of chromatin regulating proteins SUZ12 and ZNF198 induces expression of several genes, including epithelial cell adhesion molecule (EpCAM). EpCAM upregulation occurs in HBV-mediated HCCs and hepatic cancer stem cells, by a mechanism not understood. Herein we demonstrate HBx induces EpCAM expression via active DNA demethylation. In hepatocytes, EpCAM is silenced by polycomb repressive complex 2 (PRC2) and ZNF198/LSD1/Co-REST/HDAC1 chromatin-modifying complexes. Cells with stable knockdown of SUZ12, an essential PRC2 subunit, upon HBx expression demethylate a CpG dinucleotide located adjacent to NF-κB/RelA half-site. This NF-κB/RelA site is in a CpG island downstream from EpCAM transcriptional start site (TSS). Chromatin immunoprecipitation (ChIP) assays demonstrate HBx-dependent RelA occupancy of NF-κB half-site, whereas RelA knockdown suppresses CpG demethylation and EpCAM expression. Tumor necrosis factor-α activates RelA, propagating demethylation to nearby CpG sites, shown by sodium bisulfite sequencing. RelA-dependent demethylation occurring upon HBx expression requires methyltrasferase EZH2, TET2 a key factor in cytosine demethylation and inactive DNMT3L, shown by knockdown assays and sodium bisulfite sequencing. Co-immunoprecipitations and sequential ChIP assays demonstrate that RelA in the presence of HBx forms a complex with EZH2, TET2 and DNMT3L, although the role of DNMT3L remains to be understood. Interestingly, the human EpCAM gene also has a CpG island downstream from its TSS, and a NF-κB-binding site flanked by CpGs. HepG2 cells derived from human HCC exhibit demethylation of these NF-κB-flanking CpG sites, and HBV replication propagates demethylation to nearby CpG sites. DLK

  11. DNA demethylation pathways: recent insights

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DNA methylation is a major epigenetic regulatory mechanism for gene expression and cell differentiation. While methyltransferases mediate cytosine methylation, until recently, it was still less clear how unmethylated regions in mammalian genomes are protected from de novo methylation and whether ac...

  12. Uracil-DNA Glycosylase UNG Promotes Tet-mediated DNA Demethylation.

    PubMed

    Xue, Jian-Huang; Xu, Gui-Fang; Gu, Tian-Peng; Chen, Guo-Dong; Han, Bin-Bin; Xu, Zhi-Mei; Bjørås, Magnar; Krokan, Hans E; Xu, Guo-Liang; Du, Ya-Rui

    2016-01-01

    In mammals, active DNA demethylation involves oxidation of 5-methylcytosine (5mC) into 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) by Tet dioxygenases and excision of these two oxidized bases by thymine DNA glycosylase (TDG). Although TDG is essential for active demethylation in embryonic stem cells and induced pluripotent stem cells, it is hardly expressed in mouse zygotes and dispensable in pronuclear DNA demethylation. To search for other factors that might contribute to demethylation in mammalian cells, we performed a functional genomics screen based on a methylated luciferase reporter assay. UNG2, one of the glycosylases known to excise uracil residues from DNA, was found to reduce DNA methylation, thus activating transcription of a methylation-silenced reporter gene when co-transfected with Tet2 into HEK293T cells. Interestingly, UNG2 could decrease 5caC from the genomic DNA and a reporter plasmid in transfected cells, like TDG. Furthermore, deficiency in Ung partially impaired DNA demethylation in mouse zygotes. Our results suggest that UNG might be involved in Tet-mediated DNA demethylation. PMID:26620559

  13. RNA-directed DNA methylation and demethylation in plants

    PubMed Central

    Viswanathan, CHINNUSAMY; Jian-Kang, ZHU

    2011-01-01

    RNA-directed DNA methylation (RdDM) is a nuclear process in which small interfering RNAs (siRNAs) direct the cytosine methylation of DNA sequences that are complementary to the siRNAs. In plants, double stranded-RNAs (dsRNAs) generated by RNA-dependent RNA polymerase 2 (RDR2) serve as precursors for Dicer-like 3 dependent biogenesis of 24-nt siRNAs. Plant specific RNA polymerase IV (Pol IV) is presumed to generate the initial RNA transcripts that are substrates for RDR2. siRNAs are loaded onto an argonaute4-containing RISC (RNA-induced silencing complex) that targets the de novo DNA methyltransferase DRM2 to RdDM target loci. Nascent RNA transcripts from the target loci are generated by another plant-specific RNA polymerase, Pol V, and these transcripts help recruit complementary siRNAs and the associated RdDM effector complex to the target loci in a transcription-coupled DNA methylation process. Small RNA binding proteins such as ROS3 may direct target-specific DNA demethylation by the ROS1 family of DNA demethylases. Chromatin remodeling enzymes and histone modifying enzymes also participate in DNA methylation and possibly demethylation. One of the well studied functions of RdDM is transposon silencing and genome stability. In addition, RdDM is important for paramutation, imprinting, gene regulation, and plant development. Locus-specific DNA methylation and demethylation, and transposon activation under abiotic stresses suggest that RdDM is also important in stress responses of plants. Further studies will help illuminate the functions of RdDM in the dynamic control of epigenomes during development and environmental stress responses. PMID:19381459

  14. DNA-demethylating and anti-tumor activity of synthetic miR-29b mimics in multiple myeloma.

    PubMed

    Amodio, Nicola; Leotta, Marzia; Bellizzi, Dina; Di Martino, Maria Teresa; D'Aquila, Patrizia; Lionetti, Marta; Fabiani, Fernanda; Leone, Emanuela; Gullà, Anna Maria; Passarino, Giuseppe; Caraglia, Michele; Negrini, Massimo; Neri, Antonino; Giordano, Antonio; Tagliaferri, Pierosandro; Tassone, Pierfrancesco

    2012-10-01

    Aberrant DNA methylation plays a relevant role in multiple myeloma (MM) pathogenesis. MicroRNAs (miRNAs) are a class of small non-coding RNAs that recently emerged as master regulator of gene expression by targeting protein-coding mRNAs. However, miRNAs involvement in the regulation of the epigenetic machinery and their potential use as therapeutics in MM remain to be investigated. Here, we provide evidence that the expression of de novo DNA methyltransferases (DNMTs) is deregulated in MM cells. Moreover, we show that miR-29b targets DNMT3A and DNMT3B mRNAs and reduces global DNA methylation in MM cells. In vitro transfection of MM cells with synthetic miR-29b mimics significantly impairs cell cycle progression and also potentiates the growth-inhibitory effects induced by the demethylating agent 5-azacitidine. Most importantly, in vivo intratumor or systemic delivery of synthetic miR-29b mimics, in two clinically relevant murine models of human MM, including the SCID-synth-hu system, induces significant anti-tumor effects. All together, our findings demonstrate that aberrant DNMTs expression is efficiently modulated by tumor suppressive synthetic miR-29b mimics, indicating that methyloma modulation is a novel matter of investigation in miRNA-based therapy of MM. PMID:23100393

  15. DNA Demethylation Dynamics in the Human Prenatal Germline.

    PubMed

    Gkountela, Sofia; Zhang, Kelvin X; Shafiq, Tiasha A; Liao, Wen-Wei; Hargan-Calvopiña, Joseph; Chen, Pao-Yang; Clark, Amander T

    2015-06-01

    Global DNA demethylation in humans is a fundamental process that occurs in pre-implantation embryos and reversion to naive ground state pluripotent stem cells (PSCs). However, the extent of DNA methylation reprogramming in human germline cells is unknown. Here, we performed whole-genome bisulfite sequencing (WGBS) and RNA-sequencing (RNA-seq) of human prenatal germline cells from 53 to 137 days of development. We discovered that the transcriptome and methylome of human germline is distinct from both human PSCs and the inner cell mass (ICM) of human blastocysts. Using this resource to monitor the outcome of global DNA demethylation with reversion of primed PSCs to the naive ground state, we uncovered hotspots of ultralow methylation at transposons that are protected from demethylation in the germline and ICM. Taken together, the human germline serves as a valuable in vivo tool for monitoring the epigenome of cells that have emerged from a global DNA demethylation event. PMID:26004067

  16. Tissue-specific DNA demethylation is required for proper B-cell differentiation and function.

    PubMed

    Orlanski, Shari; Labi, Verena; Reizel, Yitzhak; Spiro, Adam; Lichtenstein, Michal; Levin-Klein, Rena; Koralov, Sergei B; Skversky, Yael; Rajewsky, Klaus; Cedar, Howard; Bergman, Yehudit

    2016-05-01

    There is ample evidence that somatic cell differentiation during development is accompanied by extensive DNA demethylation of specific sites that vary between cell types. Although the mechanism of this process has not yet been elucidated, it is likely to involve the conversion of 5mC to 5hmC by Tet enzymes. We show that a Tet2/Tet3 conditional knockout at early stages of B-cell development largely prevents lineage-specific programmed demethylation events. This lack of demethylation affects the expression of nearby B-cell lineage genes by impairing enhancer activity, thus causing defects in B-cell differentiation and function. Thus, tissue-specific DNA demethylation appears to be necessary for proper somatic cell development in vivo. PMID:27091986

  17. A Comparative Analysis of 5-Azacytidine- and Zebularine-Induced DNA Demethylation

    PubMed Central

    Griffin, Patrick T.; Niederhuth, Chad E.; Schmitz, Robert J.

    2016-01-01

    The nonmethylable cytosine analogs, 5-azacytidine and zebularine, are widely used to inhibit DNA methyltransferase activity and reduce genomic DNA methylation. In this study, whole-genome bisulfite sequencing is used to construct maps of DNA methylation with single base pair resolution in Arabidopsis thaliana seedlings treated with each demethylating agent. We find that both inhibitor treatments result in nearly indistinguishable patterns of genome-wide DNA methylation and that 5-azacytidine had a slightly greater demethylating effect at higher concentrations across the genome. Transcriptome analyses revealed a substantial number of upregulated genes, with an overrepresentation of transposable element genes, in particular CACTA-like elements. This demonstrates that chemical demethylating agents have a disproportionately large effect on loci that are otherwise silenced by DNA methylation. PMID:27402357

  18. Promoter DNA demethylation of Keap1 gene in diabetic cardiomyopathy

    PubMed Central

    Liu, Zhong-Zhi; Zhao, Xiang-Zhi; Zhang, Xue-Song; Zhang, Mei

    2014-01-01

    Researches have shown that the onset of diabetes is closely associated with oxidative stress and the chronic exposure leads to the development of complications such as diabetic cardiomyopathy. One of the central adaptive responses against the oxidative stresses is the activation of the nuclear transcriptional factor, NF-E2-related factor 2 (Nrf2), which then activates more than 20 different antioxidative enzymes. Kelch-like ECH associated protein 1 (Keap1) targets and binds to Nrf2 for proteosomal degradation. The aim of the present study was to investigate the status of Nrf2 mediated antioxidant system in myocardial biopsies of non-diabetic (NDM) and type-2 diabetic (DM-T2) cardiomyopathy patients. The western blot analysis of antioxidant proteins, real-time PCR analysis of Nrf2/Keap1 gene and bisulphate DNA sequencing analysis to study the methylation status of the CpG islands of Keap1 promoter DNA were performed. The immunoblot analysis showed the decreased level of antioxidant proteins other than Keap1 in the diabetic cardiopathy patients. Similarly, mRNA levels of Keap1 showed 5-fold increase in diabetic patients. Further analysis on promoter region of Keap1 gene revealed 80% demethylation in diabetic patients. Altogether, our results indicated that demethylation of the CpG islands in the Keap1 promoter will activate the expression of Keap1 protein, which then increases the targeting of Nrf2 for proteosomal degradation. Decreased Nrf2 activity represses the transcription of many antioxidant enzyme genes and alters the redox-balance up on diabetes. Thus, our study clearly demonstrates the failure of Nrf2 mediated antioxidant system revealed in biopsies of diabetic cardiomyopathy. PMID:25674242

  19. A CRISPR-based approach for targeted DNA demethylation.

    PubMed

    Xu, Xingxing; Tao, Yonghui; Gao, Xiaobo; Zhang, Lei; Li, Xufang; Zou, Weiguo; Ruan, Kangcheng; Wang, Feng; Xu, Guo-Liang; Hu, Ronggui

    2016-01-01

    In mammalian cells, DNA methylation critically regulates gene expression and thus has pivotal roles in myriad of physiological and pathological processes. Here we report a novel method for targeted DNA demethylation using the widely used clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system. Initially, modified single guide RNAs (sgRNAs) (sgRNA2.0) were constructed by inserting two copies of bacteriophage MS2 RNA elements into the conventional sgRNAs, which would facilitate the tethering of the Tet1 catalytic domain (Tet-CD), in fusion with dCas9 or MS2 coat proteins, to the targeted gene loci. Subsequently, such system was shown to significantly upregulate transcription of the target genes, including RANKL, MAGEB2 or MMP2, which was in close correlation to DNA demethylation of their neighboring CpGs in the promoters. In addition, the dCas9/sgRNA2.0-directed demethylation system appeared to afford efficient demethylation of the target genes with tenuous off-target effects. Applications of this system would not only help us understand mechanistically how DNA methylation might regulate gene expression in specific contexts, but also enable control of gene expression and functionality with potential clinical benefits. PMID:27462456

  20. A CRISPR-based approach for targeted DNA demethylation

    PubMed Central

    Xu, Xingxing; Tao, Yonghui; Gao, Xiaobo; Zhang, Lei; Li, Xufang; Zou, Weiguo; Ruan, Kangcheng; Wang, Feng; Xu, Guo-liang; Hu, Ronggui

    2016-01-01

    In mammalian cells, DNA methylation critically regulates gene expression and thus has pivotal roles in myriad of physiological and pathological processes. Here we report a novel method for targeted DNA demethylation using the widely used clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system. Initially, modified single guide RNAs (sgRNAs) (sgRNA2.0) were constructed by inserting two copies of bacteriophage MS2 RNA elements into the conventional sgRNAs, which would facilitate the tethering of the Tet1 catalytic domain (Tet-CD), in fusion with dCas9 or MS2 coat proteins, to the targeted gene loci. Subsequently, such system was shown to significantly upregulate transcription of the target genes, including RANKL, MAGEB2 or MMP2, which was in close correlation to DNA demethylation of their neighboring CpGs in the promoters. In addition, the dCas9/sgRNA2.0-directed demethylation system appeared to afford efficient demethylation of the target genes with tenuous off-target effects. Applications of this system would not only help us understand mechanistically how DNA methylation might regulate gene expression in specific contexts, but also enable control of gene expression and functionality with potential clinical benefits. PMID:27462456

  1. Identification of RING finger protein 4 (RNF4) as a modulator of DNA demethylation through a functional genomics screen.

    PubMed

    Hu, Xiaoyi V; Rodrigues, Tânia M A; Tao, Haiyan; Baker, Robert K; Miraglia, Loren; Orth, Anthony P; Lyons, Gary E; Schultz, Peter G; Wu, Xu

    2010-08-24

    DNA methylation is an important epigenetic modification involved in transcriptional regulation, nuclear organization, development, aging, and disease. Although DNA methyltransferases have been characterized, the mechanisms for DNA demethylation remain poorly understood. Using a cell-based reporter assay, we performed a functional genomics screen to identify genes involved in DNA demethylation. Here we show that RNF4 (RING finger protein 4), a SUMO-dependent ubiquitin E3-ligase previously implicated in maintaining genome stability, plays a key role in active DNA demethylation. RNF4 reactivates methylation-silenced reporters and promotes global DNA demethylation. Rnf4 deficiency is embryonic lethal with higher levels of methylation in genomic DNA. Mechanistic studies show that RNF4 interacts with and requires the base excision repair enzymes TDG and APE1 for active demethylation. This activity appears to occur by enhancing the enzymatic activities that repair DNA G:T mismatches generated from methylcytosine deamination. Collectively, our study reveals a unique function for RNF4, which may serve as a direct link between epigenetic DNA demethylation and DNA repair in mammalian cells. PMID:20696907

  2. No Evidence for AID/MBD4-Coupled DNA Demethylation in Zebrafish Embryos

    PubMed Central

    Kaneto, Reiya; Izawa, Toshiaki; Yokoi, Hayato; Hashimoto, Naohiro; Kikuchi, Yutaka

    2014-01-01

    The mechanisms responsible for active DNA demethylation remain elusive in Metazoa. A previous study that utilized zebrafish embryos provided a potent mechanism for active demethylation in which three proteins, AID, MBD4, and GADD45 are involved. We recently found age-dependent DNA hypomethylation in zebrafish, and it prompted us to examine if AID and MBD4 could be involved in the phenomenon. Unexpectedly, however, we found that most of the findings in the previous study were not reproducible. First, the injection of a methylated DNA fragment into zebrafish eggs did not affect either the methylation of genomic DNA, injected methylated DNA itself, or several loci tested or the expression level of aid, which has been shown to play a role in demethylation. Second, aberrant methylation was not observed at certain CpG islands following the injection of antisense morpholino oligonucleotides against aid and mbd4. Furthermore, we demonstrated that zebrafish MBD4 cDNA lacked a coding region for the methyl-CpG binding domain, which was assumed to be necessary for guidance to target regions. Taken together, we concluded that there is currently no evidence to support the proposed roles of AID and MBD4 in active demethylation in zebrafish embryos. PMID:25536520

  3. DNA demethylation in the PTEN gene promoter induced by 5-azacytidine activates PTEN expression in the MG-63 human osteosarcoma cell line.

    PubMed

    Song, Deye; Ni, Jiangdong; Xie, Hongming; Ding, Muliang; Wang, Jun

    2014-05-01

    This study used the MG-63 osteosarcoma cell line to investigate the demethylation of the phosphate and tension homolog (PTEN) gene promoter and the change in PTEN gene expression levels, which are caused by the methylation inhibitor 5-azacytidine (5-Zac), and the association between the two. Different concentrations of 5-Zac (0, 5 and 10 μmol/l) were added into the MG-63 cell culture medium and the cells were cultured for 72 h. The following techniques were performed on the cells: Western blot analysis to detect the PTEN protein; reverse transcription-polymerase chain reaction (PCR) to detect the mRNA transcription levels of the PTEN gene; flow cytometry to detect the cell apoptotic rate; and sodium bisulfate to deal with the DNA of each group. The genes of the PTEN promoter and the transcription factors specificity protein 1 (Sp1) and Myc were PCR amplified and transformed into Escherichia coli, then a number of clones were selected for sequencing and the methylation status of the amplified PTEN promoter fragment was detected. Following culture of the MG-63 cells with 5-Zac at concentrations of 0, 5 and 10 μmol/l for 72 h, the expression levels of PTEN protein in each group were gradually increased, presenting a concentration-dependent effect: Group 0 μmol/l compared with groups 5 and 10 μmol/l, P<0.05; and group 5 μmol/l compared with group 10 μmol/l, P=0.007. The mRNA expression levels of the PTEN gene significantly increased. The apoptotic rates of groups 0, 5 and 10 μmol/l were 0.69±0.42, 2.50±0.30 and 6.59±0.62%, and significant differences (P<0.01) were observed between every two groups. The bisulfate DNA sequencing results of three groups showed that, following the treatment with 5-Zac, the binding of the CG site to transcription factors was affected by demethylation. The average rate of demethylation indicated a statistical difference among the three groups. In conclusion, the methylation inhibitor 5-Zac leads to a significant increase in the

  4. DNA demethylation in the PTEN gene promoter induced by 5-azacytidine activates PTEN expression in the MG-63 human osteosarcoma cell line

    PubMed Central

    SONG, DEYE; NI, JIANGDONG; XIE, HONGMING; DING, MULIANG; WANG, JUN

    2014-01-01

    This study used the MG-63 osteosarcoma cell line to investigate the demethylation of the phosphate and tension homolog (PTEN) gene promoter and the change in PTEN gene expression levels, which are caused by the methylation inhibitor 5-azacytidine (5-Zac), and the association between the two. Different concentrations of 5-Zac (0, 5 and 10 μmol/l) were added into the MG-63 cell culture medium and the cells were cultured for 72 h. The following techniques were performed on the cells: Western blot analysis to detect the PTEN protein; reverse transcription-polymerase chain reaction (PCR) to detect the mRNA transcription levels of the PTEN gene; flow cytometry to detect the cell apoptotic rate; and sodium bisulfate to deal with the DNA of each group. The genes of the PTEN promoter and the transcription factors specificity protein 1 (Sp1) and Myc were PCR amplified and transformed into Escherichia coli, then a number of clones were selected for sequencing and the methylation status of the amplified PTEN promoter fragment was detected. Following culture of the MG-63 cells with 5-Zac at concentrations of 0, 5 and 10 μmol/l for 72 h, the expression levels of PTEN protein in each group were gradually increased, presenting a concentration-dependent effect: Group 0 μmol/l compared with groups 5 and 10 μmol/l, P<0.05; and group 5 μmol/l compared with group 10 μmol/l, P=0.007. The mRNA expression levels of the PTEN gene significantly increased. The apoptotic rates of groups 0, 5 and 10 μmol/l were 0.69±0.42, 2.50±0.30 and 6.59±0.62%, and significant differences (P<0.01) were observed between every two groups. The bisulfate DNA sequencing results of three groups showed that, following the treatment with 5-Zac, the binding of the CG site to transcription factors was affected by demethylation. The average rate of demethylation indicated a statistical difference among the three groups. In conclusion, the methylation inhibitor 5-Zac leads to a significant increase in the

  5. Germline DNA demethylation dynamics and imprint erasure through 5-hydroxymethylcytosine.

    PubMed

    Hackett, Jamie A; Sengupta, Roopsha; Zylicz, Jan J; Murakami, Kazuhiro; Lee, Caroline; Down, Thomas A; Surani, M Azim

    2013-01-25

    Mouse primordial germ cells (PGCs) undergo sequential epigenetic changes and genome-wide DNA demethylation to reset the epigenome for totipotency. Here, we demonstrate that erasure of CpG methylation (5mC) in PGCs occurs via conversion to 5-hydroxymethylcytosine (5hmC), driven by high levels of TET1 and TET2. Global conversion to 5hmC initiates asynchronously among PGCs at embryonic day (E) 9.5 to E10.5 and accounts for the unique process of imprint erasure. Mechanistically, 5hmC enrichment is followed by its protracted decline thereafter at a rate consistent with replication-coupled dilution. The conversion to 5hmC is an important component of parallel redundant systems that drive comprehensive reprogramming in PGCs. Nonetheless, we identify rare regulatory elements that escape systematic DNA demethylation in PGCs, providing a potential mechanistic basis for transgenerational epigenetic inheritance. PMID:23223451

  6. 5-hydroxymethylcytosine-mediated DNA demethylation in stem cells and development.

    PubMed

    Sun, Wenjia; Guan, Minxin; Li, Xuekun

    2014-05-01

    The pursuit of DNA demethylation has a colorful history, but it was not until 2009 that the stars of this story, the Ten-eleven-translocation (Tet) family of proteins, were really identified. Tet proteins convert 5-methylcytosine to 5-hydroxymethylcytosine (5hmC), which can be further oxidized to 5-formylcytosine and 5-cyboxycytosine by Tet proteins to achieve DNA demethylation. Recent studies have revealed that 5hmC-mediated DNA demethylation can play essential roles in diverse biological processes, including development and diseases. Here, we review recent discoveries in 5hmC-mediated DNA demethylation in the context of stem cells and development. PMID:24400731

  7. Neil DNA glycosylases promote substrate turnover by Tdg during DNA demethylation

    PubMed Central

    Arab, Khelifa; Kienhöfer, Sabine; von Seggern, Annika; Niehrs, Christof

    2016-01-01

    DNA 5-methylcytosine is a dynamic epigenetic mark which plays important roles in development and disease. In the Tet-Tdg demethylation pathway, methylated cytosine is iteratively oxidized by Tet dioxygenases and unmodified cytosine is restored via thymine DNA glycosylase (Tdg). Here we show that human NEIL1 and NEIL2 DNA glycosylases coordinate abasic site processing during TET–TDG DNA demethylation. NEIL1 and NEIL2 cooperate with TDG during base excision: TDG occupies the abasic site and is displaced by NEILs, which further process the baseless sugar, thereby stimulating TDG substrate turnover. In early Xenopus embryos Neil2 cooperates with Tdg to remove oxidized methylcytosines and to specify neural crest development together with Tet3. Thus, Neils function as AP lyases in the coordinated AP site hand-over during oxidative DNA demethylation. PMID:26751644

  8. DNA oxidation as triggered by H3K9me2 demethylation drives estrogen-induced gene expression.

    PubMed

    Perillo, Bruno; Ombra, Maria Neve; Bertoni, Alessandra; Cuozzo, Concetta; Sacchetti, Silvana; Sasso, Annarita; Chiariotti, Lorenzo; Malorni, Antonio; Abbondanza, Ciro; Avvedimento, Enrico V

    2008-01-11

    Modifications at the N-terminal tails of nucleosomal histones are required for efficient transcription in vivo. We analyzed how H3 histone methylation and demethylation control expression of estrogen-responsive genes and show that a DNA-bound estrogen receptor directs transcription by participating in bending chromatin to contact the RNA polymerase II recruited to the promoter. This process is driven by receptor-targeted demethylation of H3 lysine 9 at both enhancer and promoter sites and is achieved by activation of resident LSD1 demethylase. Localized demethylation produces hydrogen peroxide, which modifies the surrounding DNA and recruits 8-oxoguanine-DNA glycosylase 1 and topoisomeraseIIbeta, triggering chromatin and DNA conformational changes that are essential for estrogen-induced transcription. Our data show a strategy that uses controlled DNA damage and repair to guide productive transcription. PMID:18187655

  9. PRMT5 Protects Genomic Integrity during Global DNA Demethylation in Primordial Germ Cells and Preimplantation Embryos

    PubMed Central

    Kim, Shinseog; Günesdogan, Ufuk; Zylicz, Jan J.; Hackett, Jamie A.; Cougot, Delphine; Bao, Siqin; Lee, Caroline; Dietmann, Sabine; Allen, George E.; Sengupta, Roopsha; Surani, M. Azim

    2014-01-01

    Summary Primordial germ cells (PGCs) and preimplantation embryos undergo epigenetic reprogramming, which includes comprehensive DNA demethylation. We found that PRMT5, an arginine methyltransferase, translocates from the cytoplasm to the nucleus during this process. Here we show that conditional loss of PRMT5 in early PGCs causes complete male and female sterility, preceded by the upregulation of LINE1 and IAP transposons as well as activation of a DNA damage response. Similarly, loss of maternal-zygotic PRMT5 also leads to IAP upregulation. PRMT5 is necessary for the repressive H2A/H4R3me2s chromatin modification on LINE1 and IAP transposons in PGCs, directly implicating this modification in transposon silencing during DNA hypomethylation. PRMT5 translocates back to the cytoplasm subsequently, to participate in the previously described PIWI-interacting RNA (piRNA) pathway that promotes transposon silencing via de novo DNA remethylation. Thus, PRMT5 is directly involved in genome defense during preimplantation development and in PGCs at the time of global DNA demethylation. PMID:25457166

  10. Krüppel like factor 4 promoter undergoes active demethylation during monocyte/macrophage differentiation.

    PubMed

    Karpurapu, Manjula; Ranjan, Ravi; Deng, Jing; Chung, Sangwoon; Lee, Yong Gyu; Xiao, Lei; Nirujogi, Teja Srinivas; Jacobson, Jeffrey R; Park, Gye Young; Christman, John W

    2014-01-01

    The role of different lineage specific transcription factors in directing hematopoietic cell fate towards myeloid lineage is well established but the status of epigenetic modifications has not been defined during this important developmental process. We used non proliferating, PU.1 inducible myeloid progenitor cells and differentiating bone marrow derived macrophages to study the PU.1 dependent KLF4 transcriptional regulation and its promoter demethylation during monocyte/macrophage differentiation. Expression of KLF4 was regulated by active demethylation of its promoter and PU.1 specifically bound to KLF4 promoter oligo harboring the PU.1 consensus sequence. Methylation specific quantitative PCR and Bisulfite sequencing indicated demethylation of CpG residues most proximal to the transcription start site of KLF4 promoter. Cloned KLF4 promoter in pGL3 Luciferase and CpG free pcpgf-bas vectors showed accentuated reporter activity when co-transfected with the PU.1 expression vector. In vitro methylation of both KLF4 promoter oligo and cloned KLF4 promoter vectors showed attenuated in vitro DNA binding activity and Luciferase/mouse Alkaline phosphotase reporter activity indicating the negative influence of KLF4 promoter methylation on PU.1 binding. The Cytosine deaminase, Activation Induced Cytidine Deaminase (AICDA) was found to be critical for KLF4 promoter demethylation. More importantly, knock down of AICDA resulted in blockade of KLF4 promoter demethylation, decreased F4/80 expression and other phenotypic characters of macrophage differentiation. Our data proves that AICDA mediated active demethylation of the KLF4 promoter is necessary for transcriptional regulation of KLF4 by PU.1 during monocyte/macrophage differentiation. PMID:24695324

  11. Genistein promotes DNA demethylation of the steroidogenic factor 1 (SF-1) promoter in endometrial stromal cells

    SciTech Connect

    Matsukura, Hiroshi; Aisaki, Ken-ichi; Igarashi, Katsuhide; Matsushima, Yuko; Kanno, Jun; Muramatsu, Masaaki; Sudo, Katsuko; Sato, Noriko

    2011-08-26

    Highlights: {yields} Genistein (GEN) is a phytoestrogen found in soy products. {yields} GEN demethylated/unsilenced the steroidogenic factor 1 gene in endometrial tissue. {yields} GEN thus altered mRNA expression in uteri of ovariectomized (OVX) mice. {yields} A high-resolution melting assay was used to screen for epigenetic change. {yields} We isolated an endometrial cell clone that was epigenetically modulated by GEN. -- Abstract: It has recently been demonstrated that genistein (GEN), a phytoestrogen in soy products, is an epigenetic modulator in various types of cells; but its effect on endometrium has not yet been determined. We investigated the effects of GEN on mouse uterine cells, in vivo and in vitro. Oral administration of GEN for 1 week induced mild proliferation of the endometrium in ovariectomized (OVX) mice, which was accompanied by the induction of steroidogenic factor 1 (SF-1) gene expression. GEN administration induced demethylation of multiple CpG sites in the SF-1 promoter; these sites are extensively methylated and thus silenced in normal endometrium. The GEN-mediated promoter demethylation occurred predominantly on the luminal side, as opposed to myometrium side, indicating that the epigenetic change was mainly shown in regenerated cells. Primary cultures of endometrial stromal cell colonies were screened for GEN-mediated alterations of DNA methylation by a high-resolution melting (HRM) method. One out of 20 colony-forming cell clones showed GEN-induced demethylation of SF-1. This clone exhibited a high proliferation capacity with continuous colony formation activity through multiple serial clonings. We propose that only a portion of endometrial cells are capable of receiving epigenetic modulation by GEN.

  12. Abiotic-stress induces demethylation and transcriptional activation of a gene encoding a glycerophosphodiesterase-like protein in tobacco plants.

    PubMed

    Choi, Chang-Sun; Sano, Hiroshi

    2007-05-01

    To examine the relationship between gene expression and DNA methylation, transcriptionally activated genes were screened in hypomethylated transgenic tobacco plants expressing an anti-DNA methyltransferase sequence. Among 16 genes initially identified, one clone was found to encode a glycerophosphodiesterase-like protein (NtGPDL), earlier reported to be responsive to aluminium stress. When detached leaves from wild type tobacco plants were treated with aluminium, NtGPDL transcripts were induced within 6 h, and corresponding genomic loci were demethylated at CCGG sites within 1 h. Direct bisulfite methylation mapping revealed that CG sites in coding regions were selectively demethylated, and that promoter regions were totally unmethylated regardless of the stress. Salt and low temperature treatments also induced similar demethylation patterns. Such effects could be attributable to oxidative stress, since reactive oxygen species generated by paraquat efficiently induced the same pattern of demethylation at coding regions. Pathogen infection induced neither transcripts nor genomic demethylation. These results suggested a close correlation between methylation and expression of NtGPDL upon abiotic stresses with a cause-effect relationship. Since DNA methylation is linked to histone modification, it is conceivable that demethylation at coding regions might induce alteration of chromatin structure, thereby enhancing transcription. We propose that environmental responses of plants are partly mediated through active alteration of the DNA methylation status. PMID:17273870

  13. PDGFRβ expression and function in fibroblasts derived from pluripotent cells is linked to DNA demethylation

    PubMed Central

    Hewitt, Kyle J.; Shamis, Yulia; Knight, Elana; Smith, Avi; Maione, Anna; Alt-Holland, Addy; Sheridan, Steven D.; Haggarty, Stephen J.; Garlick, Jonathan A.

    2012-01-01

    Platelet-derived growth factor receptor-beta (PDGFRβ) is required for the development of mesenchymal cell types, and plays a diverse role in the function of fibroblasts in tissue homeostasis and regeneration. In this study, we characterized the expression of PDGFRβ in fibroblasts derived from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), and showed that this expression is important for cellular functions such as migration, extracellular matrix production and assembly in 3D self-assembled tissues. To determine potential regulatory regions predictive of expression of PDGFRβ following differentiation from ESCs and iPSCs, we analyzed the DNA methylation status of a region of the PDGFRB promoter that contains multiple CpG sites, before and after differentiation. We demonstrated that this promoter region is extensively demethylated following differentiation, and represents a developmentally regulated, differentially methylated region linked to PDGFRβ expression. Understanding the epigenetic regulation of genes such as PDGFRB, and identifying sites of active DNA demethylation, is essential for future applications of iPSC-derived fibroblasts for regenerative medicine. PMID:22344267

  14. Structural and mutation studies of two DNA demethylation related glycosylases: MBD4 and TDG

    PubMed Central

    Hashimoto, Hideharu

    2014-01-01

    Two mammalian DNA glycosylases, methyl-CpG binding domain protein 4 (MBD4) and thymine DNA glycosylase (TDG), are involved in active DNA demethylation via the base excision repair pathway. Both MBD4 and TDG excise the mismatch base from G:X, where X is uracil, thymine, and 5-hydroxymethyluracil (5hmU). In addition, TDG excises 5mC oxidized bases i.e. when X is 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) not 5-hydroxymethylcytosine (5hmC). A MBD4 inactive mutant and substrate crystal structure clearly explains how MBD4 glycosylase discriminates substrates: 5mC are not able to be directly excised, but a deamination process from 5mC to thymine is required. On the other hand, TDG is much more complicated; in this instance, crystal structures show that TDG recognizes G:X mismatch DNA containing DNA and G:5caC containing DNA from the minor groove of DNA, which suggested that TDG might recognize 5mC oxidized product 5caC like mismatch DNA. In mutation studies, a N157D mutation results in a more 5caC specific glycosylase, and a N191A mutation inhibits 5caC activity while that when X=5fC or T remains. Here I revisit the recent MBD4 glycos ylase domain co-crystal structures with DNA, as well as TDG glycosylase domain co-crystal structures with DNA in conjunction with its mutation studies.

  15. DNA demethylation in normal colon tissue predicts predisposition to multiple cancers.

    PubMed

    Kamiyama, H; Suzuki, K; Maeda, T; Koizumi, K; Miyaki, Y; Okada, S; Kawamura, Y J; Samuelsson, J K; Alonso, S; Konishi, F; Perucho, M

    2012-11-29

    Some colon cancer (CC) patients present synchronous cancers at diagnosis and others develop metachronous neoplasms, but the risk factors are unclear for non-hereditary CC. We showed previously that global DNA demethylation increased with aging and correlated with genomic damage in CC, and we show now that preferentially associates to CCs with wild-type p53. This study aimed to elucidate the extent of DNA hypomethylation in patients with single and multiple CC, its relationship with aging, and its potential as predictive tool. We compared by real-time methylation-specific PCR the relative demethylation level (RDL) of long interspersed nucleotide element-1 (LINE-1) sequences in matched cancer tissues and non-cancerous colonic mucosa (NCM) from patients with single and multiple right-sided CCs. Although no RDL difference was found in NCM from single CC patients and healthy volunteers (P=0.5), there was more demethylation (higher RDL) in NCM from synchronous cancer patients (P=1.1 × 10(-5)) multiple CCs also were more demethylated than single CCs (P=0.0014). High NCM demethylation was predictive for metachronous neoplasms (P=0.003). In multivariate logistic regression analyses RDL was the only independent predictor for metachronous (P=0.02) and multiple (P=4.9 × 10(-5)) tumors. The higher LINE-1 demethylation in NCM from patients with multiple (synchronous and metachronous) tumors (P=9.6 × 10(-7)) was also very significant in patients with tumors without (P=3.8 × 10(-6)), but not with (P=0.16) microsatellite instability. NCM demethylation increased with aging in patients with single tumors, but decreased in those with multiple tumors. Moreover, the demethylation difference between patients with single vs multiple tumors appeared higher in younger (P=3.6 × 10(-4)) than in older (P=0.0016) patients. These results predict that LINE-1 hypomethylation in NCM can be used as an epigenetic predictive biomarker for multiple CC risk. The stronger association of

  16. MicroRNA Mediates DNA De-methylation Events Triggered By Retinoic Acid During Neuroblastoma Cell Differentiation

    PubMed Central

    Das, Sudipto; Foley, Niamh; Bryan, Kenneth; Watters, Karen M; Bray, Isabella; Murphy, Derek M; Buckley, Patrick G; Stallings, Raymond L

    2010-01-01

    Neuroblastoma is an often fatal pediatric cancer arising from precursor cells of the sympathetic nervous system. 13-Cis retinoic acid is included in the treatment regime for patients with high-risk disease, and a similar derivative, all-trans retinoic acid (ATRA) causes neuroblastoma cell lines to undergo differentiation. The molecular signaling pathways involved with ATRA induced differentiation are complex, and the role that DNA methylation changes might play are unknown. The purpose of this study was to evaluate the genome-wide effects of ATRA on DNA methylation using methylated DNA immunoprecipitation applied to microarrays representing all known promoter and CpG islands. 402 gene promoters became demethylated, while 88 were hypermethylated post-ATRA. mRNA expression microarrays revealed that 82 of the demethylated genes were over-expressed by >2 fold, while 13 of the hyper methylated genes were under-expressed. Gene ontology analysis indicated that de-methylated and re-expressed genes were enriched for signal transduction pathways, including NOS1, which is required for neural cell differentiation. As a potential mechanism for the DNA methylation changes, we demonstrate the down-regulation of methyltransferases, DNMT1 and DNMT3B, along with the up-regulation of endogenous microRNAs targeting them. Ectopic over-expression of miR-152, targeting DNMT1, also negatively impacted cell invasiveness and anchorage independent growth, contributing in part to the differentiated phenotype. We conclude that functionally important, miRNA-mediated DNA de-methylation changes contribute to the process of ATRA induced differentiation resulting in the activation of NOS1, a critical determinant of neural cell differentiation. Our findings illustrate the plasticity and dynamic nature of the epigenome during cancer cell differentiation. PMID:20841484

  17. Demethylation-linked Activation of uPA is Involved in Progression of Prostate Cancer

    PubMed Central

    Pulukuri, Sai MuraliKrishna; Estes, Norman; Patel, Jitendra; Rao, Jasti S.

    2006-01-01

    Increased expression of urokinase plasminogen activator (uPA) has been reported in various malignancies including prostate cancer. However, the mechanism by which uPA is abnormally expressed in prostate cancer remains elusive. Here, we show that uPA is aberrantly expressed in a high-percentage of human prostate cancer tissues, but rarely expressed either in tumor-matched, non-neoplastic adjacent tissues (NNAT) or benign prostatic hyperplasia (BPH) samples. This aberrant expression is associated with cancer-linked demethylation of the uPA promoter. Furthermore, treatment with demethylation inhibitor S-Adenosylmethionine (Ado-Met) or stable expression of uPA shRNA significantly inhibits uPA expression and tumor cell invasion in vitro and tumor growth and incidence of lung metastasis in vivo. Collectively, these findings strongly suggest that DNA demethylation is a common mechanism underlying the abnormal expression of uPA and is a critical contributing factor to the malignant progression of human prostate tumors. PMID:17283123

  18. Damage proneness induced by genomic DNA demethylation in mammalian cells cultivated in vitro.

    PubMed

    Perticone, P; Gensabella, G; Cozzi, R

    1997-07-01

    Variations in the genomic DNA methylation level have been shown to be an epigenetic inheritable modification affecting, among other targets, the sister chromatid exchange (SCE) rate in mammalian cells in vitro. The inheritable increase in SCE rate in affected cell populations appears as a puzzling phenomenon in view of the well established relation between SCE and both mutagenesis and carcinogenesis. In the present work we demonstrate that, in a treated cell population, demethylation could be responsible for the inheritable induction of damage proneness affecting both damage induction and repair. Normal and ethionine or azacytidine treated Chinese hamster ovary cells, subclone K1 (CHO-K1), were challenged with UV light (UV) or mitomycin-C (MMC) at different times from the demethylating treatment. The SCE rate was measured with two main objects in view: (i) the induction of synergism or additivity in combined treatments, where mutagen (UV or MMC) pulse is supplied from 0 to 48 h after the end of the demethylating treatment; and (ii) the pattern of damage extinction, for the duration of up to six cell cycles after the end of the combined (demethylating agent + mutagen) treatment. Results indicate both a synergism in SCE induction by mutagens in demethylated cells even if supplied up to four cell cycles after the end of the demethylation treatment and a delay in recovery of induced damage, compared with normally methylated cells. These data are discussed in the light of the supposed mechanism of SCE increase and of the possible biological significance in terms of mutagenesis and carcinogenesis. PMID:9237771

  19. Real-time dynamics of methyl-CpG-binding domain protein 3 and its role in DNA demethylation by fluorescence correlation spectroscopy

    PubMed Central

    Cui, Yi; Cho, Il-Hoon; Chowdhury, Basudev; Irudayaraj, Joseph

    2013-01-01

    With unprecedented development in technology, epigenetics is recognized as a substantial and flexible regulatory pathway for phenotyping. Cytosine methylation and its subsequent oxidization have attracted significant attention due to their direct impact on gene regulation, in association with methyl-CpG-binding domain proteins (MBDs) and transcription related factors. In this study we record the dynamics of DNA demethylation using the recombinant MBD3-GFP protein in living cells under hypoxia and Decitabine treatment using Fluorescence Correlation Spectroscopy (FCS) by monitoring the diffusion dynamics of MBD3. Our study shows a DNA-replication-independent decrease of 5-methylcytosine (5mC)/5-hydroxymethylcytosine (5hmC) under hypoxia vs. a dependent decrease under Decitabine treatment. Further, we define a significantly faster diffusion of MBD3 in the nucleus as a precursory event for active demethylation rather than the Decitabine induced passive demethylation. By monitoring the diffusion of bound and unbound MBD3 in the nucleus we were able to identify and characterize hypoxia-sensitive cells from insensitive/tolerant cells, as well as the respective contribution to active demethylation in a time-dependent manner. Last, we quantitatively describe the concurrent decreasing trend in all of the three oxidized products of 5mC, which points to the potential involvement of ten-eleven-translocation proteins (TETs) in hypoxia induced active demethylation. Overall, for the first time we correlate the dynamic process of DNA demethylation with the biophysical properties of the corresponding DNA binding proteins in live single cells by single molecule spectroscopy. PMID:23974971

  20. TET3 Inhibits Type I IFN Production Independent of DNA Demethylation.

    PubMed

    Xue, Shengjie; Liu, Chang; Sun, Xiujie; Li, Weiyun; Zhang, Chi; Zhou, Xin; Lu, Yao; Xiao, Jun; Li, Chunyang; Xu, Xiaoyan; Sun, Bing; Xu, Guoliang; Wang, Hongyan

    2016-07-26

    Type I interferons (IFNs) play both beneficial and harmful roles in antiviral responses. Precise regulation of host type I IFNs is thus needed to prevent immune dysregulation. Here, we find that the DNA demethylase TET3 is a negative regulator of IFN-β in response to poly(I:C) stimulation or viral infection. Deletion of TET3 enhances antiviral responses, with elevated expression of IFN-β and IFN-stimulated genes. The catalytic domain of TET3 was critical for the suppression of IFN-β production, but TET3 enzymatic activity was dispensable. Instead, the catalytic domain of TET3 interacts with HDAC1 and SIN3A, thus enhancing their binding to the Ifnb1 promoter. Our study demonstrates that TET3 negatively regulates type I IFN production independent of DNA demethylation. This not only sheds light on TET3 as a signaling protein in immune cells for gene regulation but also will help to develop strategies to prevent type I IFN-related disease. PMID:27425624

  1. Epigenetic DNA Demethylation Causes Inner Ear Stem Cell Differentiation into Hair Cell-Like Cells

    PubMed Central

    Zhou, Yang; Hu, Zhengqing

    2016-01-01

    The DNA methyltransferase (DNMT) inhibitor 5-azacytidine (5-aza) causes genomic demethylation to regulate gene expression. However, it remains unclear whether 5-aza affects gene expression and cell fate determination of stem cells. In this study, 5-aza was applied to mouse utricle sensory epithelia-derived progenitor cells (MUCs) to investigate whether 5-aza stimulated MUCs to become sensory hair cells. After treatment, MUCs increased expression of hair cell genes and proteins. The DNA methylation level (indicated by percentage of 5-methylcytosine) showed a 28.57% decrease after treatment, which causes significantly repressed DNMT1 protein expression and DNMT activity. Additionally, FM1-43 permeation assays indicated that the permeability of 5-aza-treated MUCs was similar to that of sensory hair cells, which may result from mechanotransduction channels. This study not only demonstrates a possible epigenetic approach to induce tissue specific stem/progenitor cells to become sensory hair cell-like cells, but also provides a cell model to epigenetically modulate stem cell fate determination. PMID:27536218

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

    PubMed Central

    La, Honggui; Ding, Bo; Mishra, Gyan P.; Zhou, Bo; Yang, Hongmei; Bellizzi, Maria del Rosario; Chen, Songbiao; Meyers, Blake C.; Peng, Zhaohua; Zhu, Jian-Kang; Wang, Guo-Liang

    2011-01-01

    DNA 5-methylcytosine (5-meC) is an important epigenetic mark for transcriptional gene silencing in many eukaryotes. In Arabidopsis, 5-meC DNA glycosylase/lyases actively remove 5-meC to counteract transcriptional gene silencing in a locus-specific manner, and have been suggested to maintain the expression of transposons. However, it is unclear whether plant DNA demethylases can promote the transposition of transposons. Here we report the functional characterization of the DNA glycosylase/lyase DNG701 in rice. DNG701 encodes a large (1,812 amino acid residues) DNA glycosylase domain protein. Recombinant DNG701 protein showed 5-meC DNA glycosylase and lyase activities in vitro. Knockout or knockdown of DNG701 in rice plants led to DNA hypermethylation and reduced expression of the retrotransposon Tos17. Tos17 showed less transposition in calli derived from dng701 knockout mutant seeds compared with that in wild-type calli. Overexpression of DNG701 in both rice calli and transgenic plants substantially reduced DNA methylation levels of Tos17 and enhanced its expression. The overexpression also led to more frequent transposition of Tos17 in calli. Our results demonstrate that rice DNG701 is a 5-meC DNA glycosylase/lyase responsible for the demethylation of Tos17 and this DNA demethylase plays a critical role in promoting Tos17 transposition in rice calli. PMID:21896764

  3. Methylglyoxal induces endoplasmic reticulum stress and DNA demethylation in the Keap1 promoter of human lens epithelial cells and age-related cataracts

    PubMed Central

    Palsamy, Periyasamy; Bidasee, Keshore R.; Ayaki, Masahiko; Augusteyn, Robert C.; Chan, Jefferson Y.; Shinohara, Toshimichi

    2015-01-01

    Age-related cataracts are a leading cause of blindness. Previously, we have demonstrated the association of unfolded protein response with various cataractogenic stressors. However, DNA methylation alterations leading to suppression of lenticular antioxidant protection remains unclear. Here, we report the methylglyoxal-mediated sequential events responsible for Keap1 promoter DNA demethylation in human lens epithelial cells, because Keap1 is a negative regulatory protein that regulates the Nrf2 antioxidant protein. Methylglyoxal induces the ER stress and activates the unfolded protein response leading to overproduction of ROS prior to human lens epithelial cells death. Methylglyoxal also suppresses the Nrf2 and DNA methyltransferases but activates the DNA demethylation pathway enzyme, TET1. Bisulfite genomic DNA sequencing confirms the methylglyoxal-mediated Keap1 promoter DNA demethylation leading to over-expression of Keap1 mRNA and protein. Similarly, bisulfite genomic DNA sequencing of human clear lenses (n=15) slowly lose 5-methylcytosine in the Keap1 promoter throughout life, at a rate of 1% per year. By contrast, diabetic cataractous lenses (n=21) lose an average of 90% of the 5-methylcytosine regardless of the age. Over-expressed Keap1 protein is responsible for decreasing the Nrf2 by proteasomal degradation, thereby suppressing the Nrf2 dependent stress protection. This study demonstrates for the first time about the associations of unfolded protein response activation, Nrf2 dependent antioxidant system failure and loss of Keap1 promoter methylation because of altered active and passive DNA demethylation pathway enzymes in human lens epithelial cells by methylglyoxal. As an outcome, cellular redox balance is altered towards lens oxidation and cataract formation. PMID:24746615

  4. DNA Demethylation Upregulated Nrf2 Expression in Alzheimer’s Disease Cellular Model

    PubMed Central

    Cao, Huimin; Wang, Li; Chen, Beibei; Zheng, Peng; He, Yi; Ding, Yubin; Deng, Yushuang; Lu, Xi; Guo, Xiuming; Zhang, Yuping; Li, Yu; Yu, Gang

    2016-01-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is an important transcription factor in the defense against oxidative stress. Cumulative evidence has shown that oxidative stress plays a key role in the pathogenesis of Alzheimer’s disease (AD). Previous animal and clinical studies had observed decreased expression of Nrf2 in AD. However, the underlying regulation mechanisms of Nrf2 in AD remain unclear. Here, we used the DNA methyltransferases (Dnmts) inhibitor 5-aza-2′-deoxycytidine (5-Aza) to test whether Nrf2 expression was regulated by methylation in N2a cells characterizing by expressing human Swedish mutant amyloid precursor protein (N2a/APPswe). We found 5-Aza treatment increased Nrf2 at both messenger RNA and protein levels via downregulating the expression of Dnmts and DNA demethylation. In addition, 5-Aza-mediated upregulation of Nrf2 expression was concomitant with increased nuclear translocation of Nrf2 and higher expression of Nrf2 downstream target gene NAD(P)H:quinone oxidoreductas (NQO1). Our study showed that DNA demethylation promoted the Nrf2 cell signaling pathway, which may enhance the antioxidant system against AD development. PMID:26779013

  5. Cell-Wide DNA De-Methylation and Re-Methylation of Purkinje Neurons in the Developing Cerebellum.

    PubMed

    Zhou, Feng C; Resendiz, Marisol; Lo, Chiao-Ling; Chen, Yuanyuan

    2016-01-01

    Global DNA de-methylation is thought to occur only during pre-implantation and gametogenesis in mammals. Scalable, cell-wide de-methylation has not been demonstrated beyond totipotent stages. Here, we observed a large scale de-methylation and subsequent re-methylation (CDR) (including 5-methylcytosine (5mC) and 5-hydroxylmethylcytosine (5hmC)) in post-mitotic cerebellar Purkinje cells (PC) through the course of normal development. Through single cell immuno-identification and cell-specific quantitative methylation assays, we demonstrate that the CDR event is an intrinsically scheduled program, occurring in nearly every PC. Meanwhile, cerebellar granule cells and basket interneurons adopt their own DNA methylation program, independent of PCs. DNA de-methylation was further demonstrated at the gene level, on genes pertinent to PC development. The PC, being one of the largest neurons in the brain, may showcase an amplified epigenetic cycle which may mediate stage transformation including cell cycle arrest, vast axonal-dendritic growth, and synaptogenesis at the onset of neuronal specificity. This discovery is a key step toward better understanding the breadth and role of DNA methylation and de-methylation during neural ontology. PMID:27583369

  6. DNA methylation and demethylation events during meiotic prophase in the mouse testis.

    PubMed Central

    Trasler, J M; Hake, L E; Johnson, P A; Alcivar, A A; Millette, C F; Hecht, N B

    1990-01-01

    The genes encoding three different mammalian testis-specific nuclear chromatin proteins, mouse transition protein 1, mouse protamine 1, and mouse protamine 2, all of which are expressed postmeiotically, are marked by methylation early during spermatogenesis in the mouse. Analysis of DNA from the testes of prepubertal mice and isolated testicular cells revealed that transition protein 1 became progressively less methylated during spermatogenesis, while the two protamines became progressively more methylated; in contrast, the methylation of beta-actin, a gene expressed throughout spermatogenesis, did not change. These findings provide evidence that both de novo methylation and demethylation events are occurring after the completion of DNA replication, during meiotic prophase in the mouse testis. Images PMID:2320009

  7. Allele-specific FKBP5 DNA demethylation mediates gene–childhood trauma interactions

    PubMed Central

    Klengel, Torsten; Mehta, Divya; Anacker, Christoph; Rex-Haffner, Monika; Pruessner, Jens C; Pariante, Carmine M; Pace, Thaddeus W W; Mercer, Kristina B; Mayberg, Helen S; Bradley, Bekh; Nemeroff, Charles B; Holsboer, Florian; Heim, Christine M; Ressler, Kerry J; Rein, Theo; Binder, Elisabeth B

    2014-01-01

    Although the fact that genetic predisposition and environmental exposures interact to shape development and function of the human brain and, ultimately, the risk of psychiatric disorders has drawn wide interest, the corresponding molecular mechanisms have not yet been elucidated. We found that a functional polymorphism altering chromatin interaction between the transcription start site and long-range enhancers in the FK506 binding protein 5 (FKBP5) gene, an important regulator of the stress hormone system, increased the risk of developing stress-related psychiatric disorders in adulthood by allele-specific, childhood trauma–dependent DNA demethylation in functional glucocorticoid response elements of FKBP5. This demethylation was linked to increased stress-dependent gene transcription followed by a long-term dysregulation of the stress hormone system and a global effect on the function of immune cells and brain areas associated with stress regulation. This identification of molecular mechanisms of genotype-directed long-term environmental reactivity will be useful for designing more effective treatment strategies for stress-related disorders. PMID:23201972

  8. Allele-specific FKBP5 DNA demethylation mediates gene-childhood trauma interactions.

    PubMed

    Klengel, Torsten; Mehta, Divya; Anacker, Christoph; Rex-Haffner, Monika; Pruessner, Jens C; Pariante, Carmine M; Pace, Thaddeus W W; Mercer, Kristina B; Mayberg, Helen S; Bradley, Bekh; Nemeroff, Charles B; Holsboer, Florian; Heim, Christine M; Ressler, Kerry J; Rein, Theo; Binder, Elisabeth B

    2013-01-01

    Although the fact that genetic predisposition and environmental exposures interact to shape development and function of the human brain and, ultimately, the risk of psychiatric disorders has drawn wide interest, the corresponding molecular mechanisms have not yet been elucidated. We found that a functional polymorphism altering chromatin interaction between the transcription start site and long-range enhancers in the FK506 binding protein 5 (FKBP5) gene, an important regulator of the stress hormone system, increased the risk of developing stress-related psychiatric disorders in adulthood by allele-specific, childhood trauma-dependent DNA demethylation in functional glucocorticoid response elements of FKBP5. This demethylation was linked to increased stress-dependent gene transcription followed by a long-term dysregulation of the stress hormone system and a global effect on the function of immune cells and brain areas associated with stress regulation. This identification of molecular mechanisms of genotype-directed long-term environmental reactivity will be useful for designing more effective treatment strategies for stress-related disorders. PMID:23201972

  9. Prevention of Helicobacter pylori-induced gastric cancers in gerbils by a DNA demethylating agent.

    PubMed

    Niwa, Tohru; Toyoda, Takeshi; Tsukamoto, Tetsuya; Mori, Akiko; Tatematsu, Masae; Ushijima, Toshikazu

    2013-04-01

    Suppression of aberrant DNA methylation is a novel approach to cancer prevention, but, so far, the efficacy of the strategy has not been evaluated in cancers associated with chronic inflammation. Gastric cancers induced by Helicobacter pylori infection are known to involve aberrant DNA methylation and associated with severe chronic inflammation in their early stages. Here, we aimed to clarify whether suppression of aberrant DNA methylation can prevent H. pylori-induced gastric cancers using a Mongolian gerbil model. Administration of a DNA demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC), to gerbils (0.125 mg/kg for 50-55 weeks) decreased the incidence of gastric cancers induced by H. pylori infection and N-methyl-N-nitrosourea (MNU) treatment from 55.2% to 23.3% (P < 0.05). In gastric epithelial cells, DNA methylation levels of six CpG islands (HE6, HG2, SB1, SB5, SF12, and SH6) decreased to 46% to 68% (P < 0.05) of gerbils without 5-aza-dC treatment. Also, the global DNA methylation level decreased from 83.0% ± 4.5% to 80.3% ± 4.4% (mean ± SD) by 5-aza-dC treatment (P < 0.05). By 5-aza-dC treatment, Il1b and Nos2 were downregulated (42% and 58% of gerbils without, respectively) but Tnf was upregulated (187%), suggesting that 5-aza-dC treatment induced dysregulation of inflammatory responses. No obvious adverse effect of 5-aza-dC treatment was observed, besides testicular atrophy. These results showed that 5-aza-dC treatment can prevent H. pylori-induced gastric cancers and suggested that removal of induced DNA methylation and/or suppression of DNA methylation induction can become a target for prevention of chronic inflammation-associated cancers. PMID:23559452

  10. Suppression of TET1-Dependent DNA Demethylation is Essential for KRAS-Mediated Transformation

    PubMed Central

    Wu, Bo-Kuan

    2014-01-01

    Summary Hypermethylation-mediated tumor suppressor gene (TSG) silencing is a central epigenetic alteration in RAS-dependent tumorigenesis. Ten-eleven translocation (TET) enzymes can depress DNA methylation by hydroxylation of 5-methylcytosine (5mC) bases to 5-hydroxymethylcytosine (5hmC). Here we report that suppression of TET1 is required for KRAS-induced DNA hypermethylation and cellular transformation. In distinct non-malignant cell lines, oncogenic KRAS promotes transformation by inhibiting TET1 expression via the ERK signaling pathway. This reduces chromatin occupancy of TET1 at TSG promoters, lowers levels of 5hmC, and increases levels of 5mC and 5mC-dependent transcriptional silencing. Restoration of TET1 expression by ERK pathway inhibition or ectopic TET1 reintroduction in KRAS-transformed cells reactivates TSGs and inhibits colony formation. KRAS knockdown increases TET1 expression and diminishes colony-forming ability, while KRAS/TET1 double knockdown bypasses the KRAS dependence of KRAS-addicted cancer cells. Thus, suppression of TET1-dependent DNA demethylation is critical for KRAS-mediated transformation. PMID:25466250

  11. Aerosol Azacytidine Inhibits Orthotopic Lung Cancers in Mice through Its DNA Demethylation and Gene Reactivation Effects

    PubMed Central

    Qiu, Xuan; Liang, Yuanxin; Sellers, Rani S.; Perez-Soler, Roman; Zou, Yiyu

    2014-01-01

    We devised an aerosol based demethylation therapy to achieve therapeutic efficacy in premalignant or in situ lesions of lung cancer, without systemic toxicity. Optimum regimens of aerosolized azacytidine (Aza) were designed and used in orthotopic human non-small cell lung cancer xenograft models. The therapeutic efficacy and toxicity of aerosol Aza were compared with intravenously administered Aza. We observed that 80% of the droplets of the aerosol Aza measured ∼0.1–5 microns, which resulted in deposition in the lower bronchial airways. An animal model that phenocopies field carcinogeneisis in humans was developed by intratracheal inoculation of the human lung cancer cells in mice, thus resulting in their distribution throughout the entire airway space. Aerosolized Aza significantly prolonged the survival of mice bearing endo-bronchial lung tumors. The aerosol treatment did not cause any detectable lung toxicity or systemic toxicity. A pre-pharmacokinetic study in mice demonstrated that lung deposition of aerosolized Aza was significantly higher than the intravenous route. Lung tumors were resected after aerosol treatment and the methylation levels of 24 promoters of tumor-suppresser genes related to lung cancer were analyzed. Aerosol Aza significantly reduced the methylation level in 9 of these promoters and reexpressed several genes tested. In conclusion, aerosol Aza at non-cytotoxic doses appears to be effective and results in DNA demethylation and tumor suppressor gene re-expression. The therapeutic index of aerosol Aza is >100-fold higher than that of intravenous Aza. These results provide a preclinical rationale for a phase I clinical trial of aerosol Aza to be initiated at our Institution. PMID:25347303

  12. Epigenetic alteration by DNA-demethylating treatment restores apoptotic response to glucocorticoids in dexamethasone-resistant human malignant lymphoid cells

    PubMed Central

    2014-01-01

    Background Glucocorticoids (GCs) are often included in the therapy of lymphoid malignancies because they kill several types of malignant lymphoid cells. GCs activate the glucocorticoid receptor (GR), to regulate a complex genetic network, culminating in apoptosis. Normal lymphoblasts and many lymphoid malignancies are sensitive to GC-driven apoptosis. Resistance to GCs can be a significant clinical problem, however, and correlates with resistance to several other major chemotherapeutic agents. Methods We analyzed the effect of treatment with the cytosine analogue 5 aza-2’ deoxycytidine (AZA) on GC resistance in two acute lymphoblastic leukemia (T or pre-T ALL) cell lines- CEM and Molt-4- and a (B-cell) myeloma cell line, RPMI 8226. Methods employed included tissue culture, flow cytometry, and assays for clonogenicity, cytosine extension, immunochemical identification of proteins, and gene transactivation. High throughput DNA sequencing was used to confirm DNA methylation status. Conclusions Treatment of these cells with AZA resulted in altered DNA methylation and restored GC-evoked apoptosis in all 3 cell lines. In CEM cells the altered epigenetic state resulted in site-specific phosphorylation of the GR, increased GR potency, and GC-driven induction of the GR from promoters that lie in CpG islands. In RPMI 8226 cells, expression of relevant coregulators of GR function was altered. Activation of p38 mitogen-activated protein kinase (MAPK), which is central to a feed-forward mechanism of site-specific GR phosphorylation and ultimately, apoptosis, occurred in all 3 cell lines. These data show that in certain malignant hematologic B- and T-cell types, epigenetically controlled GC resistance can be reversed by cell exposure to a compound that causes DNA demethylation. The results encourage studies of application to in vivo systems, looking towards eventual clinical applications. PMID:24795534

  13. Transient accumulation of 5-carboxylcytosine indicates involvement of active demethylation in lineage specification of neural stem cells.

    PubMed

    Wheldon, Lee M; Abakir, Abdulkadir; Ferjentsik, Zoltan; Dudnakova, Tatiana; Strohbuecker, Stephanie; Christie, Denise; Dai, Nan; Guan, Shengxi; Foster, Jeremy M; Corrêa, Ivan R; Loose, Matthew; Dixon, James E; Sottile, Virginie; Johnson, Andrew D; Ruzov, Alexey

    2014-06-12

    5-Methylcytosine (5mC) is an epigenetic modification involved in regulation of gene activity during differentiation. Tet dioxygenases oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be excised from DNA by thymine-DNA glycosylase (TDG) followed by regeneration of unmodified cytosine via the base excision repair pathway. Despite evidence that this mechanism is operative in embryonic stem cells, the role of TDG-dependent demethylation in differentiation and development is currently unclear. Here, we demonstrate that widespread oxidation of 5hmC to 5caC occurs in postimplantation mouse embryos. We show that 5fC and 5caC are transiently accumulated during lineage specification of neural stem cells (NSCs) in culture and in vivo. Moreover, 5caC is enriched at the cell-type-specific promoters during differentiation of NSCs, and TDG knockdown leads to increased 5fC/5caC levels in differentiating NSCs. Our data suggest that active demethylation contributes to epigenetic reprogramming determining lineage specification in embryonic brain. PMID:24882006

  14. 5-azacytidine improves the osteogenic differentiation potential of aged human adipose-derived mesenchymal stem cells by DNA demethylation.

    PubMed

    Yan, Xueying; Ehnert, Sabrina; Culmes, Mihaela; Bachmann, Anastasia; Seeliger, Claudine; Schyschka, Lilianna; Wang, Zhiyong; Rahmanian-Schwarz, Afshin; Stöckle, Ulrich; De Sousa, Paul A; Pelisek, Jaroslav; Nussler, Andreas K

    2014-01-01

    The therapeutic value of adipose-derived mesenchymal stem cells (Ad-MSCs) for bone regeneration is critically discussed. A possible reason for reduced osteogenic potential may be an age-related deterioration of the Ad-MSCs. In long term in vitro culture, epigenomic changes in DNA methylation are known to cause gene silencing, affecting stem cell growth as well as the differentiation potential. In this study, we observed an age-related decline in proliferation of primary human Ad-MSCs. Decreased Nanog, Oct4 and Lin28A and increased Sox2 gene-expression was accompanied by an impaired osteogenic differentiation potential of Ad-MSCs isolated from old donors (>60 a) as compared to Ad-MSCs isolated from younger donors (<45 a). 5-hydroxymethylcytosine (5 hmC) and 5-methylcytonsine (5 mC) distribution as well as TET gene expression were evaluated to assess the evidence of active DNA demethylation. We observed a decrease of 5 hmC in Ad-MSCs from older donors. Incubation of these cells with 5-Azacytidine induced proliferation and improved the osteogenic differentiation potential in these cells. The increase in AP activity and matrix mineralization was associated with an increased presence of 5 hmC as well as with an increased TET2 and TET3 gene expression. Our data show, for the first time, a decrease of DNA hydroxymethylation in Ad-MSCs which correlates with donor-age and that treatment with 5-Azacytidine provides an approach which could be used to rejuvenate Ad-MSCs from aged donors. PMID:24603866

  15. 5-Azacytidine Improves the Osteogenic Differentiation Potential of Aged Human Adipose-Derived Mesenchymal Stem Cells by DNA Demethylation

    PubMed Central

    Culmes, Mihaela; Bachmann, Anastasia; Seeliger, Claudine; Schyschka, Lilianna; Wang, Zhiyong; Rahmanian-Schwarz, Afshin; Stöckle, Ulrich; De Sousa, Paul A.; Pelisek, Jaroslav; Nussler, Andreas K.

    2014-01-01

    The therapeutic value of adipose-derived mesenchymal stem cells (Ad-MSCs) for bone regeneration is critically discussed. A possible reason for reduced osteogenic potential may be an age-related deterioration of the Ad-MSCs. In long term in vitro culture, epigenomic changes in DNA methylation are known to cause gene silencing, affecting stem cell growth as well as the differentiation potential. In this study, we observed an age-related decline in proliferation of primary human Ad-MSCs. Decreased Nanog, Oct4 and Lin28A and increased Sox2 gene-expression was accompanied by an impaired osteogenic differentiation potential of Ad-MSCs isolated from old donors (>60 a) as compared to Ad-MSCs isolated from younger donors (<45 a). 5-hydroxymethylcytosine (5 hmC) and 5-methylcytonsine (5 mC) distribution as well as TET gene expression were evaluated to assess the evidence of active DNA demethylation. We observed a decrease of 5 hmC in Ad-MSCs from older donors. Incubation of these cells with 5-Azacytidine induced proliferation and improved the osteogenic differentiation potential in these cells. The increase in AP activity and matrix mineralization was associated with an increased presence of 5 hmC as well as with an increased TET2 and TET3 gene expression. Our data show, for the first time, a decrease of DNA hydroxymethylation in Ad-MSCs which correlates with donor-age and that treatment with 5-Azacytidine provides an approach which could be used to rejuvenate Ad-MSCs from aged donors. PMID:24603866

  16. Vimentin-Mediated Steroidogenesis Induced by Phthalate Esters: Involvement of DNA Demethylation and Nuclear Factor κB

    PubMed Central

    Li, Yuan; Hu, Yanhui; Dong, Congcong; Lu, Hongchao; Zhang, Chang; Hu, Qi; Li, Shifeng; Qin, Heng; Li, Zhong; Wang, Yubang

    2016-01-01

    Di-n-butyl phthalate (DBP) and its active metabolite, monobutyl phthalate (MBP) are the most common endocrine disrupting chemicals. Many studies indicate that high-doses of DBP and/or MBP exhibit toxicity on testicular function, however, little attention have been paid to the effects of low levels of DBP/MBP on steroidogenesis. As we all know, the steroidogenic acute regulatory protein (StAR) is a key regulator involved in the steroidogenesis. Here we found that, in addition to StAR, MBP/DBP increased the steroidogenesis by a cytoskeletal protein, vimentin. Briefly, in murine adrenocortical tumor (Y1) and the mouse Leydig tumor (MLTC-1) cells, vimentin regulated the secretion of progesterone. When these two cells were exposure to MBP, the DNA demethylation in the vimentin promoter was observed. In addition, MBP also induced the activation of nuclear factor kappa B (NF-κB, a transcriptional regulator of vimentin). These two processes improved the transcriptional elevation of vimentin. Knockdown of NF-κB/vimentin signaling blocked the DBP/MBP-induced steroidogenesis. These in vitro results were also confirmed via an in vivo model. By identifying a mechanism whereby DBP/MBP regulates vimentin, our results expand the understanding of the endocrine disrupting potential of phthalate esters. PMID:26745512

  17. Growth Arrest and DNA-Damage-Inducible, Beta (GADD45b)-Mediated DNA Demethylation in Major Psychosis

    PubMed Central

    Gavin, David P; Sharma, Rajiv P; Chase, Kayla A; Matrisciano, Francesco; Dong, Erbo; Guidotti, Alessandro

    2012-01-01

    Aberrant neocortical DNA methylation has been suggested to be a pathophysiological contributor to psychotic disorders. Recently, a growth arrest and DNA-damage-inducible, beta (GADD45b) protein-coordinated DNA demethylation pathway, utilizing cytidine deaminases and thymidine glycosylases, has been identified in the brain. We measured expression of several members of this pathway in parietal cortical samples from the Stanley Foundation Neuropathology Consortium (SFNC) cohort. We find an increase in GADD45b mRNA and protein in patients with psychosis. In immunohistochemistry experiments using samples from the Harvard Brain Tissue Resource Center, we report an increased number of GADD45b-stained cells in prefrontal cortical layers II, III, and V in psychotic patients. Brain-derived neurotrophic factor IX (BDNF IXabcd) was selected as a readout gene to determine the effects of GADD45b expression and promoter binding. We find that there is less GADD45b binding to the BDNF IXabcd promoter in psychotic subjects. Further, there is reduced BDNF IXabcd mRNA expression, and an increase in 5-methylcytosine and 5-hydroxymethylcytosine at its promoter. On the basis of these results, we conclude that GADD45b may be increased in psychosis compensatory to its inability to access gene promoter regions. PMID:22048458

  18. Impacts of Activated Carbon Amendment on Hg Methylation, Demethylation and Microbial Activity in Marsh Soils

    NASA Astrophysics Data System (ADS)

    Gilmour, C. C.; Ghosh, U.; Santillan, E. F. U.; Soren, A.; Bell, J. T.; Butera, D.; McBurney, A. W.; Brown, S.; Henry, E.; Vlassopoulos, D.

    2015-12-01

    In-situ sorbent amendments are a low-impact approach for remediation of contaminants in sediments, particular in habitats like wetlands that provide important ecosystem services. Laboratory microcosm trials (Gilmour et al. 2013) and early field trials show that activated carbon (AC) can effectively increase partitioning of both inorganic Hg and methylmercury to the solid phase. Sediment-water partitioning can serve as a proxy for Hg and MeHg bioavailability in soils. One consideration in using AC in remediation is its potential impact on organisms. For mercury, a critical consideration is the potential impact on net MeHg accumulation and bioavailability. In this study, we specifically evaluated the impact of AC on rates of methylmercury production and degradation, and on overall microbial activity, in 4 different Hg-contaminated salt marsh soils. The study was done over 28 days in anaerobic, sulfate-reducing slurries. A double label of enriched mercury isotopes (Me199Hg and inorganic 201Hg) was used to separately follow de novo Me201Hg production and Me199Hg degradation. AC amendments decreased both methylation and demethylation rate constants relative to un-amended controls, but the impact on demethylation was stronger. The addition of 5% (dry weight) regenerated AC to soil slurries drove demethylation rate constants to nearly zero; i.e. MeHg sorption to AC almost totally blocked its degradation. The net impact was increased solid phase MeHg concentrations in some of the soil slurries with the highest methylation rate constants. However, the net impact of AC amendments was to increase MeHg (and inorganic Hg) partitioning to the soil phase and decrease concentrations in the aqueous phase. AC significantly decreased aqueous phase inorganic Hg and MeHg concentrations after 28 days. Overall, the efficacy of AC in reducing aqueous MeHg was highest in the soils with the highest MeHg concentrations. The AC addition did not significantly impact microbial activity, as

  19. Local generation of fumarate promotes DNA repair through inhibition of histone H3 demethylation

    PubMed Central

    Jiang, Yuhui; Qian, Xu; Shen, Jianfeng; Wang, Yugang; Li, Xinjian; Liu, Rui; Xia, Yan; Chen, Qianming; Peng, Guang; Lin, Shiaw-Yih; Lu, Zhimin

    2016-01-01

    Histone methylation regulates DNA repair. However, the mechanisms that underlie the regulation of histone methylation during this repair remain to be further defined. Here, we show that ionizing radiation (IR) induces DNA-PK-dependent phosphorylation of nuclear fumarase at T236, which leads to an interaction between fumarase and the histone variant H2A.Z at DNA double-strand break (DSB) regions. Locally generated fumarate inhibits KDM2B histone demethylase activity, resulting in enhanced dimethylation of histone H3 K36; in turn, this increases the accumulation of the Ku70-containing DNA-PK at DSB regions for non-homologous end joining (NHEJ) DNA repair and cell survival. These findings reveal a feedback mechanism that underlies DNA-PK regulation by chromatin-associated fumarase and an instrumental function of fumarase in regulating histone H3 methylation and DNA repair. PMID:26237645

  20. Combination of PDT and a DNA demethylating agent produces anti-tumor immune response in a mouse tumor model

    NASA Astrophysics Data System (ADS)

    Mroz, Pawel; Hamblin, Michael R.

    2009-06-01

    Epigenetic mechanisms, which involve DNA methylation and histone modifications, result in the heritable silencing of genes without a change in their coding sequence. However, these changes must be actively maintained after each cell division rendering them a promising target for pharmacologic inhibition. DNA methyltransferase inhibitors like 5-aza-deoxycytidine (5-aza-dC) induce and/or up-regulate the expression of MAGE-type antigens in human and mice cancer cells. Photodynamic therapy (PDT) has been shown to be an effective locally ablative anti-cancer treatment that has the additional advantage of stimulating tumor-directed immune response. We studied the effects of a new therapy that combined the demethylating agent 5-aza-dC with PDT in the breast cancer model 4T1 syngenic to immunocompetent BALB/c mice. PDT was used as a locally ablating tumor treatment that is capable of eliciting strong and tumor directed immune response while 5-aza-dC pretreatment was used promote de novo induction of the expression of P1A.protein. This is the mouse homolog of human MAGE family antigens and is reported to function as a tumor rejection antigen in certain mouse tumors. This strategy led to an increase in PDT-mediated immune response and better treatment outcome. These results strongly suggest that the MAGE family antigens are important target for PDT mediated immune response but that their expression can be silenced by epigenetic mechanisms. Therefore the possibility that PDT can be combined with epigenetic strategies to elicit anti-tumor immunity in MAGE-positive tumor models is highly clinically significant and should be studied in detail.

  1. Histone H3 K4 demethylation during activation and attenuation of GAL1 transcription in Saccharomyces cerevisiae.

    PubMed

    Ingvarsdottir, Kristin; Edwards, Chris; Lee, Min Gyu; Lee, Jung Shin; Schultz, David C; Shilatifard, Ali; Shiekhattar, Ramin; Berger, Shelley L

    2007-11-01

    In mammalian cells, histone lysine demethylation is carried out by two classes of enzymes, the LSD1/BHC110 class and the jumonji class. The enzymes of the jumonji class in the yeast Saccharomyces cerevisiae have recently also been shown to have lysine demethylation activity. Here we report that the protein encoded by YJR119c (termed KDM5), coding for one of five predicted jumonji domain proteins in yeast, specifically demethylates trimethylated histone H3 lysine 4 (H3K4me3), H3K4me2, and H3K4me1 in vitro. We found that loss of KDM5 increased mono-, di-, and trimethylation of lysine 4 during activation of the GAL1 gene. Interestingly, cells deleted of KDM5 also displayed a delayed reduction of K4me3 upon reestablishment of GAL1 repression. These results indicate that K4 demethylation has two roles at GAL1, first to establish appropriate levels of K4 methylation during gene activation and second to remove K4 trimethylation during the attenuation phase of transcription. Thus, analysis of lysine demethylation in yeast provides new insight into the physiological roles of jumonji demethylase enzymes. PMID:17875926

  2. H3K4me3 demethylation by the histone demethylase KDM5C/JARID1C promotes DNA replication origin firing

    PubMed Central

    Rondinelli, Beatrice; Schwerer, Hélène; Antonini, Elena; Gaviraghi, Marco; Lupi, Alessio; Frenquelli, Michela; Cittaro, Davide; Segalla, Simona; Lemaitre, Jean-Marc; Tonon, Giovanni

    2015-01-01

    DNA replication is a tightly regulated process that initiates from multiple replication origins and leads to the faithful transmission of the genetic material. For proper DNA replication, the chromatin surrounding origins needs to be remodeled. However, remarkably little is known on which epigenetic changes are required to allow the firing of replication origins. Here, we show that the histone demethylase KDM5C/JARID1C is required for proper DNA replication at early origins. JARID1C dictates the assembly of the pre-initiation complex, driving the binding to chromatin of the pre-initiation proteins CDC45 and PCNA, through the demethylation of the histone mark H3K4me3. Fork activation and histone H4 acetylation, additional early events involved in DNA replication, are not affected by JARID1C downregulation. All together, these data point to a prominent role for JARID1C in a specific phase of DNA replication in mammalian cells, through its demethylase activity on H3K4me3. PMID:25712104

  3. PPARα activation drives demethylation of the CpG islands of the Gadd45b promoter in the mouse liver.

    PubMed

    Kim, Jung-Hwan; Wahyudi, Lilik Duwi; Kim, Kee K; Gonzalez, Frank J

    2016-08-01

    Growth arrest and DNA damage-inducible beta (GADD45b) plays a pivotal role in many intracellular events in both cell survival- and cell death-related signaling. To date, the study of GADD35b has mainly focused on investigation of its function, as well as interacting molecules. However, studies of Gadd45b gene regulation are limited. In this study, we investigated the transcriptional regulation mechanism of Gadd45b. Since Gadd45b mRNA is highly induced by the PPARα agonist Wy-14,643 in the mouse liver, we analyzed the Gadd45b promoter using an in vivo reporter assay. Interestingly, the naked Gadd45b-luciferase construct strongly induced luciferase activity without any stimulant in our in vivo system. Therefore, we investigated the epigenetic changes in the Gadd45b promoter region using mouse liver genomic DNA, the methylation-specific restriction enzyme (HpaII), and disulfide conversion. Our results showed that two possible CpG methylation sites were methylated and demethylated by Wy-14,643 treatment. This study indicates that epigenetic change at the Gadd45b promoter is critical for Gadd45b induction. PMID:27233605

  4. H2-CO2-Dependent Anaerobic O-Demethylation Activity in Subsurface Sediments and by an Isolated Bacterium

    PubMed Central

    Liu, Shi; Suflita, Joseph M.

    1993-01-01

    The ability of microorganisms in sediments from the Atlantic Coastal Plain to biodegrade methoxylated aromatic compounds was examined. O-demethylation activity was detected in deep (121- and 406-m) sediments, as well as in the surface soil. A syringate-demethylating consortium, containing at least three types of bacteria, was enriched from a deep-sediment sample in a medium containing syringate as the sole organic carbon source and with a N2-CO2 atmosphere. An isolate which demethylated syringate was obtained from the enrichment on an agar medium incubated under a H2-CO2 but not a N2-CO2 or N2 atmosphere. O demethylation of syringate of this isolate was dependent on the presence of both H2 and CO2 in the gas phase. The metabolism of syringate occurred in a sequential manner: methylgallate accumulated transiently before it was converted to gallate. Mass balance analysis suggests that the stoichiometry of the reaction in this isolate proceeds in accordance with the following generalized equation: C7H3O3(OCH3)n- + nHCO3- + nH2 → C7H3O3(OH)n- + nCH3COO- + nH2O. Images PMID:16348928

  5. DNA demethylation caused by 5-Aza-2′-deoxycytidine induces mitotic alterations and aneuploidy

    PubMed Central

    Lentini, Laura; Cilluffo, Danilo; Di Leonardo, Aldo

    2016-01-01

    Aneuploidy, the unbalanced number of chromosomes in a cell, is considered a prevalent form of genetic instability and is largely acknowledged as a condition implicated in tumorigenesis. Epigenetic alterations like DNA hypomethylation have been correlated with cancer initiation/progression. Furthermore, a growing body of evidence suggests the involvement of epigenome-wide disruption as a cause of global DNA hypomethylation in aneuploidy generation. Here, we report that the DNA hypomethylating drug 5-aza-2′-deoxycytidine (DAC), affects the correct ploidy of nearly diploid HCT-116 human cells by altering the methylation pattern of the chromosomes. Specifically, we show that a DAC-induced reduction of 5-Methyl Cytosine at the pericentromeric region of chromosomes correlates with aneuploidy and mitotic defects. Our results suggest that DNA hypomethylation leads to aneuploidy by altering the DNA methylation landscape at the centromere that is necessary to ensure proper chromosomes segregation by recruiting the proteins necessary to build up a functional kinetochore. PMID:26771138

  6. Automated Quantification of DNA Demethylation Effects in Cells via 3D Mapping of Nuclear Signatures and Population Homogeneity Assessment1

    PubMed Central

    Gertych, Arkadiusz; Wawrowsky, Kolja A.; Lindsley, Erik; Vishnevsky, Eugene; Farkas, Daniel L.; Tajbakhsh, Jian

    2009-01-01

    Background Today’s advanced microscopic imaging applies to the preclinical stages of drug discovery that employ high-throughput and high-content three-dimensional (3D) analysis of cells to more efficiently screen candidate compounds. Drug efficacy can be assessed by measuring response homogeneity to treatment within a cell population. In this study topologically quantified nuclear patterns of methylated cytosine and global nuclear DNA are utilized as signatures of cellular response to the treatment of cultured cells with the demethylating anti-cancer agents: 5-azacytidine (5-AZA) and octreotide (OCT). Methods Mouse pituitary folliculostellate TtT-GF cells treated with 5-AZA and OCT for 48 hours, and untreated populations, were studied by immunofluorescence with a specific antibody against 5-methylcytosine (MeC), and 4,6-diamidino-2-phenylindole (DAPI) for delineation of methylated sites and global DNA in nuclei (n=163). Cell images were processed utilizing an automated 3D analysis software that we developed by combining seeded watershed segmentation to extract nuclear shells with measurements of Kullback-Leibler’s (K-L) divergence to analyze cell population homogeneity in the relative nuclear distribution patterns of MeC versus DAPI stained sites. Each cell was assigned to one of the four classes: similar, likely similar, unlikely similar and dissimilar. Results Evaluation of the different cell groups revealed a significantly higher number of cells with similar or likely similar MeC/DAPI patterns among untreated cells (~100%), 5-AZA-treated cells (90%), and a lower degree of same type of cells (64%) in the OCT-treated population. The latter group contained (28%) of unlikely similar or dissimilar (7%) cells. Conclusion Our approach was successful in the assessment of cellular behavior relevant to the biological impact of the applied drugs, i.e. the reorganization of MeC/DAPI distribution by demethylation. In a comparison with other metrics, K-L divergence has

  7. Induction of flowering by 5-azacytidine in some plant species: relationship between the stability of photoperiodically induced flowering and flower-inducing effect of DNA demethylation.

    PubMed

    Kondo, Hiroshi; Miura, Takashi; Wada, Kaede C; Takeno, Kiyotoshi

    2007-11-01

    The flower-inducing effect of 5-azacytidine, a DNA demethylating reagent, was examined in several plant species with a stable or unstable photoperiodically induced flowering state under non-inductive photoperiodic conditions. The long day plant Silene armeria, whose flowering state is stable and the short day plant Pharbitis nil, whose flowering state is unstable were induced to flower by 5-azacytidine under a non-inductive condition. Thus, the replacement of photoinduction by 5-azacytidine treatment is not specific to Perilla frutescens. On the other hand, 5-azacytidine did not induce flowering in Xanthium strumarium whose flowering state is stable and Lemna paucicostata whose flowering state is unstable. Thus, epigenetics caused by DNA demethylation may be involved in the regulation of photoperiodic flowering irrespective of the stability of the photoperiodically induced flowering state. PMID:18251884

  8. High salt promotes autoimmunity by TET2-induced DNA demethylation and driving the differentiation of Tfh cells.

    PubMed

    Wu, Haijing; Huang, Xin; Qiu, Hong; Zhao, Ming; Liao, Wei; Yuan, Shuguang; Xie, Yubing; Dai, Yong; Chang, Christopher; Yoshimura, Akihiko; Lu, Qianjin

    2016-01-01

    Follicular helper T cells (Tfh) have been well documented to play a critical role in autoimmunity, such as systemic lupus erythematosus (SLE), by helping B cells. In this study, high salt (sodium chloride, NaCl), under physiological conditions, was demonstrated to increase the differentiation of Tfh. A high-salt diet markedly increased lupus features in MRL/lpr mice. The mechanism is NaCl-induced DNA demethylation via the recruitment of the hydroxytransferase Ten-Eleven Translocation 2 (TET2). Gene silencing of TET2 obviously diminished NaCl-induced Tfh cell polarization in vitro. In addition, the gene expression of sh2d1a, map3k1, spn and stat5b was enhanced after NaCl treatment, consistent with the findings in lupus CD4(+)T cells. However, only spn was directly regulated by TET2, and spn was not the sole target for NaCl. Our findings not only explain the epigenetic mechanisms of high-salt induced autoimmunity but also provide an attractive molecular target for intervention strategies of patients. PMID:27325182

  9. High salt promotes autoimmunity by TET2-induced DNA demethylation and driving the differentiation of Tfh cells

    PubMed Central

    Wu, Haijing; Huang, Xin; Qiu, Hong; Zhao, Ming; Liao, Wei; Yuan, Shuguang; Xie, Yubing; Dai, Yong; Chang, Christopher; Yoshimura, Akihiko; Lu, Qianjin

    2016-01-01

    Follicular helper T cells (Tfh) have been well documented to play a critical role in autoimmunity, such as systemic lupus erythematosus (SLE), by helping B cells. In this study, high salt (sodium chloride, NaCl), under physiological conditions, was demonstrated to increase the differentiation of Tfh. A high-salt diet markedly increased lupus features in MRL/lpr mice. The mechanism is NaCl-induced DNA demethylation via the recruitment of the hydroxytransferase Ten-Eleven Translocation 2 (TET2). Gene silencing of TET2 obviously diminished NaCl-induced Tfh cell polarization in vitro. In addition, the gene expression of sh2d1a, map3k1, spn and stat5b was enhanced after NaCl treatment, consistent with the findings in lupus CD4+T cells. However, only spn was directly regulated by TET2, and spn was not the sole target for NaCl. Our findings not only explain the epigenetic mechanisms of high-salt induced autoimmunity but also provide an attractive molecular target for intervention strategies of patients. PMID:27325182

  10. The activation of human gene MAGE-1 in tumor cells is correlated with genome-wide demethylation.

    PubMed Central

    De Smet, C; De Backer, O; Faraoni, I; Lurquin, C; Brasseur, F; Boon, T

    1996-01-01

    Human gene MAGE-1 encodes tumor-specific antigens that are recognized on melanoma cells by autologous cytolytic T lymphocytes. This gene is expressed in a significant proportion of tumors of various histological types, but not in normal tissues except male germ-line cells. We reported previously that reporter genes driven by the MAGE-1 promoter are active not only in the tumor cell lines that express MAGE-1 but also in those that do not. This suggests that the critical factor causing the activation of MAGE-1 in certain tumors is not the presence of the appropriate transcription factors. The two major MAGE-1 promoter elements have an Ets binding site, which contains a CpG dinucleotide. We report here that these CpG are demethylated in the tumor cell lines that express MAGE-1, and are methylated in those that do not express the gene. Methylation of these CpG inhibits the binding of transcription factors, as seen by mobility shift assay. Treatment with the demethylating agent 5-aza-2'-deoxycytidine activated gene MAGE-1 not only in tumor cell lines but also in primary fibroblasts. Finally, the overall level of CpG methylation was evaluated in 20 different tumor cell lines. It was inversely correlated with the expression of MAGE-1. We conclude that the activation of MAGE-1 in cancer cells is due to the demethylation of the promoter. This appears to be a consequence of a genome-wide demethylation process that occurs in many cancers and is correlated with tumor progression. Images Fig. 1 Fig. 2 Fig. 3 PMID:8692960

  11. Antiproliferative activity and synthesis of 8-prenylnaringenin derivatives by demethylation of 7-O- and 4'-O-substituted isoxanthohumols.

    PubMed

    Anioł, Mirosław; Swiderska, Anna; Stompor, Monika; Zołnierczyk, Anna Katarzyna

    2012-12-01

    Several analogues of 7-O- and 4'-O-substituted isoxanthohumol and 8-prenylnaringenin, the strongest known phytoestrogen and potential anticancerogenic agent, were synthesized. Acyl, alkyl, and allyl derivatives of isoxanthohumol underwent the demethylation process using MgI(2 )× 2Et(2)O in anhydrous THF with the yields of 61-89%. Some of the compounds approached the international criteria of antiproliferative activity (4 μg/ml) for synthetic agents against the human cancer cell lines. PMID:23087590

  12. DNA modifications: Another stable base in DNA

    NASA Astrophysics Data System (ADS)

    Brazauskas, Pijus; Kriaucionis, Skirmantas

    2014-12-01

    Oxidation of 5-methylcytosine has been proposed to mediate active and passive DNA demethylation. Tracking the history of DNA modifications has now provided the first solid evidence that 5-hydroxymethylcytosine is a stable epigenetic modification.

  13. The accumulation of DNA demethylation in Sat α in normal gastric tissues with Helicobacter pylori infection renders susceptibility to gastric cancer in some individuals.

    PubMed

    Saito, Masaaki; Suzuki, Koichi; Maeda, Takafumi; Kato, Takaharu; Kamiyama, Hidenori; Koizumi, Kei; Miyaki, Yuichiro; Okada, Shinichiro; Kiyozaki, Hirokazu; Konishi, Fumio

    2012-06-01

    Helicobacter pylori (HP) infection is widely recognized as a risk factor for gastric cancer, but only a minority of infected individuals develop gastric cancer. The aim of this study was to determine whether DNA demethylation in non-cancerous gastric mucosa (NGM) significantly enhances susceptibility to gastric cancer. A total of 165 healthy volunteers, including 83 HP-positive and 82-negative individuals, as well as 83 patients with single and 18 with synchronous double gastric cancer (GC) were enrolled in this study. The relative demethylation levels (RDLs) of repetitive sequences, including Alu, LINE-1 and Sat α, were quantified by real-time methylation-specific polymerase chain reaction. The Alu RDL did not exhibit any differences within each respective group, whereas LINE-1 RDL was significantly elevated in cancer tissues compared with the NGM in the other groups (P<0.001). Our results indicated that a gradual increase in Sat α RDL correlated with HP infection and cancer development. Sat α RDL was significantly elevated in the NGM in HP-positive compared with HP-negative (P<0.001), and significantly elevated in cancer tissues (P<0.001). Although the Sat α RDL of the NGM in the total population increased in an age-dependent manner, it was significantly increased in a fraction of younger GC patients (<45 years) compared with all of the others (45 years or older, P=0.0391). In addition, double GC exhibited a significantly higher Sat α RDL in the NGM compared with single GC (P=0.0014). In these two fractions, Sat α RDL in the NGM exhibited an inverse correlation with age. In conclusion, the present study demonstrated that the accumulation of DNA demethylation in Sat α RDL in the NGM with HP infection potentially renders susceptibility to gastric cancer in a fraction of GC patients younger than 45 years or in patients with multiple cancers. PMID:22426602

  14. DNA Demethylation Rescues the Impaired Osteogenic Differentiation Ability of Human Periodontal Ligament Stem Cells in High Glucose

    PubMed Central

    Liu, Zhi; Chen, Tian; Sun, Wenhua; Yuan, Zongyi; Yu, Mei; Chen, Guoqing; Guo, Weihua; Xiao, Jingang; Tian, Weidong

    2016-01-01

    Diabetes mellitus, characterized by abnormally high blood glucose levels, gives rise to impaired bone remodeling. In response to high glucose (HG), the attenuated osteogenic differentiation capacity of human periodontal ligament stem cells (hPDLSCs) is associated with the loss of alveolar bone. Recently, DNA methylation was reported to affect osteogenic differentiation of stem cells in pathological states. However, the intrinsic mechanism linking DNA methylation to osteogenic differentiation ability in the presence of HG is still unclear. In this study, we found that diabetic rats with increased DNA methylation levels in periodontal ligaments exhibited reduced bone mass and density. In vitro application of 5-aza-2′-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, to decrease DNA methylation levels in hPDLSCs, rescued the osteogenic differentiation capacity of hPDLSCs under HG conditions. Moreover, we demonstrated that the canonical Wnt signaling pathway was activated during this process and, under HG circumstances, the 5-aza-dC-rescued osteogenic differentiation capacity was blocked by Dickkopf-1, an effective antagonist of the canonical Wnt signaling pathway. Taken together, these results demonstrate for the first time that suppression of DNA methylation is able to facilitate the osteogenic differentiation capacity of hPDLSCs exposed to HG, through activation of the canonical Wnt signaling pathway. PMID:27273319

  15. DNA Demethylation Rescues the Impaired Osteogenic Differentiation Ability of Human Periodontal Ligament Stem Cells in High Glucose.

    PubMed

    Liu, Zhi; Chen, Tian; Sun, Wenhua; Yuan, Zongyi; Yu, Mei; Chen, Guoqing; Guo, Weihua; Xiao, Jingang; Tian, Weidong

    2016-01-01

    Diabetes mellitus, characterized by abnormally high blood glucose levels, gives rise to impaired bone remodeling. In response to high glucose (HG), the attenuated osteogenic differentiation capacity of human periodontal ligament stem cells (hPDLSCs) is associated with the loss of alveolar bone. Recently, DNA methylation was reported to affect osteogenic differentiation of stem cells in pathological states. However, the intrinsic mechanism linking DNA methylation to osteogenic differentiation ability in the presence of HG is still unclear. In this study, we found that diabetic rats with increased DNA methylation levels in periodontal ligaments exhibited reduced bone mass and density. In vitro application of 5-aza-2'-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, to decrease DNA methylation levels in hPDLSCs, rescued the osteogenic differentiation capacity of hPDLSCs under HG conditions. Moreover, we demonstrated that the canonical Wnt signaling pathway was activated during this process and, under HG circumstances, the 5-aza-dC-rescued osteogenic differentiation capacity was blocked by Dickkopf-1, an effective antagonist of the canonical Wnt signaling pathway. Taken together, these results demonstrate for the first time that suppression of DNA methylation is able to facilitate the osteogenic differentiation capacity of hPDLSCs exposed to HG, through activation of the canonical Wnt signaling pathway. PMID:27273319

  16. Transcriptional activation of the Epstein-Barr virus latency C promoter after 5-azacytidine treatment: evidence that demethylation at a single CpG site is crucial.

    PubMed Central

    Robertson, K D; Hayward, S D; Ling, P D; Samid, D; Ambinder, R F

    1995-01-01

    The Epstein-Barr Virus (EBV) latency C promoter (Cp) is the origin of transcripts for six viral proteins. The promoter is active in lymphoblastoid B-cell lines but silent in many EBV-associated tumors and tumor cell lines. In these latter cell lines, the viral episome is hypermethylated in the vicinity of this promoter. We show that in such a cell line (Rael, a Burkitt's lymphoma line), 5-azacytidine inhibits DNA methyltransferase, brings about demethylation of EBV genomes, activates Cp transcription, and induces the expression of EBNA-2. Investigation of the phenomenon demonstrates the importance of the methylation status of a particular CpG site for the regulation of the Cp: (i) genomic sequencing shows that this site is methylated when the Cp is inactive and is not methylated when the promoter is active; (ii) methylation or transition mutation at this site abolishes complex formation with a cellular binding activity (CBF2) as determined by electrophoretic mobility shift analyses, competition binding analyses, and DNase I footprinting; and (iii) a single C --> T transition mutation at this site is associated with a marked reduction (> 50-fold) of transcriptional activity in a reporter plasmid. Thus, the CBF2 binding activity is shown to be methylation sensitive and crucial to EBNA-2-mediated activation of the Cp. PMID:7565767

  17. The DNA methylation profile of activated human natural killer cells.

    PubMed

    Wiencke, John K; Butler, Rondi; Hsuang, George; Eliot, Melissa; Kim, Stephanie; Sepulveda, Manuel A; Siegel, Derick; Houseman, E Andres; Kelsey, Karl T

    2016-05-01

    Natural killer (NK) cells are now recognized to exhibit characteristics akin to cells of the adaptive immune system. The generation of adaptive memory is linked to epigenetic reprogramming including alterations in DNA methylation. The study herein found reproducible genome wide DNA methylation changes associated with human NK cell activation. Activation led predominately to CpG hypomethylation (81% of significant loci). Bioinformatics analysis confirmed that non-coding and gene-associated differentially methylated sites (DMS) are enriched for immune related functions (i.e., immune cell activation). Known DNA methylation-regulated immune loci were also identified in activated NK cells (e.g., TNFA, LTA, IL13, CSF2). Twenty-one loci were designated high priority and further investigated as potential markers of NK activation. BHLHE40 was identified as a viable candidate for which a droplet digital PCR assay for demethylation was developed. The assay revealed high demethylation in activated NK cells and low demethylation in naïve NK, T- and B-cells. We conclude the NK cell methylome is plastic with potential for remodeling. The differentially methylated region signature of activated NKs revealed similarities with T cell activation, but also provided unique biomarker candidates of NK activation, which could be useful in epigenome-wide association studies to interrogate the role of NK subtypes in global methylation changes associated with exposures and/or disease states. PMID:26967308

  18. Zinc deficiency enhanced inflammatory response by increasing immune cell activation and inducing IL6 promoter demethylation

    PubMed Central

    Wong, Carmen P.; Rinaldi, Nicole A.; Ho, Emily

    2015-01-01

    Scope Zinc deficiency results in immune dysfunction and promotes systemic inflammation. The objective of this study was to examine the effects of zinc deficiency on cellular immune activation and epigenetic mechanisms that promote inflammation. This work is potentially relevant to the aging population given that age-related immune defects, including chronic inflammation, coincide with declining zinc status. Methods and results An in vitro cell culture system and the aged mouse model were used to characterize immune activation and DNA methylation profiles that may contribute to the enhanced proinflammatory response mediated by zinc deficiency. Zinc deficiency up-regulated cell activation markers ICAM1, MHC class II, and CD86 in THP1 cells, that coincided with increased IL1β and IL6 responses following LPS stimulation. A decreased zinc status in aged mice was similarly associated with increased ICAM1 and IL6 gene expression. Reduced IL6 promoter methylation was observed in zinc deficient THP1 cells, as well as in aged mice and human lymphoblastoid cell lines derived from aged individuals. Conclusion Zinc deficiency induced inflammatory response in part by eliciting aberrant immune cell activation and altered promoter methylation. Our results suggested potential interactions between zinc status, epigenetics, and immune function, and how their dysregulation could contribute to chronic inflammation. PMID:25656040

  19. 4'-Demethyl-deoxypodophyllotoxin glucoside isolated from Podophyllum hexandrum exhibits potential anticancer activities by altering Chk-2 signaling pathway in MCF-7 breast cancer cells.

    PubMed

    Zilla, Mahesh K; Nayak, Debasis; Amin, Hina; Nalli, Yedukondalu; Rah, Bilal; Chakraborty, Souneek; Kitchlu, Surender; Goswami, Anindya; Ali, Asif

    2014-12-01

    We investigated the root of Podophyllum hexandrum as a potential source of lead bioactive metabolites with anticancer activity. The present study led to the isolation of six known aryltetralin-type lignans designated as 4'-demethyl-deoxypodophyllotoxin (1), podophyllotoxin (2), 4'-demethyl-podophyllotoxin (3), podophyllotoxin-4-O-β-d-glucopyranoside (4), 4'-demethyl-deoxypodophyllotoxin-4-O-β-d-glucopyranoside (5), 4'-demethyl-podophyllotoxin-4-O-β-d-glucopyranoside (6), along with three known flavones Kaempferol (7), Quercetin (8), Astragalin (9) from the root of P. hexandrum. Compounds (1-9) exhibited the remarkable cytotoxic potential in diverse cancer cell lines. 5 therapeutic potential was extensively studied first time which exhibiting antiproliferative and ROS generating activity than its non-glycoside analogue 1. Furthermore, 5 augmented the apoptotic cascades in MCF-7 breast cancer cells, viz. nuclear condensation, membrane blebbing, probably by destabilizing the micro-tubular protein tubulin. Strikingly, our docking study and in vitro assays demonstrate that 5 binds to and modulate checkpoint kinase-2, a key cell cycle regulatory protein in normal and cancer cells. PMID:25446499

  20. Sulfation of o-demethyl apixaban: enzyme identification and species comparison.

    PubMed

    Wang, Lifei; Raghavan, Nirmala; He, Kan; Luettgen, Joseph M; Humphreys, W Griffith; Knabb, Robert M; Pinto, Donald J; Zhang, Donglu

    2009-04-01

    Apixaban, a potent and highly selective factor Xa inhibitor, is currently under development for treatment of arterial and venous thrombotic diseases. The O-demethyl apixaban sulfate is a major circulating metabolite in humans but circulates at lower concentrations relative to parent in animals. The aim of this study was to identify the sulfotransferases (SULTs) responsible for the sulfation reaction. Apixaban undergoes O-demethylation catalyzed by cytochrome P450 enzymes to O-demethyl apixaban, and then is conjugated by SULTs to form O-demethyl apixaban sulfate. Of the five human cDNA-expressed SULTs tested, SULT1A1 and SULT1A2 exhibited significant levels of catalytic activity for formation of O-demethyl apixaban sulfate, and SULT1A3, SULT1E1, and SULT2A1 showed much lower catalytic activities. In human liver S9, quercetin, a highly selective inhibitor of SULT1A1 and SULT1E1, inhibited O-demethyl apixaban sulfate formation by 99%; 2,6-dichloro-4-nitrophenol, another inhibitor of SULT1A1, also inhibited this reaction by >90%; estrone, a competitive inhibitor for SULT1E1, had no effect on this reaction. The comparable K(m) values for formation of O-demethyl apixaban sulfate were 41.4 microM (human liver S9), 36.8 microM (SULT1A1), and 70.8 microM (SULT1A2). Because of the high level of expression of SULT1A1 in liver and its higher level of catalytic activity for formation of O-demethyl apixaban sulfate, SULT1A1 might play a major role in humans for formation of O-demethyl apixaban sulfate. O-Demethyl apixaban was also investigated in liver S9 of mice, rats, rabbits, dogs, monkeys, and humans. The results indicated that liver S9 samples from dogs, monkeys, and humans had higher activities for formation of O-demethyl apixaban sulfate than those of mice, rats, and rabbits. PMID:19131519

  1. Reactivation of a silenced H19 gene in human rhabdomyosarcoma by demethylation of DNA but not by histone hyperacetylation

    PubMed Central

    Lynch, Catherine A; Tycko, Benjamin; Bestor, Timothy H; Walsh, Colum P

    2002-01-01

    Background The active copy of the imprinted gene H19 is turned off by inappropriate methylation in several pediatric tumors including Wilms' Tumour and embryonal rhabdomyosarcoma. H19 controls in cis the linked Insulin-like Growth Factor 2 (IGF2) gene, encoding an important growth factor. Recent work has suggested that methylation of a gene may lead to deacetylation of its associated histones and that silenced genes can be reactivated by increasing histone acetylation levels. Results Treatment of a rhabdomyosarcoma cell line which has a silent, methylated H19 gene with histone deacetylase (HDAC) inhibitors under conditions which gave maximal hyperacetylation of histone 4, both globally and at the H19 gene itself could not reactivate H19 or affect the active Insulin-like Growth Factor 2 (IGF2) gene, but caused clear up-regulation of the Tissue-type Plasminogen Activator (TPA) gene, a non-imprinted gene known to respond to changes in histone acetylation. In contrast, mild treatment of the cells with the methylation inhibitor 5-AzaC-2'-deoxycytidine (AzaC) on its own was able to reactivate H19. Combining AzaC treatment with HDAC inhibitors gave a reduced rather than enhanced reactivation. These findings were confirmed in mouse primary liver and kidney explants which maintain normal imprinting, where we also found that the silent Igf2 gene could not be reactivated by HDAC inhibitors. Conclusion These results suggest that DNA methylation rather than histone acetylation is the primary determinant of silencing of H19 in rhabdomyosarcoma. PMID:12234381

  2. Fluconazole Binding and Sterol Demethylation in Three CYP51 Isoforms Indicate Differences in Active Site Topology

    SciTech Connect

    Bellamine, A.; Lepesheva, Galina I.; Waterman, Mike

    2010-11-16

    14{alpha}-Demethylase (CYP51) is a key enzyme in all sterol biosynthetic pathways (animals, fungi, plants, protists, and some bacteria), catalyzing the removal of the C-14 methyl group following cyclization of squalene. Based on mutations found in CYP51 genes from Candida albicans azole-resistant isolates obtained after fluconazole treatment of fungal infections, and using site-directed mutagenesis, we have found that fluconazole binding and substrate metabolism vary among three different CYP51 isoforms: human, fungal, and mycobacterial. In C. albicans, the Y132H mutant from isolates shows no effect on fluconazole binding, whereas the F145L mutant results in a 5-fold increase in its IC{sub 50} for fluconazole, suggesting that F145 (conserved only in fungal 14{alpha}-demethylases) interacts with this azole. In C. albicans, F145L accounts, in part, for the difference in fluconazole sensitivity reported between mammals and fungi, providing a basis for treatment of fungal infections. The C. albicans Y132H and human Y145H CYP51 mutants show essentially no effect on substrate metabolism, but the Mycobacterium tuberculosis F89H CYP51 mutant loses both its substrate binding and metabolism. Because these three residues align in the three isoforms, the results indicate that their active sites contain important structural differences, and further emphasize that fluconazole and substrate binding are uncoupled properties.

  3. Sustained exposure to the DNA demethylating agent, 2'-deoxy-5-azacytidine, leads to apoptotic cell death in chronic myeloid leukemia by promoting differentiation, senescence, and autophagy.

    PubMed

    Schnekenburger, Michael; Grandjenette, Cindy; Ghelfi, Jenny; Karius, Tommy; Foliguet, Bernard; Dicato, Mario; Diederich, Marc

    2011-02-01

    In addition to its demethylating properties, 2'-deoxy-5-azacytidine (DAC) induces cell cycle arrest, differentiation, cell sensitization to chemotherapy, and cell death. However, the mechanisms by which DAC induces antiproliferation via these processes and how they are interconnected remain unclear. In this study, we found that a clinically relevant concentration of DAC triggered erythroid and megakaryocytic differentiation in the human chronic myeloid leukemia (CML) K-562 and MEG-01 cell lines, respectively. In addition, cells showed a marked increase in cell size in both cell lines and a more adhesive cell profile for MEG-01. Furthermore, DAC treatment induced cellular senescence and autophagy as shown by β-galactosidase staining and by autophagosome formation, respectively. After prolonged DAC treatment, phosphatidyl serine exposure, nuclear morphology analysis, and caspase cleavage revealed an activation of mitochondrial-dependent apoptosis in CML cells. This activation was accompanied by a decrease of anti-apoptotic proteins and an increase of calpain activity. Finally, we showed that combinatory treatment of relatively resistant CML with DAC and either conventional apoptotic inducers or with an histone deacetylase inhibitor increased synergistically apoptosis. We therefore conclude that induction of differentiation, senescence, and autophagy in CML are a key in cell sensitization and DAC-induced apoptosis. PMID:21044612

  4. A DEMETER-like DNA demethylase governs tomato fruit ripening

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This work shows that active DNA demethylation governs ripening, an important plant developmental process. Our work defines a molecular mechanism, which has until now been missing, to explain the correlation between genomic DNA demethylation and fruit ripening. It demonstrates a direct cause-and-effe...

  5. Demethylation of methylarsonic acid by a microbial community

    PubMed Central

    Yoshinaga, Masafumi; Cai, Yong; Rosen, Barry P.

    2013-01-01

    Summary Arsenic is one of the most widespread environmental carcinogens and has created devastating human health problems worldwide, yet little is known about mechanisms of biotransformation in contaminated regions. Methylarsonic acid [MAs(V)], extensively utilized as an herbicide, is largely demethylated to more toxic inorganic arsenite, which causes environmental problems. To understand the process of demethylation of methylarsenicals, soil samples commonly used on Florida golf courses were studied. Several soil extracts were found to demethylate MAs(V) to inorganic arsenite [As(III)]. From these extracts, a bacterial isolate was capable of reducing MAs(V) to MAs(III) but not of demethylating to As(III). A second bacterial isolate was capable of demethylating MAs(III) to As(III) but not of reducing MAs(V). A mixed culture could carry out the complete process of reduction and demethylation, demonstrating that demethylation of MAs(V) to As(III) is a two-step process. Analysis of the 16S ribosomal DNA sequences of the two organisms identified the MAs(V)-reducing and the MAs(III)-demethylating isolates as belong to Burkholderia and Streptomyces species respectively. This is the first report of a novel pathway of degradation of a methylarsenical herbicide by sequential reduction and demethylation in a microbial soil community, which we propose plays a significant role in the arsenic biogeocycle. PMID:21272184

  6. Demethylating Agents in the Treatment of Cancer

    PubMed Central

    Howell, Paul M.; Liu, Zixing; Khong, Hung T.

    2010-01-01

    Gene silencing resulting from aberrant DNA methylation can lead to tumorigenesis. Therefore, drugs that inhibit or interfere with DNA methylation have been used to reactivate and induce silenced gene re-expression in malignancies. Two demethylating agents, azacitidine and decitabine, are approved for the treatment of myelodysplastic syndromes (MDS) by the U.S. Food and Drug Administration (FDA), and are now considered the standard of care in MDS. In this review, we discuss clinical data, including clinical benefits and toxicities, which led to the approval of azacitidine and decitabine. We also summarize findings from clinical trials that used these two demethylating agents in the treatment of solid tumors. Lastly, we discuss some limitations in the use of azacitidine and decitabine in cancer therapy.

  7. Evidence for conserved DNA and histone H3 methylation reprogramming in mouse, bovine and rabbit zygotes

    PubMed Central

    Lepikhov, Konstantin; Zakhartchenko, Valeri; Hao, Ru; Yang, Feikun; Wrenzycki, Christine; Niemann, Heiner; Wolf, Eckhard; Walter, Joern

    2008-01-01

    Background In mammals the parental genomes are epigenetically reprogrammed after fertilization. This reprogramming includes a rapid demethylation of the paternal (sperm-derived) chromosomes prior to DNA replication in zygotes. Such active DNA demethylation in the zygote has been documented for several mammalian species, including mouse, rat, pig, human and cow, but questioned to occur in rabbit. Results When comparing immunohistochemical patterns of antibodies against 5-methyl-cytosine, H3K4me3 and H3K9me2 modifications we observe similar pronuclear distribution and dynamics in mouse, bovine and rabbit zygotes. In rabbit DNA demethylation of the paternal chromosomes occurs at slightly advanced pronuclear stages. We also show that the rabbit oocyte rapidly demethylates DNA of donor fibroblast after nuclear transfer. Conclusion Our data reveal that major events of epigenetic reprogramming during pronuclear maturation, including mechanisms of active DNA demethylation, are apparently conserved among mammalian species. PMID:19014417

  8. Arsenic Induces Functional Re-Expression of Estrogen Receptor α by Demethylation of DNA in Estrogen Receptor-Negative Human Breast Cancer

    PubMed Central

    Liu, Hongxia; Jiang, Fei; Wang, Yubang; Hu, Chunyan; Qi, Hong; Zhong, Caiyun; Wang, Xinru; Li, Zhong

    2012-01-01

    Estrogen receptor α (ERα) is a marker predictive for response of breast cancers to endocrine therapy. About 30% of breast cancers, however, are hormone- independent because of lack of ERα expression. New strategies are needed for re-expression of ERα and sensitization of ER-negative breast cancer cells to selective ER modulators. The present report shows that arsenic trioxide induces reactivated ERα, providing a target for therapy with ER antagonists. Exposure of ER-negative breast cancer cells to arsenic trioxide leads to re-expression of ERα mRNA and functional ERα protein in in vitro and in vivo. Luciferase reporter gene assays and 3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)- 2H-tetrazolium (MTS) assays show that, upon exposure to arsenic trioxide, formerly unresponsive, ER-negative MDA-MB-231 breast cancer cells become responsive to ER antagonists, 4-hydroxytamoxifen and ICI 182,780. Furthermore, methylation- specific PCR and bisulfite-sequencing PCR assays show that arsenic trioxide induces partial demethylation of the ERα promoter. A methyl donor, S-adenosylmethionine (SAM), reduces the degree of arsenic trioxide-induced re-expression of ERα and demethylation. Moreover, Western blot and ChIP assays show that arsenic trioxide represses expression of DNMT1 and DNMT3a along with partial dissociation of DNMT1 from the ERα promoter. Thus, arsenic trioxide exhibits a previously undefined function which induces re-expression ERα in ER-negative breast cancer cells through demethylation of the ERα promoter. These findings could provide important information regarding the application of therapeutic agents targeting epigenetic changes in breast cancers and potential implication of arsenic trioxide as a new drug for the treatment of ER–negative human breast cancer. PMID:22558281

  9. A DEMETER-like DNA demethylase governs tomato fruit ripening

    PubMed Central

    Liu, Ruie; How-Kit, Alexandre; Stammitti, Linda; Teyssier, Emeline; Rolin, Dominique; Mortain-Bertrand, Anne; Halle, Stefanie; Liu, Mingchun; Kong, Junhua; Wu, Chaoqun; Degraeve-Guibault, Charlotte; Chapman, Natalie H.; Maucourt, Mickael; Hodgman, T. Charlie; Tost, Jörg; Bouzayen, Mondher; Hong, Yiguo; Seymour, Graham B.; Giovannoni, James J.; Gallusci, Philippe

    2015-01-01

    In plants, genomic DNA methylation which contributes to development and stress responses can be actively removed by DEMETER-like DNA demethylases (DMLs). Indeed, in Arabidopsis DMLs are important for maternal imprinting and endosperm demethylation, but only a few studies demonstrate the developmental roles of active DNA demethylation conclusively in this plant. Here, we show a direct cause and effect relationship between active DNA demethylation mainly mediated by the tomato DML, SlDML2, and fruit ripening— an important developmental process unique to plants. RNAi SlDML2 knockdown results in ripening inhibition via hypermethylation and repression of the expression of genes encoding ripening transcription factors and rate-limiting enzymes of key biochemical processes such as carotenoid synthesis. Our data demonstrate that active DNA demethylation is central to the control of ripening in tomato. PMID:26261318

  10. NLRP3 Activation Was Regulated by DNA Methylation Modification during Mycobacterium tuberculosis Infection

    PubMed Central

    Wei, Meili; Wang, Lu; Wu, Tao; Xi, Jun; Han, Yuze; Yang, Xingxiang; Zhang, Ding; Fang, Qiang

    2016-01-01

    Mycobacterium tuberculosis (Mtb) infection activates the NLRP3 inflammasome in macrophages and dendritic cells. Much attention has been paid to the mechanisms for regulation of NLRP3 against Mtb. However, whether epigenetic mechanisms participated in NLRP3 activation is still little known. Here we showed that NLRP3 activation was regulated by DNA methylation modification. Mtb infection promoted NLRP3 activation and inflammatory cytokines expression. NLRP3 promoter was cloned and subsequently identified by Dual-Luciferase Reporter System. The results showed that NLRP3 promoter activity was decreased after methylation by DNA methylase Sss I in vitro. Meanwhile, DNA methyltransferases inhibitor DAC could upregulate the expression of NLRP3. Furthermore, promoter region of NLRP3 gene was demethylated after Mtb H37Rv strain infection. These data revealed that DNA methylation was involved in NLRP3 inflammasome activation during Mtb infection and provided a new insight into the relationship between host and pathogens. PMID:27366746

  11. Promoter demethylation of Keap1 gene in human diabetic cataractous lenses

    SciTech Connect

    Palsamy, Periyasamy; Ayaki, Masahiko; Elanchezhian, Rajan; Shinohara, Toshimichi

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer We found significant Keap1 promoter demethylation in diabetic cataractous lenses. Black-Right-Pointing-Pointer Demethylation of Keap1 gene upregulated the expression of Keap1 mRNA and protein. Black-Right-Pointing-Pointer Elevated levels of Keap1 are known to decrease the levels of Nrf2. Black-Right-Pointing-Pointer Thereby, the levels of antioxidant enzymes are suppressed by decreased Nrf2 level. -- Abstract: Age-related cataracts (ARCs) are the major cause of visual impairments worldwide, and diabetic adults tend to have an earlier onset of ARCs. Although age is the strongest risk factor for cataracts, little is known how age plays a role in the development of ARCs. It is known that oxidative stress in the lens increases with age and more so in the lenses of diabetics. One of the central adaptive responses against the oxidative stresses is the activation of the nuclear transcriptional factor, NF-E2-related factor 2 (Nrf2), which then activates more than 20 different antioxidative enzymes. Kelch-like ECH associated protein 1 (Keap1) targets and binds to Nrf2 for proteosomal degradation. We hypothesized that hyperglycemia will lead to a dysfunction of the Nrf2-dependent antioxidative protection in the lens of diabetics. We studied the methylation status of the CpG islands in 15 clear and 21 diabetic cataractous lenses. Our results showed significant levels of demethylated DNA in the Keap1 promoter in the cataractous lenses from diabetic patients. In contrast, highly methylated DNA was found in the clear lens and tumorized human lens epithelial cell (HLEC) lines (SRA01/04). HLECs treated with a demethylation agent, 5-aza-2 Prime deoxycytidine (5-Aza), had a 10-fold higher levels of Keap1 mRNA, 3-fold increased levels of Keap1 protein, produced higher levels of ROS, and increased cell death. Our results indicated that demethylation of the CpG islands in the Keap1 promoter will activate the expression of Keap1 protein, which

  12. Long-term stability of demethylation after transient exposure to 5-aza-2'-deoxycytidine correlates with sustained RNA polymerase II occupancy.

    PubMed

    Kagey, Jacob D; Kapoor-Vazirani, Priya; McCabe, Michael T; Powell, Doris R; Vertino, Paula M

    2010-07-01

    DNA methyltransferase inhibitors are currently the standard of care for myelodysplastic syndrome and are in clinical trials for leukemias and solid tumors. However, the molecular basis underlying their activity remains poorly understood. Here, we studied the induction and long-term stability of gene reactivation at three methylated tumor suppressor loci in response to the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-azaCdR) in human breast cancer cells. At the TMS1/ASC locus, treatment with 5-azaCdR resulted in partial DNA demethylation, the reengagement of RNA polymerase II (Pol II), and a shift from a repressive chromatin profile marked with H3K9me2 and H4K20me3 to an active profile enriched in H3ac and H3K4me2. Using a single-molecule approach coupling chromatin immunoprecipitation with bisulfite sequencing, we show that H3ac, H3K4me2, and Pol II selectively associated with the demethylated alleles, whereas H3K9me2 preferentially marked alleles resistant to demethylation. H4K20me3 was unaffected by DNA demethylation and associated with both unmethylated and methylated alleles. After drug removal, TMS1 underwent partial remethylation, yet a subset of alleles remained stably demethylated for over 3 months. These alleles remained selectively associated with H3K4me2, H3ac, and Pol II and correlated with a sustained low level of gene expression. TMS1 alleles reacquired H3K9me2 over time, and those alleles that became remethylated retained H3ac. In contrast, CDH1 and ESR1 were remethylated and completely silenced within approximately 1 week of drug removal, and failed to maintain stably unmethylated alleles. Our data suggest that the ability to maintain Pol II occupancy is a critical factor in the long-term stability of drug-induced CpG island demethylation. PMID:20587535

  13. Quantification for total demethylation potential of environmental samples utilizing the EGFP reporter gene.

    PubMed

    Qian, Yan; Wang, Xiao-li; Lv, Zhan-lu; Tysklind, Mats; Guo, Chen; Liang, Bao; Wu, Jia-bing; Yang, Yong-jian; Yang, Yi-shu; Wang, Fei-fei; Duan, Xiao-li; Ma, Jin; Wei, Yong-jie; Wang, Chun-hui; Yang, Li-xin; Zhang, Jin-liang; Shi, Xiao-ming; Wang, Xian-liang

    2016-04-01

    The demethylation potential of pollutants is arguably an innate component of their toxicity in environmental samples. A method was developed for determining the total demethylation potential of food samples (TDQ). The demethylation epigenetic toxicity was determined using the Hep G2 cell line transfected with pEGFP-C3 plasmids containing a methylated promoter of the EGFP reporter gene. The total demethylation potential of the sample extracts (the 5-AZA-CdR demethylation toxic equivalency) can be quantified within one week by using a standard curve of the 5-AZA-CdR demethylation agent. To explore the applicability of TDQ for environmental samples, 17 groundwater samples were collected from heavy polluted Kuihe river and the total demethylation potentials of the sample extracts were measured successfully. Meaningful demethylation toxic equivalencies ranging from 0.00050 to 0.01747μM were found in all groundwater sample extracts. Among 19 kinds of inorganic substance, As and Cd played important roles for individual contribution to the total demethylation epigenetic toxicity. The TDQ assay is reliable and fast for quantifying the DNA demethylation potential of environmental sample extracts, which may improve epigenetic toxicity evaluations for human risk assessment, and the consistent consuming of groundwater alongside the Kuihe river pose unexpected epigenetic health risk to the local residents. PMID:26774982

  14. Pluripotency Transcription Factor Oct4 Mediates Stepwise Nucleosome Demethylation and Depletion

    PubMed Central

    Shakya, Arvind; Callister, Catherine; Goren, Alon; Yosef, Nir; Garg, Neha; Khoddami, Vahid; Nix, David; Regev, Aviv

    2015-01-01

    The mechanisms whereby the crucial pluripotency transcription factor Oct4 regulates target gene expression are incompletely understood. Using an assay system based on partially differentiated embryonic stem cells, we show that Oct4 opposes the accumulation of local H3K9me2 and subsequent Dnmt3a-mediated DNA methylation. Upon binding DNA, Oct4 recruits the histone lysine demethylase Jmjd1c. Chromatin immunoprecipitation (ChIP) time course experiments identify a stepwise Oct4 mechanism involving Jmjd1c recruitment and H3K9me2 demethylation, transient FACT (facilitates chromatin transactions) complex recruitment, and nucleosome depletion. Genome-wide and targeted ChIP confirms binding of newly synthesized Oct4, together with Jmjd1c and FACT, to the Pou5f1 enhancer and a small number of other Oct4 targets, including the Nanog promoter. Histone demethylation is required for both FACT recruitment and H3 depletion. Jmjd1c is required to induce endogenous Oct4 expression and fully reprogram fibroblasts to pluripotency, indicating that the assay system identifies functional Oct4 cofactors. These findings indicate that Oct4 sequentially recruits activities that catalyze histone demethylation and depletion. PMID:25582194

  15. Long-term stability of demethylation after transient exposure to 5-aza-2′-deoxycytidine correlates with sustained RNA polymerase II occupancy*

    PubMed Central

    Kagey, Jacob D.; Kapoor-Vazirani, Priya; McCabe, Michael T.; Powell, Doris R.; Vertino, Paula M.

    2010-01-01

    DNA methyltransferase (DNMT) inhibitors are currently the standard of care for myelodysplastic syndrome and are in clinical trials for leukemias and solid tumors. However, the molecular basis underlying their activity remains poorly understood. Here we studied the induction and long-term stability of gene reactivation at three methylated tumor suppressor loci in response to the DNMT inhibitor 5-aza-2′-deoxycytidine (5-azaCdR)in human breast cancer cells. At the TMS/ASC locus, treatment with 5-azaCdR resulted in partial DNA demethylation, the re-engagement of RNA polymerase II (Pol II), and a shift from a repressive chromatin profile marked with H3K9me2 and H4K20me3 to an active profile enriched in H3ac and H3K4me2. Using a single molecule approach coupling chromatin immunoprecipitation with bisulfite sequencing, we show that H3ac, H3K4me2, and Pol II selectively associated with the demethylated alleles, whereas H3K9me2 preferentially marked alleles resistant to demethylation. H4K20me3 was unaffected by DNA demethylation and associated with unmethylated and methylated alleles. After drug removal, TMS1 underwent partial remethylation yet a subset of alleles remained stably demethylated for over three months. These alleles remained selectively associated with H3K4me2, H3ac, and Pol II and correlated with a sustained low level of gene expression. TMS1 alleles reacquire H3K9me2over time and those alleles that became remethylated retained H3ac. In contrast, CDH1and ESR1 were remethylated and completely silenced within ~1 week of drug removal, and failed to maintain stably unmethylated alleles. Our data suggest that the ability to maintain Pol II occupancy is a critical factor in the long-term stability of drug-induced CpG island demethylation. PMID:20587535

  16. Biotic and abiotic mercury methylation and demethylation in sediments

    SciTech Connect

    Zhang, L.; Planas, D. )

    1994-05-01

    Inorganic mercury (Hg(II)) methylation and methylmercury (MeHg) demethylation may occur in the water column, sediment-water interface and subsurficial sediment of aquatic ecosystems. These transformations involve mainly microbial mechanisms, although abiotic methylation may play a more important role in the water compartment. The relative importance of biotic versus abiotic mechanisms of methylation has not been determined however, and abiotic demethylation remains unknown. Little quantitative information is available on the role of bacterial activity in mercury transformations. It has been reported that at least 16 genera of aerobic and anaerobic microorganisms are able to methylate HG(II), and that a greater number are able to demethylate MeHg. Nevertheless, not all populations of these species are capable of methyl- and demethyl-transformations. The actual concentration of MeHg in the aquatic environment is regulated by the relative production and decomposition rates. This, in turn, depends on the availability of Hg(II), MeHg, and bacteria as well as on the physico-chemical properties of the sample. The objective of this study was to compare mercury methylation and demethylation rates in sediment samples with and without active bacterial populations. We therefore performed experiments to follow bacterial evolution during the course of Hg(II) methylation and MeHg demethylation in sediment slurries containing both sterile and non-sterile sediments.

  17. Biochemistry and Occurrence of O-Demethylation in Plant Metabolism

    PubMed Central

    Hagel, Jillian M.; Facchini, Peter J.

    2010-01-01

    Demethylases play a pivitol role in numerous biological processes from covalent histone modification and DNA repair to specialized metabolism in plants and microorganisms. Enzymes that catalyze O- and N-demethylation include 2-oxoglutarate (2OG)/Fe(II)-dependent dioxygenases, cytochromes P450, Rieske-domain proteins and flavin adenine dinucleotide (FAD)-dependent oxidases. Proposed mechanisms for demethylation by 2OG/Fe(II)-dependent enzymes involve hydroxylation at the O- or N-linked methyl group followed by formaldehyde elimination. Members of this enzyme family catalyze a wide variety of reactions in diverse plant metabolic pathways. Recently, we showed that 2OG/Fe(II)-dependent dioxygenases catalyze the unique O-demethylation steps of morphine biosynthesis in opium poppy, which provides a rational basis for the widespread occurrence of demethylases in benzylisoquinoline alkaloid metabolism. PMID:21423357

  18. De-methylation of displacement loop of mitochondrial DNA is associated with increased mitochondrial copy number and nicotinamide adenine dinucleotide subunit 2 expression in colorectal cancer.

    PubMed

    Gao, Jinhang; Wen, Shilei; Zhou, Hongying; Feng, Shi

    2015-11-01

    DNA methylation occurs in the displacement loop (D-loop) region of mammals; however, D-loop regions of certain tumor tissue types were found to be de‑methylated. Whether hypomethylation of the D‑loop region is involved in the regulation of the mitochondrial DNA (mtDNA) copy number and nicotinamide adenine dinucleotide subunit 2 (ND‑2) expressions in colorectal cancer has remained elusive. In the present study, the association between methylation status of the D‑loop region, mtDNA copy number and ND‑2 expression was investigated in 65 colorectal cancer specimens and their corresponding non‑cancerous tissues. In addition, a de‑methylation experiment was performed on the Caco‑2 colorectal cancer cell line by using 5‑aza-2'-deoxycytidine (5‑Aza). The methylation rate of the D‑loop region in all 65 colorectal cancer tissues was markedly reduced when compared with that of their corresponding non‑cancerous tissues (13.8 vs. 81.5%; P<0.05). Furthermore, the methylation rate of the D‑loop region in colorectal cancer tissues was markedly decreased in clinicopathological stages III and IV compared with that in clinicopathological stages I and II (7.1 and 0% vs. 25 and 16%; P<0.05). In addition, the mean relative mtDNA copy number and ND‑2 expression in colorectal cancer tissues were increased compared with those in the corresponding non‑cancerous tissues. De‑methylation of the D‑loop region was associated with an elevated mtDNA copy number and an increased ND‑2 expression. Furthermore, the mtDNA copy number and ND‑2 expression in Caco‑2 cells were significantly increased after 5‑Aza treatment. In conclusion, de‑methylation of the D‑loop region is likely to be involved in the regulation of the mtDNA copy number and ND-2 expression. PMID:26323487

  19. Non-small-cell lung cancer-induced immunosuppression by increased human regulatory T cells via Foxp3 promoter demethylation.

    PubMed

    Ke, Xing; Zhang, Shuping; Xu, Jian; Liu, Genyan; Zhang, Lixia; Xie, Erfu; Gao, Li; Li, Daqian; Sun, Ruihong; Wang, Fang; Pan, Shiyang

    2016-05-01

    Patients with non-small-cell lung cancer (NSCLC) have immune defects that are poorly understood. Forkhead box protein P3 (Foxp3) is crucial for immunosuppression by CD4(+) regulatory T cells (Tregs). It is not well known how NSCLC induces Foxp3 expression and causes immunosuppression in tumor-bearing patients. Our study found a higher percentage of CD4(+) Tregs in the peripheral blood of NSCLC compared with healthy donors. NSCLC patients showed demethylation of eight CpG sites within the Foxp3 promoter with methylation ratios negatively correlated with CD4(+)CD25(+)Foxp3(+) T levels. Foxp3 expression in CD4(+) Tregs was directly regulated by Foxp3 promoter demethylation and was involved in immunosuppression by NSCLC. To verify the effect of tumor cells on the phenotype and function of CD4(+) Tregs, we established a coculture system using NSCLC cell line and healthy CD4(+) T cells and showed that SPC-A1 induced IL-10 and TGF-β1 secretion by affecting the function of CD4(+) Tregs. The activity of DNA methyltransferases from CD4(+) T was decreased during this process. Furthermore, eight CpG sites within the Foxp3 promoter also appeared to have undergone demethylation. Foxp3 is highly expressed in CD4(+) T cells, and this may be caused by gene promoter demethylation. These induced Tregs are highly immunosuppressive and dramatically inhibit the proliferative activity of naïve CD4(+) T cells. Our study provides one possible mechanism describing Foxp3 promoter demethylation changes by which NSCLC down-regulates immune responses and contributes to tumor progression. Foxp3 represents an important target for NSCLC anti-tumor immunotherapy. PMID:27000869

  20. Emodin enhances the demethylation by 5-Aza-CdR of pancreatic cancer cell tumor-suppressor genes P16, RASSF1A and ppENK

    PubMed Central

    PAN, FENG-PING; ZHOU, HONG-KUN; BU, HE-QI; CHEN, ZI-QIANG; ZHANG, HAO; XU, LU-PING; TANG, JIAN; YU, QING-JIANG; CHU, YONG-QUAN; PAN, JIE; FEI, YONG; LIN, SHENG-ZHANG; LIU, DIAN-LEI; CHEN, LIANG

    2016-01-01

    5-Aza-2′-deoxycytidine (5-Aza-CdR) is currently acknowledged as a demethylation drug, and causes a certain degree of demethylation in a variety of cancer cells, including pancreatic cancer cells. Emodin, a traditional Chinese medicine (TCM), is an effective monomer extracted from rhubarb and has been reported to exhibit antitumor activity in different manners in pancreatic cancer. In the present study, we examined whether emodin caused demethylation and increased the demethylation of three tumor-suppressor genes P16, RASSF1A and ppENK with a high degree of methylation in pancreatic cancer when combined with 5-Aza-CdR. Our research showed that emodin inhibited the growth of pancreatic cancer Panc-1 cells in a dose- and time-dependent manner. Dot-blot results showed that emodin combined with 5-Aza-CdR significantly suppressed the expression of genome 5mC in PANC-1 cells. In order to verify the effect of methylation, methylation-specific PCR (MSP) and bisulfite genomic sequencing PCR (BSP) combined with TA were selected for the cloning and sequencing. Results of MSP and BSP confirmed that emodin caused faint demethylation, and 5-Aza-CdR had a certain degree of demethylation. When emodin was combined with 5-Aza-CdR, the demethylation was more significant. At the same time, fluorescent quantitative PCR and western blot analysis results confirmed that when emodin was combined with 5-Aza-CdR, the expression levels of P16, RASSF1A and ppENK were increased more significantly compared to either treatment alone. In contrast, the expression levels of DNA methyltransferase 1 (DNMT1) and DNMT3a were more significantly reduced with the combination treatment than the control or either agent alone, further proving that emodin in combination with 5-Aza-CdR enhanced the demethylation effect of 5-Aza-CdR by reducing the expression of meth-yltransferases. In conclusion, the present study confirmed that emodin in combination with 5-Aza-CdR enhanced the demethylation by 5-Aza-CdR of

  1. Anaerobic O-demethylation of phenylmethylethers

    SciTech Connect

    Frazer, A.C.; Young, L.Y.

    1990-01-01

    Anaerobic O-demethylation (AOD) of phenylmethylethers is a process of both basic and applied significance. The aryl-O-methyl ethers are abundant in natural products, particularly as components of lignin. They are present as methoxylated lignin monomers in anaerobic environments and can be completely degraded there by mixed microbial populations. AOD is an essential early step in this process, and it is also a key reaction in the utilization of the O-methyl substituent as a C-one substrate by acetogens. An understanding of the AOD reaction mechanism might suggest new ways in which chemicals could be derived from lignocellulosic materials. The biochemical mechanism for the anaerobic cleavage of the aryl-O-methyl ether bond is an intriguing, but relatively unexplored process. In contrast to aerobic O-demethylating enzymes, AOD appears to involve methyl group transfer. Thus, novel biochemical information on an important biotransformation reaction will be gained from the research proposed. Recently, we have shown that AOD activity is inducible and have developed an assay for detecting AOD activity in cell-free extracts of Acetobacterium woodii. AOD activity is stimulated in vitro by the addition of ATP (1mM) and pyruvate (30 mM), the K{sub M} for vanillate being 0.4 mM. In collaboration with protein purification experts, we proposed to purify the AOD enzyme and characterize the protein(s) and the enzymatic reaction involved. 8 figs., 5 tabs.

  2. Base Excision Repair Facilitates a Functional Relationship Between Guanine Oxidation and Histone Demethylation

    PubMed Central

    Li, Jianfeng; Braganza, Andrea

    2013-01-01

    Abstract Significance: Appropriately controlled epigenetic regulation is critical for the normal development and health of an organism. Misregulation of epigenetic control via deoxyribonucleic acid (DNA) methylation or histone methylation has been associated with cancer and chromosomal instability syndromes. Recent Advances: The main function of the proteins in the base excision repair (BER) pathway is to repair DNA single-strand breaks and deamination, oxidation, and alkylation-induced DNA base damage that may result from chemotherapy, environmental exposure, or byproducts of cellular metabolism. Recent studies have suggested that one or more BER proteins may also participate in epigenetic regulation to facilitate gene expression modulation via alteration of the state of DNA methylation or via a reaction coupled to histone modification. BER proteins have also been reported to play an essential role in pluripotent stem cell reprogramming. Critical Issues: One emerging function for BER in epigenetic regulation is the repair of base lesions induced by hydrogen peroxide as a byproduct of lysine-specific demethylase 1 (LSD1) enzymatic activity (LSD1/LSD2-coupled BER) for transcriptional regulation. Future Directions: To shed light on this novel role of BER, this review focuses on the repair of oxidative lesions in nuclear DNA that are induced during LSD1-mediated histone demethylation. Further, we highlight current studies suggesting a role for BER proteins in transcriptional regulation of gene expression via BER-coupled active DNA demethylation in mammalian cells. Such efforts to address the role of BER proteins in epigenetic regulation could broaden cancer therapeutic strategies to include epigenetic modifiers combined with BER inhibitors. Antioxid. Redox Signal. 18, 2429–2443. PMID:23311711

  3. Simultaneous quantitative determination of 5-aza-2'-deoxycytidine genomic incorporation and DNA demethylation by liquid chromatography tandem mass spectrometry as exposure-response measures of nucleoside analog DNA methyltransferase inhibitors.

    PubMed

    Anders, Nicole M; Liu, Jianyong; Wanjiku, Teresia; Giovinazzo, Hugh; Zhou, Jianya; Vaghasia, Ajay; Nelson, William G; Yegnasubramanian, Srinivasan; Rudek, Michelle A

    2016-06-01

    The epigenetic and anti-cancer activities of the nucleoside analog DNA methyltransferase (DNMT) inhibitors decitabine (5-aza-2'-deoxycytidine, DAC), azacitidine, and guadecitabine are thought to require cellular uptake, metabolism to 5-aza-2'-deoxycytidine triphosphate, and incorporation into DNA. This genomic incorporation can then lead to trapping and degradation of DNMT enzymes, and ultimately, passive loss of DNA methylation. To facilitate measurement of critical exposure-response relationships of nucleoside analog DNMT inhibitors, a sensitive and reliable method was developed to simultaneously quantitate 5-aza-2'-deoxycytidine genomic incorporation and genomic 5-methylcytosine content using LC-MS/MS. Genomic DNA was extracted and digested into single nucleosides. Chromatographic separation was achieved with a Thermo Hyperpcarb porous graphite column (100mm×2.1mm, 5μm) and isocratic elution with a 10mM ammonium acetate:acetonitrile with 0.1% formic acid (70:30, v/v) mobile phase over a 5min total analytical run time. An AB Sciex 5500 triple quadrupole mass spectrometer operated in positive electrospray ionization mode was used for the detection of 5-aza-2'-deoxycytidine, 2'-deoxycytidine, and 5-methyl-2'-deoxycytidine. The assay range was 2-400ng/mL for 5-aza-2'-deoxycytidine, 50-10,000ng/mL for 2'-deoxycytidine, and was 5-1000ng/mL for 5-methyl-2'-deoxycytidine. The assay proved to be accurate (93.0-102.2%) and precise (CV≤6.3%) across all analytes. All analytes exhibited long-term frozen digest matrix stability at -70°C for at least 117 days. The method was applied for the measurement of genomic 5-aza-2'-deoxycytidine and 5-methyl-2'-deoxycytidine content following exposure of in vitro cell culture and in vivo animal models to decitabine. PMID:27082761

  4. REF6 recognizes a specific DNA sequence to demethylate H3K27me3 and regulate organ boundary formation in Arabidopsis.

    PubMed

    Cui, Xia; Lu, Falong; Qiu, Qi; Zhou, Bing; Gu, Lianfeng; Zhang, Shuaibin; Kang, Yanyuan; Cui, Xiekui; Ma, Xuan; Yao, Qingqing; Ma, Jinbiao; Zhang, Xiaoyu; Cao, Xiaofeng

    2016-06-01

    RELATIVE OF EARLY FLOWERING 6 (REF6, also known as JMJ12) counteracts Polycomb-mediated gene silencing by removing methyl groups from trimethylated histone H3 lysine 27 (H3K27me3) in hundreds of genes in Arabidopsis thaliana. Here we show that REF6 function and genome-wide targeting require its four Cys2His2 zinc fingers, which directly recognize a CTCTGYTY motif. Motifs bound by REF6 tend to cluster and reside in loci with active chromatin states. Furthermore, REF6 targets CUP-SHAPED COTYLEDON 1 (CUC1), which harbors CTCTGYTY motifs, to modulate H3K27me3 levels and activate CUC1 expression. Loss of REF6 causes CUC1 repression and defects in cotyledon separation. In contrast, REF6 does not bind CUC2, encoding a close homolog of CUC1, which lacks the CTCTGYTY motif. Collectively, these results identify a new targeting mechanism of an H3K27 demethylase to counteract Polycomb-mediated gene silencing that regulates plant development, including organ boundary formation. PMID:27111035

  5. Non-growth-associated demethylation of dimethylsulfoniopropionate by (homo)acetogenic bacteria.

    PubMed

    Jansen, M; Hansen, T A

    2001-01-01

    The demethylation of the algal osmolyte dimethylsulfoniopropionate (DMSP) to methylthiopropionate (MTPA) by (homo)acetogenic bacteria was studied. Five Eubacterium limosum strains (including the type strain), Sporomusa ovata DSM 2662(T), Sporomusa sphaeroides DSM 2875(T), and Acetobacterium woodii DSM 1030(T) were shown to demethylate DMSP stoichiometrically to MTPA. The (homo)acetogenic fermentation based on this demethylation did not result in any significant increase in biomass. The analogous demethylation of glycine betaine to dimethylglycine does support growth of acetogens. In batch cultures of E. limosum PM31 DMSP and glycine betaine were demethylated simultaneously. In mixed substrates experiments with fructose-DMSP or methanol-DMSP, DMSP was used rapidly but only after exhaustion of the fructose or the methanol. In steady-state fructose-limited chemostat cultures (at a dilution rate of 0.03 h(-1)) with DMSP as a second reservoir substrate, DMSP was biotransformed to MTPA but this did not result in higher biomass values than in cultures without DMSP; cells from such cultures demethylated DMSP at rates of approximately 50 nmol min(-1) mg of protein(-1), both after growth in the presence of DMSP and after growth in its absence. In cell extracts of glycine betaine-grown strain PM31, DMSP demethylation activities of 21 to 24 nmol min(-1) mg of protein(-1) were detected with tetrahydrofolate as a methyl acceptor; the activities seen with glycine betaine were approximately 10-fold lower. A speculative explanation for the demethylation of DMSP without an obvious benefit for the organism is that the DMSP-demethylating activity is catalyzed by the glycine betaine-demethylating enzyme and that a transport-related factor, in particular a higher energy demand for DMSP transport across the cytoplasmic membrane than for glycine betaine transport, may reduce the overall ATP yield of the fermentation to virtually zero. PMID:11133459

  6. Inhibition of DNA Methylation Suppresses Intestinal Tumor Organoids by Inducing an Anti-Viral Response

    PubMed Central

    Saito, Yoshimasa; Nakaoka, Toshiaki; Sakai, Kasumi; Muramatsu, Toshihide; Toshimitsu, Kohta; Kimura, Masaki; Kanai, Takanori; Sato, Toshiro; Saito, Hidetsugu

    2016-01-01

    Recent studies have proposed that the major anti-tumor effect of DNA methylation inhibitors is induction of interferon-responsive genes via dsRNAs-containing endogenous retroviruses. Recently, a 3D culture system for stem cells known as organoid culture has been developed. Lgr5-positive stem cells form organoids that closely recapitulate the properties of original tissues. To investigate the effect of DNA demethylation on tumor organoids, we have established organoids from intestinal tumors of ApcMin/+ (Min) mice and subjected them to 5-aza-2′-deoxycytidine (5-Aza-CdR) treatment and Dnmt1 knockdown. DNA demethylation induced by 5-Aza-CdR treatment and Dnmt1 knockdown significantly reduced the cell proliferation of the tumor organoids. Microarray analyses of the tumor organoids after 5-Aza-CdR treatment and Dnmt1 knockdown revealed that interferon-responsive genes were activated by DNA demethylation. Gene ontology and pathway analyses clearly demonstrated that these genes activated by DNA demethylation are involved in the anti-viral response. These findings indicate that DNA demethylation suppresses the proliferation of intestinal tumor organoids by inducing an anti-viral response including activation of interferon-responsive genes. Treatment with DNA methylation inhibitors to activate a growth-inhibiting immune response may be an effective therapeutic approach for colon cancers. PMID:27143627

  7. miR-29b induces SOCS-1 expression by promoter demethylation and negatively regulates migration of multiple myeloma and endothelial cells.

    PubMed

    Amodio, Nicola; Bellizzi, Dina; Leotta, Marzia; Raimondi, Lavinia; Biamonte, Lavinia; D'Aquila, Patrizia; Di Martino, Maria Teresa; Calimeri, Teresa; Rossi, Marco; Lionetti, Marta; Leone, Emanuela; Passarino, Giuseppe; Neri, Antonino; Giordano, Antonio; Tagliaferri, Pierosandro; Tassone, Pierfrancesco

    2013-12-01

    Epigenetic silencing of tumor suppressor genes frequently occurs and may account for their inactivation in cancer cells. We previously demonstrated that miR-29b is a tumor suppressor microRNA (miRNA) that targets de novo DNA methyltransferases and reduces the global DNA methylation of multiple myeloma (MM) cells. Here, we provide evidence that epigenetic activity of miR-29b leads to promoter demethylation of suppressor of cytokine signaling-1 (SOCS-1), a hypermethylated tumor suppressor gene. Enforced expression of synthetic miR-29b mimics in MM cell lines resulted in SOCS-1 gene promoter demethylation, as assessed by Sequenom MassARRAY EpiTYPER analysis, and SOCS-1 protein upregulation. miR-29b-induced SOCS-1 demethylation was associated with reduced STAT3 phosphorylation and impaired NFκB activity. Downregulation of VEGF-A and IL-8 mRNAs could be detected in MM cells transfected with miR-29b mimics as well as in endothelial (HUVEC) or stromal (HS-5) cells treated with conditioned medium from miR-29b-transfected MM cells. Notably, enforced expression of miR-29b mimics increased adhesion of MM cells to HS-5 and reduced migration of both MM and HUVEC cells. These findings suggest that miR-29b is a negative regulator of either MM or endothelial cell migration. Finally, the proteasome inhibitor bortezomib, which induces the expression of miR-29b, decreased global DNA methylation by a miR-29b-dependent mechanism and induced SOCS-1 promoter demethylation and protein upregulation. In conclusion, our data indicate that miR-29b is endowed with epigenetic activity and mediates previously unknown functions of bortezomib in MM cells. PMID:24091729

  8. miR-29b induces SOCS-1 expression by promoter demethylation and negatively regulates migration of multiple myeloma and endothelial cells

    PubMed Central

    Amodio, Nicola; Bellizzi, Dina; Leotta, Marzia; Raimondi, Lavinia; Biamonte, Lavinia; D’Aquila, Patrizia; Di Martino, Maria Teresa; Calimeri, Teresa; Rossi, Marco; Lionetti, Marta; Leone, Emanuela; Passarino, Giuseppe; Neri, Antonino; Giordano, Antonio; Tagliaferri, Pierosandro; Tassone, Pierfrancesco

    2013-01-01

    Epigenetic silencing of tumor suppressor genes frequently occurs and may account for their inactivation in cancer cells. We previously demonstrated that miR-29b is a tumor suppressor microRNA (miRNA) that targets de novo DNA methyltransferases and reduces the global DNA methylation of multiple myeloma (MM) cells. Here, we provide evidence that epigenetic activity of miR-29b leads to promoter demethylation of suppressor of cytokine signaling-1 (SOCS-1), a hypermethylated tumor suppressor gene. Enforced expression of synthetic miR-29b mimics in MM cell lines resulted in SOCS-1 gene promoter demethylation, as assessed by Sequenom MassARRAY EpiTYPER analysis, and SOCS-1 protein upregulation. miR-29b-induced SOCS-1 demethylation was associated with reduced STAT3 phosphorylation and impaired NFκB activity. Downregulation of VEGF-A and IL-8 mRNAs could be detected in MM cells transfected with miR-29b mimics as well as in endothelial (HUVEC) or stromal (HS-5) cells treated with conditioned medium from miR-29b-transfected MM cells. Notably, enforced expression of miR-29b mimics increased adhesion of MM cells to HS-5 and reduced migration of both MM and HUVEC cells. These findings suggest that miR-29b is a negative regulator of either MM or endothelial cell migration. Finally, the proteasome inhibitor bortezomib, which induces the expression of miR-29b, decreased global DNA methylation by a miR-29b-dependent mechanism and induced SOCS-1 promoter demethylation and protein upregulation. In conclusion, our data indicate that miR-29b is endowed with epigenetic activity and mediates previously unknown functions of bortezomib in MM cells. PMID:24091729

  9. Methylmercury decomposition in sediments and bacterial cultures: Involvement of methanogens and sulfate reducers in oxidative demethylation

    USGS Publications Warehouse

    Oremland, R.S.; Culbertson, C.W.; Winfrey, M.R.

    1991-01-01

    Demethylation of monomethylmercury in freshwater and estuarine sediments and in bacterial cultures was investigated with 14CH3HgI. Under anaerobiosis, results with inhibitors indicated partial involvement of both sulfate reducers and methanogens, the former dominating estuarine sediments, while both were active in freshwaters. Aerobes were the most significant demethylators in estuarine sediments, but were unimportant in freshwater sediments. Products of anaerobic demethylation were mainly 14CO2 as well as lesser amounts of 14CH4. Acetogenic activity resulted in fixation of some 14CO2 produced from 14CH3HgI into acetate. Aerobic demethylation in estuarine sediments produced only 14CH4, while aerobic demethylation in freshwater sediments produced small amounts of both 14CH4 and 14CO2. Two species of Desulfovibrio produced only traces of 14CH4 from 14CH3HgI, while a culture of a methylotrophic methanogen formed traces of 14CO2 and 14CH4 when grown on trimethylamine in the presence of the 14CH3HgI. These results indicate that both aerobes and anaerobes demethylate mercury in sediments, but that either group may dominate in a particular sediment type. Aerobic demethylation in the estuarine sediments appeared to proceed by the previously characterized organomercurial-lyase pathway, because methane was the sole product. However, aerobic demethylation in freshwater sediments as well as anaerobic demethylation in all sediments studied produced primarily carbon dioxide. This indicates the presence of an oxidative pathway, possibly one in which methylmercury serves as an analog of one-carbon substrates.

  10. Epigenetic Changes during Hepatic Stellate Cell Activation

    PubMed Central

    Götze, Silke; Schumacher, Eva C.; Kordes, Claus; Häussinger, Dieter

    2015-01-01

    Background and Aims Hepatic stellate cells (HSC), which can participate in liver regeneration and fibrogenesis, have recently been identified as liver-resident mesenchymal stem cells. During their activation HSC adopt a myofibroblast-like phenotype accompanied by profound changes in the gene expression profile. DNA methylation changes at single genes have been reported during HSC activation and may participate in the regulation of this process, but comprehensive DNA methylation analyses are still missing. The aim of the present study was to elucidate the role of DNA methylation during in vitro activation of HSC. Methods and Results The analysis of DNA methylation changes by antibody-based assays revealed a strong decrease in the global DNA methylation level during culture-induced activation of HSC. To identify genes which may be regulated by DNA methylation, we performed a genome-wide Methyl-MiniSeq EpiQuest sequencing comparing quiescent and early culture-activated HSC. Approximately 400 differentially methylated regions with a methylation change of at least 20% were identified, showing either hypo- or hypermethylation during activation. Further analysis of selected genes for DNA methylation and expression were performed revealing a good correlation between DNA methylation changes and gene expression. Furthermore, global DNA demethylation during HSC activation was investigated by 5-bromo-2-deoxyuridine assay and L-mimosine treatment showing that demethylation was independent of DNA synthesis and thereby excluding a passive DNA demethylation mechanism. Conclusions In summary, in vitro activation of HSC initiated strong DNA methylation changes, which were associated with gene regulation. These results indicate that epigenetic mechanisms are important for the control of early HSC activation. Furthermore, the data show that global DNA demethylation during activation is based on an active DNA demethylation mechanism. PMID:26065684

  11. DNA Methyltransferase Activity Assays: Advances and Challenges

    PubMed Central

    Poh, Wan Jun; Wee, Cayden Pang Pee; Gao, Zhiqiang

    2016-01-01

    DNA methyltransferases (MTases), a family of enzymes that catalyse the methylation of DNA, have a profound effect on gene regulation. A large body of evidence has indicated that DNA MTase is potentially a predictive biomarker closely associated with genetic disorders and genetic diseases like cancer. Given the attention bestowed onto DNA MTases in molecular biology and medicine, highly sensitive detection of DNA MTase activity is essential in determining gene regulation, epigenetic modification, clinical diagnosis and therapeutics. Conventional techniques such as isotope labelling are effective, but they often require laborious sample preparation, isotope labelling, sophisticated equipment and large amounts of DNA, rendering them unsuitable for uses at point-of-care. Simple, portable, highly sensitive and low-cost assays are urgently needed for DNA MTase activity screening. In most recent technological advances, many alternative DNA MTase activity assays such as fluorescent, electrochemical, colorimetric and chemiluminescent assays have been proposed. In addition, many of them are coupled with nanomaterials and/or enzymes to significantly enhance their sensitivity. Herein we review the progress in the development of DNA MTase activity assays with an emphasis on assay mechanism and performance with some discussion on challenges and perspectives. It is hoped that this article will provide a broad coverage of DNA MTase activity assays and their latest developments and open new perspectives toward the development of DNA MTase activity assays with much improved performance for uses in molecular biology and clinical practice. PMID:26909112

  12. Microarrays Made Simple: "DNA Chips" Paper Activity

    ERIC Educational Resources Information Center

    Barnard, Betsy

    2006-01-01

    DNA microarray technology is revolutionizing biological science. DNA microarrays (also called DNA chips) allow simultaneous screening of many genes for changes in expression between different cells. Now researchers can obtain information about genes in days or weeks that used to take months or years. The paper activity described in this article…

  13. Hydroxymethylcytosine and demethylation of the γ-globin gene promoter during erythroid differentiation.

    PubMed

    Ruiz, Maria Armila; Rivers, Angela; Ibanez, Vinzon; Vaitkus, Kestis; Mahmud, Nadim; DeSimone, Joseph; Lavelle, Donald

    2015-01-01

    The mechanism responsible for developmental stage-specific regulation of γ-globin gene expression involves DNA methylation. Previous results have shown that the γ-globin promoter is nearly fully demethylated during fetal liver erythroid differentiation and partially demethylated during adult bone marrow erythroid differentiation. The hypothesis that 5-hydroxymethylcytosine (5 hmC), a known intermediate in DNA demethylation pathways, is involved in demethylation of the γ-globin gene promoter during erythroid differentiation was investigated by analyzing levels of 5-methylcytosine (5 mC) and 5 hmC at a CCGG site within the 5' γ-globin gene promoter region in FACS-purified cells from baboon bone marrow and fetal liver enriched for different stages of erythroid differentiation. Our results show that 5 mC and 5 hmC levels at the γ-globin promoter are dynamically modulated during erythroid differentiation with peak levels of 5 hmC preceding and/or coinciding with demethylation. The Tet2 and Tet3 dioxygenases that catalyze formation of 5 hmC are expressed during early stages of erythroid differentiation and Tet3 expression increases as differentiation proceeds. In baboon CD34+ bone marrow-derived erythroid progenitor cell cultures, γ-globin expression was positively correlated with 5 hmC and negatively correlated with 5 mC at the γ-globin promoter. Supplementation of culture media with Vitamin C, a cofactor of the Tet dioxygenases, reduced γ-globin promoter DNA methylation and increased γ-globin expression when added alone and in an additive manner in combination with either DNA methyltransferase or LSD1 inhibitors. These results strongly support the hypothesis that the Tet-mediated 5 hmC pathway is involved in developmental stage-specific regulation of γ-globin expression by mediating demethylation of the γ-globin promoter. PMID:25932923

  14. Hydroxymethylcytosine and demethylation of the γ-globin gene promoter during erythroid differentiation

    PubMed Central

    Ruiz, Maria Armila; Rivers, Angela; Ibanez, Vinzon; Vaitkus, Kestis; Mahmud, Nadim; DeSimone, Joseph; Lavelle, Donald

    2015-01-01

    The mechanism responsible for developmental stage-specific regulation of γ-globin gene expression involves DNA methylation. Previous results have shown that the γ-globin promoter is nearly fully demethylated during fetal liver erythroid differentiation and partially demethylated during adult bone marrow erythroid differentiation. The hypothesis that 5-hydroxymethylcytosine (5hmC), a known intermediate in DNA demethylation pathways, is involved in demethylation of the γ-globin gene promoter during erythroid differentiation was investigated by analyzing levels of 5-methylcytosine (5mC) and 5hmC at a CCGG site within the 5′ γ-globin gene promoter region in FACS-purified cells from baboon bone marrow and fetal liver enriched for different stages of erythroid differentiation. Our results show that 5mC and 5hmC levels at the γ-globin promoter are dynamically modulated during erythroid differentiation with peak levels of 5hmC preceding and/or coinciding with demethylation. The Tet2 and Tet3 dioxygenases that catalyze formation of 5hmC are expressed during early stages of erythroid differentiation and Tet3 expression increases as differentiation proceeds. In baboon CD34+ bone marrow-derived erythroid progenitor cell cultures, γ-globin expression was positively correlated with 5hmC and negatively correlated with 5mC at the γ-globin promoter. Supplementation of culture media with Vitamin C, a cofactor of the Tet dioxygenases, reduced γ-globin promoter DNA methylation and increased γ-globin expression when added alone and in an additive manner in combination with either DNA methyltransferase or LSD1 inhibitors. These results strongly support the hypothesis that the Tet-mediated 5hmC pathway is involved in developmental stage-specific regulation of γ-globin expression by mediating demethylation of the γ-globin promoter. PMID:25932923

  15. Oxidative demethylation of 2-picolines on vanadium oxide catalysts

    SciTech Connect

    Suvorov, B.V.; Glubokovskikh, L.K.; Demin, V.V.; Kan, I.I.

    1988-07-10

    One of the known methods for the preparation of pyridine is based on the dealkylation of alkylpyridines in the presence of vanadium-containing catalysts, molecular oxygen and steam. By using the oxidative demethylation of 2-picoline in the presence of steam on a fused vanadium(V) oxide, pyridine can be obtained in a yield of up to 88% of theory. To lower the consumption of vanadium(V) oxide and increase the thermostability of the catalyst, they studied the possible use of V/sub 2/O/sub 5/ catalysts on various carriers (diatomite, silica gel, porcelain balls), including the industrially produced brand SVD and SVS catalysts. The SVS brand catalyst has a satisfactory activity and selectivity in the oxidation demethylation of 2-picoline into pyridine. Under optimal conditions, pyridine is formed on this catalyst in a yield of 88% of the theoretical.

  16. Characterizing Requirements for Small Ubiquitin-like Modifier (SUMO) Modification and Binding on Base Excision Repair Activity of Thymine-DNA Glycosylase in Vivo.

    PubMed

    McLaughlin, Dylan; Coey, Christopher T; Yang, Wei-Chih; Drohat, Alexander C; Matunis, Michael J

    2016-04-22

    Thymine-DNA glycosylase (TDG) plays critical roles in DNA base excision repair and DNA demethylation. It has been proposed, based on structural studies and in vitro biochemistry, that sumoylation is required for efficient TDG enzymatic turnover following base excision. However, whether sumoylation is required for TDG activity in vivo has not previously been tested. We have developed an in vivo assay for TDG activity that takes advantage of its recently discovered role in DNA demethylation and selective recognition and repair of 5-carboxylcytosine. Using this assay, we investigated the role of sumoylation in regulating TDG activity through the use of TDG mutants defective for sumoylation and Small Ubiquitin-like Modifier (SUMO) binding and by altering TDG sumoylation through SUMO and SUMO protease overexpression experiments. Our findings indicate that sumoylation and SUMO binding are not essential for TDG-mediated excision and repair of 5-carboxylcytosine bases. Moreover, in vitro assays revealed that apurinic/apyrimidinic nuclease 1 provides nearly maximum stimulation of TDG processing of G·caC substrates. Thus, under our assay conditions, apurinic/apyrimidinic nuclease 1-mediated stimulation or other mechanisms sufficiently alleviate TDG product inhibition and promote its enzymatic turnover in vivo. PMID:26917720

  17. Nuclease activity of Saccharomyces cerevisiae Dna2 inhibits its potent DNA helicase activity

    PubMed Central

    Levikova, Maryna; Klaue, Daniel; Seidel, Ralf; Cejka, Petr

    2013-01-01

    Dna2 is a nuclease-helicase involved in several key pathways of eukaryotic DNA metabolism. The potent nuclease activity of Saccharomyces cerevisiae Dna2 was reported to be required for all its in vivo functions tested to date. In contrast, its helicase activity was shown to be weak, and its inactivation affected only a subset of Dna2 functions. We describe here a complex interplay of the two enzymatic activities. We show that the nuclease of Dna2 inhibits its helicase by cleaving 5′ flaps that are required by the helicase domain for loading onto its substrate. Mutational inactivation of Dna2 nuclease unleashes unexpectedly vigorous DNA unwinding activity, comparable with that of the most potent eukaryotic helicases. Thus, the ssDNA-specific nuclease activity of Dna2 limits and controls the enzyme's capacity to unwind dsDNA. We postulate that regulation of this interplay could modulate the biochemical properties of Dna2 and thus license it to carry out its distinct cellular functions. PMID:23671118

  18. Acetylation of Werner syndrome protein (WRN): relationships with DNA damage, DNA replication and DNA metabolic activities

    PubMed Central

    Lozada, Enerlyn; Yi, Jingjie; Luo, Jianyuan; Orren, David K.

    2014-01-01

    Loss of WRN function causes Werner Syndrome, characterized by increased genomic instability, elevated cancer susceptibility and premature aging. Although WRN is subject to acetylation, phosphorylation and sumoylation, the impact of these modifications on WRN’s DNA metabolic function remains unclear. Here, we examined in further depth the relationship between WRN acetylation and its role in DNA metabolism, particularly in response to induced DNA damage. Our results demonstrate that endogenous WRN is acetylated somewhat under unperturbed conditions. However, levels of acetylated WRN significantly increase after treatment with certain DNA damaging agents or the replication inhibitor hydroxyurea. Use of DNA repair-deficient cells or repair pathway inhibitors further increase levels of acetylated WRN, indicating that induced DNA lesions and their persistence are at least partly responsible for increased acetylation. Notably, acetylation of WRN correlates with inhibition of DNA synthesis, suggesting that replication blockage might underlie this effect. Moreover, WRN acetylation modulates its affinity for and activity on certain DNA structures, in a manner that may enhance its relative specificity for physiological substrates. Our results also show that acetylation and deacetylation of endogenous WRN is a dynamic process, with sirtuins and other histone deacetylases contributing to WRN deacetylation. These findings advance our understanding of the dynamics of WRN acetylation under unperturbed conditions and following DNA damage induction, linking this modification not only to DNA damage persistence but also potentially to replication stalling caused by specific DNA lesions. Our results are consistent with proposed metabolic roles for WRN and genomic instability phenotypes associated with WRN deficiency. PMID:24965941

  19. Methylation-Sensitive Expression of a DNA Demethylase Gene Serves As an Epigenetic Rheostat

    PubMed Central

    Williams, Ben P.; Pignatta, Daniela; Henikoff, Steven; Gehring, Mary

    2015-01-01

    Genomes must balance active suppression of transposable elements (TEs) with the need to maintain gene expression. In Arabidopsis, euchromatic TEs are targeted by RNA-directed DNA methylation (RdDM). Conversely, active DNA demethylation prevents accumulation of methylation at genes proximal to these TEs. It is unknown how a cellular balance between methylation and demethylation activities is achieved. Here we show that both RdDM and DNA demethylation are highly active at a TE proximal to the major DNA demethylase gene ROS1. Unexpectedly, and in contrast to most other genomic targets, expression of ROS1 is promoted by DNA methylation and antagonized by DNA demethylation. We demonstrate that inducing methylation in the ROS1 proximal region is sufficient to restore ROS1 expression in an RdDM mutant. Additionally, methylation-sensitive expression of ROS1 is conserved in other species, suggesting it is adaptive. We propose that the ROS1 locus functions as an epigenetic rheostat, tuning the level of demethylase activity in response to methylation alterations, thus ensuring epigenomic stability. PMID:25826366

  20. Genome-wide demethylation by 5-aza-2'-deoxycytidine alters the cell fate of stem/progenitor cells.

    PubMed

    Zhou, Yang; Hu, Zhengqing

    2015-02-01

    DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) is able to cause DNA demethylation in the genome and induce the expression of silenced genes. Whether DNA demethylation can affect the gene expression of stem/progenitor cells has not been understood. Mouse utricle epithelia-derived progenitor cells (MUCs), which possess stem cell features as previously described, exhibit a potential DNA methylation status in the genome. In this study, MUCs were treated with 5-aza-CdR to determine whether DNMT inhibitor is able to induce the differentiation of MUCs. With 5-aza-CdR treatment for 72 hr, MUCs expressed epithelial genes including Cdh1, Krt8, Krt18, and Dsp. Further, hair cell genes Myo7a and Myo6 increased their expressions in response to 5-aza-CdR treatment. The decrease in the global methylated DNA values after 5-aza-CdR treatment indicated a significant DNA demethylation in the genome of MUCs, which may contribute to remarkably increased expression of epithelial genes and hair cell genes. The progenitor MUCs then turned into an epithelial-like hair cell fate with the expression of both epithelial and hair cell genes. This study suggests that stem cell differentiation can be stimulated by DNA demethylation, which may open avenues for studying stem cell fate induction using epigenetic approaches. PMID:25096638

  1. Activity and Regulation of Archaeal DNA Alkyltransferase

    PubMed Central

    Perugino, Giuseppe; Vettone, Antonella; Illiano, Giuseppina; Valenti, Anna; Ferrara, Maria C.; Rossi, Mosè; Ciaramella, Maria

    2012-01-01

    Agents that form methylation adducts in DNA are highly mutagenic and carcinogenic, and organisms have evolved specialized cellular pathways devoted to their repair, including DNA alkyltransferases. These are proteins conserved in eucarya, bacteria and archaea, acting by a unique reaction mechanism, which leads to direct repair of DNA alkylation damage and irreversible protein alkylation. The alkylated form of DNA alkyltransferases is inactive, and in eukaryotes, it is rapidly directed to degradation. We report here in vitro and in vivo studies on the DNA alkyltransferase from the thermophilic archaeon Sulfolobus solfataricus (SsOGT). The development of a novel, simple, and sensitive fluorescence-based assay allowed a careful characterization of the SsOGT biochemical and DNA binding activities. In addition, transcriptional and post-translational regulation of SsOGT by DNA damage was studied. We show that although the gene transcription is induced by alkylating agent treatment, the protein is degraded in vivo by an alkylation-dependent mechanism. These experiments suggest a striking conservation, from archaea to humans, of this important pathway safeguarding genome stability. PMID:22167184

  2. Ligand binding affinities of arctigenin and its demethylated metabolites to estrogen receptor alpha.

    PubMed

    Jin, Jong-Sik; Lee, Jong-Hyun; Hattori, Masao

    2013-01-01

    Phytoestrogens are defined as plant-derived compounds with estrogen-like activities according to their chemical structures and activities. Plant lignans are generally categorized as phytoestrogens. It was reported that (-)-arctigenin, the aglycone of arctiin, was demethylated to (-)-dihydroxyenterolactone (DHENL) by Eubacterium (E.) sp. ARC-2. Through stepwise demethylation, E. sp. ARC-2 produced six intermediates, three mono-desmethylarctigenins and three di-desmethylarctigenins. In the present study, ligand binding affinities of (-)-arctigenin and its seven metabolites, including DHENL, were investigated for an estrogen receptor alpha, and found that demethylated metabolites had stronger binding affinities than (-)-arctigenin using a ligand binding screen assay method. The IC(50) value of (2R,3R)-2-(4-hydroxy-3-methoxybenzyl)-3-(3,4-dihydroxybenzyl)-butyrolactone was 7.9 × 10⁻⁴ M. PMID:23325100

  3. A novel apoptosis-inducing mechanism of 5-aza-2'-deoxycitidine in melanoma cells: Demethylation of TNF-α and activation of FOXO1.

    PubMed

    Noguchi, Shunsuke; Mori, Takashi; Igase, Masaya; Mizuno, Takuya

    2015-12-28

    Melanoma is a poor-prognosis cancer in both humans and dogs, and so the anti-tumor effects of 5-aza-2'-deoxycitidine (5-aza) on solid tumors such as melanoma have gained much attention. However, its anti-tumor mechanism remains entirely unclear. This present study revealed a part of the anti-tumor effects of 5-aza, focusing on apoptosis induction, on human and canine melanoma cells. Treatment with 5-aza markedly induced obvious apoptosis in melanoma cells. 5-Aza-induced apoptosis was possibly due to induced expression of cytotoxic cytokines such as TNF-α. We revealed hypermethylation of the promoter region of TNF-α as a consequence of treatment with 5-aza. Concurrently, we evaluated the effect of 5-aza on the Akt/FOXO1 signaling cascade, which plays a pivotal role in the transcription of cytokine genes. As a result, 5-aza inactivated Akt and inversely activated FOXO1, which contributed to the up-regulation of TNF-α. Furthermore, up-regulation of TNF-α by 5-aza administration was found in in vivo experiments. These current data suggest a novel apoptosis-inducing mechanism of 5-aza and indicate that 5-aza could be a promising therapeutic agent for the treatment of human and canine melanomas. PMID:26335173

  4. Enzymatic DNA oxidation: mechanisms and biological significance

    PubMed Central

    Xu, Guo-Liang; Walsh, Colum P.

    2014-01-01

    DNA methylation at cytosines (5mC) is a major epigenetic modification involved in the regulation of multiple biological processes in mammals. How methylation is reversed was until recently poorly understood. The family of dioxygenases commonly known as Ten-eleven translocation (Tet) proteins are responsible for the oxidation of 5mC into three new forms, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Current models link Tet-mediated 5mC oxidation with active DNA demethylation. The higher oxidation products (5fC and 5caC) are recognized and excised by the DNA glycosylase TDG via the base excision repair pathway. Like DNA methyltransferases, Tet enzymes are important for embryonic development. We will examine the mechanism and biological significance of Tet-mediated 5mC oxidation in the context of pronuclear DNA demethylation in mouse early embryos. In contrast to its role in active demethylation in the germ cells and early embryo, a number of lines of evidence suggest that the intragenic 5hmC present in brain may act as a stable mark instead. This short review explores mechanistic aspects of TET oxidation activity, the impact Tet enzymes have on epigenome organization and their contribution to the regulation of early embryonic and neuronal development. [BMB Reports 2014; 47(11): 609-618] PMID:25341925

  5. Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease.

    PubMed

    Gilsbach, Ralf; Preissl, Sebastian; Grüning, Björn A; Schnick, Tilman; Burger, Lukas; Benes, Vladimir; Würch, Andreas; Bönisch, Ulrike; Günther, Stefan; Backofen, Rolf; Fleischmann, Bernd K; Schübeler, Dirk; Hein, Lutz

    2014-01-01

    The heart is a highly specialized organ with essential function for the organism throughout life. The significance of DNA methylation in shaping the phenotype of the heart remains only partially known. Here we generate and analyse DNA methylomes from highly purified cardiomyocytes of neonatal, adult healthy and adult failing hearts. We identify large genomic regions that are differentially methylated during cardiomyocyte development and maturation. Demethylation of cardiomyocyte gene bodies correlates strongly with increased gene expression. Silencing of demethylated genes is characterized by the polycomb mark H3K27me3 or by DNA methylation. De novo methylation by DNA methyltransferases 3A/B causes repression of fetal cardiac genes, including essential components of the cardiac sarcomere. Failing cardiomyocytes partially resemble neonatal methylation patterns. This study establishes DNA methylation as a highly dynamic process during postnatal growth of cardiomyocytes and their adaptation to pathological stress in a process tightly linked to gene regulation and activity. PMID:25335909

  6. Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease

    PubMed Central

    Gilsbach, Ralf; Preissl, Sebastian; Grüning, Björn A.; Schnick, Tilman; Burger, Lukas; Benes, Vladimir; Würch, Andreas; Bönisch, Ulrike; Günther, Stefan; Backofen, Rolf; Fleischmann, Bernd K.; Schübeler, Dirk; Hein, Lutz

    2014-01-01

    The heart is a highly specialized organ with essential function for the organism throughout life. The significance of DNA methylation in shaping the phenotype of the heart remains only partially known. Here we generate and analyse DNA methylomes from highly purified cardiomyocytes of neonatal, adult healthy and adult failing hearts. We identify large genomic regions that are differentially methylated during cardiomyocyte development and maturation. Demethylation of cardiomyocyte gene bodies correlates strongly with increased gene expression. Silencing of demethylated genes is characterized by the polycomb mark H3K27me3 or by DNA methylation. De novo methylation by DNA methyltransferases 3A/B causes repression of fetal cardiac genes, including essential components of the cardiac sarcomere. Failing cardiomyocytes partially resemble neonatal methylation patterns. This study establishes DNA methylation as a highly dynamic process during postnatal growth of cardiomyocytes and their adaptation to pathological stress in a process tightly linked to gene regulation and activity. PMID:25335909

  7. Testosterone Depletion Induces Demethylation of Murine Reelin Promoter CpG Dinucleotides: A Preliminary Study.

    PubMed

    da Silva, Victor Augusto Moraes; Dantas, Marília de Souza; Silva, Leonardo Agostinho de Castro; Carneiro, Juliana Garcia; Schamber-Reis, Bruno Luiz Fonseca

    2015-01-01

    Schizophrenia (SZ) is a debilitating mental disorder characterized by psychotic events, abnormal social behavior, false beliefs, and auditory hallucinations. Hypermethylation of the promoter region of reelin (RELN), a gene involved in regulation of neuronal positioning during telencephalic development, is strongly associated with low protein expression in several cortical structures and promoter hypermethylation in brain from postmortem SZ subjects. Recent experimental data suggests that testosterone is able to promote RELN demethylation, although no direct evidence of hormonal influence on reelin promoter methylation was obtained. We investigated if reduced levels of plasma testosterone in adult male mice lead to Reln promoter demethylation. Animals were administered with flutamide, an antiandrogenic compound, and reelin promoter methylation was assessed using methylationspecific PCR using bisulfite DNA from cerebellum. We found that flutamide was able to significantly lower plasma testosterone when compared to control mice, and treatment did not influence animal survival and body weight. We also show that low plasma testosterone was associated with demethylation of a cytosine residue located at -860 in reelin promoter region. These preliminary data suggest that androgenic hormones can influence cerebral reelin demethylation. To our knowledge, this is the first experimental approach directly linking testosterone depletion and RELN promoter methylation. PMID:26526966

  8. Harnessing Noxa demethylation to overcome Bortezomib resistance in mantle cell lymphoma

    PubMed Central

    Leshchenko, Violetta V.; Kuo, Pei-Yu; Jiang, Zewei; Weniger, Marc A.; Overbey, Jessica; Dunleavy, Kieron; Wilson, Wyndham H.; Wiestner, Adrian; Parekh, Samir

    2015-01-01

    Bortezomib (BZM) is the first proteasome inhibitor approved for relapsed Mantle Cell Lymphoma (MCL) with durable responses seen in 30%–50% of patients. Given that a large proportion of patients will not respond, BZM resistance is a significant barrier to use this agent in MCL. We hypothesized that a subset of aberrantly methylated genes may be modulating BZM response in MCL patients. Genome-wide DNA methylation analysis using a NimbleGen array platform revealed a striking promoter hypomethylation in MCL patient samples following BZM treatment. Pathway analysis of differentially methylated genes identified molecular mechanisms of cancer as a top canonical pathway enriched among hypomethylated genes in BZM treated samples. Noxa, a pro-apoptotic Bcl-2 family member essential for the cytotoxicity of BZM, was significantly hypomethylated and induced following BZM treatment. Therapeutically, we could demethylate Noxa and induce anti-lymphoma activity using BZM and the DNA methytransferase inhibitor Decitabine (DAC) and their combination in vitro and in vivo in BZM resistant MCL cells. These findings suggest a role for dynamic Noxa methylation for the therapeutic benefit of BZM. Potent and synergistic cytotoxicity between BZM and DAC in vitro and in vivo supports a strategy for using epigenetic priming to overcome BZM resistance in relapsed MCL patients. PMID:25714012

  9. DNA-based control of protein activity

    PubMed Central

    Engelen, W.; Janssen, B. M. G.

    2016-01-01

    DNA has emerged as a highly versatile construction material for nanometer-sized structures and sophisticated molecular machines and circuits. The successful application of nucleic acid based systems greatly relies on their ability to autonomously sense and act on their environment. In this feature article, the development of DNA-based strategies to dynamically control protein activity via oligonucleotide triggers is discussed. Depending on the desired application, protein activity can be controlled by directly conjugating them to an oligonucleotide handle, or expressing them as a fusion protein with DNA binding motifs. To control proteins without modifying them chemically or genetically, multivalent ligands and aptamers that reversibly inhibit their function provide valuable tools to regulate proteins in a noncovalent manner. The goal of this feature article is to give an overview of strategies developed to control protein activity via oligonucleotide-based triggers, as well as hurdles yet to be taken to obtain fully autonomous systems that interrogate, process and act on their environments by means of DNA-based protein control. PMID:26812623

  10. DNA-based control of protein activity.

    PubMed

    Engelen, W; Janssen, B M G; Merkx, M

    2016-03-01

    DNA has emerged as a highly versatile construction material for nanometer-sized structures and sophisticated molecular machines and circuits. The successful application of nucleic acid based systems greatly relies on their ability to autonomously sense and act on their environment. In this feature article, the development of DNA-based strategies to dynamically control protein activity via oligonucleotide triggers is discussed. Depending on the desired application, protein activity can be controlled by directly conjugating them to an oligonucleotide handle, or expressing them as a fusion protein with DNA binding motifs. To control proteins without modifying them chemically or genetically, multivalent ligands and aptamers that reversibly inhibit their function provide valuable tools to regulate proteins in a noncovalent manner. The goal of this feature article is to give an overview of strategies developed to control protein activity via oligonucleotide-based triggers, as well as hurdles yet to be taken to obtain fully autonomous systems that interrogate, process and act on their environments by means of DNA-based protein control. PMID:26812623

  11. Bivalent Regions of Cytosine Methylation and H3K27 Acetylation Suggest an Active Role for DNA Methylation at Enhancers.

    PubMed

    Charlet, Jessica; Duymich, Christopher E; Lay, Fides D; Mundbjerg, Kamilla; Dalsgaard Sørensen, Karina; Liang, Gangning; Jones, Peter A

    2016-05-01

    The role of cytosine methylation in the structure and function of enhancers is not well understood. In this study, we investigate the role of DNA methylation at enhancers by comparing the epigenomes of the HCT116 cell line and its highly demethylated derivative, DKO1. Unlike promoters, a portion of regular and super- or stretch enhancers show active H3K27ac marks co-existing with extensive DNA methylation, demonstrating the unexpected presence of bivalent chromatin in both cultured and uncultured cells. Furthermore, our findings also show that bivalent regions have fewer nucleosome-depleted regions and transcription factor-binding sites than monovalent regions. Reduction of DNA methylation genetically or pharmacologically leads to a decrease of the H3K27ac mark. Thus, DNA methylation plays an unexpected dual role at enhancer regions, being anti-correlated focally at transcription factor-binding sites but positively correlated globally with the active H3K27ac mark to ensure structural enhancer integrity. PMID:27153539

  12. Mercury methylation and demethylation in Hg-contaminated lagoon sediments (Marano and Grado Lagoon, Italy)

    NASA Astrophysics Data System (ADS)

    Hines, Mark E.; Poitras, Erin N.; Covelli, Stefano; Faganeli, Jadran; Emili, Andrea; Žižek, Suzana; Horvat, Milena

    2012-11-01

    Mercury (Hg) transformation activities and sulfate (SO42-) reduction were studied in sediments of the Marano and Grado Lagoons in the Northern Adriatic Sea region as part of the "MIRACLE" project. The lagoons, which are sites of clam (Tapes philippinarum) farming, have been receiving excess Hg from the Isonzo River for centuries. Marano Lagoon is also contaminated from a chlor-alkali plant. Radiotracer methods were used to measure mercury methylation (230Hg, 197Hg), methylmercury (MeHg) demethylation (14C-MeHg) and SO42- reduction (35S) in sediment cores collected in autumn, winter and summer. Mercury methylation rate constants ranged from near zero to 0.054 day-1, generally decreased with depth, and were highest in summer. Demethylation rate constants were much higher than methylation reaching values of ˜0.6 day-1 in summer. Demethylation occurred via the oxidative pathway, except in winter when the reductive pathway increased in importance in surficial sediments. Sulfate reduction was also most active in summer (up to 1600 nmol mL-1 day-1) and depth profiles reflected seasonally changing redox conditions near the surface. Methylation and demethylation rate constants correlated positively with SO42- reduction and pore-water Hg concentrations, and inversely with Hg sediment-water partition coefficients indicating the importance of SO42- reduction and Hg dissolution on Hg cycling. Hg transformation rates were calculated using rate constants and concentrations of Hg species. In laboratory experiments, methylation was inhibited by amendments of the SO42--reduction inhibitor molybdate and by nitrate. Lagoon sediments displayed a dynamic seasonal cycle in which Hg dissolution in spring/summer stimulated Hg methylation, which was followed by a net loss of MeHg in autumn from demethylation. Sulfate-reducing bacteria (SRB) tended to be responsible for methylation of Hg and the oxidative demethylation of MeHg. However, during winter in surficial sediments, iron

  13. AP endonuclease 1 prevents the extension of a T/G mismatch by DNA polymerase β to prevent mutations in CpGs during base excision repair.

    PubMed

    Lai, Yanhao; Jiang, Zhongliang; Zhou, Jing; Osemota, Emmanuel; Liu, Yuan

    2016-07-01

    Dynamics of DNA methylation and demethylation at CpG clusters are involved in gene regulation. CpG clusters have been identified as hot spots of mutagenesis because of their susceptibility to oxidative DNA damage. Damaged Cs and Gs at CpGs can disrupt a normal DNA methylation pattern through modulation of DNA methylation and demethylation, leading to mutations and deregulation of gene expression. DNA base excision repair (BER) plays a dual role of repairing oxidative DNA damage and mediating an active DNA demethylation pathway on CpG clusters through removal of a T/G mismatch resulting from deamination of a 5mC adjacent to a guanine that can be simultaneously damaged by oxidative stress. However, it remains unknown how BER processes clustered lesions in CpGs and what are the consequences from the repair of these lesions. In this study, we examined BER of an abasic lesion next to a DNA demethylation intermediate, the T/G mismatch in a CpG dinucleotide, and its effect on the integrity of CpGs. Surprisingly, we found that the abasic lesion completely abolished the activity of thymine DNA glycosylase (TDG) for removing the mismatched T. However, we found that APE1 could still efficiently incise the abasic lesion leaving a 3-terminus mismatched T, which was subsequently extended by pol β. This in turn resulted in a C to T transition mutation. Interestingly, we also found that APE1 3'-5' exonuclease activity efficiently removed the mismatched T, thereby preventing pol β extension of the mismatched nucleotide and the resulting mutation. Our results demonstrate a crucial role of APE1 3'-5' exonuclease activity in combating mutations in CpG clusters caused by an intermediate of DNA demethylation during BER. PMID:27183823

  14. Demethylation profile of the TNF-α promoter gene is associated with high expression of this cytokine in Dengue virus patients.

    PubMed

    Gomes, Alessandra Vilas Boas Terra; de Souza Morais, Stella Maria; Menezes-Filho, Sergio Luiz; de Almeida, Luiz Gustavo Nogueira; Rocha, Raissa Prado; Ferreira, Jaqueline Maria Siqueira; Dos Santos, Luciana Lara; Malaquias, Luiz Cosme Cotta; Coelho, Luiz Felipe Leomil

    2016-08-01

    Dengue is the most prevalent arthropod-borne viral illness in humans. The overexpression of cytokines by Dengue virus (DENV) infected cells is associated with the most severe forms of the disease. Unmethylated CpG islands are related to a transcriptionally active structure, whereas methylated DNA recruits methyl-binding proteins that inhibit gene expression. Several studies have described the importance of epigenetic events in the regulation and expression of many cytokines. The purpose of the present study was to evaluate the methylation status of the IFN-γ and TNF-α promoters in DNA extracted from dengue infected patients using methylation-specific polymerase chain reaction. A high frequency of demethylation was observed in the TNF-α promoter of DENV infected patients when compared to non-infected controls. The patients with an unmethylated profile showed higher expression of TNF-α mRNA than patients with the methylated status. No difference was found in the methylation frequency between the two analyzed groups regarding the IFN-γ promoter or in the expression of IFN-γ transcripts. The present study provides the first association of TNF-α promoter demethylation in DENV infected individuals and demonstrates a correlation between the methylation status of the region analyzed and the expression of TNF-α transcripts in DENV infected patients. J. Med. Virol. 88:1297-1302, 2016. © 2016 Wiley Periodicals, Inc. PMID:26792115

  15. Global identification of genes regulated by estrogen signaling and demethylation in MCF-7 breast cancer cells

    SciTech Connect

    Putnik, Milica; Zhao, Chunyan; Gustafsson, Jan-Ake; Dahlman-Wright, Karin

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer Estrogen signaling and demethylation can both control gene expression in breast cancers. Black-Right-Pointing-Pointer Cross-talk between these mechanisms is investigated in human MCF-7 breast cancer cells. Black-Right-Pointing-Pointer 137 genes are influenced by both 17{beta}-estradiol and demethylating agent 5-aza-2 Prime -deoxycytidine. Black-Right-Pointing-Pointer A set of genes is identified as targets of both estrogen signaling and demethylation. Black-Right-Pointing-Pointer There is no direct molecular interplay of mediators of estrogen and epigenetic signaling. -- Abstract: Estrogen signaling and epigenetic modifications, in particular DNA methylation, are involved in regulation of gene expression in breast cancers. Here we investigated a potential regulatory cross-talk between these two pathways by identifying their common target genes and exploring underlying molecular mechanisms in human MCF-7 breast cancer cells. Gene expression profiling revealed that the expression of approximately 140 genes was influenced by both 17{beta}-estradiol (E2) and a demethylating agent 5-aza-2 Prime -deoxycytidine (DAC). Gene ontology (GO) analysis suggests that these genes are involved in intracellular signaling cascades, regulation of cell proliferation and apoptosis. Based on previously reported association with breast cancer, estrogen signaling and/or DNA methylation, CpG island prediction and GO analysis, we selected six genes (BTG3, FHL2, PMAIP1, BTG2, CDKN1A and TGFB2) for further analysis. Tamoxifen reverses the effect of E2 on the expression of all selected genes, suggesting that they are direct targets of estrogen receptor. Furthermore, DAC treatment reactivates the expression of all selected genes in a dose-dependent manner. Promoter CpG island methylation status analysis revealed that only the promoters of BTG3 and FHL2 genes are methylated, with DAC inducing demethylation, suggesting DNA methylation directs repression of

  16. Production of Hydrolysable Tannin-Like Structures During the Microbial Demethylation of lignin: An Assessment Using13C-Labeled Tetramethylammonium Hydroxide Thermochemolysis.

    NASA Astrophysics Data System (ADS)

    Filley, T.; Blanchette, R.; Nierop, K.; Gamblin, D.

    2003-12-01

    Phenolic compounds in soils are important mediators of microbial activity, metal mobility, soil redox, and soil organic matter building processes. Direct tannin input and the microbial decomposition of lignin in litter and soil are important contributors to this pool of phenols. The ability to accurately assess the relative differences in lignin decay (which are initiated by demethylation and side chain oxidation) among synapyl, coniferyl, and p-coumaryl components of detrital lignin requires the ability to determine microbial demethylation within the complex soil residues. Differentiating between hydrolysable tannins and contributions from advanced lignin decay can be problematic for many of the most common molecular techniques such as alkaline CuO oxidation, pyrolysis GC, and tetramethylammonium hydroxide thermochemolysis because of either the masking effects of derivatizing agents, oxidative damage to ortho-phenols or low volatility of lignin monomers. In this study we investigate lignin demethylation and polyhydroxyl-aromatic production in BC and C horizons of sandy forest soils dominated by oak, the A horizon from a red spruce forest, and controlled microbial inoculation studies of woody tissue using in-line 13C-labeled tetramethylammonium hydroxide thermochemolysis. Both white-rot and brown-rot decay resulted in syringyl demethylation, with the latter exhibiting more aggressive demethylation chemistry, while coniferyl monomer demethylation was essentially restricted to brown-rot decay. In a typical brown-rot sequence demethylation of syringyl components occurs more rapidly than coniferyl units within the same tissue and lower molecular weight fragments are likewise more demethylated than lignin monomers containing the full glycerol side chain. Demethylation of both methoxyl groups in the syringyl monomer is evident in soil horizons as well as laboratory inoculations. The latter may suggest demethylation after lignin depolymerization. Low molecular weight

  17. A DNA polymerase activity is associated with Cauliflower Mosaic Virus.

    PubMed Central

    Menissier, J; Laquel, P; Lebeurier, G; Hirth, L

    1984-01-01

    A DNA polymerase activity is found within the Cauliflower Mosaic Virus (CaMV) particle. Analysis of the reaction product reveals that the linear form of the virion DNA is preferentially labelled. The molecular weight of the DNA polymerase as determined on an "activity gel" is 76 kDa. Images PMID:6514573

  18. A phase I study of hydralazine to demethylate and reactivate the expression of tumor suppressor genes

    PubMed Central

    Zambrano, Pilar; Segura-Pacheco, Blanca; Perez-Cardenas, Enrique; Cetina, Lucely; Revilla-Vazquez, Alma; Taja-Chayeb, Lucía; Chavez-Blanco, Alma; Angeles, Enrique; Cabrera, Gustavo; Sandoval, Karina; Trejo-Becerril, Catalina; Chanona-Vilchis, Jose; Duenas-González, Alfonso

    2005-01-01

    Background The antihypertensive compound hydralazine is a known demethylating agent. This phase I study evaluated the tolerability and its effects upon DNA methylation and gene reactivation in patients with untreated cervical cancer. Methods Hydralazine was administered to cohorts of 4 patients at the following dose levels: I) 50 mg/day, II) 75 mg/day, III) 100 mg/day and IV) 150 mg/day. Tumor biopsies and peripheral blood samples were taken the day before and after treatment. The genes APC, MGMT; ER, GSTP1, DAPK, RARβ, FHIT and p16 were evaluated pre and post-treatment for DNA promoter methylation and gene expression by MSP (Methylation-Specific PCR) and RT-PCR respectively in each of the tumor samples. Methylation of the imprinted H19 gene and the "normally methylated" sequence clone 1.2 was also analyzed. Global DNA methylation was analyzed by capillary electrophoresis and cytosine extension assay. Toxicity was evaluated using the NCI Common Toxicity Criteria. Results Hydralazine was well tolerated. Toxicities were mild being the most common nausea, dizziness, fatigue, headache and palpitations. Overall, 70% of the pretreatment samples and all the patients had at least one methylated gene. Rates of demethylation at the different dose levels were as follows: 50 mg/day, 40%; 75 mg/day, 52%, 100 mg/day, 43%, and 150 mg/day, 32%. Gene expression analysis showed only 12 informative cases, of these 9 (75%) re-expressed the gene. There was neither change in the methylation status of H19 and clone 1.2 nor changes in global DNA methylation. Conclusion Hydralazine at doses between 50 and 150 mg/day is well tolerated and effective to demethylate and reactivate the expression of tumor suppressor genes without affecting global DNA methylation PMID:15862127

  19. Demethylation and cleavage of dimethylsulfoniopropionate in marine intertidal sediments

    USGS Publications Warehouse

    Visscher, P.T.; Kiene, R.P.; Taylor, B.F.

    1994-01-01

    Demethylation and cleavage of dimethylsulfoniopropionate (DMSP) was measured in three different types of,intertidal marine sediments: a cyanobacterial mat, a diatom-covered tidal flat and a carbonate sediment. Consumption rates of added DMSP were highest in cyanobacterial mat slurries (59 ?? mol DMSP l-1 slurry h-1) and lower in slurries from a diatom mat and a carbonate tidal sediment (24 and 9 ??mol DMSP l-1 h-1, respectively). Dimethyl sulfide (DMS) and 3-mercaptopropionate (MPA) were produced simultaneously during DMSP consumption, indicating that cleavage and demethylation occurred at the same time. Viable counts of DMSP-utilizing bacteria revealed a population of 2 x 107 cells cm-3 sediment (90% of these cleaved DMSP to DMS, 10% demethylated DMSP to MPA) in the cyanobacterial mat, 7 x 105 cells cm-3 in the diatom mat (23% cleavers, 77% demethylators), and 9 x 104 cells cm-3 (20% cleavers and 80% demethylators) in the carbonate sediment. In slurries of the diatom mat, the rate of MPA production from added 3-methiolpropionate (MMPA) was 50% of the rate of MPA formation from DMSP. The presence of a large population of demethylating bacteria and the production of MPA from DMSP suggest that the demethylation pathway, in addition to cleavage, contributes significantly to DMSP consumption in coastal sediments.

  20. Constitutive activation of the ATM/BRCA1 pathway prevents DNA damage-induced apoptosis in 5-azacytidine-resistant cell lines.

    PubMed

    Imanishi, Satoshi; Umezu, Tomohiro; Ohtsuki, Kazushige; Kobayashi, Chiaki; Ohyashiki, Kazuma; Ohyashiki, Junko H

    2014-06-01

    5-Azacytidine (AZA) exerts its anti-tumor effects by exerting cytotoxicity via its incorporation into RNA and DNA, which causes the reactivation of aberrantly silenced growth-regulatory genes by promoter demethylation, as well as DNA damage. AZA is used for patients with myelodysplastic syndrome and acute myeloid leukemia. However, some patients demonstrate resistance to AZA, the mechanisms of which are not fully elucidated. We therefore sought to better characterize the molecular mechanism of AZA resistance using an in vitro model of AZA resistance. We established AZA-resistant cell lines by exposing the human leukemia cell lines U937 and HL-60 to clinical concentrations of AZA, and characterized these cells. AZA-resistant cells showed a down-regulation of the DNMT3A protein, in correlation with their marked genome-wide DNA hypomethylation. Furthermore, genes involved in pyrimidine metabolism were down-regulated in both AZA-resistant cell lines; AZA sensitivity was restored by inhibition of CTP synthase. Of note is that the DNA damage response pathway is constitutively activated in the AZA-resistant cell lines, but not in the parental cell lines. Inhibition of the DNA damage response pathway canceled the AZA resistance, in association with an increase in apoptotic cells. We found that the molecular mechanism underlying AZA resistance involves pyrimidine metabolism and the DNA damage response through ATM kinase. This study therefore sheds light on the mechanisms underlying AZA resistance, and will enable better understanding of AZA resistance in patients undergoing AZA treatment. PMID:24680865

  1. Lidocaine Sensitizes the Cytotoxicity of Cisplatin in Breast Cancer Cells via Up-Regulation of RARβ2 and RASSF1A Demethylation

    PubMed Central

    Li, Kehan; Yang, Jianxue; Han, Xuechang

    2014-01-01

    It has been reported that lidocaine is toxic to various types of cells. And a recent study has confirmed that lidocaine exerts a demethylation effect and regulates the proliferation of human breast cancer cell lines. To recognize a potential anti-tumor effect of lidocaine, we evaluated the DNA demethylation by lidocaine in human breast cancer lines, MCF-7 and MDA-MB-231 cells, and determined the influence of demethylation on the toxicity to these cells of cisplatin, which is a commonly utilized anti-tumor agent for breast cancer. Results demonstrated that lidocaine promoted a significant global genomic demethylation, and particularly in the promoters of tumor suppressive genes (TSGs), RARβ2 and RASSF1A. Further, the lidocaine treatment increased cisplatin-induced apoptosis and enhanced cisplatin-induced cytotoxicity. The combined treatment with both lidocaine and cisplatin promoted a significantly higher level of MCF-7 cell apoptosis than singular lidocaine or cisplatin treatment. Moreover, the abrogation of RARβ2 or RASSF1A expression inhibited such apoptosis. In conclusion, the present study confirms the demethylation effect of lidocaine in breast cancer cells, and found that the demethylation of RARβ2 and RASSF1A sensitized the cytotoxicity of cisplatin in breast cancer cells. PMID:25526566

  2. Purification of total DNA extracted from activated sludge.

    PubMed

    Shan, Guobin; Jin, Wenbiao; Lam, Edward K H; Xing, Xinhui

    2008-01-01

    Purification of the total DNA extracted from activated sludge samples was studied. The effects of extraction buffers and lysis treatments (lysozyme, sodium dodecyl sulfate (SDS), sonication, mechanical mill and thermal shock) on yield and purity of the total DNA extracted from activated sludge were investigated. It was found that SDS and mechanical mill were the most effective ways for cell lysis, and both gave the highest DNA yields, while by SDS and thermal shock, the purest DNA extract could be obtained. The combination of SDS with other lysis treatment, such as sonication and thermal shock, could apparently increase the DNA yields but also result in severe shearing. For the purification of the crude DNA extract, polyvinyl polypyrrolidone was used for the removal of humic contaminants. Cetyltrimethyl ammonium bromide, potassium acetate and phenol/chloroform were used to remove proteins and polysaccharides from crude DNA. Crude DNA was further purified by isopropanol precipitation. Thus, a suitable protocol was proposed for DNA extraction, yielding about 49.9 mg (total DNA)/g volatile suspended solids, and the DNA extracts were successfully used in PCR amplifications for 16S rDNA and 16S rDNA V3 region. The PCR products of 16S rDNA V3 region allowed the DGGE analysis (denatured gradient gel electrophoresis) to be possible. PMID:18572527

  3. Integrity and Biological Activity of DNA after UV Exposure

    NASA Astrophysics Data System (ADS)

    Lyon, Delina Y.; Monier, Jean-Michel; Dupraz, Sébastien; Freissinet, Caroline; Simonet, Pascal; Vogel, Timothy M.

    2010-04-01

    The field of astrobiology lacks a universal marker with which to indicate the presence of life. This study supports the proposal to use nucleic acids, specifically DNA, as a signature of life (biosignature). In addition to its specificity to living organisms, DNA is a functional molecule that can confer new activities and characteristics to other organisms, following the molecular biology dogma, that is, DNA is transcribed to RNA, which is translated into proteins. Previous criticisms of the use of DNA as a biosignature have asserted that DNA molecules would be destroyed by UV radiation in space. To address this concern, DNA in plasmid form was deposited onto different surfaces and exposed to UVC radiation. The surviving DNA was quantified via the quantitative polymerase chain reaction (qPCR). Results demonstrate increased survivability of DNA attached to surfaces versus non-adsorbed DNA. The DNA was also tested for biological activity via transformation into the bacterium Acinetobacter sp. and assaying for antibiotic resistance conferred by genes encoded by the plasmid. The success of these methods to detect DNA and its gene products after UV exposure (254 nm, 3.5 J/m2s) not only supports the use of the DNA molecule as a biosignature on mineral surfaces but also demonstrates that the DNA retained biological activity.

  4. Integrity and biological activity of DNA after UV exposure.

    PubMed

    Lyon, Delina Y; Monier, Jean-Michel; Dupraz, Sébastien; Freissinet, Caroline; Simonet, Pascal; Vogel, Timothy M

    2010-04-01

    The field of astrobiology lacks a universal marker with which to indicate the presence of life. This study supports the proposal to use nucleic acids, specifically DNA, as a signature of life (biosignature). In addition to its specificity to living organisms, DNA is a functional molecule that can confer new activities and characteristics to other organisms, following the molecular biology dogma, that is, DNA is transcribed to RNA, which is translated into proteins. Previous criticisms of the use of DNA as a biosignature have asserted that DNA molecules would be destroyed by UV radiation in space. To address this concern, DNA in plasmid form was deposited onto different surfaces and exposed to UVC radiation. The surviving DNA was quantified via the quantitative polymerase chain reaction (qPCR). Results demonstrate increased survivability of DNA attached to surfaces versus non-adsorbed DNA. The DNA was also tested for biological activity via transformation into the bacterium Acinetobacter sp. and assaying for antibiotic resistance conferred by genes encoded by the plasmid. The success of these methods to detect DNA and its gene products after UV exposure (254 nm, 3.5 J/m(2)s) not only supports the use of the DNA molecule as a biosignature on mineral surfaces but also demonstrates that the DNA retained biological activity. PMID:20446869

  5. Escherichia coli DnaE Polymerase Couples Pyrophosphatase Activity to DNA Replication

    PubMed Central

    Lapenta, Fabio; Montón Silva, Alejandro; Brandimarti, Renato; Lanzi, Massimiliano; Gratani, Fabio Lino; Vellosillo Gonzalez, Perceval; Perticarari, Sofia; Hochkoeppler, Alejandro

    2016-01-01

    DNA Polymerases generate pyrophosphate every time they catalyze a step of DNA elongation. This elongation reaction is generally believed as thermodynamically favoured by the hydrolysis of pyrophosphate, catalyzed by inorganic pyrophosphatases. However, the specific action of inorganic pyrophosphatases coupled to DNA replication in vivo was never demonstrated. Here we show that the Polymerase-Histidinol-Phosphatase (PHP) domain of Escherichia coli DNA Polymerase III α subunit features pyrophosphatase activity. We also show that this activity is inhibited by fluoride, as commonly observed for inorganic pyrophosphatases, and we identified 3 amino acids of the PHP active site. Remarkably, E. coli cells expressing variants of these catalytic residues of α subunit feature aberrant phenotypes, poor viability, and are subject to high mutation frequencies. Our findings indicate that DNA Polymerases can couple DNA elongation and pyrophosphate hydrolysis, providing a mechanism for the control of DNA extension rate, and suggest a promising target for novel antibiotics. PMID:27050298

  6. Engineered DNA ligases with improved activities in vitro.

    PubMed

    Wilson, Robert H; Morton, Susan K; Deiderick, Heather; Gerth, Monica L; Paul, Hayden A; Gerber, Ilana; Patel, Ankita; Ellington, Andrew D; Hunicke-Smith, Scott P; Patrick, Wayne M

    2013-07-01

    The DNA ligase from bacteriophage T4 is one of the most widely used enzymes in molecular biology. It has evolved to seal single-stranded nicks in double-stranded DNA, but not to join double-stranded fragments with cohesive or blunt ends. Its poor activity in vitro, particularly with blunt-ended substrates, can lead to failed or sub-optimal experimental outcomes. We have fused T4 DNA ligase to seven different DNA-binding proteins, including eukaryotic transcription factors, bacterial DNA repair proteins and archaeal DNA-binding domains. Representatives from each of these classes improved the activity of T4 DNA ligase, by up to 7-fold, in agarose gel-based screens for cohesive- and blunt-ended fragment joining. Overall, the most active variants were p50-ligase (i.e. NF-κB p50 fused to T4 DNA ligase) and ligase-cTF (T4 DNA ligase fused to an artificial, chimeric transcription factor). Ligase-cTF out-performed T4 DNA ligase by ∼160% in blunt end 'vector + insert' cloning assays, and p50-ligase showed an improvement of a similar magnitude when it was used to construct a library for Illumina sequencing. The activity of the Escherichia coli DNA ligase was also enhanced by fusion to p50. Together, these results suggest that our protein design strategy is a generalizable one for engineering improved DNA ligases. PMID:23754529

  7. Homologous DNA strand exchange activity of the human mitochondrial DNA helicase TWINKLE

    PubMed Central

    Sen, Doyel; Patel, Gayatri; Patel, Smita S.

    2016-01-01

    A crucial component of the human mitochondrial DNA replisome is the ring-shaped helicase TWINKLE—a phage T7-gene 4-like protein expressed in the nucleus and localized in the human mitochondria. Our previous studies showed that despite being a helicase, TWINKLE has unique DNA annealing activity. At the time, the implications of DNA annealing by TWINKLE were unclear. Herein, we report that TWINKLE uses DNA annealing function to actively catalyze strand-exchange reaction between the unwinding substrate and a homologous single-stranded DNA. Using various biochemical experiments, we demonstrate that the mechanism of strand-exchange involves active coupling of unwinding and annealing reactions by the TWINKLE. Unlike strand-annealing, the strand-exchange reaction requires nucleotide hydrolysis and greatly stimulated by short region of homology between the recombining DNA strands that promote joint molecule formation to initiate strand-exchange. Furthermore, we show that TWINKLE catalyzes branch migration by resolving homologous four-way junction DNA. These four DNA modifying activities of TWINKLE: strand-separation, strand-annealing, strand-exchange and branch migration suggest a dual role of TWINKLE in mitochondrial DNA maintenance. In addition to playing a major role in fork progression during leading strand DNA synthesis, we propose that TWINKLE is involved in recombinational repair of the human mitochondrial DNA. PMID:26887820

  8. Homologous DNA strand exchange activity of the human mitochondrial DNA helicase TWINKLE.

    PubMed

    Sen, Doyel; Patel, Gayatri; Patel, Smita S

    2016-05-19

    A crucial component of the human mitochondrial DNA replisome is the ring-shaped helicase TWINKLE-a phage T7-gene 4-like protein expressed in the nucleus and localized in the human mitochondria. Our previous studies showed that despite being a helicase, TWINKLE has unique DNA annealing activity. At the time, the implications of DNA annealing by TWINKLE were unclear. Herein, we report that TWINKLE uses DNA annealing function to actively catalyze strand-exchange reaction between the unwinding substrate and a homologous single-stranded DNA. Using various biochemical experiments, we demonstrate that the mechanism of strand-exchange involves active coupling of unwinding and annealing reactions by the TWINKLE. Unlike strand-annealing, the strand-exchange reaction requires nucleotide hydrolysis and greatly stimulated by short region of homology between the recombining DNA strands that promote joint molecule formation to initiate strand-exchange. Furthermore, we show that TWINKLE catalyzes branch migration by resolving homologous four-way junction DNA. These four DNA modifying activities of TWINKLE: strand-separation, strand-annealing, strand-exchange and branch migration suggest a dual role of TWINKLE in mitochondrial DNA maintenance. In addition to playing a major role in fork progression during leading strand DNA synthesis, we propose that TWINKLE is involved in recombinational repair of the human mitochondrial DNA. PMID:26887820

  9. Dynamic regulation of the angiotensinogen gene by DNA methylation, which is influenced by various stimuli experienced in daily life.

    PubMed

    Demura, Masashi; Demura, Yosiki; Takeda, Yoshiyu; Saijoh, Kiyofumi

    2015-08-01

    Angiotensinogen (AGT) has a central role in maintaining blood pressure and fluid balance. DNA methylation is an epigenomic modification maintaining a steady pattern in somatic cells. Herein we summarize the link between AGT regulation and DNA methylation. DNA methylation negatively regulates AGT expression and dynamically changes in response to continuous AGT promoter stimulation. High-salt intake and excess circulating aldosterone cause DNA demethylation around the CCAAT enhancer-binding protein-binding sites, thereby converting the phenotype of AGT expression from an inactive to an active state in visceral adipose tissue. Salt-dependent hypertension may be partially affected by increased adipose AGT expression. Because angiotensin II is a well-established aldosterone-releasing hormone, stimulation of adipose AGT by aldosterone creates a positive feedback loop. This effect is pathologically associated with obesity-related hypertension, although it would be physiologically favorable for humans to efficiently retain their body fluid. The clear difference in DNA demethylation patterns between aldosterone and cortisol indicates a difference in the respective target DNA-binding sites between mineralocorticoid and glucocorticoid receptors in the AGT promoter. Stimulation-induced interactions between transcription factors and target DNA-binding sites trigger DNA demethylation. Dynamic changes in DNA methylation occur in relaxed chromatin regions both where transcription factors actively interact and where transcription is initiated. In contrast to rapid histone modifications, DNA demethylation and remethylation will progress relatively slowly over days or years. A wide variety of stimuli in daily life will continue to slowly and dynamically change DNA methylation patterns throughout life. Wise choices of beneficial stimuli will improve health. PMID:25809578

  10. Regioselective demethylation of quinoline derivatives. A DFT rationalization

    NASA Astrophysics Data System (ADS)

    Belferdi, Fatiha; Merabet, Naima; Belkhiri, Lotfi; Douara, Bachir

    2016-08-01

    Demethylation of compound 2,7-dimethoxyquinoline-3-carbaldehyde 1, is carried out using BBr3. However, all attempts led, either to the starting material or to the regioselective demethylation at position 2 affording the product 4a. The nature (donor or acceptor) and the position of the R (CHO or CN) group is likely to play a role in the preventing the demethylation at position 7. To address this phenomena, the demethylation of 2-chloro-7-methoxyquinoline-3-carbaldehyde 2 and 2,7-dimethoxyquinoline-3-carbaldehyde 3 has been carried out. To support the results obtained, theoretical computations at DFT level (vide infra) have been carried out upon compound 1. The exploration of how the gas-phase demethylation process on Quinoline can be affected at a position 7 center by stepwise substation effects using different electro-donor and attractor groups, show that demethylation process seems to be more favorable when substituent is an electro-donor. This is sustained by bond energy and thermodynamic analyses (vide infra).

  11. Inhibitors of Histone Deacetylase and DNA Methyltransferase Synergistically Activate the Methylated Metallothionein I Promoter by Activating the Transcription Factor MTF-1 and Forming an Open Chromatin Structure

    PubMed Central

    Ghoshal, Kalpana; Datta, Jharna; Majumder, Sarmila; Bai, Shoumei; Dong, Xiaocheng; Parthun, Mark; Jacob, Samson T.

    2002-01-01

    Inhibitors of DNA methyltransferase (Dnmt) and histone deacetylases (HDAC) synergistically activate the methylated metallothionein I gene (MT-I) promoter in mouse lymphosarcoma cells. The cooperative effect of these two classes of inhibitors on MT-I promoter activity was robust following demethylation of only a few CpG dinucleotides by brief exposure to 5-azacytidine (5-AzaC) but persisted even after prolonged treatment with the nucleoside analog. HDAC inhibitors (trichostatin A [TSA] and depsipeptide) either alone or in combination with 5-AzaC did not facilitate demethylation of the MT-I promoter. Treatment of cells with HDAC inhibitors increased accumulation of multiply acetylated forms of H3 and H4 histones that remained unaffected after treatment with 5-AzaC. Chromatin immunoprecipitation (ChIP) assay showed increased association of acetylated histone H4 and lysine 9 (K9)-acetyl H3 with the MT-I promoter after treatment with TSA, which was not affected following treatment with 5-AzaC. In contrast, the association of K9-methyl histone H3 with the MT-I promoter decreased significantly after treatment with 5-AzaC and TSA. ChIP assay with antibodies specific for methyl-CpG binding proteins (MBDs) demonstrated that only methyl-CpG binding protein 2 (MeCP2) was associated with the MT-I promoter, which was significantly enhanced after TSA treatment. Association of histone deacetylase 1 (HDAC1) with the promoter decreased after treatment with TSA or 5-AzaC and was abolished after treatment with both inhibitors. Among the DNA methyltransferases, both Dnmt1 and Dnmt3a were associated with the MT-I promoter in the lymphosarcoma cells, and association of Dnmt1 decreased with time after treatment with 5-AzaC. Treatment of these cells with HDAC inhibitors also increased expression of the MTF-1 (metal transcription factor-1) gene as well as its DNA binding activity. In vivo genomic footprinting studies demonstrated increased occupancy of MTF-1 to metal response elements of

  12. cis-active elements from mouse chromosomal DNA suppress simian virus 40 DNA replication.

    PubMed Central

    Hartl, M; Willnow, T; Fanning, E

    1990-01-01

    Simian virus 40 (SV40)-containing DNA was rescued after the fusion of SV40-transformed VLM cells with permissive COS1 monkey cells and cloned, and prototype plasmid clones were characterized. A 2-kilobase mouse DNA fragment fused with the rescued SV40 DNA, and derived from mouse DNA flanking the single insert of SV40 DNA in VLM cells, was sequenced. Insertion of the intact rescued mouse sequence, or two nonoverlapping fragments of it, into wild-type SV40 plasmid DNA suppressed replication of the plasmid in TC7 monkey cells, although the plasmids expressed replication-competent T antigen. Rat cells were transformed with linearized wild-type SV40 plasmid DNA with or without fragments of the mouse DNA in cis. Although all of the rat cell lines expressed approximately equal amounts of T antigen and p53, transformants carrying SV40 DNA linked to either of the same two replication suppressor fragments produced significantly less free SV40 DNA after fusion with permissive cells than those transformed by SV40 DNA without a cellular insert or with a cellular insert lacking suppressor activity. The results suggest that two independent segments of cellular DNA act in cis to suppress SV40 replication in vivo, either as a plasmid or integrated in chromosomal DNA. Images PMID:2159549

  13. Simulated vibrational spectra of aflatoxins and their demethylated products and the estimation of the energies of the demethylation reactions

    NASA Astrophysics Data System (ADS)

    Billes, Ferenc; Móricz, Ágnes M.; Tyihák, Ernő; Mikosch, Hans

    2006-06-01

    The structure of four natural mycotoxins, the aflatoxin B 1, B 2, G 1 and G 2 and their demethylated products were optimized with quantum chemical method. The energies and the thermodynamic functions of the molecules were calculated and applied to calculation of the reaction energies of the demethylations. Further results of the calculations are the vibrational force constants, the infrared spectra of the molecules and the assignments of the spectral bands.

  14. Effect of. gamma. -irradiated DNA on the activity of DNA polymerase. [/sup 60/Co

    SciTech Connect

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

    1981-06-01

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

  15. Tissue specific expression of avian vitellogenin gene is correlated with DNA hypomethylation and in vivo specific protein-DNA interactions.

    PubMed

    Jost, J P; Saluz, H P; McEwan, I; Feavers, I M; Hughes, M; Reiber, S; Liang, H M; Vaccaro, M

    1990-01-30

    The avian vitellogenin gene is expressed only in the liver of egg-laying hens. It can, however, be activated in immature chicks or roosters by oestradiol. Parallel to the onset of transcription, there is a demethylation of specific mCpGs in the promoter region and in the oestrogen response element (ERE). The methylation pattern in the promoter region is hormone and expression specific, whereas in the ERE it is only hormone and not organ specific. The demethylation occurring in the promoter region is correlated with the appearance of DNase I hypersensitivity sites and changes in the specific protein-DNA interactions. In vivo genomic footprinting of the ERE with varying concentrations of dimethylsulphate revealed, upon gene activation, only minor changes in the protein-DNA interaction. We present evidence that there is another protein that binds with high affinity to the ERE, besides the oestrogen receptor. PMID:1968660

  16. Simultaneous determination of the novel tyrosine kinase inhibitor meditinib and its active metabolite demethylation meditinib in monkey plasma by liquid chromatography-tandem mass spectrometry and its application to pharmacokinetic studies.

    PubMed

    Liang, Feng; Kong, Qi; Guo, Yongqi; Wang, Yu; Sun, Dejie; Liu, Shi; Cai, Jinling; Guan, Yongbiao; Ding, Rigao

    2015-08-01

    Meditinib (ME) is a novel tyrosine kinase inhibitor used as an antichronic myeloid leukemia drug. A simple, sensitive and specific LC/MS/MS method was developed and validated for the analysis of ME and its metabolite demethylation meditinib (PI) in monkey plasma using naltrexone as the internal standard. Sample preparation involved protein precipitation with methanol. The analysis was carried out on an Agilent C8 column (3.5 µm, 2.1 × 50 mm). Elution was achieved with a mobile phase gradient varying the proportion of a water solution containing 0.1% formic acid (solvent A) and a 0.1% formic acid in methanol solution (solvent B) at a flow rate of 300 μL/min. The method had a linear calibration curve over the concentration range of 2-1000 ng/mL for ME and 2-1000 ng/mL for PI. The lower limits of quantification of ME and PI were 2 and 2 ng/mL, respectively. The intra- and inter-day precision values were <15% and accuracy values were within ±10.0%. The mean recoveries of ME and PI from plasma were >85%. The assay has been successfully used for pharmacokinetic evaluation of ME and PI using the monkey as an animal model, and those data are reported for the first time. PMID:25616210

  17. A DNA enzyme with N-glycosylase activity

    NASA Technical Reports Server (NTRS)

    Sheppard, T. L.; Ordoukhanian, P.; Joyce, G. F.

    2000-01-01

    In vitro evolution was used to develop a DNA enzyme that catalyzes the site-specific depurination of DNA with a catalytic rate enhancement of about 10(6)-fold. The reaction involves hydrolysis of the N-glycosidic bond of a particular deoxyguanosine residue, leading to DNA strand scission at the apurinic site. The DNA enzyme contains 93 nucleotides and is structurally complex. It has an absolute requirement for a divalent metal cation and exhibits optimal activity at about pH 5. The mechanism of the reaction was confirmed by analysis of the cleavage products by using HPLC and mass spectrometry. The isolation and characterization of an N-glycosylase DNA enzyme demonstrates that single-stranded DNA, like RNA and proteins, can form a complex tertiary structure and catalyze a difficult biochemical transformation. This DNA enzyme provides a new approach for the site-specific cleavage of DNA molecules.

  18. Enzymatic Activities and DNA Substrate Specificity of Mycobacterium tuberculosis DNA Helicase XPB

    PubMed Central

    Balasingham, Seetha V.; Zegeye, Ephrem Debebe; Homberset, Håvard; Rossi, Marie L.; Laerdahl, Jon K.; Bohr, Vilhelm A.; Tønjum, Tone

    2012-01-01

    XPB, also known as ERCC3 and RAD25, is a 3′→5′ DNA repair helicase belonging to the superfamily 2 of helicases. XPB is an essential core subunit of the eukaryotic basal transcription factor complex TFIIH. It has two well-established functions: in the context of damaged DNA, XPB facilitates nucleotide excision repair by unwinding double stranded DNA (dsDNA) surrounding a DNA lesion; while in the context of actively transcribing genes, XPB facilitates initiation of RNA polymerase II transcription at gene promoters. Human and other eukaryotic XPB homologs are relatively well characterized compared to conserved homologs found in mycobacteria and archaea. However, more insight into the function of bacterial helicases is central to understanding the mechanism of DNA metabolism and pathogenesis in general. Here, we characterized Mycobacterium tuberculosis XPB (Mtb XPB), a 3′→5′ DNA helicase with DNA-dependent ATPase activity. Mtb XPB efficiently catalyzed DNA unwinding in the presence of significant excess of enzyme. The unwinding activity was fueled by ATP or dATP in the presence of Mg2+/Mn2+. Consistent with the 3′→5′ polarity of this bacterial XPB helicase, the enzyme required a DNA substrate with a 3′ overhang of 15 nucleotides or more. Although Mtb XPB efficiently unwound DNA model substrates with a 3′ DNA tail, it was not active on substrates containing a 3′ RNA tail. We also found that Mtb XPB efficiently catalyzed ATP-independent annealing of complementary DNA strands. These observations significantly enhance our understanding of the biological roles of Mtb XPB. PMID:22615856

  19. Anaerobic O-demethylation of phenylmethylethers. [und Acetobacterium woodii :a3

    SciTech Connect

    Frazer, A.C.; Young, L.Y.

    1991-08-01

    Assay of O-demethylation in cell-free extracts of Acetobacterium woodii: we have shown that THF and ATP are necessary for enzyme activity and thus are probably reactants; apparent Km values were 0.65 mM for the methoxylated substrate, 0.27 mM for ATP, and 0.17 mM for DL-THF. The enzyme activity is present in the cytosol, rather than being membrane bound, and is sensitive to oxygen. There is evidence to suggest that the enzyme system involves more than one protein component. Studies using suspensions of whole cells, suggest that there are several inducible AOD systems with distinguishable substrate specificities in A. woodii. A similar phenomenon has previously been suggested for the related acetogen, Eubacterium limosum. We have developed a system for obtaining mutants that are deficient in O-demethylation (AOD{sup {minus}}) in E. limosum, by using transposon mutagenesis with Tn916. In an ancillary study, A. woodii and E. limosum, were compared with respect to their capacity to O-demethylate guaiacol and chloroguaiacols. 8 refs., 1 fig., 1 tab.

  20. Phosphorothioate primers improve the amplification of DNA sequences by DNA polymerases with proofreading activity.

    PubMed Central

    Skerra, A

    1992-01-01

    Two thermostable DNA polymerases with proofreading activity--Vent DNA polymerase and Pfu DNA polymerase--have attracted recent attention, mainly because of their enhanced fidelities during amplification of DNA sequences by the polymerase chain reaction. A severe disadvantage for their practical application, however, results from the observation that due to their 3' to 5' exonuclease activities these enzymes degrade the oligodeoxynucleotides serving as primers for the DNA synthesis. It is demonstrated that this exonucleolytic attack on the primer molecules can be efficiently prevented by the introduction of single phosphorothioate bonds at their 3' termini. This strategy, which can be easily accomplished using routine DNA synthesis methodology, may open the way to a widespread use of these novel enzymes in the polymerase chain reaction. Images PMID:1641322

  1. Promoted Interaction of Nuclear Factor-κB With Demethylated Purinergic P2X3 Receptor Gene Contributes to Neuropathic Pain in Rats With Diabetes.

    PubMed

    Zhang, Hong-Hong; Hu, Ji; Zhou, You-Lang; Qin, Xin; Song, Zhen-Yuan; Yang, Pan-Pan; Hu, Shufen; Jiang, Xinghong; Xu, Guang-Yin

    2015-12-01

    Painful diabetic neuropathy is a common complication of diabetes produced by mechanisms that as yet are incompletely defined. The aim of this study was to investigate the roles of nuclear factor-κB (NF-κB) in the regulation of purinergic receptor P2X ligand-gated ion channel 3 (P2X3R) plasticity in dorsal root ganglion (DRG) neurons of rats with painful diabetes. Here, we showed that hindpaw pain hypersensitivity in streptozocin-induced diabetic rats was attenuated by treatment with purinergic receptor antagonist suramin or A-317491. The expression and function of P2X3Rs was markedly enhanced in hindpaw-innervated DRG neurons in diabetic rats. The CpG (cytosine guanine dinucleotide) island in the p2x3r gene promoter region was significantly demethylated, and the expression of DNA methyltransferase 3b was remarkably downregulated in DRGs in diabetic rats. The binding ability of p65 (an active form of NF-κB) with the p2x3r gene promoter region and p65 expression were enhanced significantly in diabetes. The inhibition of p65 signaling using the NF-κB inhibitor pyrrolidine dithiocarbamate or recombinant lentiviral vectors designated as lentiviral vector-p65 small interfering RNA remarkably suppressed P2X3R activities and attenuated diabetic pain hypersensitivity. Insulin treatment significantly attenuated pain hypersensitivity and suppressed the expression of p65 and P2X3Rs. Our findings suggest that the p2x3r gene promoter DNA demethylation and enhanced interaction with p65 contributes to P2X3R sensitization and diabetic pain hypersensitivity. PMID:26130762

  2. The p16-specific reactivation and inhibition of cell migration through demethylation of CpG islands by engineered transcription factors.

    PubMed

    Zhang, Baozhen; Xiang, Shengyan; Zhong, Qiming; Yin, Yanru; Gu, Liankun; Deng, Dajun

    2012-10-01

    Methylation of CpG islands inactivates transcription of tumor suppressor genes including p16 (CDKN2A). Inhibitors of DNA methylation and histone deacylation are recognized as useful cancer therapeutic chemicals through reactivation of the expression of methylated genes. However, these inhibitors are not target gene-specific, so that they lead to serious side effects as regular cytotoxic chemotherapy agents. To explore the feasibility of methylated gene-specific reactivation by artificial transcription factors, we engineered a set of Sp1-like seven-finger zinc-finger proteins (7ZFPs) targeted to a 21-bp sequence of the p16 promoter and found that these 7ZFPs could bind specifically to the target p16 promoter probe. Then the p16-specific artificial transcription factors (p16ATFs) were made from these 7ZFPs and the transcription activator VP64. Results showed that transient transfection of some p16ATFs selectively up-regulated the endogenous p16 expression in the p16-active 293T cells. Moreover, the transient transfection of the representative p16ATF-6I specifically reactivated p16 expression in the p16-methylated H1299 and AGS cells pretreated with a nontoxic amount of 5'-aza-deoxycytidine (20 and 80 nM, respectively). In addition, stable transfection of the p16ATF induced demethylation of p16 CpG island and trimethylation of histone H3K4, and inhibited recruitment of DNA methyltransferase 1 and trimethylation of H3K9 and H3K27 in the p16 promoter in H1299 cells without 5'-aza-deoxycytidine pretreatment. Notably, inhibition of cell migration and invasion was observed in these p16-reactivated cells induced by transient and stable p16ATF transfection. These results demonstrate that p16ATF not only specifically reactivates p16 expression through demethylation of CpG islands, but also restores methylated p16 function. PMID:22738793

  3. An arcane role of DNA in transcription activation.

    PubMed Central

    Ryu, S; Garges, S; Adhya, S

    1994-01-01

    The mechanism by which the cAMP receptor protein (CRP) activates transcription has been investigated using the lac promoter of Escherichia coli. For transcription activation, an interaction between DNA-bound CRP and RNA polymerase is not sufficient. CRP must bind to a site in the same DNA and close to the promoter. CRP action requires an intact spacer DNA to provide a rigid support in building a CRP-RNA polymerase protein bridge or to allow a conformational change in the DNA to be transmitted to the lac promoter using the protein bridge as a structural support. Images PMID:7811325

  4. Anaerobic Mercury Methylation and Demethylation by Geobacter bemidjiensis Bem

    DOE PAGESBeta

    Lu, Xia; Liu, Yurong; Johs, Alexander; Zhao, Linduo; Wang, Tieshan; Yang, Ziming; Lin, Hui; Elias, Dwayne A.; Pierce, Eric M.; Liang, Liyuan; et al

    2016-03-28

    Two competing processes controlling the net production and bioaccumulation of neurotoxic methylmercury (MeHg) in natural ecosystems are microbial methylation and demethylation. Though mercury (Hg) methylation by anaerobic microorganisms and demethylation by aerobic Hg-resistant bacteria have both been extensively studied, little attention has been given to MeHg degradation by anaerobic bacteria, particularly the iron-reducing bacterium Geobacter bemidjensis Bem. Here we report, for the first time, that the strain G. bemidjensis Bem can methylate inorganic Hg and degrade MeHg concurrently under anoxic conditions. Our results suggest that G. bemidjensis cells utilize a reductive demethylation pathway to degrade MeHg, with elemental Hg(0) asmore » the major reaction product, possibly due to the presence of homologs encoding both organo-mercurial lyase (MerB) and mercuric reductase (MerA) in this organism. In addition, the cells can mediate multiple reactions including Hg/MeHg sorption, Hg reduction and oxidation, resulting in both time and concentration dependent Hg species transformations. Moderate concentrations (10 500 M) of Hg-binding ligands such as cysteine enhance Hg(II) methylation but inhibit MeHg degradation. These findings indicate a cycle of methylation and demethylation among anaerobic bacteria and suggest that mer-mediated demethylation may play a role in the net balance of MeHg production in anoxic water and sediments.« less

  5. Cytochrome P450 3A Enzymes Catalyze the O6-Demethylation of Thebaine, a Key Step in Endogenous Mammalian Morphine Biosynthesis.

    PubMed

    Kramlinger, Valerie M; Alvarado Rojas, Mónica; Kanamori, Tatsuyuki; Guengerich, F Peter

    2015-08-14

    Morphine, first characterized in opium from the poppy Papaver somniferum, is one of the strongest known analgesics. Endogenous morphine has been identified in several mammalian cells and tissues. The synthetic pathway of morphine in the opium poppy has been elucidated. The presence of common intermediates in plants and mammals suggests that biosynthesis occurs through similar pathways (beginning with the amino acid L-tyrosine), and the pathway has been completely delineated in plants. Some of the enzymes in the mammalian pathway have been identified and characterized. Two of the latter steps in the morphine biosynthesis pathway are demethylation of thebaine at the O(3)- and the O(6)-positions, the latter of which has been difficult to demonstrate. The plant enzymes responsible for both the O(3)-demethylation and the O(6)-demethylation are members of the Fe(II)/α-ketoglutarate-dependent dioxygenase family. Previous studies showed that human cytochrome P450 (P450) 2D6 can catalyze thebaine O(3)-demethylation. We report that demethylation of thebaine at the O(6)-position is selectively catalyzed by human P450s 3A4 and 3A5, with the latter being more efficient, and rat P450 3A2. Our results do not support O(6)-demethylation of thebaine by an Fe(II)/α-ketoglutarate-dependent dioxygenase. In rat brain microsomes, O(6)-demethylation was inhibited by ketoconazole, but not sulfaphenazole, suggesting that P450 3A enzymes are responsible for this activity in the brain. An alternate pathway to morphine, oripavine O(6)-demethylation, was not detected. The major enzymatic steps in mammalian morphine synthesis have now been identified. PMID:26157146

  6. A DNA-recombinogenic activity in human cells.

    PubMed Central

    Kenne, K; Ljungquist, S

    1984-01-01

    A DNA recombining protein has been partly purified from cell lines derived from patients suffering from the hereditary disease, Bloom's syndrome. The protein induces the formation of displacement loops in phi X174 RFI DNA molecules after the addition of single-stranded DNA fragments. A filter binding method and electron microscopy were used to determine the reaction. The recombinogenic protein is dependent on divalent cations and ATP for activity. Images PMID:6232501

  7. Reversible Condensation of DNA using a Redox-Active Surfactant

    PubMed Central

    Hays, Melissa E.; Jewell, Christopher M.; Lynn, David M.; Abbott, Nicholas L.

    2008-01-01

    We report characterization of aqueous solutions of dilute Lambda phage DNA containing the redox-active surfactant (11-ferrocenylundecyl)trimethylammonium bromide (FTMA) as a function of the oxidation state of the FTMA. FTMA undergoes a reversible one-electron oxidation from a reduced state that forms micelles in aqueous solution to an oxidized state (containing the ferrocenium cation) that does not selfassociate in solution. This investigation sought to test the hypothesis that FTMA can be used to achieve reversible control over the conformation of DNA-surfactant complexes in solution. Whereas DNA adopts extended coil conformations in aqueous solutions, our measurements revealed that addition of reduced FTMA (2–5μM) to aqueous solutions of DNA (5 μM in nucleotide units) resulted in coexistence of extended coils and compact globules in solution. At higher concentrations of reduced FTMA (up to 30μM), the DNA was present as compact globules only. In contrast, oxidized FTMA had no measurable effect on the conformation of DNA, allowing DNA to maintain an extended coil state up to a concentration of 75μM oxidized FTMA. We further demonstrate that it is possible to chemically or electrochemically transform the oxidation state of FTMA in preformed complexes of FTMA and DNA, thus achieving in situ control over the conformations of the DNA in solution. These results provide guidance for the design of surfactant systems that permit active control of DNA-surfactant interactions. PMID:17428073

  8. Hepatitis B virus: DNA polymerase activity of deletion mutants.

    PubMed

    Kim, Y; Hong, Y B; Jung, G

    1999-02-01

    The hepadnavirus P gene product is a multifunctional protein with priming, DNA- and RNA-dependent DNA polymerase, and RNase H activities. Nested N- or C-terminal deletion mutations and deletions of domain(s) in human HBV polymerase have been made. Wild-type and deletion forms of MBP-fused HBV polymerase were expressed in E. coli, purified by amylose column chromatography, and the DNA-dependent DNA polymerase activities of the purified proteins were compared. Deletion of the terminal protein or spacer regions reduced enzyme activity to 70%, respectively. However, deletion of the RNase H domain affected polymerase activity more than that of the terminal protein or spacer region. The polymerase domain alone or the N-terminal deletion of the polymerase domain still exhibited enzymatic activity. In this report, it is demonstrated that the minimal domain for the polymerizing activity of the HBV polymerase is smaller than the polymerase domain. PMID:10205676

  9. Sphingosine, a modulator of human translesion DNA polymerase activity.

    PubMed

    Kamath-Loeb, Ashwini S; Balakrishna, Sharath; Whittington, Dale; Shen, Jiang-Cheng; Emond, Mary J; Okabe, Takayoshi; Masutani, Chikahide; Hanaoka, Fumio; Nishimura, Susumu; Loeb, Lawrence A

    2014-08-01

    Translesion (TLS) DNA polymerases are specialized, error-prone enzymes that synthesize DNA across bulky, replication-stalling DNA adducts. In so doing, they facilitate the progression of DNA synthesis and promote cell proliferation. To potentiate the effect of cancer chemotherapeutic regimens, we sought to identify inhibitors of TLS DNA polymerases. We screened five libraries of ∼ 3000 small molecules, including one comprising ∼ 600 nucleoside analogs, for their effect on primer extension activity of DNA polymerase η (Pol η). We serendipitously identified sphingosine, a lipid-signaling molecule that robustly stimulates the activity of Pol η by ∼ 100-fold at low micromolar concentrations but inhibits it at higher concentrations. This effect is specific to the Y-family DNA polymerases, Pols η, κ, and ι. The addition of a single phosphate group on sphingosine completely abrogates this effect. Likewise, the inclusion of other sphingolipids, including ceramide and sphingomyelin to extension reactions does not elicit this response. Sphingosine increases the rate of correct and incorrect nucleotide incorporation while having no effect on polymerase processivity. Endogenous Pol η activity is modulated similarly as the recombinant enzyme. Importantly, sphingosine-treated cells exhibit increased lesion bypass activity, and sphingosine tethered to membrane lipids mimics the effects of free sphingosine. Our studies have uncovered sphingosine as a modulator of TLS DNA polymerase activity; this property of sphingosine may be associated with its known role as a signaling molecule in regulating cell proliferation in response to cellular stress. PMID:24928506

  10. Metabolic activation of carcinogenic ethylbenzene leads to oxidative DNA damage.

    PubMed

    Midorikawa, Kaoru; Uchida, Takafumi; Okamoto, Yoshinori; Toda, Chitose; Sakai, Yoshie; Ueda, Koji; Hiraku, Yusuke; Murata, Mariko; Kawanishi, Shosuke; Kojima, Nakao

    2004-12-01

    Ethylbenzene is carcinogenic to rats and mice, while it has no mutagenic activity. We have investigated whether ethylbenzene undergoes metabolic activation, leading to DNA damage. Ethylbenzene was metabolized to 1-phenylethanol, acetophenone, 2-ethylphenol and 4-ethylphenol by rat liver microsomes. Furthermore, 2-ethylphenol and 4-ethylphenol were metabolically transformed to ring-dihydroxylated metabolites such as ethylhydroquinone and 4-ethylcatechol, respectively. Experiment with 32P-labeled DNA fragment revealed that both ethylhydroquinone and 4-ethylcatechol caused DNA damage in the presence of Cu(II). These dihydroxylated compounds also induced the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in calf thymus DNA in the presence of Cu(II). Catalase, methional and Cu(I)-specific chelator, bathocuproine, significantly (P<0.05) inhibited oxidative DNA damage, whereas free hydroxyl radical scavenger and superoxide dismutase did not. These results suggest that Cu(I) and H2O2 produced via oxidation of ethylhydroquinone and 4-ethylcatechol are involved in oxidative DNA damage. Addition of an endogenous reductant NADH dramatically enhanced 4-ethylcatechol-induced oxidative DNA damage, whereas ethylhydroquinone-induced DNA damage was slightly enhanced. Enhancing effect of NADH on oxidative DNA damage by 4-ethylcatechol may be explained by assuming that reactive species are generated from the redox cycle. In conclusion, these active dihydroxylated metabolites would be involved in the mechanism of carcinogenesis by ethylbenzene. PMID:15560893

  11. DNA-gold nanoparticles network based electrochemical biosensors for DNA MTase activity.

    PubMed

    Hong, Lu; Wan, Jing; Zhang, Xiaojun; Wang, Guangfeng

    2016-05-15

    In this work, a highly sensitive electrochemical DNA methyltransferase (MTase) activity assay was fabricated with DNA-gold nanoparticles (Au NPs) network as signal amplification unit and an easy assembly method by the linkage of benzenedithiol bridge. By two complementary AuNPs modified single-stranded DNA, DNA-gold nanoparticles network was self-assembled. With the linkage of benzenedithiol bridge, the DNA network structure was immobilized on the surface of gold electrode through the covalent Au-S bond. In the presence of Dam MTase, the special sites of DNA-AuNPs network were methylated and could not be digested by restriction endonuclease Mbo I. Thus the loaded electrochemical indicator Methylene blue (MB) was MB molecules still remained on the DNA-Au NPs network. The electrochemical response depended on the methylated degree, which could be used to detect MTase activity. By the differential pulse voltammetry (DPV), it was demonstrated that a linear relationship between the DPV response and logarithm of Dam concentration ranged from 0.075 to 30U/mL, achieving a low detection limit of 0.02U/mL. The use of benzenedithiol avoided the direct incubation of the solid electrode with the capture DNA probe under complex and harsh conditions. Therefore the immobilization of DNA-AuNPs network was easy to be carried out, which is favorable for the specially high stability and reproducibility of the electrochemical biosensor. PMID:26992515

  12. Inhibition of DNA damage repair by artificial activation of PARP with siDNA.

    PubMed

    Croset, Amelie; Cordelières, Fabrice P; Berthault, Nathalie; Buhler, Cyril; Sun, Jian-Sheng; Quanz, Maria; Dutreix, Marie

    2013-08-01

    One of the major early steps of repair is the recruitment of repair proteins at the damage site, and this is coordinated by a cascade of modifications controlled by phosphatidylinositol 3-kinase-related kinases and/or poly (ADP-ribose) polymerase (PARP). We used short interfering DNA molecules mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) to promote DNA-dependent protein kinase (DNA-PK) and PARP activation. Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP. Therefore, comparative study of the two molecules allows analysis of the respective roles of the two signaling pathways: both recruit proteins involved in single-strand break repair (PARP, XRCC1 and PCNA) and prevent their recruitment at chromosomal damage. Dbait, but not Pbait, also inhibits recruitment of proteins involved in double-strand break repair (53BP1, NBS1, RAD51 and DNA-PK). By these ways, Pbait and Dbait disorganize DNA repair, thereby sensitizing cells to various treatments. Single-strand breaks repair inhibition depends on direct trapping of the main proteins on both molecules. Double-strand breaks repair inhibition may be indirect, resulting from the phosphorylation of double-strand breaks repair proteins and chromatin targets by activated DNA-PK. The DNA repair inhibition by both molecules is confirmed by their synthetic lethality with BRCA mutations. PMID:23761435

  13. Inactivation efficiencies of radical reactions with biologically active DNA

    NASA Astrophysics Data System (ADS)

    Lafleur, M. V. M.; Retèl, J.; Loman, H.

    Dilute aqueous solutions of biologically active θX174 DNA may serve as a simplified model system of the cell. Damage to the DNA after irradiation with γ-rays, may be ascribed to reactions with .OH, .H and e -aq or secondary radicals, arising from reactions of water radicals with added scavengers. Conversion of primary (water) radicals into secondary (scavenger) radicals leads to a considerable protection of the DNA, which, however, would have been larger if these secondary radicals did not contribute to DNA inactivation. The inactivation yield due to isopropanol or formate (secondary) radicals depends on dose rate as well as DNA concentration. Furthermore the inactivation efficiencies of the reactions of both the primary and the secondary radicals with single-stranded DNA could be established.

  14. Activation and Regulation of DNA-Driven Immune Responses

    PubMed Central

    2015-01-01

    SUMMARY The innate immune system provides early defense against infections and also plays a key role in monitoring alterations of homeostasis in the body. DNA is highly immunostimulatory, and recent advances in this field have led to the identification of the innate immune sensors responsible for the recognition of DNA as well as the downstream pathways that are activated. Moreover, information on how cells regulate DNA-driven immune responses to avoid excessive inflammation is now emerging. Finally, several reports have demonstrated how defects in DNA sensing, signaling, and regulation are associated with susceptibility to infections or inflammatory diseases in humans and model organisms. In this review, the current literature on DNA-stimulated innate immune activation is discussed, and important new questions facing this field are proposed. PMID:25926682

  15. Cytotoxic activity and DNA-binding properties of isoeuxanthone derivatives.

    PubMed

    Wang, Hui Fang; Yan, Hong; Gao, Xianghua; Niu, Baolong; Guo, Ruijie; Wei, Liqiao; Xu, Bingshe; Tang, Ning

    2014-01-01

    In this study, the interactions of different groups substituted isoeuxanthone derivatives with calf thymus DNA (ct DNA) were investigated by spectrophotometric methods and viscosity measurements. Results indicated that the xanthone derivatives could intercalate into the DNA base pairs by the plane of xanthone ring and the various substituents may influence the binding affinity with DNA according to the calculated quenching constant values. Furthermore, two tumor cell lines including the human cervical cancer cell line (HeLa) and human hepatocellular liver carcinoma cell line (HepG2) were used to evaluate the cytotoxic activities of xanthone derivatives by acid phosphatase assay. Analyses showed that the oxiranylmethoxy substituted xanthone exhibited more effective cytotoxic activity against the cancer cells than the other substituted xanthones. The effects on the inhibition of tumor cells in vitro agreed with the studies of DNA-binding. PMID:24583780

  16. DNA hybridization activity of single-stranded DNA-conjugated gold nanoparticles used as probes for DNA detection

    NASA Astrophysics Data System (ADS)

    Kira, Atsushi; Matsuo, Kosuke; Nakajima, Shin-ichiro

    2016-02-01

    Colloidal nanoparticles (NPs) have potential applications in bio-sensing technologies as labels or signal enhancers. In order to meet demands for a development of biomolecular assays by a quantitative understanding of single-molecule, it is necessary to regulate accuracy of the NPs probes modified with biomolecules to optimize the characteristics of NPs. However, to our knowledge, there is little information about the structural effect of conjugated biomolecules to the NPs. In this study, we investigated the contribution of a density of single-stranded DNA (ssDNA) conjugating gold NP to hybridization activity. Hybridization activity decreased in accordance with increases in the density of attached ssDNAs, likely due to electrostatic repulsion generated by negatively charged phosphate groups in the ssDNA backbone. These results highlight the importance of controlling the density of ssDNAs attached to the surface of NPs used as DNA detection probes.

  17. Rapid restriction enzyme free detection of DNA methyltransferase activity based on DNA-templated silver nanoclusters.

    PubMed

    Kermani, Hanie Ahmadzade; Hosseini, Morteza; Dadmehr, Mehdi; Ganjali, Mohammad Reza

    2016-06-01

    DNA methylation has significant roles in gene regulation. DNA methyltransferase (MTase) enzyme characterizes DNA methylation and also induces an aberrant methylation pattern that is related to many diseases, especially cancers. Thus, it is required to develop a method to detect the DNA MTase activity. In this study, we developed a new sensitive and reliable method for methyltransferase activity assay by employing DNA-templated silver nanoclusters (DNA/Ag NCs) without using restriction enzymes. The Ag NCs have been utilized for the determination of M.SssI MTase activity and its inhibition. We designed an oligonucleotide probe which contained an inserted six-cytosine loop as Ag NCs formation template. The changes in fluorescence intensity were monitored to quantify the M.SssI activity. The fluorescence spectra showed a linear decrease in the range of 0.4 to 20 U/ml with a detection limit of 0.1 U/ml, which was significant compared with previous reports. The proposed method was applied successfully for demonstrating the Gentamicin effect as MTase inhibitor. The proposed method showed convenient reproducibility and sensitivity indicating its potential for the determination of methyltransferase activity. PMID:27052776

  18. A Specific LSD1/KDM1A Isoform Regulates Neuronal Differentiation through H3K9 Demethylation

    PubMed Central

    Laurent, Benoit; Ruitu, Lv; Murn, Jernej; Hempel, Kristina; Ferrao, Ryan; Xiang, Yang; Liu, Shichong; Garcia, Benjamin A.; Wu, Hao; Wu, Feizhen; Steen, Hanno; Shi, Yang

    2015-01-01

    Lysine-specific demethylase 1 (LSD1) has been reported to repress and activate transcription by mediating histone H3K4me1/2 and H3K9me1/2 demethylation, respectively. The molecular mechanism that underlies this dual substrate specificity has remained unknown. Here we report that an isoform of LSD1, LSD1+8a, does not have the intrinsic capability to demethylate H3K4me2. Instead, LSD1+8a mediates H3K9me2 demethylation in collaboration with supervillin (SVIL), a new LSD1+8a interacting protein. LSD1+8a knockdown increases H3K9me2, but not H3K4me2, levels at its target promoters and compromises neuronal differentiation. Importantly, SVIL co-localizes to LSD1+8a-bound promoters, and its knockdown mimics the impact of LSD1+8a loss, supporting SVIL as a cofactor for LSD1+8a in neuronal cells. These findings provide insight into mechanisms by which LSD1 mediates H3K9me demethylation and highlight alternative splicing as a means by which LSD1 acquires selective substrate specificities (H3K9 versus H3K4) to differentially control specific gene expression programs in neurons. PMID:25684206

  19. Lipoxygenase-mediated hydrogen peroxide-dependent N-demethylation of N,N-dimethylaniline and related compounds.

    PubMed

    Hover, C G; Kulkarni, A P

    2000-02-01

    To date, studies of xenobiotic N-demethylation have focused on heme-proteins such as P450 and peroxidases. In this study we investigated the ability of non-heme iron proteins, namely soybean lipoxygenase (SLO) and human term placental lipoxygenase (HTPLO) to mediate N-demethylation of N,N-dimethylaniline (DMA) and related compounds in the presence of hydrogen peroxide. In addition to being hydrogen peroxide dependent, the reaction was also dependent on incubation time, concentration of enzyme and DMA and the pH of the medium. Using Nash reagent to estimate formaldehyde production, we determined the specific activity for SLO mediated N-demethylation of DMA to be 200 + 18 nmol HCHO/min per mg protein or 23 +/- 2 nmol/min per nmol of enzyme, while that of HTPLO was 33 +/- 4 nmol HCHO/min per mg protein. Nordihydroguaiaretic acid (NDGA), a classical inhibitor of lipoxygenase (LO), as well as antioxidants and free radical reducing agents, caused a marked reduction in the rate of production of formaldehyde from DMA by SLO. Besides N,N-dimethylaniline, N-methylaniline, N,N,N',N'-tetramethylbenzidine, N,N-dimethyl-p-phenylenediamine, N,N-dimethyl-3-nitroaniline and N,N-dimethyl-p-toluidine were also demethylated by SLO. The formation of a DMA N-oxide was not detected. Preliminary experiments suggested SLO-mediated hydrogen peroxide-dependent S-dealkylation of methiocarb or O-dealkylation of 4-nitroanisole does not occur. PMID:10728778

  20. MES16, a member of the methylesterase protein family, specifically demethylates fluorescent chlorophyll catabolites during chlorophyll breakdown in Arabidopsis.

    PubMed

    Christ, Bastien; Schelbert, Silvia; Aubry, Sylvain; Süssenbacher, Iris; Müller, Thomas; Kräutler, Bernhard; Hörtensteiner, Stefan

    2012-02-01

    During leaf senescence, chlorophyll (Chl) is broken down to nonfluorescent chlorophyll catabolites (NCCs). These arise from intermediary fluorescent chlorophyll catabolites (FCCs) by an acid-catalyzed isomerization inside the vacuole. The chemical structures of NCCs from Arabidopsis (Arabidopsis thaliana) indicate the presence of an enzyme activity that demethylates the C13(2)-carboxymethyl group present at the isocyclic ring of Chl. Here, we identified this activity as methylesterase family member 16 (MES16; At4g16690). During senescence, mes16 leaves exhibited a strong ultraviolet-excitable fluorescence, which resulted from large amounts of different FCCs accumulating in the mutants. As confirmed by mass spectrometry, these FCCs had an intact carboxymethyl group, which slowed down their isomerization to respective NCCs. Like a homologous protein cloned from radish (Raphanus sativus) and named pheophorbidase, MES16 catalyzed the demethylation of pheophorbide, an early intermediate of Chl breakdown, in vitro, but MES16 also demethylated an FCC. To determine the in vivo substrate of MES16, we analyzed pheophorbide a oxygenase1 (pao1), which is deficient in pheophorbide catabolism and accumulates pheophorbide in the chloroplast, and a mes16pao1 double mutant. In the pao1 background, we additionally mistargeted MES16 to the chloroplast. Normally, MES16 localizes to the cytosol, as shown by analysis of a MES16-green fluorescent protein fusion. Analysis of the accumulating pigments in these lines revealed that pheophorbide is only accessible for demethylation when MES16 is targeted to the chloroplast. Together, these data demonstrate that MES16 is an integral component of Chl breakdown in Arabidopsis and specifically demethylates Chl catabolites at the level of FCCs in the cytosol. PMID:22147518

  1. DNA binding, antiviral activities and cytotoxicity of new furochromone and benzofuran derivatives.

    PubMed

    Abdelhafez, Omaima Mohamed; Abedelatif, Nehad Ahmed; Badria, Farid A

    2011-10-01

    Bromination of visnagin (1) afforded 9-bromovisnagin (2) which on its alkaline hydrolysis afforded the 3-acetyl benzofuran derivative (3). The condensation of (3) with hydrazine hydrate, phenylhydrazine and/or hydroxylamine hydrochloride afforded the corresponding pyrazole derivatives (4a, b) and isoxazole derivative (4c). On the other hand, when compound 3 was condensed with some aromatic aldehydes, this yielded corresponding α, β-unsaturated keto derivatives (5a-e). Furthermore, when 1 was subjected to chlorosulfonation, the visnaginsulfonylchloride derivative 6 was afforded, which on amidation using morpholine, a sulonamido derivative (7) was obtained. Alkaline hydrolysis of the latter compound yielded 7-N-morpholinosulsamidobenzofuran (8) which was condensed with some aromatic aldehydes to yield the corresponding chalcone compounds (9a-e). Demethylation of visnagin afforded norvisnagin (10). The reaction of 10 with ethylbromoacetate in dry acetone yielded the ester benzopyran derivative (11) which reacted with hydrazine hydrate to afford the corresponding hydrazide derivative (12) and this was condensed with 3,4,5-trimethoxybenzaldehyde to give the corresponding hydrazone (13). A thaizolidinone derivative (14) was obtained by condensation of (13) with thioglycolic acid. Chloromethylation of norvisnagin afforded a 4-chloromethyl derivative (15) which reacted with different primary and secondary amines to yield the corresponding ethylamino derivative (16a, b). Moreover, mannich bases (16a, b) and (17a-c) were obtained by reacting norvisnagin with different primary and secondary amines in the presence of formalin but benzoylation of (16a, b) and (17a-c) afforded 4-oxybenzoyl derivative (18a-e). The prepared compounds were tested for their interaction with DNA; bromovisnagin 2 showed the highest affinity and compounds 6, 15, 8a, > 14, > 16b, 17a, and 16a showed moderate activity in decreasing potency. Moreover, compound 2 also was the most active as antiviral

  2. Bacterial infection remodels the DNA methylation landscape of human dendritic cells

    PubMed Central

    Pacis, Alain; Tailleux, Ludovic; Morin, Alexander M.; Lambourne, John; MacIsaac, Julia L.; Yotova, Vania; Dumaine, Anne; Danckaert, Anne; Luca, Francesca; Grenier, Jean-Christophe; Hansen, Kasper D.; Gicquel, Brigitte; Yu, Miao; Pai, Athma; He, Chuan; Tung, Jenny; Pastinen, Tomi; Kobor, Michael S.; Pique-Regi, Roger; Gilad, Yoav; Barreiro, Luis B.

    2015-01-01

    DNA methylation is an epigenetic mark thought to be robust to environmental perturbations on a short time scale. Here, we challenge that view by demonstrating that the infection of human dendritic cells (DCs) with a live pathogenic bacteria is associated with rapid and active demethylation at thousands of loci, independent of cell division. We performed an integrated analysis of data on genome-wide DNA methylation, histone mark patterns, chromatin accessibility, and gene expression, before and after infection. We found that infection-induced demethylation rarely occurs at promoter regions and instead localizes to distal enhancer elements, including those that regulate the activation of key immune transcription factors. Active demethylation is associated with extensive epigenetic remodeling, including the gain of histone activation marks and increased chromatin accessibility, and is strongly predictive of changes in the expression levels of nearby genes. Collectively, our observations show that active, rapid changes in DNA methylation in enhancers play a previously unappreciated role in regulating the transcriptional response to infection, even in nonproliferating cells. PMID:26392366

  3. Importance of tetrahydrofolate and ATP in the anaerobic O-demethylation reaction for phenylmethylethers.

    PubMed

    Berman, M H; Frazer, A C

    1992-03-01

    DL-Tetrahydrofolate (THF) and ATP were necessary for the anaerobic O-demethylation of phenylmethylethers in cell extracts of the type strain (ATCC 29683) of the homoacetogen Acetobacterium woodii. The reactants for this enzymatic activity have not been previously demonstrated in any system, nor has the mediating enzyme been studied. An assay using reaction mixtures containing 1 mM THF, 2 mM ATP, and 2 mM hydroferulate (i.e., 4-hydroxy,3-methoxyphenylpropionate) was developed and was performed under stringent anaerobic conditions. Pyridine nucleotides and several other possible cofactors were tested but had no effect on the activity. After centrifugation of disrupted cells at 27,000 x g, the activity was found primarily in the supernatant, which had a specific activity of 14.2 +/- 0.5 nmol/min/mg of protein. At saturating levels of each of the other two substrates, apparent Km values for the variable substrate were 0.65 mM hydroferulate, 0.27 mM ATP, and 0.17 mM THF. Activity was significantly decreased when extract was preincubated at 60 degrees C and was completely lost after preincubation in air for 30 min. Thus, the soluble anaerobic O-demethylating enzyme system of A. woodii is oxygen sensitive. The THF- and ATP-dependent activity measurable in the soluble fraction of cell extracts constituted about 34% of the activity seen with intact cells. PMID:1575495

  4. Importance of tetrahydrofolate and ATP in the anaerobic O-demethylation reaction for phenylmethylethers

    SciTech Connect

    Berman, M.H.; Frazer, A.C. )

    1992-03-01

    DL-Tetrahydrofolate (THF) and ATP were necessary for the anaerobic O-demethylation of phenylmethylethers in cell extracts of the type strain (ATCC 29683) of the homoacetogen Acetobacterium woodii. The reactants for this enzymatic activity have not been previously demonstrated in any system, nor has the mediating enzyme been studied. An assay using reaction mixtures containing 1 mM THF, 2 mM ATP, and 2 mM hydroferulate (i.e., 4-hydroxy,3-methoxyphenylpropionate) was developed and was performed under stringent anaerobic conditions. Pyridine nucleotides and several other possible cofactors were tested but had no effect on the activity. After centrifugation of disrupted cells at 27,000 x g, the activity was found primarily in the supernatant, which had a specific activity of 14.2 {plus minus} 0.5 nmol/min/mg of protein. At saturating levels of each of the other two substrates, apparent K{sub m} values for the variable substrate were 0.65 mM hydroferulate, 0.27 mM ATP, and 0.17 mM THF. Activity was significantly decreased when extract was preincubated at 60C and was completely lost after preincubation in air for 30 min. Thus, the soluble anaerobic O-demethylating enzyme system of A. woodii is oxygen sensitive. The THF- and ATP-dependent activity measurable in the soluble fraction of cell extracts constituted about 34% of the activity seen with intact cells.

  5. Demethylation and expression of murine mammary tumor proviruses in mouse thymoma cell lines.

    PubMed Central

    Mermod, J J; Bourgeois, S; Defer, N; Crépin, M

    1983-01-01

    Murine mammary tumor virus (MMTV) expression is analyzed in a T-lymphoid cell line (T1M1) sensitive to the killing effect of glucocorticoids and in two of its variants, one resistant (T1M1r) and one supersensitive (T1M1ss) to glucocorticoid-induced lymphocytolysis. In the T1M1 line, MMTV is expressed and induced approximately 10-fold by short treatment with dexamethasone. Southern blot analyses of restriction enzyme digests of DNA from T1M1 cells reveal three proviruses similar to those of normal C57BL mouse tissue. In the T1M1ss line, which has retained functional glucocorticoid receptors, MMTV mRNA is inducible by glucocorticoids, while induction is reduced in the T1M1r line defective in glucocorticoid receptors. Moreover, the T1M1r line expresses a strikingly elevated basal level of MMTV mRNA in the absence of hormone. No rearrangements or superinfection have occurred in the variants, but all the regions containing 5'-long terminal repeats are demethylated in the T1M1r variant although other sites of the provirus remain methylated. Because this variant was selected by prolonged treatment with dexamethasone, these observations raise the possibility that the continuous transcription of MMTV that occurred during this selection can result in glucocorticoid-induced demethylation of long-terminal-repeat sequences. Images PMID:6296860

  6. Hyperosmotic stress memory in Arabidopsis is mediated by distinct epigenetically labile sites in the genome and is restricted in the male germline by DNA glycosylase activity

    PubMed Central

    Wibowo, Anjar; Becker, Claude; Marconi, Gianpiero; Durr, Julius; Price, Jonathan; Hagmann, Jorg; Papareddy, Ranjith; Putra, Hadi; Kageyama, Jorge; Becker, Jorg; Weigel, Detlef; Gutierrez-Marcos, Jose

    2016-01-01

    Inducible epigenetic changes in eukaryotes are believed to enable rapid adaptation to environmental fluctuations. We have found distinct regions of the Arabidopsis genome that are susceptible to DNA (de)methylation in response to hyperosmotic stress. The stress-induced epigenetic changes are associated with conditionally heritable adaptive phenotypic stress responses. However, these stress responses are primarily transmitted to the next generation through the female lineage due to widespread DNA glycosylase activity in the male germline, and extensively reset in the absence of stress. Using the CNI1/ATL31 locus as an example, we demonstrate that epigenetically targeted sequences function as distantly-acting control elements of antisense long non-coding RNAs, which in turn regulate targeted gene expression in response to stress. Collectively, our findings reveal that plants use a highly dynamic maternal ‘short-term stress memory’ with which to respond to adverse external conditions. This transient memory relies on the DNA methylation machinery and associated transcriptional changes to extend the phenotypic plasticity accessible to the immediate offspring. DOI: http://dx.doi.org/10.7554/eLife.13546.001 PMID:27242129

  7. Hyperosmotic stress memory in Arabidopsis is mediated by distinct epigenetically labile sites in the genome and is restricted in the male germline by DNA glycosylase activity.

    PubMed

    Wibowo, Anjar; Becker, Claude; Marconi, Gianpiero; Durr, Julius; Price, Jonathan; Hagmann, Jorg; Papareddy, Ranjith; Putra, Hadi; Kageyama, Jorge; Becker, Jorg; Weigel, Detlef; Gutierrez-Marcos, Jose

    2016-01-01

    Inducible epigenetic changes in eukaryotes are believed to enable rapid adaptation to environmental fluctuations. We have found distinct regions of the Arabidopsis genome that are susceptible to DNA (de)methylation in response to hyperosmotic stress. The stress-induced epigenetic changes are associated with conditionally heritable adaptive phenotypic stress responses. However, these stress responses are primarily transmitted to the next generation through the female lineage due to widespread DNA glycosylase activity in the male germline, and extensively reset in the absence of stress. Using the CNI1/ATL31 locus as an example, we demonstrate that epigenetically targeted sequences function as distantly-acting control elements of antisense long non-coding RNAs, which in turn regulate targeted gene expression in response to stress. Collectively, our findings reveal that plants use a highly dynamic maternal 'short-term stress memory' with which to respond to adverse external conditions. This transient memory relies on the DNA methylation machinery and associated transcriptional changes to extend the phenotypic plasticity accessible to the immediate offspring. PMID:27242129

  8. Hepatic microsomal N-oxidation and N-demethylation of N,N-dimethylaniline in red-winged blackbird compared with rat and other birds

    USGS Publications Warehouse

    Pan, H.P.; Fouts, J.R.; Devereux, T.R.

    1975-01-01

    Hepatic microsomes prepared from red-winged blackbirds and albino rats were incubated with N,N-dimethylaniline (DMA)_in complete incubation mixtures at pH 7.9 and 37?C for 10 min. Formaldehyde and N,N-dimethylaniline--oxide produced from DMA were measured. Redwings were found to have significantly lower N-demethylation activities than rats, and redwings had only marginal or no N-oxidation activities. Hepatic microsomes from redwings did not further metabolize the N-oxide. The N-oxidation and N-demethylation activities of brown-headed cowbirds, common grackles, and starlings were similar to those of redwings.

  9. Inaccurate DNA Synthesis in Cell Extracts of Yeast Producing Active Human DNA Polymerase Iota

    PubMed Central

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

    2011-01-01

    Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn2+ ions, can bypass some DNA lesions and misincorporates “G” opposite template “T” more frequently than incorporates the correct “A.” We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of “G” versus “A” method of Gening, abbreviated as “misGvA”). We provide unambiguous proof of the “misGvA” approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The “misGvA” activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts. PMID:21304950

  10. Activation of the DNA Damage Response by RNA Viruses.

    PubMed

    Ryan, Ellis L; Hollingworth, Robert; Grand, Roger J

    2016-01-01

    RNA viruses are a genetically diverse group of pathogens that are responsible for some of the most prevalent and lethal human diseases. Numerous viruses introduce DNA damage and genetic instability in host cells during their lifecycles and some species also manipulate components of the DNA damage response (DDR), a complex and sophisticated series of cellular pathways that have evolved to detect and repair DNA lesions. Activation and manipulation of the DDR by DNA viruses has been extensively studied. It is apparent, however, that many RNA viruses can also induce significant DNA damage, even in cases where viral replication takes place exclusively in the cytoplasm. DNA damage can contribute to the pathogenesis of RNA viruses through the triggering of apoptosis, stimulation of inflammatory immune responses and the introduction of deleterious mutations that can increase the risk of tumorigenesis. In addition, activation of DDR pathways can contribute positively to replication of viral RNA genomes. Elucidation of the interactions between RNA viruses and the DDR has provided important insights into modulation of host cell functions by these pathogens. This review summarises the current literature regarding activation and manipulation of the DDR by several medically important RNA viruses. PMID:26751489

  11. Using DNA devices to track anticancer drug activity.

    PubMed

    Kahanda, Dimithree; Chakrabarti, Gaurab; Mcwilliams, Marc A; Boothman, David A; Slinker, Jason D

    2016-06-15

    It is beneficial to develop systems that reproduce complex reactions of biological systems while maintaining control over specific factors involved in such processes. We demonstrated a DNA device for following the repair of DNA damage produced by a redox-cycling anticancer drug, beta-lapachone (β-lap). These chips supported ß-lap-induced biological redox cycle and tracked subsequent DNA damage repair activity with redox-modified DNA monolayers on gold. We observed drug-specific changes in square wave voltammetry from these chips at therapeutic ß-lap concentrations of high statistical significance over drug-free control. We also demonstrated a high correlation of this change with the specific ß-lap-induced redox cycle using rational controls. The concentration dependence of ß-lap revealed significant signal changes at levels of high clinical significance as well as sensitivity to sub-lethal levels of ß-lap. Catalase, an enzyme decomposing peroxide, was found to suppress DNA damage at a NQO1/catalase ratio found in healthy cells, but was clearly overcome at a higher NQO1/catalase ratio consistent with cancer cells. We found that it was necessary to reproduce key features of the cellular environment to observe this activity. Thus, this chip-based platform enabled tracking of ß-lap-induced DNA damage repair when biological criteria were met, providing a unique synthetic platform for uncovering activity normally confined to inside cells. PMID:26901461

  12. Activation of the DNA Damage Response by RNA Viruses

    PubMed Central

    Ryan, Ellis L.; Hollingworth, Robert; Grand, Roger J.

    2016-01-01

    RNA viruses are a genetically diverse group of pathogens that are responsible for some of the most prevalent and lethal human diseases. Numerous viruses introduce DNA damage and genetic instability in host cells during their lifecycles and some species also manipulate components of the DNA damage response (DDR), a complex and sophisticated series of cellular pathways that have evolved to detect and repair DNA lesions. Activation and manipulation of the DDR by DNA viruses has been extensively studied. It is apparent, however, that many RNA viruses can also induce significant DNA damage, even in cases where viral replication takes place exclusively in the cytoplasm. DNA damage can contribute to the pathogenesis of RNA viruses through the triggering of apoptosis, stimulation of inflammatory immune responses and the introduction of deleterious mutations that can increase the risk of tumorigenesis. In addition, activation of DDR pathways can contribute positively to replication of viral RNA genomes. Elucidation of the interactions between RNA viruses and the DDR has provided important insights into modulation of host cell functions by these pathogens. This review summarises the current literature regarding activation and manipulation of the DDR by several medically important RNA viruses. PMID:26751489

  13. Norcantharidin blocks Wnt/β-catenin signaling via promoter demethylation of WIF-1 in glioma.

    PubMed

    Xie, Dajiang; Xie, Jixi; Wan, Yingfeng; Ma, Li; Qi, Xuchen; Wang, Kun; Yang, Shuxu

    2016-04-01

    Glioma is one of the most common primary intracranial tumors, and the prognosis is poor even though much treatment management is employed. Wnt/β-catenin signaling has been reported to be associated with glioma. Norcantharidin (NCTD) is the demethylated analog of cantharidin isolated from blister beetles, and it is reported to possess anticancer activity but less nephrotoxicity than cantharidin. Accordingly, we aimed to investigate NCTD as an anti-neoplastic drug that inhibits the Wnt/β‑catenin pathway via promoter demethylation of Wnt inhibitory factor-1 (WIF-1) in glioma growth in vitro. In the present study, we report that NCTD inhibited cell proliferation, induced apoptosis and cell cycle arrest, and suppressed cell migration and invasion in vitro. Moreover, we observed that the expression levels of WIF-1 mRNA and protein in the NCTD-treated cells were increased significantly compared with these levels in the negative control (NC) cells. Promoter demethylation was observed in the NCTD‑treated cells. In contrast, aberrant methylation was observed in the NC cells. Additionally, more investigation revealed that NCTD suppressed activity of Wnt/β-catenin signaling and transcription of β-catenin/TCF-4. Furthermore, the expression of apoptosis-related proteins Bcl-2 and cleaved caspase-3 indicated significant cell apoptosis. We provide initial evidence that NCTD reactivates WIF-1 from a methylation state, and downregulates canonical Wnt/β-catenin signaling. Our findings revealed that NCTD is effective for glioma in vitro and may be a new therapeutic option in vivo. PMID:26781164

  14. Requirement for the Kinase Activity of Human DNA-Dependent Protein Kinase Catalytic Subunit in DNA Strand Break Rejoining

    PubMed Central

    Kurimasa, Akihiro; Kumano, Satoshi; Boubnov, Nikolai V.; Story, Michael D.; Tung, Chang-Shung; Peterson, Scott R.; Chen, David J.

    1999-01-01

    The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an enormous, 470-kDa protein serine/threonine kinase that has homology with members of the phosphatidylinositol (PI) 3-kinase superfamily. This protein contributes to the repair of DNA double-strand breaks (DSBs) by assembling broken ends of DNA molecules in combination with the DNA-binding factors Ku70 and Ku80. It may also serve as a molecular scaffold for recruiting DNA repair factors to DNA strand breaks. This study attempts to better define the role of protein kinase activity in the repair of DNA DSBs. We constructed a contiguous 14-kb human DNA-PKcs cDNA and demonstrated that it can complement the DNA DSB repair defects of two mutant cell lines known to be deficient in DNA-PKcs (M059J and V3). We then created deletion and site-directed mutations within the conserved PI 3-kinase domain of the DNA-PKcs gene to test the importance of protein kinase activity for DSB rejoining. These DNA-PKcs mutant constructs are able to express the protein but fail to complement the DNA DSB or V(D)J recombination defects of DNA-PKcs mutant cells. These results indicate that the protein kinase activity of DNA-PKcs is essential for the rejoining of DNA DSBs in mammalian cells. We have also determined a model structure for the DNA-PKcs kinase domain based on comparisons to the crystallographic structure of a cyclic AMP-dependent protein kinase. This structure gives some insight into which amino acid residues are crucial for the kinase activity in DNA-PKcs. PMID:10207111

  15. Plasma-activated air mediates plasmid DNA delivery in vivo.

    PubMed

    Edelblute, Chelsea M; Heller, Loree C; Malik, Muhammad A; Bulysheva, Anna; Heller, Richard

    2016-01-01

    Plasma-activated air (PAA) provides a noncontact DNA transfer platform. In the current study, PAA was used for the delivery of plasmid DNA in a 3D human skin model, as well as in vivo. Delivery of plasmid DNA encoding luciferase to recellularized dermal constructs was enhanced, resulting in a fourfold increase in luciferase expression over 120 hours compared to injection only (P < 0.05). Delivery of plasmid DNA encoding green fluorescent protein (GFP) was confirmed in the epidermal layers of the construct. In vivo experiments were performed in BALB/c mice, with skin as the delivery target. PAA exposure significantly enhanced luciferase expression levels 460-fold in exposed sites compared to levels obtained from the injection of plasmid DNA alone (P < 0.001). Expression levels were enhanced when the plasma reactor was positioned more distant from the injection site. Delivery of plasmid DNA encoding GFP to mouse skin was confirmed by immunostaining, where a 3-minute exposure at a 10 mm distance displayed delivery distribution deep within the dermal layers compared to an exposure at 3 mm where GFP expression was localized within the epidermis. Our findings suggest PAA-mediated delivery warrants further exploration as an alternative approach for DNA transfer for skin targets. PMID:27110584

  16. Plasma-activated air mediates plasmid DNA delivery in vivo

    PubMed Central

    Edelblute, Chelsea M; Heller, Loree C; Malik, Muhammad A; Bulysheva, Anna; Heller, Richard

    2016-01-01

    Plasma-activated air (PAA) provides a noncontact DNA transfer platform. In the current study, PAA was used for the delivery of plasmid DNA in a 3D human skin model, as well as in vivo. Delivery of plasmid DNA encoding luciferase to recellularized dermal constructs was enhanced, resulting in a fourfold increase in luciferase expression over 120 hours compared to injection only (P < 0.05). Delivery of plasmid DNA encoding green fluorescent protein (GFP) was confirmed in the epidermal layers of the construct. In vivo experiments were performed in BALB/c mice, with skin as the delivery target. PAA exposure significantly enhanced luciferase expression levels 460-fold in exposed sites compared to levels obtained from the injection of plasmid DNA alone (P < 0.001). Expression levels were enhanced when the plasma reactor was positioned more distant from the injection site. Delivery of plasmid DNA encoding GFP to mouse skin was confirmed by immunostaining, where a 3-minute exposure at a 10 mm distance displayed delivery distribution deep within the dermal layers compared to an exposure at 3 mm where GFP expression was localized within the epidermis. Our findings suggest PAA-mediated delivery warrants further exploration as an alternative approach for DNA transfer for skin targets. PMID:27110584

  17. Redox-active quinones induces genome-wide DNA methylation changes by an iron-mediated and Tet-dependent mechanism

    PubMed Central

    Zhao, Bailin; Yang, Ying; Wang, Xiaoli; Chong, Zechen; Yin, Ruichuan; Song, Shu-Hui; Zhao, Chao; Li, Cuiping; Huang, Hua; Sun, Bao-Fa; Wu, Danni; Jin, Kang-Xuan; Song, Maoyong; Zhu, Ben-Zhan; Jiang, Guibin; Rendtlew Danielsen, Jannie M.; Xu, Guo-Liang; Yang, Yun-Gui; Wang, Hailin

    2014-01-01

    DNA methylation has been proven to be a critical epigenetic mark important for various cellular processes. Here, we report that redox-active quinones, a ubiquitous class of chemicals found in natural products, cancer therapeutics and environment, stimulate the conversion of 5mC to 5hmC in vivo, and increase 5hmC in 5751 genes in cells. 5hmC increase is associated with significantly altered gene expression of 3414 genes. Interestingly, in quinone-treated cells, labile iron-sensitive protein ferritin light chain showed a significant increase at both mRNA and protein levels indicating a role of iron regulation in stimulating Tet-mediated 5mC oxidation. Consistently, the deprivation of cellular labile iron using specific chelator blocked the 5hmC increase, and a delivery of labile iron increased the 5hmC level. Moreover, both Tet1/Tet2 knockout and dimethyloxalylglycine-induced Tet inhibition diminished the 5hmC increase. These results suggest an iron-regulated Tet-dependent DNA demethylation mechanism mediated by redox-active biomolecules. PMID:24214992

  18. Brain feminization requires active repression of masculinization via DNA methylation

    PubMed Central

    Nugent, Bridget M.; Wright, Christopher L.; Shetty, Amol C.; Hodes, Georgia E.; Lenz, Kathryn M.; Mahurkar, Anup; Russo, Scott J.; Devine, Scott E.; McCarthy, Margaret M.

    2015-01-01

    The developing mammalian brain is destined for a female phenotype unless exposed to gonadal hormones during a perinatal sensitive period. It has been assumed that the undifferentiated brain is masculinized by direct induction of transcription by ligand-activated nuclear steroid receptors. We found that a primary effect of gonadal steroids in the highly sexually-dimorphic preoptic area (POA) is to reduce activity of DNA methyltransferase (Dnmt) enzymes, thereby decreasing DNA methylation and releasing masculinizing genes from epigenetic repression. Pharmacological inhibition of Dnmts mimicked gonadal steroids, resulting in masculinized neuronal markers and male sexual behavior in females. Conditional knockout of the de novo Dnmt isoform, Dnmt3a, also masculinized sexual behavior in female mice. RNA sequencing revealed gene and isoform variants modulated by methylation that may underlie the divergent reproductive behaviors of males versus females. Our data show that brain feminization is maintained by the active suppression of masculinization via DNA methylation. PMID:25821913

  19. 5-Azacytidine-induced reactivation of the human X chromosome-linked PGK1 gene is associated with a large region of cytosine demethylation in the 5' CpG island.

    PubMed Central

    Hansen, R S; Gartler, S M

    1990-01-01

    Hamster-human cell hybrids containing an inactive human X chromosome were treated with 5-azacytidine and derived clones were examined for phosphoglycerate kinase activity and cytosine methylation in the human PGK1 (X chromosome-linked phosphoglycerate kinase) gene. Comparisons between expressing and nonexpressing clones indicated that demethylation of several methylation-sensitive restriction sites outside of the 5' CpG island were unnecessary for expression. High-resolution polyacrylamide gel analysis of 25 Hpa II, Hha I, and Tha I sites revealed that all clones expressing PGK1 were unmethylated in a large region of the CpG island that includes the transcription start site and 400 base pairs upstream. Many nonexpressing clones had discontinuous patterns of demethylation. Remethylation was often observed in subclones of nonexpressing hybrids. These data suggest that a specific zone of methylation-free DNA within the PGK1 promoter is required for transcription. In addition, the presence of neighboring methylcytosines appears to decrease the heritable stability of unmethylated CpGs in this region. Images PMID:1693431

  20. Inhibition of thrombin activity with DNA-aptamers.

    PubMed

    Dobrovolsky, A B; Titaeva, E V; Khaspekova, S G; Spiridonova, V A; Kopylov, A M; Mazurov, A V

    2009-07-01

    The effects of two DNA aptamers (oligonucleotides) 15TBA and 31TBA (15- and 31-mer thrombin-binding aptamers, respectively) on thrombin activity were studied. Both aptamers added to human plasma dose-dependently increased thrombin time (fibrin formation upon exposure to exogenous thrombin), prothrombin time (clotting activation by the extrinsic pathway), and activated partial thromboplastin time (clotting activation by the intrinsic pathway). At the same time, these aptamers did not modify amidolytic activity of thrombin evaluated by cleavage of synthetic chromogenic substrate. Aptamers also inhibited thrombin-induced human platelet aggregation. The inhibitory effects of 31TBA manifested at lower concentrations than those of 15TBA in all tests. These data indicate that the studied antithrombin DNA aptamers effectively suppress its two key reactions, fibrin formation and stimulation of platelet aggregation, without modifying active center of the thrombin molecule. PMID:19902090

  1. DNA Methylation Suppresses Leptin Gene in 3T3-L1 Adipocytes

    PubMed Central

    Kuroda, Masashi; Tominaga, Ayako; Nakagawa, Kasumi; Nishiguchi, Misa; Sebe, Mayu; Miyatake, Yumiko; Kitamura, Tadahiro; Tsutsumi, Rie; Harada, Nagakatsu; Nakaya, Yutaka; Sakaue, Hiroshi

    2016-01-01

    Leptin is a key regulator of energy intake and expenditure. This peptide hormone is expressed in mouse white adipose tissue, but hardly expressed in 3T3-L1 adipocytes. Using bisulfite sequencing, we found that CpG islands in the leptin promoter are highly methylated in 3T3-L1cells. 5-azacytidine, an inhibitor of DNA methyltransferase, markedly increased leptin expression as pre-adipocytes matured into adipocytes. Remarkably, leptin expression was stimulated by insulin in adipocytes derived from precursor cells exposed to 5-azacytidine, but suppressed by thiazolidinedione and dexamethasone. In contrast, adipocytes derived from untreated precursor cells were unresponsive to both 5-azacytidine and hormonal stimuli, although lipid accumulation was sufficient to boost leptin expression in the absence of demethylation. Taken together, the results suggest that leptin expression in 3T3-L1 cells requires DNA demethylation prior to adipogenesis, transcriptional activation during adipogenesis, and lipid accumulation after adipogenesis. PMID:27494408

  2. Active, motor-driven mechanics in a DNA gel

    PubMed Central

    Bertrand, Olivier J. N.; Fygenson, Deborah Kuchnir; Saleh, Omar A.

    2012-01-01

    Cells are capable of a variety of dramatic stimuli-responsive mechanical behaviors. These capabilities are enabled by the pervading cytoskeletal network, an active gel composed of structural filaments (e.g., actin) that are acted upon by motor proteins (e.g., myosin). Here, we describe the synthesis and characterization of an active gel using noncytoskeletal components. We use methods of base-pair-templated DNA self assembly to create a hybrid DNA gel containing stiff tubes and flexible linkers. We then activate the gel by adding the motor FtsK50C, a construct derived from the bacterial protein FtsK that, in vitro, has a strong and processive DNA contraction activity. The motors stiffen the gel and create stochastic contractile events that affect the positions of attached beads. We quantify the fluctuations of the beads and show that they are comparable both to measurements of cytoskeletal systems and to theoretical predictions for active gels. Thus, we present a DNA-based active gel whose behavior highlights the universal aspects of nonequilibrium, motor-driven networks. PMID:23045635

  3. RING finger protein 4 (RNF4) derepresses gene expression from DNA methylation.

    PubMed

    Wang, Yu

    2014-12-01

    RNF4 is an E3 ubiquitin ligase originally identified as a transcription co-activator. The mechanism by which RNF4 promotes transcription remains unclear. In this study, I found that RNF4 antagonizes transcriptional repression mediated by DNA methylation. RNF4 does not promote DNA demethylation, but mediates the ubiquitination of MeCP2, a methyl-CpG-binding domain (MBD) protein. Removal of MeCP2 from gene promoters activates transcription. This study thus not only uncovers how RNF4 functions as a transcription activator, but also reveals the mechanism by which MeCP2 protein stability is regulated. PMID:25355316

  4. Anthocyanidins modulate the activity of human DNA topoisomerases I and II and affect cellular DNA integrity.

    PubMed

    Habermeyer, Michael; Fritz, Jessica; Barthelmes, Hans U; Christensen, Morten O; Larsen, Morten K; Boege, Fritz; Marko, Doris

    2005-09-01

    In the present study, we investigated the effect of anthocyanidins on human topoisomerases I and II and its relevance for DNA integrity within human cells. Anthocyanidins bearing vicinal hydroxy groups at the B-ring (delphinidin, DEL; cyanidin, CY) were found to potently inhibit the catalytic activity of human topoisomerases I and II, without discriminating between the IIalpha and the IIbeta isoforms. However, in contrast to topoisomerase poisons, DEL and CY did not stabilize the covalent DNA-topoisomerase intermediates (cleavable complex) of topoisomerase I or II. Using recombinant topoisomerase I, the presence of CY or DEL (> or = 1 microM) effectively prohibited the stabilization of the cleavable complex by the topoisomerase I poison camptothecin. We furthermore investigated whether the potential protective effect vs topoisomerase I poisons is reflected also on the cellular level, affecting the DNA damaging properties of camptothecin. Indeed, in HT29 cells, low micromolar concentrations of DEL (1-10 microM) significantly diminished the DNA strand breaking effect of camptothecin (100 microM). However, at concentrations > or = 50 microM, all anthocyanidins tested (delphinidin, cyanidin, malvidin, pelargonidin, and paeonidin), including those not interfering with topoisomerases, were found to induce DNA strand breaks in the comet assay. All of these analogues were able to compete with ethidium bromide for the intercalation into calf thymus DNA and to replace the minor groove binder Hoechst 33258. These data indicate substantial affinity to double-stranded DNA, which might contribute at least to the DNA strand breaking effect of anthocyanidins at higher concentrations (> or = 50 microM). PMID:16167831

  5. Electrophilicities and Protein Covalent Binding of Demethylation Metabolites of Colchicine.

    PubMed

    Guo, Xiucai; Lin, Dongju; Li, Weiwei; Wang, Kai; Peng, Ying; Zheng, Jiang

    2016-03-21

    Colchicine, an alkaloid existing in plants of Liliaceous colchicum, has been widely used in the treatment of gout and familial Mediterranean fever. The administration of colchicine was found to cause liver injury in humans. The mechanisms of colchicine-induced liver toxicity remain unknown. The objectives of this study were to determine the electrophilicities of demethylation metabolites of colchicine and investigate the protein adductions derived from the reactive metabolites of colchicine. Four demethylated colchicine (1-, 2-, 3-, and 10-DMCs), namely, M1-M4, were detected in colchicine-fortified microsomal incubations. Four N-acetyl cysteine (NAC) conjugates (M5-M8) derived from colchicine were detected in the microsomes in the presence of NAC. M5 and M6 were derived from 10-DMC. M7 resulted from the reaction of 2-DMC or 3-DMC with NAC, and M8 originated from 10-DMC. Microsomal protein covalent binding was observed after exposure to colchicine. Two cysteine adducts (CA-1 and CA-2) derived from 10-DMC were found in proteolytically digested microsomal protein samples after incubation with colchicine. The findings allow us to define the chemical property of demethylation metabolites of colchicine and the interaction between protein and the reactive metabolites of colchicine generated in situ. PMID:26845511

  6. Inhibiting DNA methylation switches adipogenesis to osteoblastogenesis by activating Wnt10a

    PubMed Central

    Chen, Yii-Shyuan; Wu, Rui; Yang, Xiaosong; Kou, Shuping; MacDougald, Ormond A.; Yu, Liqing; Shi, Hang; Xue, Bingzhong

    2016-01-01

    Both adipocytes and osteoblasts share the mesodermal lineage that derives from mesenchymal stem cells. Most studies investigating the mechanisms underlying the regulation of adipogenic or osteoblastogenic development focus on transcriptional pathways; little is known about the epigenetic mechanisms in this process. We thus determined the role of 5-aza-2′-deoxycytidine (5-Aza-dC), an inhibitor of DNA methylation, in the lineage determination between adipogenesis and osteoblastogenesis. Inhibiting DNA methylation in 3T3-L1 preadipocytes by 5-Aza-dC significantly inhibited adipogenesis whereas promoted osteoblastogenesis. This dual effect of 5-Aza-dC was associated with up-regulation of Wnt10a, a key factor determining the fate of the mesenchymal lineage towards osteoblasts. Consistently, IWP-2, an inhibitor of Wnt proteins, was found to prevent the anti-adipogenic effect of 5-Aza-dC in 3T3-L1 preadipocytes and block the osteoblastogenic effect of 5-Aza-dC in ST2 mesenchymal stem cell line. Finally, the Wnt10a 5′-region is enriched with CpG sites, whose methylation levels were markedly reduced by 5-Aza-dC. Thus we conclude that inhibiting DNA methylation by 5-Aza-dC mutual-exclusively regulates the lineage determination of adipogenesis and osteoblastogenesis by demethylating Wnt10a gene and upregulating its expression. Our study defines DNA methylation as a novel mechanism underlying adipocyte and bone cell development. PMID:27136753

  7. Construction of DNA recognition sites active in Haemophilus transformation.

    PubMed Central

    Danner, D B; Smith, H O; Narang, S A

    1982-01-01

    Competent Haemophilus cells recognize and preferentially take up Haemophilus DNA during genetic transformation. This preferential uptake is correlated with the presence on incoming DNA of an 11-base-pair (bp) sequence, 5'-A-A-G-T-G-C-G-G-T-C-A-3'. To prove that this sequence is the recognition site that identifies Haemophilus DNA to the competent cell, we have now constructed a series of plasmids, each of which contains the 11-bp sequence. Using two different assay systems we have tested the ability of fragments from these plasmids to compete with cloned Haemophilus DNA fragments that naturally contain the 11-bp sequence. We find that the addition of the 11-bp sequence to a DNA fragment is necessary and sufficient for preferential uptake of that fragment. However, plasmid DNAs containing this sequence may vary as much as 48-fold in uptake activity, and this variation correlates with the A+T-richness of the DNA flanking the 11-mer. Images PMID:6285382

  8. Dietary compound isoliquiritigenin prevents mammary carcinogenesis by inhibiting breast cancer stem cells through WIF1 demethylation

    PubMed Central

    Wang, Yu; Xie, Xiaoming; Shen, Jiangang; Peng, Cheng; You, Jieshu; Peng, Fu; Tang, Hailin; Guan, Xinyuan; Chen, Jianping

    2015-01-01

    Breast cancer stem cells (CSCs) are considered as the root of mammary tumorigenesis. Previous studies have demonstrated that ISL efficiently limited the activities of breast CSCs. However, the cancer prevention activities of ISL and its precise molecular mechanisms remain largely unknown. Here, we report a novel function of ISL as a natural demethylation agent targeting WIF1 to prevent breast cancer. ISL administration suppressed in vivo breast cancer initiation and progression, accompanied by reduced CSC-like populations. A global gene expression profile assay further identified WIF1 as the main response gene of ISL treatment, accompanied by the simultaneous downregulation of β-catenin signaling and G0/G1 phase arrest in breast CSCs. In addition, WIF1 inhibition significantly relieved the CSC-limiting effects of ISL and methylation analysis further revealed that ISL enhanced WIF1 gene expression via promoting the demethylation of its promoter, which was closely correlated with the inhibition of DNMT1 methyltransferase. Molecular docking analysis finally revealed that ISL could stably dock into the catalytic domain of DNMT1. Taken together, our findings not only provide preclinical evidence to demonstrate the use of ISL as a dietary supplement to inhibit mammary carcinogenesis but also shed novel light on WIF1 as an epigenetic target for breast cancer prevention. PMID:25918249

  9. Blockage of TGFβ-SMAD2 by demethylation-activated miR-148a is involved in caffeic acid-induced inhibition of cancer stem cell-like properties in vitro and in vivo

    PubMed Central

    Li, Yuan; Jiang, Fei; Chen, Lijun; Yang, Ye; Cao, Shuyuan; Ye, Yuting; Wang, Xingxing; Mu, Juan; Li, Zhong; Li, Lei

    2015-01-01

    Current standard practices for treatment of cancers are less than satisfactory because of recurrence mediated by cancer stem cells (CSCs). Caffeic acid (CaA) is a novel anti-tumor agent that inhibits proliferation, migration, and invasion in human cancer cells. However, little is known about the functions of CaA in regulating CSCs-like properties and the potential molecular mechanisms. Here, we found that CaA attenuated the CSCs-like properties by the microRNA-148a (miR-148a)-mediated inhibition of transforming growth factor beta (TGFβ)-SMAD2 signaling pathway both in vitro and in vivo. CaA enhanced the expression of miR-148a by inducing DNA methylation. MiR-148a, which targeted the SMAD2-3′UTR, decreased the expression of SMAD2. Knockdown of miR-148a abolished the CaA-induced inhibition of TGFβ-SMAD2 signal pathway and the CSCs-like properties. Our study found a novel mechanism that CaA inhibits the CSCs-like properties via miR-148a-mediated inhibition of TGFβ-SMAD2 signaling pathway, which may help to identify a new approach for the treatment of human cancers. PMID:26106521

  10. Genetically encoded optical activation of DNA recombination in human cells.

    PubMed

    Luo, J; Arbely, E; Zhang, J; Chou, C; Uprety, R; Chin, J W; Deiters, A

    2016-06-30

    We developed two tightly regulated, light-activated Cre recombinase enzymes through site-specific incorporation of two genetically-encoded photocaged amino acids in human cells. Excellent optical off to on switching of DNA recombination was achieved. Furthermore, we demonstrated precise spatial control of Cre recombinase through patterned illumination. PMID:27277957

  11. ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method.

    PubMed

    Yang, Mu; Wang, Ganggang

    2016-09-15

    The DnaB helicase from Bacillus stearothermophilus (DnaBBst) was a model protein for studying the bacterial DNA replication. In this work, a non-radioactive method for measuring ATPase activity of DnaBBst helicase was described. The working parameters and conditions were optimized. Furthermore, this method was applied to investigate effects of DnaG primase, ssDNA and helicase loader protein (DnaI) on ATPase activity of DnaBBst. Our results showed this method was sensitive and efficient. Moreover, it is suitable for the investigation of functional interaction between DnaB and related factors. PMID:27372608

  12. 75 FR 42114 - Office of Biotechnology Activities; Recombinant DNA Research: Proposed Action Under the NIH...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-20

    ... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA Research: Proposed Action Under the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH... transgenic rodents by recombinant DNA technology must be registered with the Institutional...

  13. Directed enzymatic activation of 1-D DNA tiles.

    PubMed

    Garg, Sudhanshu; Chandran, Harish; Gopalkrishnan, Nikhil; LaBean, Thomas H; Reif, John

    2015-02-24

    The tile assembly model is a Turing universal model of self-assembly where a set of square shaped tiles with programmable sticky sides undergo coordinated self-assembly to form arbitrary shapes, thereby computing arbitrary functions. Activatable tiles are a theoretical extension to the Tile assembly model that enhances its robustness by protecting the sticky sides of tiles until a tile is partially incorporated into a growing assembly. In this article, we experimentally demonstrate a simplified version of the Activatable tile assembly model. In particular, we demonstrate the simultaneous assembly of protected DNA tiles where a set of inert tiles are activated via a DNA polymerase to undergo linear assembly. We then demonstrate stepwise activated assembly where a set of inert tiles are activated sequentially one after another as a result of attachment to a growing 1-D assembly. We hope that these results will pave the way for more sophisticated demonstrations of activated assemblies. PMID:25625898

  14. Binding-activated localization microscopy of DNA structures.

    PubMed

    Schoen, Ingmar; Ries, Jonas; Klotzsch, Enrico; Ewers, Helge; Vogel, Viola

    2011-09-14

    Many nucleic acid stains show a strong fluorescence enhancement upon binding to double-stranded DNA. Here we exploit this property to perform superresolution microscopy based on the localization of individual binding events. The dynamic labeling scheme and the optimization of fluorophore brightness yielded a resolution of ∼14 nm (fwhm) and a spatial sampling of 1/nm. We illustrate our approach with two different DNA-binding dyes and apply it to visualize the organization of the bacterial chromosome in fixed Escherichia coli cells. In general, the principle of binding-activated localization microscopy (BALM) can be extended to other dyes and targets such as protein structures. PMID:21838238

  15. DNA methylation does not stably lock gene expression but instead serves as a molecular mark for gene silencing memory

    PubMed Central

    Raynal, Noël J.-M.; Si, Jiali; Taby, Rodolphe F.; Gharibyan, Vazganush; Ahmed, Saira; Jelinek, Jaroslav; Estécio, Marcos R.H.; Issa, Jean-Pierre J.

    2012-01-01

    DNA methylation is commonly thought of as a "molecular lock" that leads to permanent gene silencing. To investigate this notion, we tested 24 different HDAC inhibitors (HDACi) on colon cancer cells that harbor a GFP locus stably integrated and silenced by a hypermethylated CMV promoter. We found that HDACi efficiently reactivated expression of GFP and many other endogenous genes silenced by DNA hypermethylation. After treatment, all promoters were marked with active chromatin, yet DNA hypermethylation did not change. Thus, DNA methylation could not prevent gene reactivation by drug-induced resetting of the chromatin state. In evaluating the relative contribution of DNA methylation and histone modifications to stable gene silencing, we followed expression levels of GFP and other genes silenced by DNA hypermethylation over time after treatment with HDACi or DNA demethylating drugs. Reactivation of methylated loci by HDACi was detectable for only 2 weeks, whereas DNA demethylating drugs induced permanent epigenetic reprogramming. Therefore, DNA methylation cannot be considered as a lock for gene expression, but rather as a memory signal for long-term maintenance of gene silencing. These findings define chromatin as an important druggable target for cancer epigenetic therapy and suggest that removal of DNA methylation signals is required to achieve long-term gene reactivation. PMID:22219169

  16. DNA repair by the cryptic endonuclease activity of Mu transposase.

    PubMed

    Choi, Wonyoung; Harshey, Rasika M

    2010-06-01

    Phage Mu transposes by two distinct pathways depending on the specific stage of its life cycle. A common strand transfer intermediate is resolved differentially in the two pathways. During lytic growth, the intermediate is resolved by replication of Mu initiated within the flanking target DNA; during integration of infecting Mu, it is resolved without replication, by removal and repair of DNA from a previous host that is still attached to the ends of the incoming Mu genome. We have discovered that the cryptic endonuclease activity reported for the isolated C-terminal domain of the transposase MuA [Wu Z, Chaconas G (1995) A novel DNA binding and nuclease activity in domain III of Mu transposase: Evidence for a catalytic region involved in donor cleavage. EMBO J 14:3835-3843], which is not observed in the full-length protein or in the assembled transpososome in vitro, is required in vivo for removal of the attached host DNA or "5'flap" after the infecting Mu genome has integrated into the E. coli chromosome. Efficient flap removal also requires the host protein ClpX, which is known to interact with the C-terminus of MuA to remodel the transpososome for replication. We hypothesize that ClpX constitutes part of a highly regulated mechanism that unmasks the cryptic nuclease activity of MuA specifically in the repair pathway. PMID:20167799

  17. Selenite cataracts: Activation of endoplasmic reticulum stress and loss of Nrf2/Keap1-dependent stress protection

    PubMed Central

    Palsamy, Periyasamy; Bidasee, Keshore R.; Shinohara, Toshimichi

    2014-01-01

    Cataract-induced by sodium selenite in suckling rats is one of the suitable animal models to study the basic mechanism of human cataracts formation. The aim of this present investigation is to study the endoplasmic reticulum (ER) stress-mediated activation of unfolded protein response (UPR), overproduction of reactive oxygen species (ROS), and suppression of Nrf2/Keap1-dependent antioxidant protection through endoplasmic reticulum-associated degradation (ERAD) pathway and Keap1 promoter DNA demethylation in human lens epithelial cells (HLECs) treated with sodium selenite. Lenses enucleated from sodium selenite injected rats generated overproduction of ROS in lens epithelial cells and newly formed lens fiber cells resulting in massive lens epithelial cells death after 1–5 days. All these lenses developed nuclear cataracts after 4–5 days. Sodium selenite treated HLECs induced ER stress and activated the UPR leading to release of Ca2+ from ER, ROS overproduction and finally HLECs death. Sodium selenite also activated the mRNA expressions of passive DNA demethylation pathway enzymes such as Dnmt1, Dnmt3a, and Dnmt3b, and active DNA demethylation pathway enzyme, Tet1 leading to DNA demethylation in the Keap1 promoter of HLECs. This demethylated Keap1 promoter results in overexpression of Keap1 mRNA and protein. Overexpression Keap1 protein suppresses the Nrf2 protein through ERAD leading to suppression of Nrf2/Keap1 dependent antioxidant protection in the HLECs treated with sodium selenite. As an outcome, the cellular redox status is altered towards lens oxidation and results in cataract formation. PMID:24997453

  18. DNA-binding activity of rat DNA topoisomerase II α C-terminal domain contributes to efficient DNA catenation in vitro.

    PubMed

    Kawano, Shinji; Kato, Yuri; Okada, Natsumi; Sano, Kuniaki; Tsutsui, Ken; Tsutsui, Kimiko M; Ikeda, Shogo

    2016-03-01

    DNA topoisomerase IIα (topo IIα) is an essential enzyme for resolution of DNA topologies arising in DNA metabolic reactions. In proliferating cells, topo II activities of DNA catenation or decatenation are required for condensation of chromosomes and segregation of chromatids. Recent studies suggest that the C-terminal domain (CTD) of human topo IIα is required for localization to mitotic chromosomes. Here, we show that the CTD of topo IIα is also associated with efficient DNA catenation in vitro, based on comparison of wild-type (WT) rat topo IIα and its deletion mutants. Unlike WT, the CTD truncated mutant (ΔCTD) lacked linear DNA binding activity, but could bind to negatively supercoiled DNA similarly to WT. The CTD alone showed linear DNA-binding activity. ΔCTD mediated formation of a DNA catenane in the presence of polyethylene glycol, which enhances macromolecular association. These results indicate that DNA-binding activity in the CTD of topo IIα concentrates the enzyme in the vicinity of condensed DNA and allows topo IIα to efficiently form a DNA catenane. PMID:26527691

  19. DNA methylation dynamics in mouse preimplantation embryos revealed by mass spectrometry.

    PubMed

    Okamoto, Yoshinori; Yoshida, Naoko; Suzuki, Toru; Shimozawa, Nobuhiro; Asami, Maki; Matsuda, Tomonari; Kojima, Nakao; Perry, Anthony C F; Takada, Tatsuyuki

    2016-01-01

    Following fertilization in mammals, paternal genomic 5-methyl-2'-deoxycytidine (5 mC) content is thought to decrease via oxidation to 5-hydroxymethyl-2'-deoxycytidine (5 hmC). This reciprocal model of demethylation and hydroxymethylation is inferred from indirect, non-quantitative methods. We here report direct quantification of genomic 5 mC and 5 hmC in mouse embryos by small scale liquid chromatographic tandem mass spectrometry (SMM). Profiles of absolute 5 mC levels in embryos produced by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) were almost identical. By 10 h after fertilization, 5 mC levels had declined by ~40%, consistent with active genomic DNA demethylation. Levels of 5 mC in androgenotes (containing only a paternal genome) and parthenogenotes (containing only a maternal genome) underwent active 5 mC loss in the first 6 h, showing that both parental genomes can undergo demethylation independently. We found no evidence for net loss of 5 mC 10-48 h after fertilization, implying that any passive 'demethylation' following DNA replication was balanced by active 5 mC maintenance methylation. However, levels of 5 mC declined during development after 48 h, to 1% (measured as a fraction of G-residues) in blastocysts (~96 h). 5 hmC levels were consistently low (<0.2% of G-residues) throughout development in normal diploid embryos. This work directly quantifies the dynamics of global genomic DNA modification in mouse preimplantation embryos, suggesting that SMM will be applicable to other biomedical situations with limiting sample sizes. PMID:26750605

  20. Cooperative demethylation by JMJD2C and LSD1 promotes androgen receptor-dependent gene expression.

    PubMed

    Wissmann, Melanie; Yin, Na; Müller, Judith M; Greschik, Holger; Fodor, Barna D; Jenuwein, Thomas; Vogler, Christine; Schneider, Robert; Günther, Thomas; Buettner, Reinhard; Metzger, Eric; Schüle, Roland

    2007-03-01

    Posttranslational modifications of histones, such as methylation, regulate chromatin structure and gene expression. Recently, lysine-specific demethylase 1 (LSD1), the first histone demethylase, was identified. LSD1 interacts with the androgen receptor and promotes androgen-dependent transcription of target genes by ligand-induced demethylation of mono- and dimethylated histone H3 at Lys 9 (H3K9) only. Here, we identify the Jumonji C (JMJC) domain-containing protein JMJD2C as the first histone tridemethylase regulating androgen receptor function. JMJD2C interacts with androgen receptor in vitro and in vivo. Assembly of ligand-bound androgen receptor and JMJD2C on androgen receptor-target genes results in demethylation of trimethyl H3K9 and in stimulation of androgen receptor-dependent transcription. Conversely, knockdown of JMJD2C inhibits androgen-induced removal of trimethyl H3K9, transcriptional activation and tumour cell proliferation. Importantly, JMJD2C colocalizes with androgen receptor and LSD1 in normal prostate and in prostate carcinomas. JMJD2C and LSD1 interact and both demethylases cooperatively stimulate androgen receptor-dependent gene transcription. In addition, androgen receptor, JMJD2C and LSD1 assemble on chromatin to remove methyl groups from mono, di and trimethylated H3K9. Thus, our data suggest that specific gene regulation requires the assembly and coordinate action of demethylases with distinct substrate specificities. PMID:17277772

  1. Dynamic and selective HERV RNA expression in neuroblastoma cells subjected to variation in oxygen tension and demethylation.

    PubMed

    Hu, Lijuan; Uzhameckis, Dmitrijs; Hedborg, Fredrik; Blomberg, Jonas

    2016-01-01

    We studied HERV expression in cell lines after hypoxia, mitogenic stimulation, and demethylation, to better understand if hypoxia may play a role in ERV activation also within the nervous system, as represented by neuroblastoma cell lines. The level of RNA of four human ERV groups (HERVs) (HERVE, I/T, H, and W), and three housekeeping genes, of different cell lines including A549, COS-1, Namalwa, RD-L and Vero-E6, as well as human neuroblastoma cell lines SH-SY5Y, SK-N-DZ, and SK-N-AS were studied using reverse transcription and real-time quantitative PCR (QPCR). During the course of recovery from hypoxia a pronounced and selective activation of RNA expression of HERVW-like sequences, but not of HERVE, I/T, H, and three housekeeping genes, was found in the neuroblastoma cell lines, most pronounced in SK-N-DZ. In the SK-N-DZ cell line, we also tested the expression of HERVs after chemical treatments. HERVW-like sequences were selectively upregulated by 5-azacytidine, a demethylating agent. Some HERVW loci seem especially responsive to hypoxia and demethylation. HERV expression in neuroblastoma cells is selectively and profoundly influenced by some physiological and chemical stimuli. PMID:26818268

  2. Genome-wide Bisulfite Sequencing in Zygotes Identifies Demethylation Targets and Maps the Contribution of TET3 Oxidation

    PubMed Central

    Peat, Julian R.; Dean, Wendy; Clark, Stephen J.; Krueger, Felix; Smallwood, Sébastien A.; Ficz, Gabriella; Kim, Jong Kyoung; Marioni, John C.; Hore, Timothy A.; Reik, Wolf

    2014-01-01

    Summary Fertilization triggers global erasure of paternal 5-methylcytosine as part of epigenetic reprogramming during the transition from gametic specialization to totipotency. This involves oxidation by TET3, but our understanding of its targets and the wider context of demethylation is limited to a small fraction of the genome. We employed an optimized bisulfite strategy to generate genome-wide methylation profiles of control and TET3-deficient zygotes, using SNPs to access paternal alleles. This revealed that in addition to pervasive removal from intergenic sequences and most retrotransposons, gene bodies constitute a major target of zygotic demethylation. Methylation loss is associated with zygotic genome activation and at gene bodies is also linked to increased transcriptional noise in early development. Our data map the primary contribution of oxidative demethylation to a subset of gene bodies and intergenic sequences and implicate redundant pathways at many loci. Unexpectedly, we demonstrate that TET3 activity also protects certain CpG islands against methylation buildup. PMID:25497087

  3. Hemi-methylated DNA regulates DNA methylation inheritance through allosteric activation of H3 ubiquitylation by UHRF1.

    PubMed

    Harrison, Joseph S; Cornett, Evan M; Goldfarb, Dennis; DaRosa, Paul A; Li, Zimeng M; Yan, Feng; Dickson, Bradley M; Guo, Angela H; Cantu, Daniel V; Kaustov, Lilia; Brown, Peter J; Arrowsmith, Cheryl H; Erie, Dorothy A; Major, Michael B; Klevit, Rachel E; Krajewski, Krzysztof; Kuhlman, Brian; Strahl, Brian D; Rothbart, Scott B

    2016-01-01

    The epigenetic inheritance of DNA methylation requires UHRF1, a histone- and DNA-binding RING E3 ubiquitin ligase that recruits DNMT1 to sites of newly replicated DNA through ubiquitylation of histone H3. UHRF1 binds DNA with selectivity towards hemi-methylated CpGs (HeDNA); however, the contribution of HeDNA sensing to UHRF1 function remains elusive. Here, we reveal that the interaction of UHRF1 with HeDNA is required for DNA methylation but is dispensable for chromatin interaction, which is governed by reciprocal positive cooperativity between the UHRF1 histone- and DNA-binding domains. HeDNA recognition activates UHRF1 ubiquitylation towards multiple lysines on the H3 tail adjacent to the UHRF1 histone-binding site. Collectively, our studies are the first demonstrations of a DNA-protein interaction and an epigenetic modification directly regulating E3 ubiquitin ligase activity. They also define an orchestrated epigenetic control mechanism involving modifications both to histones and DNA that facilitate UHRF1 chromatin targeting, H3 ubiquitylation, and DNA methylation inheritance. PMID:27595565

  4. Hemi-methylated DNA regulates DNA methylation inheritance through allosteric activation of H3 ubiquitylation by UHRF1

    PubMed Central

    Harrison, Joseph S; Cornett, Evan M; Goldfarb, Dennis; DaRosa, Paul A; Li, Zimeng M; Yan, Feng; Dickson, Bradley M; Guo, Angela H; Cantu, Daniel V; Kaustov, Lilia; Brown, Peter J; Arrowsmith, Cheryl H; Erie, Dorothy A; Major, Michael B; Klevit, Rachel E; Krajewski, Krzysztof; Kuhlman, Brian; Strahl, Brian D; Rothbart, Scott B

    2016-01-01

    The epigenetic inheritance of DNA methylation requires UHRF1, a histone- and DNA-binding RING E3 ubiquitin ligase that recruits DNMT1 to sites of newly replicated DNA through ubiquitylation of histone H3. UHRF1 binds DNA with selectivity towards hemi-methylated CpGs (HeDNA); however, the contribution of HeDNA sensing to UHRF1 function remains elusive. Here, we reveal that the interaction of UHRF1 with HeDNA is required for DNA methylation but is dispensable for chromatin interaction, which is governed by reciprocal positive cooperativity between the UHRF1 histone- and DNA-binding domains. HeDNA recognition activates UHRF1 ubiquitylation towards multiple lysines on the H3 tail adjacent to the UHRF1 histone-binding site. Collectively, our studies are the first demonstrations of a DNA-protein interaction and an epigenetic modification directly regulating E3 ubiquitin ligase activity. They also define an orchestrated epigenetic control mechanism involving modifications both to histones and DNA that facilitate UHRF1 chromatin targeting, H3 ubiquitylation, and DNA methylation inheritance. DOI: http://dx.doi.org/10.7554/eLife.17101.001 PMID:27595565

  5. Effect of nitroso-chloramphenicol on mitochondrial DNA polymerase activity

    SciTech Connect

    Lim, L.O.; Abou-Khalil, W.H.; Yunis, A.A.; Abou-Khalil, S.

    1984-08-01

    A study was made of the effects of nitroso-chloramphenicol, chloramphenicol, amino-chloramphenicol, and thiamphenicol on the activity of mitochondrial DNA polymerase of rat liver. /sup 3/H-thymidine triphosphate incorporation into DNA was used to measure the DNA polymerase activity in the mitochondrial matrix fraction. This fraction was in the supernatant of sonicated mitochondria obtained by ultracentrifugation. Under standard experimental conditions, thymidine triphosphate incorporation was time dependent up to 10 minutes. This activity was enhanced by ..beta..-mercaptoethanol and was blocked by the known polymerase inhibitors ethidium bromide and 2',3'-dideoxythymidine 5'-triphosphate. Chloramphenicol and its analogues, amino-chloramphenicol and thiamphenicol, did not have a significant effect on the polymerase activity, whereas nitroso-chloramphenicol was inhibitory. The degree of inhibition was dependent on the experimental conditions. Thus, in the absence of ..beta..-mercaptoethanol, nitroso-chloramphenicol was inhibitory. The degree of inhibition was dependent on the experimental conditions. Under similar conditions, the addition of dithiothreitol also provided partial protection. On the other hand, the inhibition by nitroso-chloramphenicol was significantly enhanced with its preincubation in the mitochondrial matrix fraction before the addition of nucleotides and DNA; thus after 40 minutes of preincubation, nitroso-chloramphenicol at a concentration of 200 ..mu..mol/L gave 53% inhibition, and produced total inhibition at 600 ..mu..mol/L. The addition of NADH or NADPH to the preincubation medium produced substantial protection against nitroso-chloramphenicol, whereas nicotinamide-adenine dinucleotide had no effect. These results suggest that mitochondrial DNA polymerase may be a target for nitroso-chloramphenicol action.

  6. DNA methylation dynamics in muscle development and disease

    PubMed Central

    Carrió, Elvira; Suelves, Mònica

    2015-01-01

    DNA methylation is an essential epigenetic modification for mammalian development and is crucial for the establishment and maintenance of cellular identity. Traditionally, DNA methylation has been considered as a permanent repressive epigenetic mark. However, the application of genome-wide approaches has allowed the analysis of DNA methylation in different genomic contexts revealing a more dynamic regulation than originally thought, since active DNA methylation and demethylation occur during cellular differentiation and tissue specification. Satellite cells are the primary stem cells in adult skeletal muscle and are responsible for postnatal muscle growth, hypertrophy, and muscle regeneration. This review outlines the published data regarding DNA methylation changes along the skeletal muscle program, in both physiological and pathological conditions, to better understand the epigenetic mechanisms that control myogenesis. PMID:25798107

  7. DNA damage processing by human 8-oxoguanine-DNA glycosylase mutants with the occluded active site.

    PubMed

    Lukina, Maria V; Popov, Alexander V; Koval, Vladimir V; Vorobjev, Yuri N; Fedorova, Olga S; Zharkov, Dmitry O

    2013-10-01

    8-Oxoguanine-DNA glycosylase (OGG1) removes premutagenic lesion 8-oxoguanine (8-oxo-G) from DNA and then nicks the nascent abasic (apurinic/apyrimidinic) site by β-elimination. Although the structure of OGG1 bound to damaged DNA is known, the dynamic aspects of 8-oxo-G recognition are not well understood. To comprehend the mechanisms of substrate recognition and processing, we have constructed OGG1 mutants with the active site occluded by replacement of Cys-253, which forms a wall of the base-binding pocket, with bulky leucine or isoleucine. The conformational dynamics of OGG1 mutants were characterized by single-turnover kinetics and stopped-flow kinetics with fluorescent detection. Additionally, the conformational mobility of wild type and the mutant OGG1 substrate complex was assessed using molecular dynamics simulations. Although pocket occlusion distorted the active site and greatly decreased the catalytic activity of OGG1, it did not fully prevent processing of 8-oxo-G and apurinic/apyrimidinic sites. Both mutants were notably stimulated in the presence of free 8-bromoguanine, indicating that this base can bind to the distorted OGG1 and facilitate β-elimination. The results agree with the concept of enzyme plasticity, suggesting that the active site of OGG1 is flexible enough to compensate partially for distortions caused by mutation. PMID:23955443

  8. Principles Governing DNA Methylation during Neuronal Lineage and Subtype Specification

    PubMed Central

    Sharma, Ali; Klein, Shifra S.; Barboza, Luendreo; Lohdi, Niraj

    2016-01-01

    Although comprehensively described during early neuronal development, the role of DNA methylation/demethylation in neuronal lineage and subtype specification is not well understood. By studying two distinct neuronal progenitors as they differentiate to principal neurons in mouse hippocampus and striatum, we uncovered several principles governing neuronal DNA methylation during brain development. (1) The program consists of three stages: an initial genome-wide methylation during progenitor proliferation is followed by loss of methylation during the transition of regional progenitors to “young” hippocampal/striatal neurons, which is then reversed by gain in methylation during maturation to subtype-specific neurons. (2) At the first two stages, gain and loss of methylation are limited to CpGs, whereas during the third maturation stage, methylation also occurs at non-CpG sites in both lineages. (3) Methylation/demethylation, similar to transcription, are initially highly similar in the two lineages, whereas diversification in methylation and transcription during maturation creates subtype-specific methylation differences. (4) Initially, methylation targets all genomic locations, whereas later, during early and late differentiation, the preferred targets are intronic/intergenic sequences with enhancer-like activity. (5) Differentially methylated genes are enriched in sequential neurodevelopmental functions (such as progenitor proliferation, migration, neuritogenesis, and synaptic transmission); upregulated genes represent current and consecutive stage-specific functions, and downregulated genes represent preceding functions that are no longer required. The main conclusion of our work is that the neuronal methylation/demethylation program is predominantly developmental with minimal lineage specificity, except in the final stage of development when neuron subtype-specific differences also emerge. SIGNIFICANCE STATEMENT Our work is the first to describe a set of

  9. Hypomethylation of host cell DNA synthesized after infection or transformation of cells by herpes simplex virus

    SciTech Connect

    Macnab, J.C.M.; Adams, R.L.P.; Rinaldi, A.; Orr, A.; Clark, L.

    1988-04-01

    Infection of rat embryo cells with herpes simplex virus type 2 caused undermethylation of host cell DNA synthesized during infection. DNA made prior to infection was not demethylated, but some of its degradation products, including methyl dCMP, were incorporated into viral DNA. The use of mutant virus showed that some viral DNA synthesis appears to be required for the inhibition of methylation. Inhibition of methylation cannot be explained by an absence of DNA methyltransferase as the activity of this enzyme did not change during the early period of infection. Inhibition of host cell DNA methylation may be an important step in the transformation of cells by herpesviruses, and various transformed cell lines tested showed reduced levels of DNA methylation.

  10. PSEN1 promoter demethylation in hyperhomocysteinemic TgCRND8 mice is the culprit, not the consequence.

    PubMed

    Fuso, Andrea; Cavallaro, Rosaria A; Cavallaroa, Rosaria A; Nicolia, Vincenzina; Scarpa, Sigfrido

    2012-06-01

    In recent years, in parallel with the growing awareness of the multifactorial nature of Late Onset Alzheimer's Disease, the possibility that epigenetic mechanisms could be involved in the onset and/or progression of the pathology assumed an increasingly intriguing and leading role in Alzheimer's research. Today, many scientific reports indicate the existence of an epigenetic drift during ageing, in particular in Alzheimer's subjects. At the same time, experimental evidences are provided with the aim to demonstrate the causative or consequential role of epigenetic mechanisms. Our research group was involved in the last ten years in studying DNA methylation, the main epigenetic modification, in relationship to altered one-carbon metabolism (namely high homocysteine and low B vitamins levels), in Alzheimer's experimental models. Our previous findings about the demethylation of Presenilin1 gene promoter in nutritionally-induced hyperhomocysteinemia in a transgenic mouse model clearly demonstrated that Presenilin1 is regulated by DNA methylation. One of the open questions raised by our studies was if the observed demethylation was solely due to the induced imbalance of one-carbon metabolism or could be a response to the massive deposition of amyloid plaques in transgenic mice. Here we analyzed old (10 months) mice under standard diet in order to evidence possible changes in Presenilin1 promoter methylation in transgenic (TgCRND8 mice, carrying a double-mutated human APP transgene) vs. wt mice (129Sv) after prolonged exposure to amyloid. We found no differences in Presenilin1 methylation despite a slight increase in gene expression; these results suggest that amyloid production is not responsible for Presenilin1 demethylation in TgCRND8 mice brain. PMID:22272624

  11. Redistribution of demethylated RNA helicase A during foot-and-mouth disease virus infection: Role of Jumonji C-domain containing protein 6 in RHA demethylation

    SciTech Connect

    Lawrence, Paul; Conderino, Joseph S.; Rieder, Elizabeth

    2014-03-15

    Previously, RNA helicase A (RHA) re-localization from the nucleus to the cytoplasm in foot-and-mouth disease virus (FMDV) infected cells was shown to coincide with loss of RHA methylated arginine residues at its C-terminus. The potential interaction between RHA and Jumonji C-domain (JmjC) protein 6 (JMJD6) arginine demethylase in infected cells was investigated. Treatment with N-oxalylglycine (NOG) inhibitor of JmjC demethylases prevented FMDV-induced RHA demethylation and re-localization, and also decreased viral protein synthesis and virus titers. Physical interaction between JMJD6 and RHA was demonstrated via reciprocal co-immunoprecipitation, where RHA preferentially bound JMJD6 monomers. Nuclear efflux of demethylated RHA (DM-RHA) coincided with nuclear influx of JMJD6, which was not observed using another picornavirus. A modified biochemical assay demonstrated JMJD6 induced dose-dependent demethylation of RHA and two RHA-derived isoforms, which could be inhibited by NOG. We propose a role for JMJD6 in RHA demethylation stimulated by FMDV, that appears to facilitate virus replication. - Highlights: • We examined the role of JMJD6 in FMDV-induced RHA demethylation process. • Using an arginine demethylation assay showed that JMJD6 is involved in RHA demethylation. • A demethylases inhibitor reduced cytoplasmic accumulation of RHA and FMDV titers.

  12. Dss1 Release Activates DNA Binding Potential in Brh2

    PubMed Central

    Zhou, Qingwen; Kojic, Milorad; Holloman, William K.

    2013-01-01

    Dss1 is an intrinsically unstructured polypeptide that partners with the much larger Brh2 protein, the BRCA2 ortholog in Ustilago maydis, to form a tight complex. Mutants lacking Dss1 have essentially the same phenotype as mutants defective in Brh2, implying that through physical interaction Dss1 serves as a positive activator of Brh2. Dss1 associates with Brh2 through an interaction surface in the carboxy-terminal region. Certain derivatives of Brh2 lacking this interaction surface remain highly competent in DNA repair as long as a DNA-binding domain is present. However, the Dss1-independent activity raises the question of what function might be met in the native protein by having Brh2 under Dss1 control. Using a set of Brh2 fusions and truncated derivatives, we show here that Dss1 is capable of exerting control when there is a cognate Dss1-interacting surface present. We find that association of Dss1 attenuates the DNA binding potential of Brh2 and that the amino-terminal domain of Brh2 helps evict Dss1 from its carboxy-terminal interaction surface. The findings presented here add to the notion that Dss1 serves in a regulatory capacity to dictate order in association of Brh2’s amino-terminal and carboxy-terminal domains with DNA. PMID:23094644

  13. Prediction of fine-tuned promoter activity from DNA sequence

    PubMed Central

    Siwo, Geoffrey; Rider, Andrew; Tan, Asako; Pinapati, Richard; Emrich, Scott; Chawla, Nitesh; Ferdig, Michael

    2016-01-01

    The quantitative prediction of transcriptional activity of genes using promoter sequence is fundamental to the engineering of biological systems for industrial purposes and understanding the natural variation in gene expression. To catalyze the development of new algorithms for this purpose, the Dialogue on Reverse Engineering Assessment and Methods (DREAM) organized a community challenge seeking predictive models of promoter activity given normalized promoter activity data for 90 ribosomal protein promoters driving expression of a fluorescent reporter gene. By developing an unbiased modeling approach that performs an iterative search for predictive DNA sequence features using the frequencies of various k-mers, inferred DNA mechanical properties and spatial positions of promoter sequences, we achieved the best performer status in this challenge. The specific predictive features used in the model included the frequency of the nucleotide G, the length of polymeric tracts of T and TA, the frequencies of 6 distinct trinucleotides and 12 tetranucleotides, and the predicted protein deformability of the DNA sequence. Our method accurately predicted the activity of 20 natural variants of ribosomal protein promoters (Spearman correlation r = 0.73) as compared to 33 laboratory-mutated variants of the promoters (r = 0.57) in a test set that was hidden from participants. Notably, our model differed substantially from the rest in 2 main ways: i) it did not explicitly utilize transcription factor binding information implying that subtle DNA sequence features are highly associated with gene expression, and ii) it was entirely based on features extracted exclusively from the 100 bp region upstream from the translational start site demonstrating that this region encodes much of the overall promoter activity. The findings from this study have important implications for the engineering of predictable gene expression systems and the evolution of gene expression in naturally occurring

  14. The structure and duplex context of DNA interstrand crosslinks affects the activity of DNA polymerase η

    PubMed Central

    Roy, Upasana; Mukherjee, Shivam; Sharma, Anjali; Frank, Ekaterina G.; Schärer, Orlando D.

    2016-01-01

    Several important anti-tumor agents form DNA interstrand crosslinks (ICLs), but their clinical efficiency is counteracted by multiple complex DNA repair pathways. All of these pathways require unhooking of the ICL from one strand of a DNA duplex by nucleases, followed by bypass of the unhooked ICL by translesion synthesis (TLS) polymerases. The structures of the unhooked ICLs remain unknown, yet the position of incisions and processing of the unhooked ICLs significantly influence the efficiency and fidelity of bypass by TLS polymerases. We have synthesized a panel of model unhooked nitrogen mustard ICLs to systematically investigate how the state of an unhooked ICL affects pol η activity. We find that duplex distortion induced by a crosslink plays a crucial role in translesion synthesis, and length of the duplex surrounding an unhooked ICL critically affects polymerase efficiency. We report the synthesis of a putative ICL repair intermediate that mimics the complete processing of an unhooked ICL to a single crosslinked nucleotide, and find that it provides only a minimal obstacle for DNA polymerases. Our results raise the possibility that, depending on the structure and extent of processing of an ICL, its bypass may not absolutely require TLS polymerases. PMID:27257072

  15. Effects of fish CYP inducers on difloxacin N-demethylation in kidney cell of Chinese idle (Ctenopharyngodon idellus).

    PubMed

    Yu, Ling Zhi; Yang, Xian Le; Wang, Xiang Ling; Yu, Wen Juan; Hu, Kun

    2010-09-01

    A drug-drug interaction occurs when the effect of one drug is altered by the presence of another drug which is generally associated with the induction of cytochrome P450s (CYPs) activity. Thus, unexpected treatment failures often happen resulting from inappropriate coadministration in fisheries. However, little information is available about CYP induction in fish. The reaction of difloxacin (DIF) biotransformation to sarafloxacin (SAR) belongs to N-demethylation catalyzed mainly by CYP(s). In order to supply useful information on CYP induction, the present study assessed the effects of fish-specific CYP inducers on DIF N-demethylation and enzyme kinetics in kidney cell of Chinese idle (CIK; grass carp (Ctenopharyngodon idellus)) by RP-HPLC. Results demonstrated that the amounts of SAR formation and enzymatic parameters Clint and Vmax were significantly increased due to beta-naphthoflavone (BNF) pretreatment. Therefore, we suggest that CYP1A may be involved in DIF N-demethylation in CIK. This study provides instructive information to ensure treatment success via avoiding CYP induction in fisheries. PMID:19685219

  16. The dynamic changes of DNA methylation in primordial germ cell differentiation.

    PubMed

    Duan, Lian; Liu, Yongmei; Wang, Jie; Liao, Jiangquan; Hu, Junyuan

    2016-10-15

    The discovery of DNA methylation has provided a new perspective on how DNA may be dynamically regulated in the mammalian genome. DNA methylation is a dynamic process with a demethylation and de novo methylation from primordial germ cell to differentiation. DNA methylation and demethylation have been proposed to play important roles in somatic cell reprogramming. Some essential components were discussed, such as hydroxymethylation which has recently been confirmed as a modification of developmental importance. PMID:27320728

  17. A DNA enzyme with Mg(2+)-Dependent RNA Phosphoesterase Activity

    NASA Technical Reports Server (NTRS)

    Breaker, Ronald R.; Joyce, Gerald F.

    1995-01-01

    Previously we demonstrated that DNA can act as an enzyme in the Pb(2+)-dependent cleavage of an RNA phosphoester. This is a facile reaction, with an uncatalyzed rate for a typical RNA phosphoester of approx. 10(exp -4)/ min in the presence of 1 mM Pb(OAc)2 at pH 7.0 and 23 C. The Mg(2+) - dependent reaction is more difficult, with an uncatalyzed rate of approx. 10(exp -7)/ min under comparable conditions. Mg(2+) - dependent cleavage has special relevance to biology because it is compatible with intracellular conditions. Using in vitro selection, we sought to develop a family of phosphoester-cleaving DNA enzymes that operate in the presence of various divalent metals, focusing particularly on the Mg(2+) - dependent reaction. Results: We generated a population of greater than 10(exp 13) DNAs containing 40 random nucleotides and carried out repeated rounds of selective amplification, enriching for molecules that cleave a target RNA phosphoester in the presence of 1 mM Mg(2+), Mn(2+), Zn(2+) or Pb(2+). Examination of individual clones from the Mg(2+) lineage after the sixth round revealed a catalytic motif comprised of a three-stem junction.This motif was partially randomized and subjected to seven additional rounds of selective amplification, yielding catalysts with a rate of 0.01/ min. The optimized DNA catalyst was divided into separate substrate and enzyme domains and shown to have a similar level of activity under multiple turnover conditions. Conclusions: We have generated a Mg(2+) - dependent DNA enzyme that cleaves a target RNA phosphoester with a catalytic rate approx. 10(exp 5) - fold greater than that of the uncatalyzed reaction. This activity is compatible with intracellular conditions, raising the possibility that DNA enzymes might be made to operate in vivo.

  18. Sequence-specific DNA primer effects on telomerase polymerization activity.

    PubMed Central

    Lee, M S; Blackburn, E H

    1993-01-01

    The ribonucleoprotein enzyme telomerase synthesizes one strand of telomeric DNA by copying a template sequence within the RNA moiety of the enzyme. Kinetic studies of this polymerization reaction were used to analyze the mechanism and properties of the telomerase from Tetrahymena thermophila. This enzyme synthesizes TTGGGG repeats, the telomeric DNA sequence of this species, by elongating a DNA primer whose 3' end base pairs with the template-forming domain of the RNA. The enzyme was found to act nonprocessively with short (10- to 12-nucleotide) primers but to become processive as TTGGGG repeats were added. Variation of the 5' sequences of short primers with a common 3' end identified sequence-specific effects which are distinct from those involving base pairing of the 3' end of the primer with the RNA template and which can markedly induce enzyme activity by increasing the catalytic rate of the telomerase polymerization reaction. These results identify an additional mechanistic basis for telomere and DNA end recognition by telomerase in vivo. Images PMID:8413255

  19. Powdered Activated Carbon: An Alternative Approach to Genomic DNA Purification.

    PubMed

    Barbarić, Lucija; Bačić, Ivana; Grubić, Zorana

    2015-07-01

    Forensic evidence samples are routinely found as stains on various substrates, which may contain substances known to inhibit polymerase chain reaction (PCR). The goal of this study was to evaluate post-Chelex(®) 100 purification using powdered activated carbon (PAC). Mock crime scene DNA extracts were analyzed using quantitative PCR and short tandem repeat (STR) profiling to test the DNA recovery and inhibitor removal using PAC with those of the Amicon(®) Ultra 100K. For extracted bloodstains on soil and wood substrates, PAC and Amicon(®) Ultra 100K generated similar DNA yield and quality. Moreover, the two methods significantly decreased the concentration of humic substances and tannins compared to nonpurified extracts (p < 0.001). In instances where extracts contained indigo dye (bloodstains on denim), Amicon(®) Ultra 100K performed better than PAC due to improved amplifiability. Efficient adsorption of humic substances and tannins, which are common inhibitors, indicates PAC's potential application in the purification of high-template DNA extracts. PMID:25929735

  20. Replication stress activates DNA repair synthesis in mitosis.

    PubMed

    Minocherhomji, Sheroy; Ying, Songmin; Bjerregaard, Victoria A; Bursomanno, Sara; Aleliunaite, Aiste; Wu, Wei; Mankouri, Hocine W; Shen, Huahao; Liu, Ying; Hickson, Ian D

    2015-12-10

    Oncogene-induced DNA replication stress has been implicated as a driver of tumorigenesis. Many chromosomal rearrangements characteristic of human cancers originate from specific regions of the genome called common fragile sites (CFSs). CFSs are difficult-to-replicate loci that manifest as gaps or breaks on metaphase chromosomes (termed CFS 'expression'), particularly when cells have been exposed to replicative stress. The MUS81-EME1 structure-specific endonuclease promotes the appearance of chromosome gaps or breaks at CFSs following replicative stress. Here we show that entry of cells into mitotic prophase triggers the recruitment of MUS81 to CFSs. The nuclease activity of MUS81 then promotes POLD3-dependent DNA synthesis at CFSs, which serves to minimize chromosome mis-segregation and non-disjunction. We propose that the attempted condensation of incompletely duplicated loci in early mitosis serves as the trigger for completion of DNA replication at CFS loci in human cells. Given that this POLD3-dependent mitotic DNA synthesis is enhanced in aneuploid cancer cells that exhibit intrinsically high levels of chromosomal instability (CIN(+)) and replicative stress, we suggest that targeting this pathway could represent a new therapeutic approach. PMID:26633632

  1. Demethylation of Veratrole by Cytochrome P-450 in Streptomyces setonii

    PubMed Central

    Sutherland, John B.

    1986-01-01

    The actinomycete Streptomyces setonii 75Vi2 demethylates vanillic acid and guaiacol to protocatechuic acid and catechol, respectively, and then metabolizes the products by the β-ketoadipate pathway. UV spectroscopy showed that this strain could also metabolize veratrole (1,2-dimethoxybenzene). When grown in veratrole-containing media supplemented with 2,2′-dipyridyl to inhibit cleavage of the aromatic ring, S. setonii accumulated catechol, which was detected by both liquid chromatography and gas chromatography. Reduced cell extracts from veratrole-grown cultures, but not sodium succinate-grown cultures, produced a carbon monoxide difference spectrum with a peak at 450 nm that indicated the presence of soluble cytochrome P-450. Addition of veratrole or guaiacol to oxidized cell extracts from veratrole-grown cultures produced difference spectra that indicated that these compounds were substrates for cytochrome P-450. My results suggest that S. setonii produces a cytochrome P-450 that is involved in the demethylation of veratrole and guaiacol to catechol, which is then catabolized by the β-ketoadipate pathway. PMID:16347120

  2. DNA-AuNPs based signal amplification for highly sensitive detection of DNA methylation, methyltransferase activity and inhibitor screening.

    PubMed

    Jing, Xiaoying; Cao, Xianqing; Wang, Li; Lan, Tian; Li, Yiyan; Xie, Guoming

    2014-08-15

    A sensitive and selective electrochemical method was developed for the detection of DNA methylation, determination of DNA methyltransferase (MTase) activity and screening of MTase inhibitor. Methylene blue (MB) was employed as electrochemical indicator and DNA-modified gold nanoparticles (AuNPs) were used as signal amplification unit because the DNA strands in this composite have strong adsorption ability for MB. First, the thiolated single-stranded DNA S1 was self-assembled on gold electrode, hybridization between the lower portion of DNA S1 and its complementary DNA S2 formed an identical double-stranded tetranucleotide target sequence for both DNA adenine methylation (Dam) MTase and methylation-resistant endonuclease Mbo I, then the upper portion of DNA S1 was hybridized with its complementary DNA S3 modified on AuNPs to bring the DNA S3-AuNPs amplification units onto the electrode. The DNA S1/S2/S3-AuNPs bioconjugate has lots of DNA strands, and they can adsorb abundant MB. Mbo I endounuclease could not cleave the identical target sequence after it was methylated by Dam MTase. On the contrary, the sequence without methylation could be cleaved, which would decrease the amount of adsorbed MB. The presence of redox-active MB was detected electrochemically by differential pulse voltammetry (DPV). Thus, the activity of Dam MTase and methylation status were sensitively converted to the DNA S3-AuNPs amplified DPV signals. The DPV signal demonstrated a linear relationship with logarithm of Dam concentration ranging from 0.075 to 30U/mL, achieving a detection limit of 0.02U/mL (S/N=3). Also, screening of Dam MTase inhibitor 5-fluorouracil was successfully investigated using this fabricated sensor. PMID:24613968

  3. DNA methylation as a regulatory mechanism in rat gamma-crystallin gene expression.

    PubMed Central

    Peek, R; Niessen, R W; Schoenmakers, J G; Lubsen, N H

    1991-01-01

    We have investigated the methylation state of the rat gamma-crystallin genes in DNA from lens cells at different developmental stages as well as from kidney and heart cells. A clear correlation between the extent of demethylation of the promoter and 5' gene regions and the expression of these genes was observed. No change in the methylation state of the far upstream or 3' regions of the genes was seen. The demethylation of the promoter region was shown to occur during the differentiation from the lens epithelial to the lens fiber cell. The effect of cytosine methylation on gamma-crystallin promoter activity was tested by measuring gamma-crystallin promoter/chloramphenicol acetyltransferase fusion gene expression after in vitro primed repair synthesis of the promoter region in the presence of either dCTP or 5mdCTP. The hemimethylated promoter was no longer capable of promoting high CAT activity after introduction into lens-like cells. Taken together, our data suggest that DNA demethylation may be the determining step in the developmental stage-specific expression of the rat gamma-crystallin genes. Images PMID:2011513

  4. Fission Yeast Pxd1 Promotes Proper DNA Repair by Activating Rad16XPF and Inhibiting Dna2

    PubMed Central

    Zhang, Jia-Min; Liu, Xiao-Man; Ding, Yue-He; Xiong, Liang-Yao; Ren, Jing-Yi; Zhou, Zhi-Xiong; Wang, Hai-Tao; Zhang, Mei-Jun; Yu, Yang; Dong, Meng-Qiu; Du, Li-Lin

    2014-01-01

    Structure-specific nucleases play crucial roles in many DNA repair pathways. They must be precisely controlled to ensure optimal repair outcomes; however, mechanisms of their regulation are not fully understood. Here, we report a fission yeast protein, Pxd1, that binds to and regulates two structure-specific nucleases: Rad16XPF-Swi10ERCC1 and Dna2-Cdc24. Strikingly, Pxd1 influences the activities of these two nucleases in opposite ways: It activates the 3′ endonuclease activity of Rad16-Swi10 but inhibits the RPA-mediated activation of the 5′ endonuclease activity of Dna2. Pxd1 is required for Rad16-Swi10 to function in single-strand annealing, mating-type switching, and the removal of Top1-DNA adducts. Meanwhile, Pxd1 attenuates DNA end resection mediated by the Rqh1-Dna2 pathway. Disabling the Dna2-inhibitory activity of Pxd1 results in enhanced use of a break-distal repeat sequence in single-strand annealing and a greater loss of genetic information. We propose that Pxd1 promotes proper DNA repair by differentially regulating two structure-specific nucleases. PMID:25203555

  5. Detection of DNA methyltransferase activity using allosteric molecular beacons.

    PubMed

    Zhang, Weiting; Zu, Xiaolong; Song, Yanling; Zhu, Zhi; Yang, Chaoyong James

    2016-01-21

    Abnormal DNA methylation patterns caused by altered DNA methyltransferase (MTase) activity are closely associated with cancer. Herein, using DNA adenine methylation methyltransferase (Dam MTase) as a model analyte, we designed an allosteric molecular beacon (aMB) for sensitive detection of Dam MTase activity. When the specific site in an aMB is methylated by Dam MTase, the probe can be cut by the restriction nuclease DpnI to release a fluorophore labeled aptamer specific for streptavidin (SA) which will bind to SA beads to generate highly fluorescent beads for easy signal readout by a microscope or flow cytometer. However, aMBs maintain a hairpin structure without the binding ability to SA beads in the absence of Dam MTase, leading to weakly fluorescent SA beads. Unlike the existing signal amplified assays, our method is simpler and more convenient. The high performance of the aptamer and the easy bead separation process make this probe superior to other methods for the detection of MTase in complex biological systems. Overall, the proposed method with a detection limit of 0.57 U mL(-1) for Dam MTase shows great potential for further applications in the detection of other MTases, screening of MTase inhibitors, and early diagnosis of cancer. PMID:26478921

  6. Hydrogen atom reactions in coal liquefaction. [Demethylation of methylnaphthalene by hydrogen

    SciTech Connect

    Bockrath, B.C.; Schroeder, K.T.; Keldsen, G.L.

    1985-06-01

    Hydrogen atom reactions were investigated in the demethylation of methylnaphthalenes at 450/sup 0/C. Demethylation by the hydrogen atom at the 1-position was about 4 times faster than at the 2-position. The methylnaphthalenes were somewhat more reactive toward hydrocracking than was bibenzyl. The extent of hydrocracking was a function of hydrogen pressure and initiator concentration. 3 refs., 2 figs., 1 tab.

  7. Phylogenomics of caspase-activated DNA fragmentation factor

    SciTech Connect

    Eckhart, Leopold . E-mail: leopold.eckhart@meduniwien.ac.at; Fischer, Heinz; Tschachler, Erwin

    2007-04-27

    The degradation of nuclear DNA by DNA fragmentation factor (DFF) is a key step in apoptosis of mammalian cells. Using comparative genomics, we have here determined the evolutionary history of the genes encoding the two DFF subunits, DFFA (also known as ICAD) and DFFB (CAD). Orthologs of DFFA and DFFB were identified in Nematostella vectensis, a representative of the primitive metazoan clade cnidarians, and in various vertebrates and insects, but not in representatives of urochordates, echinoderms, and nematodes. The domains mediating the interaction of DFFA and DFFB, a caspase cleavage site in DFFA, and the amino acid residues critical for endonuclease activity of DFFB were conserved in Nematostella. These findings suggest that DFF has been a part of the primordial apoptosis system of the eumetazoan common ancestor and that the ancient cell death machinery has degenerated in several evolutionary lineages, including the one leading to the prototypical apoptosis model, Caenorhabditis elegans.

  8. Metabolites and DNA adduct formation from flavoenzyme-activated porfiromycin.

    PubMed

    Pan, S S; Iracki, T

    1988-08-01

    Porfiromycin was reductively metabolized by NADPH cytochrome P-450 reductase and xanthine oxidase under anaerobic conditions. The production of metabolites varied with the pH and the contents of the reaction buffer. In Tris buffer, two major metabolites were produced at pH 7.5 and above, whereas one major metabolite was produced at pH 6.5. The three major metabolites were separated and isolated by HPLC. Identification by californium-252 plasma desorption mass spectrometry showed that the two major metabolites from pH 7.5 were (trans) and (cis)-forms of 7-amino-1-hydroxyl-2-methylaminomitosene and the major metabolite from pH 6.5 was 7-amino-2-methylaminomitosene. All three major metabolites showed substitutions at the C-1 position. DNA was alkylated readily by enzyme-activated porfiromycin. Digestion of porfiromycin-alkylated DNA by DNase, snake venom phosphodiesterase, and alkaline phosphatase resulted in an insoluble nuclease-resistant fraction and a soluble fraction. The nuclease-resistant fraction reflected a high content of cross-linked adducts. Upon HPLC analysis, the solubilized fraction contained two monofunctionally linked porfiromycin adducts and a possibly cross-linked dinucleotide. The major adduct was isolated by HPLC and identified by NMR, as N2-(2'-deoxyguanosyl)-7-amino-2-methylaminomitosene. The N2 position of deoxyguanosine appeared as the major monofunctional alkylating site for DNA alkylation by porfiromycin. Thus, mitomycin C and porfiromycin (which differs from mitomycin C only by the addition of a methyl group to the aziridine nitrogen) share the same enzymatic activating mechanism that leads to the formation of the same types of metabolites and the same specificity of DNA alkylation. PMID:3412325

  9. Chromium reduces the in vitro activity and fidelity of DNA replication mediated by the human cell DNA synthesome

    SciTech Connect

    Dai Heqiao; Liu Jianying; Malkas, Linda H.; Catalano, Jennifer; Alagharu, Srilakshmi

    2009-04-15

    Hexavalent chromium Cr(VI) is known to be a carcinogenic metal ion, with a complicated mechanism of action. It can be found within our environment in soil and water contaminated by manufacturing processes. Cr(VI) ion is readily taken up by cells, and is recognized to be both genotoxic and cytotoxic; following its reduction to the stable trivalent form of the ion, chromium(Cr(III)), within cells. This form of the ion is known to impede the activity of cellular DNA polymerase and polymerase-mediated DNA replication. Here, we report the effects of chromium on the activity and fidelity of the DNA replication process mediated by the human cell DNA synthesome. The DNA synthesome is a functional multiprotein complex that is fully competent to carry-out each phase of the DNA replication process. The IC{sub 50} of Cr(III) toward the activity of DNA synthesome-associated DNA polymerases {alpha}, {delta} and {epsilon} is 15, 45 and 125 {mu}M, respectively. Cr(III) inhibits synthesome-mediated DNA synthesis (IC{sub 50} = 88 {mu}M), and significantly reduces the fidelity of synthesome-mediated DNA replication. The mutation frequency induced by the different concentrations of Cr(III) ion used in our assays ranges from 2-13 fold higher than that which occurs spontaneously, and the types of mutations include single nucleotide substitutions, insertions, and deletions. Single nucleotide substitutions are the predominant type of mutation, and they occur primarily at GC base-pairs. Cr(III) ion produces a lower number of transition and a higher number of transversion mutations than occur spontaneously. Unlike Cr(III), Cr(VI) ion has little effect on the in vitro DNA synthetic activity and fidelity of the DNA synthesome, but does significantly inhibit DNA synthesis in intact cells. Cell growth and proliferation is also arrested by increasing concentrations of Cr(VI) ion. Our studies provide evidence indicating that the chromium ion induced decrease in the fidelity and activity of

  10. Design and synthesis of novel 4'-demethyl-4-deoxypodophyllotoxin derivatives as potential anticancer agents.

    PubMed

    Zhu, Xiong; Fu, Junjie; Tang, Yan; Gao, Yuan; Zhang, Shijin; Guo, Qinglong

    2016-02-15

    A group of podophyllotoxin (PPT) derivatives (7a-j) were synthesized by conjugating aryloxyacetanilide moieties to the 4'-hydroxyl of 4'-demethyl-4-deoxypodophyllotoxin (DDPT), and their anticancer activity was evaluated. It was found that the most potent compound 7d inhibited the proliferation of three cancer cell lines with sub to low micromolar IC50 values. Furthermore, it was demonstrated that 7d induced cell cycle arrest in G2/M phase in MGC-803 cells, and regulated the expression of cell cycle check point proteins, such as cyclin A, cyclin B, CDK1, cdc25c, and p21. Finally, 4 mg/kg of 7d reduced the weights and volumes of HepG2 xenografts in mice. Our findings suggest that 7d might be a potential anticancer agent. PMID:26804229

  11. Crystal Structure of Dicamba Monooxygenase: A Rieske Nonheme Oxygenase that Catalyzes Oxidative Demethylation

    SciTech Connect

    Dumitru, Razvan; Jiang, Wen Zhi; Weeks, Donald P.; Wilson, Mark A.

    2009-08-28

    Dicamba (3,6-dichloro-2-methoxybenzoic acid) is a widely used herbicide that is efficiently degraded by soil microbes. These microbes use a novel Rieske nonheme oxygenase, dicamba monooxygenase (DMO), to catalyze the oxidative demethylation of dicamba to 3,6-dichlorosalicylic acid (DCSA) and formaldehyde. We have determined the crystal structures of DMO in the free state, bound to its substrate dicamba, and bound to the product DCSA at 2.10-1.75 {angstrom} resolution. The structures show that the DMO active site uses a combination of extensive hydrogen bonding and steric interactions to correctly orient chlorinated, ortho-substituted benzoic-acid-like substrates for catalysis. Unlike other Rieske aromatic oxygenases, DMO oxygenates the exocyclic methyl group, rather than the aromatic ring, of its substrate. This first crystal structure of a Rieske demethylase shows that the Rieske oxygenase structural scaffold can be co-opted to perform varied types of reactions on xenobiotic substrates.

  12. Suppression of Gluconeogenic Gene Expression by LSD1-Mediated Histone Demethylation

    PubMed Central

    Pan, Dongning; Mao, Chunxiao; Wang, Yong-Xu

    2013-01-01

    Aberrant gluconeogenic gene expression is associated with diabetes, glycogen storage disease, and liver cancer. However, little is known how these genes are regulated at the chromatin level. In this study, we investigated in HepG2 cells whether histone demethylation is a potential mechanism. We found that knockdown or pharmacological inhibition of histone demethylase LSD1 causes remarkable transcription activation of two gluconeogenic genes, FBP1 and G6Pase, and consequently leads to increased de novo glucose synthesis and decreased intracellular glycogen content. Mechanistically, LSD1 occupies the promoters of FBP1 and G6Pase, and modulates their H3K4 dimethylation levels. Thus, our work identifies an epigenetic pathway directly governing gluconeogenic gene expression, which might have important implications in metabolic physiology and diseases. PMID:23755305

  13. Demethylation of the aryl hydrocarbon receptor repressor as a biomarker for nascent smokers

    PubMed Central

    Philibert, Robert A.; Beach, Steven R.H.; Brody, Gene H.

    2012-01-01

    Epigenetic modifications to peripheral white blood cell DNA occur in response to a wide variety of exposures. In prior work, we and others have shown that broad changes in DNA methylation, particularly at the aryl hydrocarbon receptor repressor (AHRR) locus, occur in samples from subjects with long histories of smoking. However, given the large number of epigenetic changes that occur in response to prolonged smoking, the primacy of the response at AHRR and the sensitivity of these changes to low levels of smoking are not known. Therefore, we examined the association of smoking to genome lymphocyte DNA methylation status in a representative sample of 399 African American youths living in the rural South that includes 72 subjects with less than one half-pack year of exposure. Consistent with our prior findings, we found a stepwise effect of smoking on DNA methylation among youth with relatively brief exposure histories at a CpG residue in AHRR (cg05575921) (FDR corrected p values; 3 × 10−7 and 0.09 in the male and female samples, respectively) that was identified in previous studies and at which the effects of smoking were significant, even in those subjects with less than one half pack year exposure. We conclude that AHRR demethylation at cg05575921 in peripheral cells may serve as an early, sensitive biomarker for even low levels of exposure to tobacco smoke, providing a non-self-report alternative for nascent exposure to tobacco smoke. We also suggest that the AHRR/AHR pathway may be functional in the response of peripheral white blood cells to tobacco smoke exposure. PMID:23070629

  14. Suppression of Wnt signaling by the miR-29 family is mediated by demethylation of WIF-1 in non-small-cell lung cancer

    SciTech Connect

    Tan, Min; Wu, Junjie; Cai, Yong

    2013-09-06

    Highlights: •Dnmt3A and Dnmt3B are involved in the down-regulation of WIF-1 expression in non-small-cell lung cancer. •MiR-29 family members could restore WIF-1 expression through demethylation. •MiR-29s suppress Wnt/β-catenin signaling pathway and inhibit tumor growth. •The expression of miR-29a and miR-29b could be regulated partially in a positive feedback loop. -- Abstract: Wnt inhibitory factor-1 (WIF-1) silencing induced by promoter hypermethylation is a common mechanism of aberrant activation of the Wnt signaling pathway in non-small-cell lung cancer (NSCLC). However, the activity of regulators associated with the methylation of the WIF-1 gene remains unclear. Here, we investigated the role of three DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) in the expression of WIF-1. The three DNMTs were up-regulated in NSCLC tumor tissues and suppression of DNMT3A and DNMT3B restored the expression of WIF-1 in NSCLC cells. The miR-29 family (miR-29a, -29b, and -29c), which negatively regulates DNMT3A and DNMT3B, was examined in association with the Wnt/β-catenin signaling pathway. A positive correlation between the expression of WIF-1 and that of MiR-29s was observed in NSCLC tissues. Methylation-specific PCR and Western blotting indicated that miR-29s positively regulate WIF-1 expression by inhibiting the methylation of its promoter. Furthermore, miR-29 overexpression downregulated β-catenin expression, inhibited cell proliferation and induced apoptosis. The expression of miR-29a and miR-29b was partially regulated by DNMT3A and DNMT3B in a positive feedback loop. Taken together, our findings show that miR-29s suppress the Wnt signaling pathway through demethylation of WIF-1 in NSCLC.

  15. Nanoplasmonic molecular ruler for nuclease activity and DNA footprinting

    DOEpatents

    Chen, Fanqing Frank; Liu, Gang L; Lee, Luke P

    2013-10-29

    This invention provides a nanoplasmonic molecular ruler, which can perform label-free and real-time monitoring of nucleic acid (e.g., DNA) length changes and perform nucleic acid footprinting. In various embodiments the ruler comprises a nucleic acid attached to a nanoparticle, such that changes in the nucleic acid length are detectable using surface plasmon resonance. The nanoplasmonic ruler provides a fast and convenient platform for mapping nucleic acid-protein interactions, for nuclease activity monitoring, and for other footprinting related methods.

  16. Synthesis, characterization and chemoprotective activity of polyoxovanadates against DNA alkylation.

    PubMed

    Nunes, Giovana G; Bonatto, Ana C; de Albuquerque, Carla G; Barison, Andersson; Ribeiro, Ronny R; Back, Davi F; Andrade, André Vitor C; de Sá, Eduardo L; Pedrosa, Fábio de O; Soares, Jaísa F; de Souza, Emanuel M

    2012-03-01

    The alkylation of pUC19 plasmid DNA has been employed as a model reaction for the first studies on chemoprotective action by a mixed-valence (+IV/+V) polyoxovanadate. A new, non-hydrothermal route for the high yield preparation of the test compound is described. The deep green, microcrystalline solid A was isolated after a three-day reaction in water at 80°C and 1 atm, while the reaction at 100°C gave green crystals of B. Both solids were structurally characterized by X-ray diffractometry and FTIR, EPR, NMR and Raman spectroscopies. Product A was identified as (NH(4))(2)V(3)O(8), while B corresponds to the spherical polyoxoanion [V(15)O(36)(Cl)](6-), isolated as the NMe(4)(+) salt. The lack of solubility of A in water and buffers prevented its use in DNA interaction studies, which were then carried out with B. Complex B was also tested for its ability to react with DNA alkylating agents by incubation with diethylsulphate (DES) and dimethylsulphate (DMS) in both the absence and presence of pUC19. For DMS, the best results were obtained with 10 mM of B (48% protection); with DES, this percentage increased to 70%. The direct reaction of B with increasing amounts of DMS in both buffered (PIPES 50 mM) and non-buffered aqueous solutions revealed the sequential formation of several vanadium(IV), vanadium(V) and mixed-valence aggregates of different nuclearities, whose relevance to the DNA-protecting activity is discussed. PMID:22265837

  17. Assays for Hepatitis B Virus DNA-and RNA-Dependent DNA Polymerase Activities.

    PubMed

    Shaw, T; Locarnini, S A

    2000-01-01

    Genomes of the hepatitis B viruses (HBVs) consist of approx 3.2 kb of partly double-stranded DNA containing three or four overlapping open reading frames, the largest of which encodes the viral polymerase (Pol) protein. After entry into the cell and uncoating, the viral genome is transported to the nucleus where it is converted into a covalently closed circular (CCC) or supercoiled molecule by cellular repair mechanisms. The viral CCC DNA is transcribed, presumably by host cell RNA polymerase II, into unspliced, capped polyadenylated mRNA species from which viral proteins are transcribed. In addition, terminally redundant 3.5-kb RNA transcripts, which function as pregenomes, are produced and exported to the cytoplasm where they are packaged into viral core particles in which reverse transcription, pregenome degradation, and duplication occurs, reproducing the partly double-stranded HBV genome (for recent review, see ref. 1). Besides its essential role in HBV genome replication, HBV Pol is also involved in virus assembly, and because hepadnaviruses do not encode enzymes functionally equivalent to deoxynucleoside kinases (2), functions associated with HBV Pol are probably the only virus-specific targets for antiviral activity of nucleoside analogs. In vitro assays for inhibition of HBV Pol functions by deoxynucleoside triphosphate (dNTP) analogs are useful indicators but, because of restrictions imposed by hepatocyte enzymology, provide no guarantee of potential anti-HBV activity of the parent (deoxy)nucleoside analogs in intact cells (2). PMID:21331902

  18. A rapid, comprehensive system for assaying DNA repair activity and cytotoxic effects of DNA-damaging reagents.

    PubMed

    Jia, Nan; Nakazawa, Yuka; Guo, Chaowan; Shimada, Mayuko; Sethi, Mieran; Takahashi, Yoshito; Ueda, Hiroshi; Nagayama, Yuji; Ogi, Tomoo

    2015-01-01

    DNA repair systems protect cells from genomic instability and carcinogenesis. Therefore, assays for measuring DNA repair activity are valuable, not only for clinical diagnoses of DNA repair deficiency disorders but also for basic research and anticancer drug development. Two commonly used assays are UDS (unscheduled DNA synthesis, requiring a precise measurement of an extremely small amount of repair DNA synthesis) and RRS (recovery of RNA synthesis after DNA damage). Both UDS and RRS are major endpoints for assessing the activity of nucleotide excision repair (NER), the most versatile DNA repair process. Conventional UDS and RRS assays are laborious and time-consuming, as they measure the incorporation of radiolabeled nucleosides associated with NER. Here we describe a comprehensive protocol for monitoring nonradioactive UDS and RRS by studying the incorporation of alkyne-conjugated nucleoside analogs followed by a fluorescent azide-coupling click-chemistry reaction. The system is also suitable for quick measurement of cell sensitivity to DNA-damaging reagents and for lentivirus-based complementation assays, which can be used to systematically determine the pathogenic genes associated with DNA repair deficiency disorders. A typical UDS or RRS assay using primary fibroblasts, including a virus complementation test, takes 1 week to complete. PMID:25474029

  19. Reversible lysine acetylation is involved in DNA replication initiation by regulating activities of initiator DnaA in Escherichia coli

    PubMed Central

    Zhang, Qiufen; Zhou, Aiping; Li, Shuxian; Ni, Jinjing; Tao, Jing; Lu, Jie; Wan, Baoshan; Li, Shuai; Zhang, Jian; Zhao, Shimin; Zhao, Guo-Ping; Shao, Feng; Yao, Yu-Feng

    2016-01-01

    The regulation of chromosomal replication is critical and the activation of DnaA by ATP binding is a key step in replication initiation. However, it remains unclear whether and how the process of ATP-binding to DnaA is regulated. Here, we show that DnaA can be acetylated, and its acetylation level varies with cell growth and correlates with DNA replication initiation frequencies in E. coli. Specifically, the conserved K178 in Walker A motif of DnaA can be acetylated and its acetylation level reaches the summit at the stationary phase, which prevents DnaA from binding to ATP or oriC and leads to inhibition of DNA replication initiation. The deacetylation process of DnaA is catalyzed by deacetylase CobB. The acetylation process of DnaA is mediated by acetyltransferase YfiQ, and nonenzymatically by acetyl-phosphate. These findings suggest that the reversible acetylation of DnaA ensures cells to respond promptly to environmental changes. Since Walker A motif is universally distributed across organisms, acetylation of Walker A motif may present a novel regulatory mechanism conserved from bacteria to eukaryotes. PMID:27484197

  20. Metal-based biologically active compounds: synthesis, characterization, DNA interaction, antibacterial, cytotoxic and SOD mimic activities.

    PubMed

    Patel, Mohan N; Patel, Chintan R; Joshi, Hardik N

    2013-02-01

    The square pyramidal copper(II) complexes of N, O- donor ligand and ciprofloxacin have been synthesized. Synthesized complexes were characterized by physicochemical parameters like elemental analysis, electronic, FT-IR and LC-MS spectra. The complexes were screened for their antimicrobial activity against Gram(+Ve), i.e. Staphylococcus aureus, Bacillus subtilis, and Gram(-Ve), i.e. Serratia marcescens, Pseudomonas aeruginosa and Escherichia coli, microorganisms in terms of minimum inhibitory concentration and colony-forming unit. To determine the binding mode of complexes with Herring Sperm DNA, absorption titration and viscosity measurement were employed. DNA cleavage activity was carried out by gel electrophoresis experiment using supercoiled form of pUC19 DNA. The complexes were tested for their superoxide dismutase mimic activity in terms of IC(50) value. Synthesized complexes were also screened for their cytotoxicity using brine shrimp lethality assay method. PMID:23306896

  1. The Dynamics of DNA Methylation in Schizophrenia and Related Psychiatric Disorders

    PubMed Central

    Grayson, Dennis R; Guidotti, Alessandro

    2013-01-01

    Major psychiatric disorders such as schizophrenia (SZ) and bipolar disorder (BP) with psychosis (BP+) express a complex symptomatology characterized by positive symptoms, negative symptoms, and cognitive impairment. Postmortem studies of human SZ and BP+ brains show considerable alterations in the transcriptome of a variety of cortical structures, including multiple mRNAs that are downregulated in both inhibitory GABAergic and excitatory pyramidal neurons compared with non-psychiatric subjects (NPS). Several reports show increased expression of DNA methyltransferases in telencephalic GABAergic neurons. Accumulating evidence suggests a critical role for altered DNA methylation processes in the pathogenesis of SZ and related psychiatric disorders. The establishment and maintenance of CpG site methylation is essential during central nervous system differentiation and this methylation has been implicated in synaptic plasticity, learning, and memory. Atypical hypermethylation of candidate gene promoters expressed in GABAergic neurons is associated with transcriptional downregulation of the corresponding mRNAs, including glutamic acid decarboxylase 67 (GAD67) and reelin (RELN). Recent reports indicate that the methylation status of promoter proximal CpG dinucleotides is in a dynamic balance between DNA methylation and DNA hydroxymethylation. Hydroxymethylation and subsequent DNA demethylation is more complex and involves additional proteins downstream of 5-hydroxymethylcytosine, including members of the base excision repair (BER) pathway. Recent advances in our understanding of altered CpG methylation, hydroxymethylation, and active DNA demethylation provide a framework for the identification of new targets, which may be exploited for the pharmacological intervention of the psychosis associated with SZ and possibly BP+. PMID:22948975

  2. Genome-Wide Screening of Genes Regulated by DNA Methylation in Colon Cancer Development

    PubMed Central

    Galamb, Orsolya; Wichmann, Barna; Sipos, Ferenc; Péterfia, Bálint; Csabai, István; Kovalszky, Ilona; Semsey, Szabolcs; Tulassay, Zsolt; Molnár, Béla

    2012-01-01

    Tumorigenesis is accompanied by changes in the DNA methylation pattern. Our aim was to test a novel approach for identification of transcripts at whole transcript level which are regulated by DNA methylation. Our approach is based on comparison of data obtained from transcriptome profiling of primary human samples and in vitro cell culture models. Epithelial cells were collected by LCM from normal, adenoma, and tumorous colonic samples. Using gene expression analysis, we identified downregulated genes in the tumors compared to normal tissues. In parallel 3000 upregulated genes were determined in HT-29 colon adenocarcinoma cell culture model after DNA demethylation treatment. Of the 2533 transcripts showing reduced expression in the tumorous samples, 154 had increased expression as a result of DNA demethylation treatment. Approximately 2/3 of these genes had decreased expression already in the adenoma samples. Expression of five genes (GCG, NMES-1, LRMP, FAM161B and PTGDR), was validated using RT-PCR. PTGDR showed ambiguous results, therefore it was further studied to verify the extent of DNA methylation and its effect on the protein level. Results confirmed that our approach is suitable for genome-wide screening of genes which are regulated or inactivated by DNA methylation. Activity of these genes possibly interferes with tumor progression, therefore genes identified can be key factors in the formation and in the progression of the disease. PMID:23049694

  3. Allele-specific DNA methylation reinforces PEAR1 enhancer activity.

    PubMed

    Izzi, Benedetta; Pistoni, Mariaelena; Cludts, Katrien; Akkor, Pinar; Lambrechts, Diether; Verfaillie, Catherine; Verhamme, Peter; Freson, Kathleen; Hoylaerts, Marc F

    2016-08-18

    Genetic variation in the PEAR1 locus is linked to platelet reactivity and cardiovascular disease. The major G allele of rs12041331, an intronic cytosine guanine dinucleotide-single-nucleotide polymorphism (CpG-SNP), is associated with higher PEAR1 expression in platelets and endothelial cells than the minor A allele. The molecular mechanism underlying this difference remains elusive. We have characterized the histone modification profiles of the intronic region surrounding rs12041331 and identified H3K4Me1 enhancer-specific enrichment for the region that covers the CpG-SNP. Interestingly, methylation studies revealed that the CpG site is fully methylated in leukocytes of GG carriers. Nuclear protein extracts from megakaryocytes, endothelial cells, vs control HEK-293 cells show a 3-fold higher affinity for the methylated G allele compared with nonmethylated G or A alleles in a gel electrophoretic mobility shift assay. To understand the positive relationship between methylation and gene expression, we studied DNA methylation at 4 different loci of PEAR1 during in vitro megakaryopoiesis. During differentiation, the CpG-SNP remained fully methylated, while we observed rapid methylation increases at the CpG-island overlapping the first 5'-untranslated region exon, paralleling the increased PEAR1 expression. In the same region, A-allele carriers of rs12041331 showed significantly lower DNA methylation at CGI1 compared with GG homozygote. This CpG-island contains binding sites for the methylation-sensitive transcription factor CTCF, whose binding is known to play a role in enhancer activation and/or repression. In conclusion, we report the molecular characterization of the first platelet function-related CpG-SNP, a genetic predisposition that reinforces PEAR1 enhancer activity through allele-specific DNA methylation. PMID:27313330

  4. Decitabine-induced demethylation of 5' CpG island in GADD45A leads to apoptosis in osteosarcoma cells.

    PubMed

    Al-Romaih, Khaldoun; Sadikovic, Bekim; Yoshimoto, Maisa; Wang, Yuzhuo; Zielenska, Maria; Squire, Jeremy A

    2008-05-01

    GADD45 genes are epigenetically inactivated in various types of cancer and tumor cell lines. To date, defects of the GADD45 gene family have not been implicated in osteosarcoma (OS) oncogenesis, and the role of this pathway in regulating apoptosis in this tumor is unknown. The therapeutic potential of Gadd45 in OS emerged when our previous studies showed that GADD45A was reexpressed by treatment with the demethylation drug decitabine. In this study, we analyze the OS cell lines MG63 and U2OS and show that on treatment with decitabine, a significant loss of DNA methylation of GADD45A was associated with elevated expression and induction of apoptosis. In vivo affects of decitabine treatment in mice showed that untreated control xenografts exhibited low nuclear staining for Gadd45a protein, whereas the nuclei from xenografts in decitabine-treated mice exhibited increased amounts of protein and elevated apoptosis. To show the specificity of this gene for decitabine-induced apoptosis in OS, GADD45A mRNAs were disrupted using short interference RNA, and the ability of the drug to induce apoptosis was reduced. Understanding the role of demethylation of GADD45A in reexpression of this pathway and restoration of apoptotic control is important for understanding OS oncogenesis and for more targeted therapeutic approaches. PMID:18472964

  5. DNA Fingerprinting Using PCR: A Practical Forensic Science Activity

    ERIC Educational Resources Information Center

    Choi, Hyun-Jung; Ahn, Jung Hoon; Ko, Minsu

    2008-01-01

    This paper describes a forensic science simulation programme applicable for use in colleges. Students were asked to find a putative suspect by DNA fingerprinting using a simple protocol developed in this study. DNA samples were obtained from a hair root and a drop of blood, common sources of DNA in forensic science. The DNA fingerprinting protocol…

  6. DNA ligase III is the major high molecular weight DNA joining activity in SV40-transformed human fibroblasts: normal levels of DNA ligase III activity in Bloom syndrome cells.

    PubMed Central

    Tomkinson, A E; Starr, R; Schultz, R A

    1993-01-01

    The phenotypes of cultured cell lines established from individuals with Bloom syndrome (BLM), including an elevated spontaneous frequency of sister chromatid exchanges (SCEs), are consistent with a defect in DNA joining. We have investigated the levels of DNA ligase I and DNA ligase III in an SV40-transformed control and BLM fibroblast cell line, as well as clonal derivatives of the BLM cell line complemented or not for the elevated SCE phenotype. No differences in either DNA ligase I or DNA ligase III were detected in extracts from these cell lines. Furthermore, the data indicate that in dividing cultures of SV40-transformed fibroblasts, DNA ligase III contributes > 85% of high molecular weight DNA joining activity. This observation contrasts with previous studies in which DNA ligase I was reported to be the major DNA joining activity in extracts from proliferating mammalian cells. Images PMID:8265359

  7. Mild Glucose Starvation Induces KDM2A-Mediated H3K36me2 Demethylation through AMPK To Reduce rRNA Transcription and Cell Proliferation

    PubMed Central

    Tanaka, Yuji; Yano, Hirohisa; Ogasawara, Sachiko; Yoshioka, Sho-ichi; Imamura, Hiromi; Okamoto, Kengo

    2015-01-01

    Environmental conditions control rRNA transcription. Previously, we found that serum and glucose deprivation induces KDM2A-mediated H3K36me2 demethylation in the rRNA gene (rDNA) promoter and reduces rRNA transcription in the human breast cancer cell line MCF-7. However, the molecular mechanism and biological significance are still unclear. In the present study, we found that glucose starvation alone induced the KDM2A-dependent reduction of rRNA transcription. The treatment of cells with 2-deoxy-d-glucose, an inhibitor of glycolysis, reduced rRNA transcription and H3K36me2 in the rDNA promoter, both of which were completely dependent on KDM2A in low concentrations of 2-deoxy-d-glucose, that is, mild starvation conditions. The mild starvation induced these KDM2A activities through AMP-activated kinase (AMPK) but did not affect another AMPK effector of rRNA transcription, TIF-IA. In the triple-negative breast cancer cell line MDA-MB-231, the mild starvation also reduced rRNA transcription in a KDM2A-dependent manner. We detected KDM2A in breast cancer tissues irrespective of their estrogen receptor, progesterone receptor, and HER2 status, including triple-negative cancer tissues. In both MCF-7 and MDA-MB-231 cells, mild starvation reduced cell proliferation, and KDM2A knockdown suppressed the reduction of cell proliferation. These results suggest that under mild glucose starvation AMPK induces KDM2A-dependent reduction of rRNA transcription to control cell proliferation. PMID:26416883

  8. Conceptual links between DNA methylation reprogramming in the early embryo and primordial germ cells.

    PubMed

    Seisenberger, Stefanie; Peat, Julian R; Reik, Wolf

    2013-06-01

    DNA methylation is a carrier of important regulatory information that undergoes global reprogramming in the mammalian germ line, including pre-implantation embryos and primordial germ cells (PGCs). A flurry of recent studies have employed technical advances to generate global profiles of methylation and hydroxymethylation in these cells, unravelling the dynamics of methylation erasure at single locus resolution. Active demethylation in the zygote, involving extensive oxidation, is followed by passive loss over early cell divisions. Certain gamete-contributed methylation marks appear to have evolved non-canonical mechanisms for targeted maintenance of methylation in the face of these processes. These protected sequences include the imprinting control regions (ICRs) required for parental imprinting but also a surprising number of other regions. Such targeted maintenance mechanisms may also operate at certain sequences during early PGC migration when global passive demethylation occurs. In later gonadal PGCs, imprints must be reset and this may be achieved through the targeting of active mechanisms including oxidation. Thus, emerging evidence paints a complex picture whereby active and passive demethylation pathways operate synergistically and in parallel to ensure robust erasure in the early embryo and PGCs. PMID:23510682

  9. Detecting the movement and spawning activity of bigheaded carps with environmental DNA

    USGS Publications Warehouse

    Erickson, Richard A.; Rees, Christopher B.; Coulter, Alison A.; Merkes, Christopher; McCalla, Sunnie; Touzinsky, Katherine F; Walleser, Liza R.; Goforth, Reuben R.; Amberg, Jon

    2016-01-01

    Bigheaded carps are invasive fishes threatening to invade the Great Lakes basin and establish spawning populations, and have been monitored using environmental DNA (eDNA). Not only does eDNA hold potential for detecting the presence of species, but may also allow for quantitative comparisons like relative abundance of species across time or space. We examined the relationships among bigheaded carp movement, hydrography, spawning and eDNA on the Wabash River, IN, USA. We found positive relationships between eDNA and movement and eDNA and hydrography. We did not find a relationship between eDNA and spawning activity in the form of drifting eggs. Our first finding demonstrates how eDNA may be used to monitor species abundance, whereas our second finding illustrates the need for additional research into eDNA methodologies. Current applications of eDNA are widespread, but the relatively new technology requires further refinement.

  10. Detecting the movement and spawning activity of bigheaded carps with environmental DNA.

    PubMed

    Erickson, Richard A; Rees, Christopher B; Coulter, Alison A; Merkes, Christopher M; McCalla, Sunnie G; Touzinsky, Katherine F; Walleser, Liza; Goforth, Reuben R; Amberg, Jon J

    2016-07-01

    Bigheaded carps are invasive fishes threatening to invade the Great Lakes basin and establish spawning populations, and have been monitored using environmental DNA (eDNA). Not only does eDNA hold potential for detecting the presence of species, but may also allow for quantitative comparisons like relative abundance of species across time or space. We examined the relationships among bigheaded carp movement, hydrography, spawning and eDNA on the Wabash River, IN, USA. We found positive relationships between eDNA and movement and eDNA and hydrography. We did not find a relationship between eDNA and spawning activity in the form of drifting eggs. Our first finding demonstrates how eDNA may be used to monitor species abundance, whereas our second finding illustrates the need for additional research into eDNA methodologies. Current applications of eDNA are widespread, but the relatively new technology requires further refinement. PMID:27087387

  11. Phosphorylation-independent stimulation of DNA topoisomerase II alpha activity.

    PubMed

    Kimura, K; Saijo, M; Tanaka, M; Enomoto, T

    1996-05-01

    It has been suggested that casein kinase II phosphorylates DNA topoisomerase II alpha (topo II alpha) in mouse FM3A cells, by comparison of phosphopeptide maps of topo II alpha labeled in intact cells and of topo II alpha phosphorylated by various kinases in vitro. The phosphorylation of purified topo II alpha by casein kinase II, which attached a maximum of two phosphate groups per topo II alpha molecule, had no effect on the activity of topo II alpha. Dephosphorylation of purified topo II alpha by potato acid phosphatase, which almost completely dephosphorylated the topo II alpha, did not reduce the activity of topo II alpha. The incubation itself, regardless of phosphorylation or dephosphorylation status, stimulated the enzyme activity in both reactions. Topo II alpha activity was stimulated by incubation in a medium containing low concentrations of glycerol but not in that containing high concentrations of glycerol, such as the 50% in which purified topo II alpha is stored. The stimulation of topo II alpha activity by incubation was dependent on the concentration of topo II alpha, requiring a relatively high concentration of topo II alpha. PMID:8631919

  12. Length-dependent binding of human XLF to DNA and stimulation of XRCC4.DNA ligase IV activity.

    PubMed

    Lu, Haihui; Pannicke, Ulrich; Schwarz, Klaus; Lieber, Michael R

    2007-04-13

    An XRCC4-like factor, called XLF or Cernunnos, was recently identified as another important factor in the non-homologous DNA end joining (NHEJ) process. NHEJ is the major pathway for the repair of double-strand DNA breaks. The similarity in the putative secondary structures of XLF and XRCC4 as well as the association of XLF with XRCC4.DNA ligase IV in vivo suggested a role in the final ligation step of NHEJ. Here, we find that purified XLF directly interacts with purified XRCC4.DNA ligase IV complex and stimulates the ligase complex in a direct assay for ligation activity. Purified XLF has DNA binding activity, but this binding is dependent on DNA length in a manner most consistent with orientation of the C-terminal alpha helices parallel to the DNA helix. To better understand the function of XLF, we purified an XLF mutant (R57G), which was identified in patients with NHEJ deficiency and severe combined immunodeficiency. Surprisingly, the mutant protein retained its ability to stimulate XRCC4.DNA ligase IV but failed to translocate to the nucleus, and this appears to be the basis for the NHEJ defect in this patient. PMID:17317666

  13. Activation of an Mg2+-dependent DNA endonuclease of avian myeloblastosis virus alpha beta DNA polymerase by in vitro proteolytic cleavage.

    PubMed Central

    Grandgenett, D P; Golomb, M; Vora, A C

    1980-01-01

    Partial chymotryptic digestion of purified avian myeloblastosis virus alpha beta DNA polymerase resulted in the activation of a Mg2+-dependent DNA endonuclease activity. Incubation of the polymerase-protease mixture in the presence of super-coiled DNA and Mg2+ permitted detection of the cleaved polymerase fragment possessing DNA nicking activity. Protease digestion conditions were established permitting selective cleavage of beta to alpha, which contained DNA polymerase and RNase H activity and to a family of polypeptides ranging in size from 30,000 to 34,000 daltons. These latter beta-unique fragments were purified by polyuridylate-Sepharose 4B chromatography and were shown to contain both DNA binding and DNA endonuclease activities. We have demonstrated that this group of polymerase fragments derived by chymotryptic digestion of alpha beta DNA polymerase is similar to the in vivo-isolated avian myeloblastosis virus p32pol in size, sequence, and DNA endonuclease activity. Images PMID:6154149

  14. Interplay of RNA Pol IV and ROS1 during post-embryonic 5S rDNA chromatin remodeling.

    PubMed

    Douet, Julien; Blanchard, Bertrand; Cuvillier, Claudine; Tourmente, Sylvette

    2008-12-01

    We have investigated the chromatin structure of 5S rDNA, a heterochromatic pericentromeric tandemly repeated family, at 2, 3, 4 and 5 days post-germination. Our results revealed a large-scale reorganization of 5S rDNA chromatin that occurs during the first days of development. Unexpectedly, there is a decondensation followed by a 're'condensation of 5S rDNA chromatin, to obtain almost mature nuclei 5 d post-germination. The reorganization of 5S rDNA chromatin is accompanied by a rapid and active demethylation of 5S rDNA mediated by the ROS1 (repressor of silencing 1) demethylase, whereas the plant-specific RNA polymerase IV (Pol IV) is essential to the 5S chromatin 're'condensation. In conclusion, Pol IV and ROS1 collaborate to unlock the 5S rDNA chromatin inherited from the seed, and establish adult features. PMID:18845569

  15. Investigation of DNA binding, DNA photocleavage, topoisomerase I inhibition and antioxidant activities of water soluble titanium(IV) phthalocyanine compounds.

    PubMed

    Özel, Arzu; Barut, Burak; Demirbaş, Ümit; Biyiklioglu, Zekeriya

    2016-04-01

    The binding mode of water soluble peripherally tetra-substituted titanium(IV) phthalocyanine (Pc) compounds Pc1, Pc2 and Pc3 with calf thymus (CT) DNA was investigated by using UV-Vis spectroscopy and thermal denaturation studies in this work. The results of DNA binding constants (Kb) and the changes in the thermal denaturation profile of DNA with the addition of Pc compounds indicated that Pc1, Pc2 and Pc3 are able to bind to CT-DNA with different binding affinities. DNA photocleavage studies of Pc compounds were performed in the absence and presence of oxidizing agents such as hydrogen peroxide (H2O2), ascorbic acid (AA) and 2-mercaptoethanol (ME) using the agarose gel electrophoresis method at irradiation 650nm. According to the results of electrophoresis studies, Pc1, Pc2 and Pc3 cleaved of supercoiled pBR322 DNA via photocleavage pathway. The Pc1, Pc2 and Pc3 compounds were examined for topoisomerase I inhibition by measuring the relaxation of supercoiled pBR322 DNA. The all of Pc compounds inhibited topoisomerase I at 20μM concentration. A series of antioxidant assays, including 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, superoxide radical scavenging (SOD) assay and metal chelating effect assay were performed for Pc1, Pc2 and Pc3 compounds. The results of antioxidant assays indicated that Pc1, Pc2 and Pc3 compounds have remarkable superoxide radical scavenging activities, moderate 2,2-diphenyl-1-picrylhydrazyl activities and metal chelating effect activities. All the experimental studies showed that Pc1, Pc2 and Pc3 compounds bind to CT-DNA via minor groove binding, cleave of supercoiled pBR322 DNA via photocleavage pathway, inhibit topoisomerase I and have remarkable superoxide radical scavenging activities. Thanks to these properties the Pc1, Pc2 and Pc3 compounds are suitable agents for photo dynamic therapy. PMID:26882290

  16. Hairpin assembly-triggered cyclic activation of a DNA machine for label-free and ultrasensitive chemiluminescence detection of DNA.

    PubMed

    Chen, Jia; Qiu, Hongdeng; Zhang, Mingliang; Gu, Tongnian; Shao, Shijun; Huang, Yong; Zhao, Shulin

    2015-06-15

    DNA plays important regulatory roles in many life activities. Here, we have developed a novel label-free, ultrasensitive and specific chemiluminescence (CL) assay protocol for DNA detection based on hairpin assembly-triggered cyclic activation of a DNA machine. The system involves two hairpin structures, H1 and H2. Firstly, a target DNA binds with and opens the hairpin structure of H1. Then, H2 hybridizes with H1 and displaces the target DNA, which is used to trigger another new hybridization cycle between H1 and H2, leading to the generation of numerous H1-H2 complexes. The generated H1-H2 complexes are further activated with the help of polymerase and nicking enzyme, continuously yielding a large amount of G-riched DNA fragments. The G-riched DNA fragment products interact with hemin to form the activated HRP-mimicking DNAzymes that can catalyze the oxidation of luminol by H2O2 to produce strong CL signal resulting in an amplified sensing process. Our newly proposed homogeneous assay enables the quantitative measurement of p53 DNA (as a model) with a detection limit of 0.85 fM, which is at least 5 orders of magnitude lower than that of traditional unamplified homogeneous optical approaches. Moreover, this assay exhibits high discrimination ability even against a single base mismatch. In addition, this strategy is also capable of detecting p53 DNA in complex biological samples. The proposed sensing approach might hold a great promise for further applications in biomedical research and early clinical diagnosis. PMID:25638797

  17. Single-Molecule Electronic Monitoring of DNA Polymerase Activity

    NASA Astrophysics Data System (ADS)

    Marushchak, Denys O.; Pugliese, Kaitlin M.; Turvey, Mackenzie W.; Choi, Yongki; Gul, O. Tolga; Olsen, Tivoli J.; Rajapakse, Arith J.; Weiss, Gregory A.; Collins, Philip G.

    Single-molecule techniques can reveal new spatial and kinetic details of the conformational changes occurring during enzymatic catalysis. Here, we investigate the activity of DNA polymerases using an electronic single-molecule technique based on carbon nanotube transistors. Single molecules of the Klenow fragment (KF) of polymerase I were conjugated to the transistors and then monitored via fluctuations in electrical conductance. Continuous, long-term monitoring recorded single KF molecules incorporating up to 10,000 new bases into single-stranded DNA templates. The duration of individual incorporation events was invariant across all analog and native nucleotides, indicating that the precise structure of different base pairs has no impact on the timing of incorporation. Despite similar timings, however, the signal magnitudes generated by certain analogs reveal alternate conformational states that do not occur with native nucleotides. The differences induced by these analogs suggest that the electronic technique is sensing KF's O-helix as it tests the stability of nascent base pairs.

  18. Activated platelets rescue apoptotic cells via paracrine activation of EGFR and DNA-dependent protein kinase

    PubMed Central

    Au, A E-L; Sashindranath, M; Borg, R J; Kleifeld, O; Andrews, R K; Gardiner, E E; Medcalf, R L; Samson, A L

    2014-01-01

    Platelet activation is a frontline response to injury, not only essential for clot formation but also important for tissue repair. Indeed, the reparative influence of platelets has long been exploited therapeutically where application of platelet concentrates expedites wound recovery. Despite this, the mechanisms of platelet-triggered cytoprotection are poorly understood. Here, we show that activated platelets accumulate in the brain to exceptionally high levels following injury and release factors that potently protect neurons from apoptosis. Kinomic microarray and subsequent kinase inhibitor studies showed that platelet-based neuroprotection relies upon paracrine activation of the epidermal growth factor receptor (EGFR) and downstream DNA-dependent protein kinase (DNA-PK). This same anti-apoptotic cascade stimulated by activated platelets also provided chemo-resistance to several cancer cell types. Surprisingly, deep proteomic profiling of the platelet releasate failed to identify any known EGFR ligand, indicating that activated platelets release an atypical activator of the EGFR. This study is the first to formally associate platelet activation to EGFR/DNA-PK – an endogenous cytoprotective cascade. PMID:25210793

  19. Structural Mechanism of Demethylation and Inactivation of Protein Phosphatase 2A

    SciTech Connect

    Xing,Y.; Li, Z.; Chen, Y.; Stock, J.; Jeffrey, P.; Shi, Y.

    2008-01-01

    Protein phosphatase 2A (PP2A) is an important serine/threonine phosphatase that plays a role in many biological processes. Reversible carboxyl methylation of the PP2A catalytic subunit is an essential regulatory mechanism for its function. Demethylation and negative regulation of PP2A is mediated by a PP2A-specific methylesterase PME-1, which is conserved from yeast to humans. However, the underlying mechanism of PME-1 function remains enigmatic. Here we report the crystal structures of PME-1 by itself and in complex with a PP2A heterodimeric core enzyme. The structures reveal that PME-1 directly binds to the active site of PP2A and that this interaction results in the activation of PME-1 by rearranging the catalytic triad into an active conformation. Strikingly, these interactions also lead to inactivation of PP2A by evicting the manganese ions that are required for the phosphatase activity of PP2A. These observations identify a dual role of PME-1 that regulates PP2A activation, methylation, and holoenzyme assembly in cells.

  20. Crystal Structure of a Bacterial Type IB DNA Topoisomerase Reveals a Preassembled Active Site in the Absence of DNA

    SciTech Connect

    Patel, Asmita; Shuman, Stewart; Mondragon, Alfonso

    2010-03-08

    Type IB DNA topoisomerases are found in all eukarya, two families of eukaryotic viruses (poxviruses and mimivirus), and many genera of bacteria. They alter DNA topology by cleaving and resealing one strand of duplex DNA via a covalent DNA-(3-phosphotyrosyl)-enzyme intermediate. Bacterial type IB enzymes were discovered recently and are described as poxvirus-like with respect to their small size, primary structures, and bipartite domain organization. Here we report the 1.75-{angstrom} crystal structure of Deinococcus radiodurans topoisomerase IB (DraTopIB), a prototype of the bacterial clade. DraTopIB consists of an amino-terminal (N) {beta}-sheet domain (amino acids 1-90) and a predominantly {alpha}-helical carboxyl-terminal (C) domain (amino acids 91-346) that closely resemble the corresponding domains of vaccinia virus topoisomerase IB. The five amino acids of DraTopIB that comprise the catalytic pentad (Arg-137, Lys-174, Arg-239, Asn-280, and Tyr-289) are preassembled into the active site in the absence of DNA in a manner nearly identical to the pentad configuration in human topoisomerase I bound to DNA. This contrasts with the apoenzyme of vaccinia topoisomerase, in which three of the active site constituents are either displaced or disordered. The N and C domains of DraTopIB are splayed apart in an 'open' conformation, in which the surface of the catalytic domain containing the active site is exposed for DNA binding. A comparison with the human topoisomerase I-DNA cocrystal structure suggests how viral and bacterial topoisomerase IB enzymes might bind DNA circumferentially via movement of the N domain into the major groove and clamping of a disordered loop of the C domain around the helix.

  1. Method for recovering and using lignin in adhesive resins by extracting demethylated lignin

    DOEpatents

    Schroeder, Herbert A.

    1991-01-01

    Lignin, or a lignin derived material, which has been significantly demethylated (e.g., the demethylated lignin found in the raffinate produced as a by-product of dimethyl sulfide production which can be carried out using the spent liquor from wood pulping operations) can be isolated by a process wherein an organic solvent is added to a lignin-containing aqueous solution. The organic solvent is typically a polar, and at least a partially water-immiscible substance such as, for example, ethyl acetate. The resulting lignin-containing aqueous solution/organic solvent mixture is acidified to produce a water layer which is discarded and an organic solvent layer which contains the demethylated lignin. Upon its recovery, the demethylated lignin is preferably dried and stored until it is used (along with an alkali, an aldehyde and an adhesive filler) in compounding an adhesive of the type generally used in the manufacture of plywood.

  2. Method for recovering and using lignin in adhesive resins by extracting demethylated lignin

    DOEpatents

    Schroeder, Herbert A.

    1991-01-01

    Lignin, or a lignin derived material, which has been significantly demethylated (e.g., the demethylated lignin found in the raffinate produced as a by-product of dimethyl sulfide production which can be carried out using the spent liquor from wood pulping operations) can be isolated by a process wherein an organic solvent is added to a lignin-containing aqueous solution. The organic solvent is typically a polar, and at least a partially water-immiscible substance such as, for example, ethyl acetate. The resulting lignin-containing aqueous solution/organic solvent mixture is acidified to produce a water layer which is discarded and an organic solvent layer which contains the demethylated lignin. Upon its recovery, the demethylated lignin is dissolved in an alkaline solution to which an aldehyde source is added to produce a resol-type resin. The aldehyde source may be formaldehyde in solution, paraformaldehyde, hexamethylenetetramine, or other aldehydes including acetaldehyde, furfural, and their derivatives.

  3. 76 FR 44339 - Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-25

    ... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines... attenuated strains of bacteria and viruses that are frequently used in recombinant DNA research. OBA is...

  4. 75 FR 69687 - Office of Biotechnology Activities Recombinant DNA Research: Proposed Actions Under the NIH...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-15

    ... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities Recombinant DNA Research: Proposed Actions Under the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines... the NIH Recombinant DNA Advisory Committee (RAC) and specifically approved by the NIH Director as...

  5. Single-molecule imaging of DNA polymerase I (Klenow fragment) activity by atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Chao, J.; Zhang, P.; Wang, Q.; Wu, N.; Zhang, F.; Hu, J.; Fan, C. H.; Li, B.

    2016-03-01

    We report a DNA origami-facilitated single-molecule platform that exploits atomic force microscopy to study DNA replication. We imaged several functional activities of the Klenow fragment of E. coli DNA polymerase I (KF) including binding, moving, and dissociation from the template DNA. Upon completion of these actions, a double-stranded DNA molecule was formed. Furthermore, the direction of KF activities was captured and then confirmed by shifting the KF binding sites on the template DNA.We report a DNA origami-facilitated single-molecule platform that exploits atomic force microscopy to study DNA replication. We imaged several functional activities of the Klenow fragment of E. coli DNA polymerase I (KF) including binding, moving, and dissociation from the template DNA. Upon completion of these actions, a double-stranded DNA molecule was formed. Furthermore, the direction of KF activities was captured and then confirmed by shifting the KF binding sites on the template DNA. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06544e

  6. Heterogeneous nuclear ribonucleoprotein B1 protein impairs DNA repair mediated through the inhibition of DNA-dependent protein kinase activity

    SciTech Connect

    Iwanaga, Kentaro; Sueoka, Naoko; Sato, Akemi; Hayashi, Shinichiro; Sueoka, Eisaburo . E-mail: sueokae@post.saga-med.ac.jp

    2005-08-05

    Heterogeneous nuclear ribonucleoprotein B1, an RNA binding protein, is overexpressed from the early stage of lung cancers; it is evident even in bronchial dysplasia, a premalignant lesion. We evaluated the proteins bound with hnRNP B1 and found that hnRNP B1 interacted with DNA-dependent protein kinase (DNA-PK) complex, and recombinant hnRNP B1 protein dose-dependently inhibited DNA-PK activity in vitro. To test the effect of hnRNP B1 on DNA repair, we performed comet assay after irradiation, using normal human bronchial epithelial (HBE) cells treated with siRNA for hnRNP A2/B1: reduction of hnRNP B1 treated with siRNA for hnRNP A2/B1 induced faster DNA repair in normal HBE cells. Considering these results, we assume that overexpression of hnRNP B1 occurring in the early stage of carcinogenesis inhibits DNA-PK activity, resulting in subsequent accumulation of erroneous rejoining of DNA double-strand breaks, causing tumor progression.

  7. Norcantharidin inhibits Wnt signal pathway via promoter demethylation of WIF-1 in human non-small cell lung cancer.

    PubMed

    Xie, Junran; Zhang, Yaping; Hu, Xuming; Lv, Ran; Xiao, Dongju; Jiang, Li; Bao, Qi

    2015-05-01

    Wingless-type (Wnt) family of secreted glycoproteins is a group of signal molecules implicated in oncogenesis. Abnormal activation of Wnt signal pathway is associated with a variety of human cancers, including non-small cell lung cancer (NSCLC). Wnt antagonists, such as the secreted frizzled-related protein (SFRP) family, Wnt inhibitory factor-1 (WIF-1) and cerberus, inhibit Wnt signal pathway by directly binding to Wnt molecules. Norcantharidin (NCTD) is known to possess anticancer activity but less nephrotoxicity than cantharidin. In this study, we found that NCTD inhibited cell proliferation, induced apoptosis, arrested cell cycle and suppressed cell invasion/migration in vitro. Additionally, Wnt signal pathway transcription was also suppressed. NCTD treatment blocked cytoplasmic translocation of beta-catenin into the nucleus. Alterations of apoptosis-related proteins, such as Bax, cleaved caspase-3 (pro-apoptotic) and Bcl-2 (anti-apoptotic), had been detected. Furthermore, the expression levels of WIF-1 and SFRP1 were significantly increased in NCTD-treated groups compared with negative control (NC) groups. Abnormal methylation was observed in NC groups, while NCTD treatment promoted WIF-1 demethylation. The present study revealed that NCTD activated WIF-1 via promoter demethylation, inhibiting the canonical Wnt signal pathway in NSCLC, which may present a new therapeutic target in vivo. PMID:25814287

  8. The activation-induced cytidine deaminase (AID) efficiently targets DNA in nucleosomes but only during transcription

    PubMed Central

    Shen, Hong Ming; Poirier, Michael G.; Allen, Michael J.; North, Justin; Lal, Ratnesh; Widom, Jonathan

    2009-01-01

    The activation-induced cytidine deaminase (AID) initiates somatic hypermutation, class-switch recombination, and gene conversion of immunoglobulin genes. In vitro, AID has been shown to target single-stranded DNA, relaxed double-stranded DNA, when transcribed, or supercoiled DNA. To simulate the in vivo situation more closely, we have introduced two copies of a nucleosome positioning sequence, MP2, into a supercoiled AID target plasmid to determine where around the positioned nucleosomes (in the vicinity of an ampicillin resistance gene) cytidine deaminations occur in the absence or presence of transcription. We found that without transcription nucleosomes prevented cytidine deamination by AID. However, with transcription AID readily accessed DNA in nucleosomes on both DNA strands. The experiments also showed that AID targeting any DNA molecule was the limiting step, and they support the conclusion that once targeted to DNA, AID acts processively in naked DNA and DNA organized within transcribed nucleosomes. PMID:19380635

  9. Arsenic Demethylation by a C·As Lyase in Cyanobacterium Nostoc sp. PCC 7120.

    PubMed

    Yan, Yu; Ye, Jun; Xue, Xi-Mei; Zhu, Yong-Guan

    2015-12-15

    Arsenic, a ubiquitous toxic substance, exists mainly as inorganic forms in the environment. It is perceived that organoarsenicals can be demethylated and degraded into inorganic arsenic by microorganisms. Few studies have focused on the mechanism of arsenic demethylation in bacteria. Here, we investigated arsenic demethylation in a typical freshwater cyanobacterium Nostoc sp. PCC 7120. This bacterium was able to demethylate monomethylarsenite [MAs(III)] rapidly to arsenite [As(III)] and also had the ability to demethylate monomethylarsenate [MAs(V)] to As(III). The NsarsI encoding a C·As lyase responsible for MAs(III) demethylation was cloned from Nostoc sp. PCC 7120 and heterologously expressed in an As-hypersensitive strain Escherichia coli AW3110 (ΔarsRBC). Expression of NsarsI was shown to confer MAs(III) resistance through arsenic demethylation. The purified NsArsI was further identified and functionally characterized in vitro. NsArsI existed mainly as the trimeric state, and the kinetic data were well-fit to the Hill equation with K0.5 = 7.55 ± 0.33 μM for MAs(III), Vmax = 0.79 ± 0.02 μM min(-1), and h = 2.7. Both of the NsArsI truncated derivatives lacking the C-terminal 10 residues (ArsI10) or 23 residues (ArsI23) had a reduced ability of MAs(III) demethylation. These results provide new insights for understanding the important role of cyanobacteria in arsenic biogeochemical cycling in the environment. PMID:26544154

  10. Mre11 Nuclease Activity has Essential Roles in DNA Repair and Genomic Stability Distinct from ATM Activation

    PubMed Central

    Buis, Jeffrey; Wu, Yipin; Deng, Yibin; Leddon, Jennifer; Westfield, Gerwin; Eckersdorff, Mark; Sekiguchi, JoAnn M.; Chang, Sandy; Ferguson, David O.

    2008-01-01

    Summary The Mre11/Rad50/NBS1 complex (MRN) maintains genomic stability by bridging DNA ends and initiating DNA damage signaling through activation of the ATM kinase. Mre11 possesses DNA nuclease activities that are highly conserved in evolution, but play unknown roles in mammals. To define functions of Mre11 we engineered targeted mouse alleles which either abrogate nuclease activities or inactivate the entire MRN complex. Mre11 nuclease deficiency causes a striking array of phenotypes indistinguishable from absence of MRN, including early embryonic lethality and dramatic genomic instability. We identify a crucial role for the nuclease activities in homology directed double strand break repair, and a contributing role in activating the ATR kinase. However, nuclease activities are not required to activate ATM after DNA damage or telomere deprotection. Therefore, nucleolytic processing by Mre11 is an essential function of fundamental importance in DNA repair distinct from MRN control of ATM signaling. PMID:18854157

  11. RCC1-dependent activation of Ran accelerates cell cycle and DNA repair, inhibiting DNA damage-induced cell senescence.

    PubMed

    Cekan, Pavol; Hasegawa, Keisuke; Pan, Yu; Tubman, Emily; Odde, David; Chen, Jin-Qiu; Herrmann, Michelle A; Kumar, Sheetal; Kalab, Petr

    2016-04-15

    The coordination of cell cycle progression with the repair of DNA damage supports the genomic integrity of dividing cells. The function of many factors involved in DNA damage response (DDR) and the cell cycle depends on their Ran GTPase-regulated nuclear-cytoplasmic transport (NCT). The loading of Ran with GTP, which is mediated by RCC1, the guanine nucleotide exchange factor for Ran, is critical for NCT activity. However, the role of RCC1 or Ran⋅GTP in promoting cell proliferation or DDR is not clear. We show that RCC1 overexpression in normal cells increased cellular Ran⋅GTP levels and accelerated the cell cycle and DNA damage repair. As a result, normal cells overexpressing RCC1 evaded DNA damage-induced cell cycle arrest and senescence, mimicking colorectal carcinoma cells with high endogenous RCC1 levels. The RCC1-induced inhibition of senescence required Ran and exportin 1 and involved the activation of importin β-dependent nuclear import of 53BP1, a large NCT cargo. Our results indicate that changes in the activity of the Ran⋅GTP-regulated NCT modulate the rate of the cell cycle and the efficiency of DNA repair. Through the essential role of RCC1 in regulation of cellular Ran⋅GTP levels and NCT, RCC1 expression enables the proliferation of cells that sustain DNA damage. PMID:26864624

  12. RCC1-dependent activation of Ran accelerates cell cycle and DNA repair, inhibiting DNA damage–induced cell senescence

    PubMed Central

    Cekan, Pavol; Hasegawa, Keisuke; Pan, Yu; Tubman, Emily; Odde, David; Chen, Jin-Qiu; Herrmann, Michelle A.; Kumar, Sheetal; Kalab, Petr

    2016-01-01

    The coordination of cell cycle progression with the repair of DNA damage supports the genomic integrity of dividing cells. The function of many factors involved in DNA damage response (DDR) and the cell cycle depends on their Ran GTPase–regulated nuclear–cytoplasmic transport (NCT). The loading of Ran with GTP, which is mediated by RCC1, the guanine nucleotide exchange factor for Ran, is critical for NCT activity. However, the role of RCC1 or Ran⋅GTP in promoting cell proliferation or DDR is not clear. We show that RCC1 overexpression in normal cells increased cellular Ran⋅GTP levels and accelerated the cell cycle and DNA damage repair. As a result, normal cells overexpressing RCC1 evaded DNA damage–induced cell cycle arrest and senescence, mimicking colorectal carcinoma cells with high endogenous RCC1 levels. The RCC1-induced inhibition of senescence required Ran and exportin 1 and involved the activation of importin β–dependent nuclear import of 53BP1, a large NCT cargo. Our results indicate that changes in the activity of the Ran⋅GTP–regulated NCT modulate the rate of the cell cycle and the efficiency of DNA repair. Through the essential role of RCC1 in regulation of cellular Ran⋅GTP levels and NCT, RCC1 expression enables the proliferation of cells that sustain DNA damage. PMID:26864624

  13. Analysis of DNA structure and sequence requirements for Pseudomonas aeruginosa MutL endonuclease activity.

    PubMed

    Correa, Elisa M E; De Tullio, Luisina; Vélez, Pablo S; Martina, Mariana A; Argaraña, Carlos E; Barra, José L

    2013-12-01

    The hallmark of the mismatch repair system in bacterial and eukaryotic organisms devoid of MutH is the presence of a MutL homologue with endonuclease activity. The aim of this study was to analyse whether different DNA structures affect Pseudomonas aeruginosa MutL (PaMutL) endonuclease activity and to determine if a specific nucleotide sequence is required for this activity. Our results showed that PaMutL was able to nick covalently closed circular plasmids but not linear DNA at high ionic strengths, while the activity on linear DNA was only found below 60 mM salt. In addition, single strand DNA, ss/ds DNA boundaries and negatively supercoiling degree were not required for PaMutL nicking activity. Finally, the analysis of the incision sites revealed that PaMutL, as well as Bacillus thuringiensis MutL homologue, did not show DNA sequence specificity. PMID:23969026

  14. Detection of DNA polymerase activities associated with purified duck hepatitis B virus core particles by using an activity gel assay.

    PubMed Central

    Oberhaus, S M; Newbold, J E

    1993-01-01

    Replication of hepadnaviruses involves reverse transcription of an intermediate RNA molecule. It is generally accepted that this replication scheme is carried out by a virally encoded, multifunctional polymerase which has DNA-dependent DNA polymerase, reverse transcriptase, and RNase H activities. Biochemical studies of the polymerase protein(s) have been limited by the inability to purify useful quantities of functional enzyme from virus particles and, until recently, to express enzymatically active polymerase proteins in heterologous systems. An activity gel assay which detects in situ catalytic activities of DNA polymerases after electrophoresis in partially denaturing polyacrylamide gels was used by M.R. Bavand and O. Laub (J. Virol. 62:626-628, 1988) to show the presence of DNA- and RNA-dependent DNA polymerase activities associated with hepatitis B virus particles produced in vitro. This assay has provided the only means by which hepadnavirus polymerase proteins have been detected in association with enzymatic activities. Since conventional methods have not allowed purification of useful quantities of enzymatically active polymerase protein(s), we have devised a protocol for purifying large quantities of duck hepatitis B virus (DHBV) core particles to near homogeneity. These immature virus particles contain DNA- and RNA-dependent DNA polymerase activities, as shown in the endogenous DNA polymerase assay. We have used the activity gel assay to detect multiple DNA- and RNA-dependent DNA polymerase proteins associated with these purified DHBV core particles. These enzymatically active proteins appear larger than, approximately the same size as, and smaller than an unmodified DHBV polymerase protein predicted from the polymerase open reading frame. This is the first report of the detection of active hepadnavirus core-associated DNA polymerase proteins derived from a natural host. Images PMID:8411359

  15. Regulatory T Cell DNA Methyltransferase Inhibition Accelerates Resolution of Lung Inflammation

    PubMed Central

    Singer, Benjamin D.; Mock, Jason R.; Aggarwal, Neil R.; Garibaldi, Brian T.; Sidhaye, Venkataramana K.; Florez, Marcus A.; Chau, Eric; Gibbs, Kevin W.; Mandke, Pooja; Tripathi, Ashutosh; Yegnasubramanian, Srinivasan; King, Landon S.

    2015-01-01

    Acute respiratory distress syndrome (ARDS) is a common and often fatal inflammatory lung condition without effective targeted therapies. Regulatory T cells (Tregs) resolve lung inflammation, but mechanisms that enhance Tregs to promote resolution of established damage remain unknown. DNA demethylation at the forkhead box protein 3 (Foxp3) locus and other key Treg loci typify the Treg lineage. To test how dynamic DNA demethylation affects lung injury resolution, we administered the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (DAC) to wild-type (WT) mice beginning 24 hours after intratracheal LPS-induced lung injury. Mice that received DAC exhibited accelerated resolution of their injury. Lung CD4+CD25hiFoxp3+ Tregs from DAC-treated WT mice increased in number and displayed enhanced Foxp3 expression, activation state, suppressive phenotype, and proliferative capacity. Lymphocyte-deficient recombinase activating gene-1–null mice and Treg-depleted (diphtheria toxin-treated Foxp3DTR) mice did not resolve their injury in response to DAC. Adoptive transfer of 2 × 105 DAC-treated, but not vehicle-treated, exogenous Tregs rescued Treg-deficient mice from ongoing lung inflammation. In addition, in WT mice with influenza-induced lung inflammation, DAC rescue treatment facilitated recovery of their injury and promoted an increase in lung Treg number. Thus, DNA methyltransferase inhibition, at least in part, augments Treg number and function to accelerate repair of experimental lung injury. Epigenetic pathways represent novel manipulable targets for the treatment of ARDS. PMID:25295995

  16. Kinetic mechanism for formation of the active, dimeric UvrD helicase-DNA complex.

    PubMed

    Maluf, Nasib K; Ali, Janid A; Lohman, Timothy M

    2003-08-22

    Escherichia coli UvrD protein is a 3' to 5' SF1 helicase required for DNA repair as well as DNA replication of certain plasmids. We have shown previously that UvrD can self-associate to form dimers and tetramers in the absence of DNA, but that a UvrD dimer is required to form an active helicase-DNA complex in vitro. Here we have used pre-steady state, chemical quenched flow methods to examine the kinetic mechanism for formation of the active, dimeric helicase-DNA complex. Experiments were designed to examine the steps leading to formation of the active complex, separate from the subsequent DNA unwinding steps. The results show that the active dimeric complex can form via two pathways. The first, faster path involves direct binding to the DNA substrate of a pre-assembled UvrD dimer (dimer path), whereas the second, slower path proceeds via sequential binding to the DNA substrate of two UvrD monomers (monomer path), which then assemble on the DNA to form the dimeric helicase. The rate-limiting step within the monomer pathway involves dimer assembly on the DNA. These results show that UvrD dimers that pre-assemble in the absence of DNA are intermediates along the pathway to formation of the functional dimeric UvrD helicase. PMID:12788954

  17. Arginine demethylation is catalysed by a subset of JmjC histone lysine demethylases.

    PubMed

    Walport, Louise J; Hopkinson, Richard J; Chowdhury, Rasheduzzaman; Schiller, Rachel; Ge, Wei; Kawamura, Akane; Schofield, Christopher J

    2016-01-01

    While the oxygen-dependent reversal of lysine N(ɛ)-methylation is well established, the existence of bona fide N(ω)-methylarginine demethylases (RDMs) is controversial. Lysine demethylation, as catalysed by two families of lysine demethylases (the flavin-dependent KDM1 enzymes and the 2-oxoglutarate- and oxygen-dependent JmjC KDMs, respectively), proceeds via oxidation of the N-methyl group, resulting in the release of formaldehyde. Here we report detailed biochemical studies clearly demonstrating that, in purified form, a subset of JmjC KDMs can also act as RDMs, both on histone and non-histone fragments, resulting in formaldehyde release. RDM catalysis is studied using peptides of wild-type sequences known to be arginine-methylated and sequences in which the KDM's methylated target lysine is substituted for a methylated arginine. Notably, the preferred sequence requirements for KDM and RDM activity vary even with the same JmjC enzymes. The demonstration of RDM activity by isolated JmjC enzymes will stimulate efforts to detect biologically relevant RDM activity. PMID:27337104

  18. Arginine demethylation is catalysed by a subset of JmjC histone lysine demethylases

    PubMed Central

    Walport, Louise J.; Hopkinson, Richard J.; Chowdhury, Rasheduzzaman; Schiller, Rachel; Ge, Wei; Kawamura, Akane; Schofield, Christopher J.

    2016-01-01

    While the oxygen-dependent reversal of lysine Nɛ-methylation is well established, the existence of bona fide Nω-methylarginine demethylases (RDMs) is controversial. Lysine demethylation, as catalysed by two families of lysine demethylases (the flavin-dependent KDM1 enzymes and the 2-oxoglutarate- and oxygen-dependent JmjC KDMs, respectively), proceeds via oxidation of the N-methyl group, resulting in the release of formaldehyde. Here we report detailed biochemical studies clearly demonstrating that, in purified form, a subset of JmjC KDMs can also act as RDMs, both on histone and non-histone fragments, resulting in formaldehyde release. RDM catalysis is studied using peptides of wild-type sequences known to be arginine-methylated and sequences in which the KDM's methylated target lysine is substituted for a methylated arginine. Notably, the preferred sequence requirements for KDM and RDM activity vary even with the same JmjC enzymes. The demonstration of RDM activity by isolated JmjC enzymes will stimulate efforts to detect biologically relevant RDM activity. PMID:27337104

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

    SciTech Connect

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

    2004-02-01

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

  20. The human DNA-activated protein kinase, DNA-PK: Substrate specificity

    SciTech Connect

    Anderson, C.W.; Connelly, M.A.; Zhang, H.; Sipley, J.A.; Lees-Miller, S.P.; Lintott, L.G.; Sakaguchi, Kazuyasu; Appella, E.

    1994-11-05

    Although much has been learned about the structure and function of p53 and the probable sequence of subsequent events that lead to cell cycle arrest, little is known about how DNA damage is detected and the nature of the signal that is generated by DNA damage. Circumstantial evidence suggests that protein kinases may be involved. In vitro, human DNA-PK phosphorylates a variety of nuclear DNA-binding, regulatory proteins including the tumor suppressor protein p53, the single-stranded DNA binding protein RPA, the heat shock protein hsp90, the large tumor antigen (TAg) of simian virus 40, a variety of transcription factors including Fos, Jun, serum response factor (SRF), Myc, Sp1, Oct-1, TFIID, E2F, the estrogen receptor, and the large subunit of RNA polymerase II (reviewed in Anderson, 1993; Jackson et al., 1993). However, for most of these proteins, the sites that are phosphorylated by DNA-PK are not known. To determine if the sites that were phosphorylated in vitro also were phosphorylated in vivo and if DNA-PK recognized a preferred protein sequence, the authors identified the sites phosphorylated by DNA-PK in several substrates by direct protein sequence analysis. Each phosphorylated serine or threonine is followed immediately by glutamine in the polypeptide chain; at no other positions are the amino acid residues obviously constrained.

  1. Copper(II) facilitates bleomycin-mediated unwinding of plasmid DNA

    SciTech Connect

    Levy, M.J.; Hecht, S.M.

    1988-04-19

    The unwinding of plasmid DNA by bleomycin A/sub 2/ (BLM A/sub 2/) was investigated by use of two-dimensional gel electrophoresis. It was found that Cu/sup 2 +/ ions greatly facilitated the unwinding of topoisomers of plasmid DNA by BLM A/sub 2/ at concentrations where cupric ions along had no effect on DNA supercoiling. The concentration of BLM A/sub 2/ required for observable unwinding was reduced at least 100-fold in the presence of equimolar Cu/sup 2 +/. A plot of (Cu/sup 2 +/) vs extent of DNA unwinding in the presence of 10/sup -4/ M BLM A/sub 2/ gave a curve consistent with the action of cupric ions on BLM in an allosteric fashion, possibly rearranging the drug into a conformation that facilitates DNA unwinding. The participation of the metal center in enhancing DNA unwinding via direct ionic interaction with one or more negatively charged groups on the DNA duplex also seems possible. Further analysis of the structural factors required for BLM-mediated DNA unwinding was carried out with Cu/sup 2 +/ + BLM demethyl A/sub 2/, the latter of which differs from BLM A/sub 2/ only in that it lacks a methyl group, and associated positive charge, at the C-terminus. Cu(II) x BLM demethyl A/sub 2/ was found to be much less effective than Cu(II) x BLM A/sub 2/ as a DNA unwinding agent, emphasizing the strong dependence of this process on the presence of positively charged groups within the BLM molecule. These findings constitute the first direct evidence that the metal center of BLM can participate in DNA interaction, as well as in the previously recognized role of oxygen binding and activation.

  2. Analysis of TET Expression/Activity and 5mC Oxidation during Normal and Malignant Germ Cell Development

    PubMed Central

    Nettersheim, Daniel; Heukamp, Lukas C.; Fronhoffs, Florian; Grewe, Marc J.; Haas, Natalie; Waha, Anke; Honecker, Friedemann; Waha, Andreas; Kristiansen, Glen; Schorle, Hubert

    2013-01-01

    During mammalian development the fertilized zygote and primordial germ cells lose their DNA methylation within one cell cycle leading to the concept of active DNA demethylation. Recent studies identified the TET hydroxylases as key enzymes responsible for active DNA demethylation, catalyzing the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine. Further oxidation and activation of the base excision repair mechanism leads to replacement of a modified cytosine by an unmodified one. In this study, we analyzed the expression/activity of TET1-3 and screened for the presence of 5mC oxidation products in adult human testis and in germ cell cancers. By analyzing human testis sections, we show that levels of 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine are decreasing as spermatogenesis proceeds, while 5-methylcytosine levels remain constant. These data indicate that during spermatogenesis active DNA demethylation becomes downregulated leading to a conservation of the methylation marks in mature sperm. We demonstrate that all carcinoma in situ and the majority of seminomas are hypomethylated and hypohydroxymethylated compared to non-seminomas. Interestingly, 5-formylcytosine and 5-carboxylcytosine were detectable in all germ cell cancer entities analyzed, but levels did not correlate to the 5-methylcytosine or 5-hydroxymethylcytosine status. A meta-analysis of gene expression data of germ cell cancer tissues and corresponding cell lines demonstrates high expression of TET1 and the DNA glycosylase TDG, suggesting that germ cell cancers utilize the oxidation pathway for active DNA demethylation. During xenograft experiments, where seminoma-like TCam-2 cells transit to an embryonal carcinoma-like state DNMT3B and DNMT3L where strongly upregulated, which correlated to increasing 5-methylcytosine levels. Additionally, 5-hydroxymethylcytosine levels were elevated, demonstrating that de novo methylation and active demethylation accompanies this transition

  3. DNA polymerase-α regulates the activation of type I interferons through cytosolic RNA:DNA synthesis.

    PubMed

    Starokadomskyy, Petro; Gemelli, Terry; Rios, Jonathan J; Xing, Chao; Wang, Richard C; Li, Haiying; Pokatayev, Vladislav; Dozmorov, Igor; Khan, Shaheen; Miyata, Naoteru; Fraile, Guadalupe; Raj, Prithvi; Xu, Zhe; Xu, Zigang; Ma, Lin; Lin, Zhimiao; Wang, Huijun; Yang, Yong; Ben-Amitai, Dan; Orenstein, Naama; Mussaffi, Huda; Baselga, Eulalia; Tadini, Gianluca; Grunebaum, Eyal; Sarajlija, Adrijan; Krzewski, Konrad; Wakeland, Edward K; Yan, Nan; de la Morena, Maria Teresa; Zinn, Andrew R; Burstein, Ezra

    2016-05-01

    Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations that disrupt nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts the expression of POLA1, which encodes the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency resulted in increased production of type I interferons. This enzyme is necessary for the synthesis of RNA:DNA primers during DNA replication and, strikingly, we found that POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Together this work identifies POLA1 as a critical regulator of the type I interferon response. PMID:27019227

  4. Heme activation by DNA: isoguanine pentaplexes, but not quadruplexes, bind heme and enhance its oxidative activity

    PubMed Central

    Shumayrikh, Nisreen; Huang, Yu Chuan; Sen, Dipankar

    2015-01-01

    Guanine-rich, single-stranded, DNAs and RNAs are able to fold to form G-quadruplexes that are held together by guanine base quartets. G-quadruplexes are known to bind ferric heme [Fe(III)-protoporphyrin IX] and to strongly activate such bound hemes toward peroxidase (1-electron oxidation) as well as oxygenase/peroxygenase (2-electron oxidation) activities. However, much remains unknown about how such activation is effected. Herein, we investigated whether G-quadruplexes were strictly required for heme activation or whether related multi-stranded DNA/RNA structures such as isoguanine (iG) quadruplexes and pentaplexes could also bind and activate heme. We found that iG-pentaplexes did indeed bind and activate heme comparably to G-quadruplexes; however, iG-quadruplexes did neither. Earlier structural and computational studies had suggested that while the geometry of backbone-unconstrained iG-quintets templated by cations such as Na+ or NH4+ was planar, that of iG-quartets deviated from planarity. We hypothesize that the binding as well as activation of heme by DNA or RNA is strongly supported by the planarity of the nucleobase quartet or quintet that interacts directly with the heme. PMID:25824944

  5. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  6. Redistribution of demethylated RNA helicase A during foot-and-mouth disease virus infection: role of jumonji C-domain containing protein 6 in RHA demethylation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We previously reported that RNA Helicase A (RHA) re-localized from the nucleus to the cytoplasm in foot-and-mouth disease virus (FMDV) infected cells, coincident with a reduction in methylation of arginine residues in the RHA C-terminus. To further define the mechanism of RHA demethylation in FMDV-...

  7. Demethylation of a model homogalacturonan with the salt-independent pectin methylesterase from citrus: I. effect of pH on demethylated block size and distribution

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A model homogalacturonan (HG); composed of galacturonic acid (94-97 %) and galactose (3-6 %), having a 94 % degree of esterification (DE) was used to produce a demethylated HG series by reacting it with the salt-independent pectin methylesterase (PME) from citrus fruit at pH 4.5 and 7.5. HGs with a...

  8. DNA methylation dynamics in mouse preimplantation embryos revealed by mass spectrometry

    PubMed Central

    Okamoto, Yoshinori; Yoshida, Naoko; Suzuki, Toru; Shimozawa, Nobuhiro; Asami, Maki; Matsuda, Tomonari; Kojima, Nakao; Perry, Anthony C. F.; Takada, Tatsuyuki

    2016-01-01

    Following fertilization in mammals, paternal genomic 5-methyl-2′-deoxycytidine (5 mC) content is thought to decrease via oxidation to 5-hydroxymethyl-2′-deoxycytidine (5 hmC). This reciprocal model of demethylation and hydroxymethylation is inferred from indirect, non-quantitative methods. We here report direct quantification of genomic 5 mC and 5 hmC in mouse embryos by small scale liquid chromatographic tandem mass spectrometry (SMM). Profiles of absolute 5 mC levels in embryos produced by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) were almost identical. By 10 h after fertilization, 5 mC levels had declined by ~40%, consistent with active genomic DNA demethylation. Levels of 5 mC in androgenotes (containing only a paternal genome) and parthenogenotes (containing only a maternal genome) underwent active 5 mC loss in the first 6 h, showing that both parental genomes can undergo demethylation independently. We found no evidence for net loss of 5 mC 10–48 h after fertilization, implying that any passive ‘demethylation’ following DNA replication was balanced by active 5 mC maintenance methylation. However, levels of 5 mC declined during development after 48 h, to 1% (measured as a fraction of G-residues) in blastocysts (~96 h). 5 hmC levels were consistently low (<0.2% of G-residues) throughout development in normal diploid embryos. This work directly quantifies the dynamics of global genomic DNA modification in mouse preimplantation embryos, suggesting that SMM will be applicable to other biomedical situations with limiting sample sizes. PMID:26750605

  9. In vitro susceptibility of Helicobacter pylori to trospectomycin, pirlimycin (U-57930E), mirincamycin (U-24729A) and N-demethyl clindamycin (U-26767A).

    PubMed

    Westblom, T U; Midkiff, B R; Czinn, S J

    1993-07-01

    The in vitro activity of trospectomycin, pirlimycin, mirincamycin and N-demethyl clindamycin was measured against 46 clinical isolates of Helicobacter pylori using an agar dilution technique. The MIC50 and MIC90 were 4 and 64 micrograms/ml for pirlimycin and N-demethyl clindamycin, and 32 and 128 micrograms/ml for mirincamycin, respectively. All 46 strains were sensitive to trospectomycin with an MIC50 of 8 micrograms/ml and an MIC90 of 16 micrograms/ml. Of seven strains with the highest trospectomycin MICs (8 or 16 micrograms/ml) 100% were found to be resistant to metronidazole. Among ten strains with low trospectomycin MICs (2 micrograms/ml or less) 100% were sensitive to metronidazole. Possible explantations for the apparent correlation between the MICs of the two drugs are discussed. Since all metronidazole resistant strains were sensitive to trospectomycin, this drug may be useful in treating infection with metronidazole resistant Helicobacter pylori. PMID:8404921

  10. Modulation of UvrD helicase activity by covalent DNA-protein cross-links.

    PubMed

    Kumari, Anuradha; Minko, Irina G; Smith, Rebecca L; Lloyd, R Stephen; McCullough, Amanda K

    2010-07-01

    UvrD (DNA helicase II) has been implicated in DNA replication, DNA recombination, nucleotide excision repair, and methyl-directed mismatch repair. The enzymatic function of UvrD is to translocate along a DNA strand in a 3' to 5' direction and unwind duplex DNA utilizing a DNA-dependent ATPase activity. In addition, UvrD interacts with many other proteins involved in the above processes and is hypothesized to facilitate protein turnover, thus promoting further DNA processing. Although UvrD interactions with proteins bound to DNA have significant biological implications, the effects of covalent DNA-protein cross-links on UvrD helicase activity have not been characterized. Herein, we demonstrate that UvrD-catalyzed strand separation was inhibited on a DNA strand to which a 16-kDa protein was covalently bound. Our sequestration studies suggest that the inhibition of UvrD activity is most likely due to a translocation block and not helicase sequestration on the cross-link-containing DNA substrate. In contrast, no inhibition of UvrD-catalyzed strand separation was apparent when the protein was linked to the complementary strand. The latter result is surprising given the earlier observations that the DNA in this covalent complex is severely bent ( approximately 70 degrees ), with both DNA strands making multiple contacts with the cross-linked protein. In addition, UvrD was shown to be required for replication of plasmid DNAs containing covalent DNA-protein complexes. Combined, these data suggest a critical role for UvrD in the processing of DNA-protein cross-links. PMID:20444702

  11. Goatpoxvirus ATPase activity is increased by dsDNA and decreased by zinc ion.

    PubMed

    Lee, Ming-Liang; Hsu, Wei-Li; Wang, Chi-Young; Chen, Hui-Yu; Lin, Fong-Yuan; Chang, Ming-Huang; Chang, Hong-You; Wong, Min-Liang; Chan, Kun-Wei

    2016-10-01

    Viral-encoded ATPase can act as a part of molecular motor in genome packaging of DNA viruses, such as vaccinia virus and adenovirus, by ATP hydrolysis and interaction with DNA. Poxviral ATPase (also called A32) is involved in genomic double-stranded DNA (dsDNA) encapsidation, and inhibition of the expression of A32 causes formation of immature virions lacking viral DNA. However, the role of A32 in goatpoxvirus genome packaging and its dsDNA binding property are not known. In this study, purified recombinant goatpoxvirus A32 protein (rA32) was examined for its dsDNA binding property as well as the effect of dsDNA on ATP hydrolysis. We found that rA32 could bind dsDNA, and its ATPase activity was significant increased with dsDNA binding. Effects of magnesium and calcium ions on ATP hydrolysis were investigated also. The ATPase activity was dramatically enhanced by dsDNA in the presence of Mg(2+); in contrast, ATPase function was not altered by Ca(2+). Furthermore, the enzyme activity of rA32 was completely blocked by Zn(2+). Regarding DNA-protein interaction, the rA32-ATP-Mg(2+) showed lower dsDNA binding affinity than that of rA32-ATP-Ca(2+). The DNA-protein binding was stronger in the presence of zinc ion. Our results implied that A32 may play a role in viral genome encapsidation and DNA condensation. PMID:27146321

  12. Suppression of Wnt signaling by the miR-29 family is mediated by demethylation of WIF-1 in non-small-cell lung cancer.

    PubMed

    Tan, Min; Wu, Junjie; Cai, Yong

    2013-09-01

    Wnt inhibitory factor-1 (WIF-1) silencing induced by promoter hypermethylation is a common mechanism of aberrant activation of the Wnt signaling pathway in non-small-cell lung cancer (NSCLC). However, the activity of regulators associated with the methylation of the WIF-1 gene remains unclear. Here, we investigated the role of three DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) in the expression of WIF-1. The three DNMTs were up-regulated in NSCLC tumor tissues and suppression of DNMT3A and DNMT3B restored the expression of WIF-1 in NSCLC cells. The miR-29 family (miR-29a, -29b, and -29c), which negatively regulates DNMT3A and DNMT3B, was examined in association with the Wnt/β-catenin signaling pathway. A positive correlation between the expression of WIF-1 and that of MiR-29s was observed in NSCLC tissues. Methylation-specific PCR and Western blotting indicated that miR-29s positively regulate WIF-1 expression by inhibiting the methylation of its promoter. Furthermore, miR-29 overexpression downregulated β-catenin expression, inhibited cell proliferation and induced apoptosis. The expression of miR-29a and miR-29b was partially regulated by DNMT3A and DNMT3B in a positive feedback loop. Taken together, our findings show that miR-29s suppress the Wnt signaling pathway through demethylation of WIF-1 in NSCLC. PMID:23939044

  13. 76 FR 3150 - Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-19

    ...). On July 20, 2010 the NIH Office of Biotechnology Activities (OBA) published a proposed action (75 FR... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH...

  14. Pseudomonas aeruginosa phage PaP1 DNA polymerase is an A-family DNA polymerase demonstrating ssDNA and dsDNA 3'-5' exonuclease activity.

    PubMed

    Liu, Binyan; Gu, Shiling; Liang, Nengsong; Xiong, Mei; Xue, Qizhen; Lu, Shuguang; Hu, Fuquan; Zhang, Huidong

    2016-08-01

    Most phages contain DNA polymerases, which are essential for DNA replication and propagation in infected host bacteria. However, our knowledge on phage-encoded DNA polymerases remains limited. This study investigated the function of a novel DNA polymerase of PaP1, which is the lytic phage of Pseudomonas aeruginosa. PaP1 encodes its sole DNA polymerase called Gp90 that was predicted as an A-family DNA polymerase with polymerase and 3'-5' exonuclease activities. The sequence of Gp90 is homologous but not identical to that of other A-family DNA polymerases, such as T7 DNA polymerases (Pol) and DNA Pol I. The purified Gp90 demonstrated a polymerase activity. The processivity of Gp90 in DNA replication and its efficiency in single-dNTP incorporation are similar to those of T7 Pol with processive thioredoxin (T7 Pol/trx). Gp90 can degrade ssDNA and dsDNA in 3'-5' direction at a similar rate, which is considerably lower than that of T7 Pol/trx. The optimized conditions for polymerization were a temperature of 37 °C and a buffer consisting of 40 mM Tris-HCl (pH 8.0), 30 mM MgCl2, and 200 mM NaCl. These studies on DNA polymerase encoded by PaP1 help advance our knowledge on phage-encoded DNA polymerases and elucidate PaP1 propagation in infected P. aeruginosa. PMID:27052734

  15. Histone demethylation and steroid receptor function in cancer.

    PubMed

    Stratmann, Antje; Haendler, Bernard

    2012-01-01

    Steroid receptors recruit various cofactors to form multi-protein complexes which locally alter chromatin structure and control DNA accessibility in order to regulate gene transcription. Some of these factors are enzymes that add or remove histone marks in the vicinity of regulatory regions of target genes. Numerous histone modifications added by specific writer enzymes and removed by eraser enzymes have been identified. Histone methylation is a modification with a complex outcome, as it can lead to gene activation or repression, depending on the modified residue and the context. Methylation marks are added by different enzyme families displaying exquisite substrate specificity. Lysine methylation is reversible and two different demethylase families have been identified in humans, the Jumonji C and the lysine-specific demethylase families. A regulatory role of histone demethylases in fine-tuning the function of steroid receptors, especially the androgen receptor and estrogen receptor, has emerged in recent years. This is mostly inferred from in vitro studies, but more recently first in vivo data have further supported this concept. This and the deregulated expression observed for several histone demethylases suggest a role in tumours such as prostate and breast cancer. PMID:21958694

  16. DNA-based hybridization chain reaction amplification for assaying the effect of environmental phenolic hormone on DNA methyltransferase activity.

    PubMed

    Xu, Zhenning; Yin, Huanshun; Han, Yunxiang; Zhou, Yunlei; Ai, Shiyun

    2014-06-01

    In this work, a novel electrochemical protocol with signal amplification for determination of DNA methylation and methyltransferase activity using DNA-based hybridization chain reaction (HCR) was proposed. After the gold electrode was modified with dsDNA, it was treated with M.SssI MTase, HpaII endonuclease, respectively. And then the HCR was initiated by the target DNA and two hairpin helper DNAs, which lead to the formation of extended dsDNA polymers on the electrode surface. The signal was amplified by the labeled biotin on the hairpin probes. As a result, the streptavidin-alkaline phosphatase (S-ALP) conjugated on the electrode surface through the specific interaction between biotin and S-ALP. ALP could convert 1-naphthyl phosphate into 1-naphthol and the latter could be electrochemically oxidized, which was used to monitor the methylation event and MTase activity. The HCR assay presents good electrochemical responses for the determination of M.SssI MTase at a concentration as low as 0.0067 uni tmL(-1). Moreover, the effects of anti-cancer drug and environmental phenolic hormone on M.SssI MTase activity were also investigated. The results indicated that 5-fluorouracil and daunorubicin hydrochloride could inhibit the activity, and the opposite results were obtained with bisphenol A and nonylphenol. Therefore, this method can not only provide a platform to screen the inhibitors of DNA MTase and develop new anticancer drugs, but also offer a novel technique to investigate the possible carcinogenesis mechanism. PMID:24856396

  17. Activation of DNA damage repair pathways in response to nitrogen mustard-induced DNA damage and toxicity in skin keratinocytes

    PubMed Central

    Inturi, Swetha; Tewari-Singh, Neera; Agarwal, Chapla; White, Carl W.; Agarwal, Rajesh

    2014-01-01

    Nitrogen mustard (NM), a structural analog of chemical warfare agent sulfur mustard (SM), forms adducts and crosslinks with DNA, RNA and proteins. Here we studied the mechanism of NM-induced skin toxicity in response to double strand breaks (DSBs) resulting in cell cycle arrest to facilitate DNA repair, as a model for developing countermeasures against vesicant-induced skin injuries. NM exposure of mouse epidermal JB6 cells decreased cell growth and caused S-phase arrest. Consistent with these biological outcomes, NM exposure also increased comet tail extent moment and the levels of DNA DSB repair molecules phospho H2A.X Ser139 and p53 Ser15 indicating NM-induced DNA DSBs. Since DNA DSB repair occurs via non homologous end joining pathway (NHEJ) or homologous recombination repair (HRR) pathways, next we studied these two pathways and noted their activation as defined by an increase in phospho- and total DNA-PK levels, and the formation of Rad51 foci, respectively. To further analyze the role of these pathways in the cellular response to NM-induced cytotoxicity, NHEJ and HRR were inhibited by DNA-PK inhibitor NU7026 and Rad51 inhibitor BO2, respectively. Inhibition of NHEJ did not sensitize cells to NM-induced decrease in cell growth and cell cycle arrest. However, inhibition of the HRR pathway caused a significant increase in cell death, and prolonged G2M arrest following NM exposure. Together, our findings, indicating that HRR is the key pathway involved in the repair of NM-induced DNA DSBs, could be useful in developing new therapeutic strategies against vesicant-induced skin injury. PMID:24732344

  18. Activation of DNA damage repair pathways in response to nitrogen mustard-induced DNA damage and toxicity in skin keratinocytes.

    PubMed

    Inturi, Swetha; Tewari-Singh, Neera; Agarwal, Chapla; White, Carl W; Agarwal, Rajesh

    2014-01-01

    Nitrogen mustard (NM), a structural analog of chemical warfare agent sulfur mustard (SM), forms adducts and crosslinks with DNA, RNA and proteins. Here we studied the mechanism of NM-induced skin toxicity in response to double strand breaks (DSBs) resulting in cell cycle arrest to facilitate DNA repair, as a model for developing countermeasures against vesicant-induced skin injuries. NM exposure of mouse epidermal JB6 cells decreased cell growth and caused S-phase arrest. Consistent with these biological outcomes, NM exposure also increased comet tail extent moment and the levels of DNA DSB repair molecules phospho H2A.X Ser139 and p53 Ser15 indicating NM-induced DNA DSBs. Since DNA DSB repair occurs via non homologous end joining pathway (NHEJ) or homologous recombination repair (HRR) pathways, next we studied these two pathways and noted their activation as defined by an increase in phospho- and total DNA-PK levels, and the formation of Rad51 foci, respectively. To further analyze the role of these pathways in the cellular response to NM-induced cytotoxicity, NHEJ and HRR were inhibited by DNA-PK inhibitor NU7026 and Rad51 inhibitor BO2, respectively. Inhibition of NHEJ did not sensitize cells to NM-induced decrease in cell growth and cell cycle arrest. However, inhibition of the HRR pathway caused a significant increase in cell death, and prolonged G2M arrest following NM exposure. Together, our findings, indicating that HRR is the key pathway involved in the repair of NM-induced DNA DSBs, could be useful in developing new therapeutic strategies against vesicant-induced skin injury. PMID:24732344

  19. Dithiocarbamate/piperazine bridged pyrrolobenzodiazepines as DNA-minor groove binders: synthesis, DNA-binding affinity and cytotoxic activity.

    PubMed

    Kamal, Ahmed; Sreekanth, Kokkonda; Shankaraiah, Nagula; Sathish, Manda; Nekkanti, Shalini; Srinivasulu, Vunnam

    2015-04-01

    A new series of C8-linked dithiocarbamate/piperazine bridged pyrrolo[2,1-c][1,4]benzodiazepine conjugates (5a-c, 6a,b) have been synthesized and evaluated for their cytotoxic potential and DNA-binding ability. The representative conjugates 5a and 5b have been screened for their cytotoxicity against a panel of 60 human cancer cell lines. Compound 5a has shown promising cytotoxic activity on selected cancer cell lines that display melanoma, leukemia, CNS, ovarian, breast and renal cancer phenotypes. The consequence of further replacement of the 3-cyano-3,3-diphenylpropyl 1-piperazinecarbodithioate in 5b and 5c with 4-methylpiperazine-1-carbodithioate yielded new conjugates 6a and 6b respectively. In addition, the compounds 5c and 6a,b have been evaluated for their in vitro cytotoxicity on some of the selected human cancer cell lines and these conjugates have exhibited significant cytotoxic activity. Further, the DNA-binding ability of these new conjugates has been evaluated by using thermal denaturation (ΔTm) studies. The correlation between structure and DNA-binding ability has been investigated by molecular modeling studies which predicted that 6b exhibits superior DNA-binding ability and these are in agreement with the experimental DNA-binding studies. PMID:25665519

  20. Regulation of Xenopus laevis DNA topoisomerase I activity by phosphorylation in vitro

    SciTech Connect

    Kaiserman, H.B.; Ingebritsen, T.S.; Benbow, R.M.

    1988-05-03

    DNA topoisomerase I has been purified to electrophoretic homogeneity from ovaries of the frog Xenopus laevis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most purified fraction revealed a single major band at 110 kDa and less abundant minor bands centered at 62 kDa. Incubation of the most purified fraction with immobilized calf intestinal alkaline phosphatase abolished all DNA topoisomerase enzymatic activity in a time-dependent reaction. Treatment of the dephosphorylated X. laevis DNA topoisomerase I with a X. laevis casein kinase type II activity and ATP restored DNA topoisomerase activity to a level higher than that observed in the most purified fraction. In vitro labeling experiments which employed the most purified DNA topoisomerase I fraction, (..gamma..-/sup 32/P)ATP, and the casein kinase type II enzyme showed that both the 110- and 62-kDa bands became phosphorylated in approximately molar proportions. Phosphoamino acid analysis showed that only serine residues became phosphorylated. Phosphorylation was accompanied by an increase in DNA topoisomerase activity in vitro. Dephosphorylation of DNA topoisomerase I appears to block formation of the initial enzyme-substrate complex on the basis of the failure of the dephosphorylated enzyme to nick DNA in the presence of camptothecin. The authors conclude that X. laevis DNA topoisomerase I is partially phosphorylated as isolated and that this phosphorylation is essential for expression of enzymatic activity in vitro. On the basis of the ability of the casein kinase type II activity to reactivate dephosphorylated DNA topoisomerase I, they speculate that this kinase may contribute to the physiological regulation of DNA topoisomerase I activity.

  1. HIPK2 restricts SIRT1 activity upon severe DNA damage by a phosphorylation-controlled mechanism.

    PubMed

    Conrad, E; Polonio-Vallon, T; Meister, M; Matt, S; Bitomsky, N; Herbel, C; Liebl, M; Greiner, V; Kriznik, B; Schumacher, S; Krieghoff-Henning, E; Hofmann, T G

    2016-01-01

    Upon severe DNA damage a cellular signalling network initiates a cell death response through activating tumour suppressor p53 in association with promyelocytic leukaemia (PML) nuclear bodies. The deacetylase Sirtuin 1 (SIRT1) suppresses cell death after DNA damage by antagonizing p53 acetylation. To facilitate efficient p53 acetylation, SIRT1 function needs to be restricted. How SIRT1 activity is regulated under these conditions remains largely unclear. Here we provide evidence that SIRT1 activity is limited upon severe DNA damage through phosphorylation by the DNA damage-responsive kinase HIPK2. We found that DNA damage provokes interaction of SIRT1 and HIPK2, which phosphorylates SIRT1 at Serine 682 upon lethal damage. Furthermore, upon DNA damage SIRT1 and HIPK2 colocalize at PML nuclear bodies, and PML depletion abrogates DNA damage-induced SIRT1 Ser682 phosphorylation. We show that Ser682 phosphorylation inhibits SIRT1 activity and impacts on p53 acetylation, apoptotic p53 target gene expression and cell death. Mechanistically, we found that DNA damage-induced SIRT1 Ser682 phosphorylation provokes disruption of the complex between SIRT1 and its activator AROS. Our findings indicate that phosphorylation-dependent restriction of SIRT1 activity by HIPK2 shapes the p53 response. PMID:26113041

  2. HIPK2 restricts SIRT1 activity upon severe DNA damage by a phosphorylation-controlled mechanism

    PubMed Central

    Conrad, E; Polonio-Vallon, T; Meister, M; Matt, S; Bitomsky, N; Herbel, C; Liebl, M; Greiner, V; Kriznik, B; Schumacher, S; Krieghoff-Henning, E; Hofmann, T G

    2016-01-01

    Upon severe DNA damage a cellular signalling network initiates a cell death response through activating tumour suppressor p53 in association with promyelocytic leukaemia (PML) nuclear bodies. The deacetylase Sirtuin 1 (SIRT1) suppresses cell death after DNA damage by antagonizing p53 acetylation. To facilitate efficient p53 acetylation, SIRT1 function needs to be restricted. How SIRT1 activity is regulated under these conditions remains largely unclear. Here we provide evidence that SIRT1 activity is limited upon severe DNA damage through phosphorylation by the DNA damage-responsive kinase HIPK2. We found that DNA damage provokes interaction of SIRT1 and HIPK2, which phosphorylates SIRT1 at Serine 682 upon lethal damage. Furthermore, upon DNA damage SIRT1 and HIPK2 colocalize at PML nuclear bodies, and PML depletion abrogates DNA damage-induced SIRT1 Ser682 phosphorylation. We show that Ser682 phosphorylation inhibits SIRT1 activity and impacts on p53 acetylation, apoptotic p53 target gene expression and cell death. Mechanistically, we found that DNA damage-induced SIRT1 Ser682 phosphorylation provokes disruption of the complex between SIRT1 and its activator AROS. Our findings indicate that phosphorylation-dependent restriction of SIRT1 activity by HIPK2 shapes the p53 response. PMID:26113041

  3. Structure-Specific nuclease activities of Artemis and the Artemis: DNA-PKcs complex

    PubMed Central

    Chang, Howard H.Y.; Lieber, Michael R.

    2016-01-01

    Artemis is a vertebrate nuclease with both endo- and exonuclease activities that acts on a wide range of nucleic acid substrates. It is the main nuclease in the non-homologous DNA end-joining pathway (NHEJ). Not only is Artemis important for the repair of DNA double-strand breaks (DSBs) in NHEJ, it is essential in opening the DNA hairpin intermediates that are formed during V(D)J recombination. Thus, humans with Artemis deficiencies do not have T- or B-lymphocytes and are diagnosed with severe combined immunodeficiency (SCID). While Artemis is the only vertebrate nuclease capable of opening DNA hairpins, it has also been found to act on other DNA substrates that share common structural features. Here, we discuss the key structural features that all Artemis DNA substrates have in common, thus providing a basis for understanding how this structure-specific nuclease recognizes its DNA targets. PMID:27198222

  4. Synthesis, DNA Binding, and Antiproliferative Activity of Novel Acridine-Thiosemicarbazone Derivatives.

    PubMed

    de Almeida, Sinara Mônica Vitalino; Lafayette, Elizabeth Almeida; da Silva, Lúcia Patrícia Bezerra Gomes; Amorim, Cézar Augusto da Cruz; de Oliveira, Tiago Bento; Ruiz, Ana Lucia Tasca Gois; de Carvalho, João Ernesto; de Moura, Ricardo Olímpio; Beltrão, Eduardo Isidoro Carneiro; de Lima, Maria do Carmo Alves; de Carvalho Júnior, Luiz Bezerra

    2015-01-01

    In this work, the acridine nucleus was used as a lead-compound for structural modification by adding different substituted thiosemicarbazide moieties. Eight new (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide derivatives (3a-h) were synthesized, their antiproliferative activities were evaluated, and DNA binding properties were performed with calf thymus DNA (ctDNA) by electronic absorption and fluorescence spectroscopies. Both hyperchromic and hypochromic effects, as well as red or blue shifts were demonstrated by addition of ctDNA to the derivatives. The calculated binding constants ranged from 1.74 × 10(4) to 1.0 × 10(6) M(-1) and quenching constants from -0.2 × 10(4) to 2.18 × 10(4) M(-1) indicating high affinity to ctDNA base pairs. The most efficient compound in binding to ctDNA in vitro was (Z)-2-(acridin-9-ylmethylene)-N- (4-chlorophenyl) hydrazinecarbothioamide (3f), while the most active compound in antiproliferative assay was (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide (3a). There was no correlation between DNA-binding and in vitro antiproliferative activity, but the results suggest that DNA binding can be involved in the biological activity mechanism. This study may guide the choice of the size and shape of the intercalating part of the ligand and the strategic selection of substituents that increase DNA-binding or antiproliferative properties. PMID:26068233

  5. Synthesis, DNA Binding, and Antiproliferative Activity of Novel Acridine-Thiosemicarbazone Derivatives

    PubMed Central

    de Almeida, Sinara Mônica Vitalino; Lafayette, Elizabeth Almeida; Gomes da Silva, Lúcia Patrícia Bezerra; Amorim, Cézar Augusto da Cruz; de Oliveira, Tiago Bento; Gois Ruiz, Ana Lucia Tasca; de Carvalho, João Ernesto; de Moura, Ricardo Olímpio; Beltrão, Eduardo Isidoro Carneiro; de Lima, Maria do Carmo Alves; de Carvalho Júnior, Luiz Bezerra

    2015-01-01

    In this work, the acridine nucleus was used as a lead-compound for structural modification by adding different substituted thiosemicarbazide moieties. Eight new (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide derivatives (3a–h) were synthesized, their antiproliferative activities were evaluated, and DNA binding properties were performed with calf thymus DNA (ctDNA) by electronic absorption and fluorescence spectroscopies. Both hyperchromic and hypochromic effects, as well as red or blue shifts were demonstrated by addition of ctDNA to the derivatives. The calculated binding constants ranged from 1.74 × 104 to 1.0 × 106 M−1 and quenching constants from −0.2 × 104 to 2.18 × 104 M−1 indicating high affinity to ctDNA base pairs. The most efficient compound in binding to ctDNA in vitro was (Z)-2-(acridin-9-ylmethylene)-N-(4-chlorophenyl) hydrazinecarbothioamide (3f), while the most active compound in antiproliferative assay was (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide (3a). There was no correlation between DNA-binding and in vitro antiproliferative activity, but the results suggest that DNA binding can be involved in the biological activity mechanism. This study may guide the choice of the size and shape of the intercalating part of the ligand and the strategic selection of substituents that increase DNA-binding or antiproliferative properties. PMID:26068233

  6. Cyclic GMP-AMP Synthase is Activated by Double-stranded DNA-Induced Oligomerization

    PubMed Central

    Li, Xin; Shu, Chang; Yi, Guanghui; Chaton, Catherine T.; Shelton, Catherine L.; Diao, Jiasheng; Zuo, Xiaobing; Kao, C Cheng; Herr, Andrew B.; Li, Pingwei

    2013-01-01

    Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor mediating innate antimicrobial immunity. It catalyzes the synthesis of a noncanonical cyclic dinucleotide 2′,5′ cGAMP that binds to STING and mediates the activation of TBK1 and IRF-3. Activated IRF-3 translocates to the nucleus and initiates the transcription of the IFN-β gene. The structure of mouse cGAS bound to an 18 bp dsDNA revealed that cGAS interacts with dsDNA through two binding sites, forming a 2:2 complex. Enzyme assays and IFN-β reporter assays of cGAS mutants demonstrated that interactions at both DNA binding sites are essential for cGAS activation. Mutagenesis and DNA binding studies showed that the two sites bind dsDNA cooperatively and site B plays a critical role in DNA binding. The structure of mouse cGAS bound to dsDNA and 2′,5′ cGAMP provided insight into the catalytic mechanism of cGAS. These results demonstrated that cGAS is activated by dsDNA-induced oligomerization. PMID:24332030

  7. DNA polymerase beta reveals enhanced activity and processivity in reverse micelles.

    PubMed

    Anarbaev, Rashid O; Rogozina, Anastasia L; Lavrik, Olga I

    2009-04-01

    Water is essential for the stability and functions of proteins and DNA. Reverse micelles are simple model systems where the structure and dynamics of water are controlled. We have estimated the size of complex reverse micelles by light scattering technique and examined the local microenvironment using fluorescein as molecular probe. The micelle size and water polarity inside reverse micelles depend on water volume fraction. We have investigated the different hydration and confinement effects on activity, processivity, and stability of mammalian DNA polymerase beta in reverse micelles. The enzyme displays high processivity on primed single-stranded M13mp19 DNA with maximal activity at 10% of water content. The processivity and activity of DNA polymerase strongly depend on the protein concentration. The enzyme reveals also the enhanced stability in the presence of template-primer and at high protein concentration. The data provide direct evidence for strong influence of microenvironment on DNA polymerase activity. PMID:19138815

  8. Priming of Human Neutrophils Is Necessary for Their Activation by Extracellular DNA.

    PubMed

    Prikhodko, A S; Vitushkina, M V; Zinovkina, L A; Popova, E N; Zinovkin, R A

    2016-06-01

    Extracellular plasma DNA is thought to act as a damage-associated molecular pattern causing activation of immune cells. However, purified preparations of mitochondrial and nuclear DNA were unable to induce neutrophil activation in vitro. Thus, we examined whether granulocyte-macrophage colony-stimulating factor (GM-CSF) acting as a neutrophil priming agent can promote the activation of neutrophils by different types of extracellular DNA. GM-CSF pretreatment greatly increased p38 MAPK phosphorylation and promoted CD11b/CD66b expression in human neutrophils treated with mitochondrial and, to a lesser extent, with nuclear DNA. Our experiments clearly indicate that GM-CSF-induced priming of human neutrophils is necessary for their subsequent activation by extracellular DNA. PMID:27301289

  9. Black Raspberry-Derived Anthocyanins Demethylate Tumor Suppressor Genes Through the Inhibition of DNMT1 and DNMT3B in Colon Cancer Cells

    PubMed Central

    Wang, Li-Shu; Kuo, Chieh-Ti; Cho, Seung-Ju; Seguin, Claire; Siddiqui, Jibran; Stoner, Kristen; Weng, Yu-I; Huang, Tim H.-M.; Tichelaar, Jay; Yearsley, Martha; Stoner, Gary D.; Huang, Yi-Wen

    2013-01-01

    We previously reported that oral administration of black raspberry powder decreased promoter methylation of tumor suppressor genes in tumors from patients with colorectal cancer. The anthocyanins (ACs) in black raspberries are responsible, at least in part, for their cancer-inhibitory effects. In the present study, we asked if ACs are responsible for the demethylation effects observed in colorectal cancers. Three days of treatment of ACs at 0.5, 5, and 25 μg/ml suppressed activity and protein expression of DNMT1 and DNMT3B in HCT116, Caco2 and SW480 cells. Promoters of CDKN2A, and SFRP2, SFRP5, and WIF1, upstream of Wnt pathway, were demethylated by ACs. mRNA expression of some of these genes was increased. mRNA expression of β-catenin and c-Myc, downstream of Wnt pathway, and cell proliferation were decreased; apoptosis was increased. ACs were taken up into HCT116 cells and were differentially localized with DNMT1 and DNMT3B in the same cells visualized using confocal laser scanning microscopy. Although it was reported that DNMT3B is regulated by c-Myc in mouse lymphoma, DNMT3B did not bind with c-Myc in HCT116 cells. In conclusion, our results suggest that ACs are responsible, at least in part, for the demethylation effects of whole black raspberries in colorectal cancers. PMID:23368921

  10. Metal-based netropsin mimics showing AT-selective DNA binding and DNA cleavage activity at red light.

    PubMed

    Patra, Ashis K; Bhowmick, Tuhin; Ramakumar, Suryanarayanarao; Chakravarty, Akhil R

    2007-10-29

    Copper(II) bis-arginate [Cu(l-arg)2](NO3)2 (1) and [Cu(l-arg)(phen)Cl]Cl (2) as mimics of the minor-groove-binding natural antibiotic netropsin show preferential binding to the AT-rich region of double-stranded DNA. The complexes with a d-d band near 600 nm display oxidative DNA cleavage activity on photoirradiation at UV-A light of 365 nm and at red light of 647.1 nm (Ar-Kr laser) in a metal-assisted photoexcitation process forming singlet oxygen (1O2) species in a type-2 pathway. PMID:17880211

  11. Novel Organotin(IV)-Schiff Base Complexes: Synthesis, Characterization, Antimicrobial Activity, and DNA Interaction Studies

    PubMed Central

    Prasad, K. Shiva; Kumar, L. Shiva; Prasad, Melvin; Revanasiddappa, Hosakere D.

    2010-01-01

    Four organotin(IV) complexes with 2-(2-hydroxybenzylideneamino)isoindoline-1,3-dione (L1), and 4-(4-hydroxy-3-methoxybenzylideneamino-N-(pyrimidin-2-yl)benzenesulfonamide (L2) were synthesized and well characterized by analytical and spectral studies. The synthesized compounds were tested for antimicrobial activity by disc diffusion method. The DNA binding of the complexes 1 and 3 with CT-DNA has been performed with absorption spectroscopy, which showed that both the complexes are avid binders of CT-DNA. Also the nuclease activity of complexes 1 and 3 with plasmid DNA (pUC19) was studied using agarose gel electrophoresis. The complex 1 can act as effective DNA cleaving agent when compared to complex 3 resulting in the nicked form of DNA under physiological conditions. The gel was run both in the absence and presence of the oxidizing agent. PMID:21253533

  12. Endonucleolytic activity directed towards 8-(2-hydroxy-2-propyl) purines in double-stranded DNA.

    PubMed Central

    Livneh, Z; Elad, D; Sperling, J

    1979-01-01

    Photoalkylation of circular covalently closed DNA from phage PM2 with isopropyl alcohol by using a free radical photoinitiator and UV light of lambda greater than 305 nm led to the specific 8-substitution of purine moieties in the DNA, yielding 8-(2-hydroxy-2-propyl)adenine and 8-(2-hydroxy-2-propyl)guanine as the only detectable damage in the DNA. Using this specifically photoalkylated DNA as a substrate, we discovered in extracts of Micrococcus luteus an endonucleolytic activity that is directed towards 8-(2-hydroxy-2-propyl) purines in DNA. The activity is not a combination of a DNA-glycosylase and an apurinic site endonuclease. It is not inhibited by single-stranded DNA, by UV- or gamma-irradiated single-stranded DNA, or by normal or depurinated double-stranded DNA. however, gamma- or UV-(254 nm) irradiated double-stranded DNAs to inhibit the activity, hinting at the possibility of a common type of lesion in these damaged DNAs. Divalent cations are not required for the incising activity, and it is fully active in 1 mM EDTA, whereas caffeine and ATP cause inhibition. Extracts of mutant M. luteus lacking pyrimidine-dimer-directed endonucleases were found to contain the endonucleolytic activity in levels comparable to those present in the wild type. After the incision, we could demonstrate the specific excision of the 8-alkylated purines from the damaged DNA. The special conformational consequences of the 8-alkylation of purines, at the nucleotide level, namely their nonregular syn conformation, suggest that it is the distortion in the DNA that is recognized by the endonuclease. PMID:293658

  13. Endonucleolytic activity directed towards 8-(2-hydroxy-2-propyl) purines in double-stranded DNA.

    PubMed

    Livneh, Z; Elad, D; Sperling, J

    1979-11-01

    Photoalkylation of circular covalently closed DNA from phage PM2 with isopropyl alcohol by using a free radical photoinitiator and UV light of lambda greater than 305 nm led to the specific 8-substitution of purine moieties in the DNA, yielding 8-(2-hydroxy-2-propyl)adenine and 8-(2-hydroxy-2-propyl)guanine as the only detectable damage in the DNA. Using this specifically photoalkylated DNA as a substrate, we discovered in extracts of Micrococcus luteus an endonucleolytic activity that is directed towards 8-(2-hydroxy-2-propyl) purines in DNA. The activity is not a combination of a DNA-glycosylase and an apurinic site endonuclease. It is not inhibited by single-stranded DNA, by UV- or gamma-irradiated single-stranded DNA, or by normal or depurinated double-stranded DNA. however, gamma- or UV-(254 nm) irradiated double-stranded DNAs to inhibit the activity, hinting at the possibility of a common type of lesion in these damaged DNAs. Divalent cations are not required for the incising activity, and it is fully active in 1 mM EDTA, whereas caffeine and ATP cause inhibition. Extracts of mutant M. luteus lacking pyrimidine-dimer-directed endonucleases were found to contain the endonucleolytic activity in levels comparable to those present in the wild type. After the incision, we could demonstrate the specific excision of the 8-alkylated purines from the damaged DNA. The special conformational consequences of the 8-alkylation of purines, at the nucleotide level, namely their nonregular syn conformation, suggest that it is the distortion in the DNA that is recognized by the endonuclease. PMID:293658

  14. Inhibition of tumorigenesis by a cytosine–DNA, methyltransferase, antisense oligodeoxynucleotide

    PubMed Central

    Ramchandani, Shyam; MacLeod, A. Robert; Pinard, Marc; von Hofe, Eric; Szyf, Moshe

    1997-01-01

    This paper tests the hypothesis that cytosine DNA methyltransferase (DNA MeTase) is a candidate target for anticancer therapy. Several observations have suggested recently that hyperactivation of DNA MeTase plays a critical role in initiation and progression of cancer and that its up-regulation is a component of the Ras oncogenic signaling pathway. We show that a phosphorothioate-modified, antisense oligodeoxynucleotide directed against the DNA MeTase mRNA reduces the level of DNA MeTase mRNA, inhibits DNA MeTase activity, and inhibits anchorage independent growth of Y1 adrenocortical carcinoma cells ex vivo in a dose-dependent manner. Injection of DNA MeTase antisense oligodeoxynucleotides i.p. inhibits the growth of Y1 tumors in syngeneic LAF1 mice, reduces the level of DNA MeTase, and induces demethylation of the adrenocortical-specific gene C21 and its expression in tumors in vivo. These results support the hypothesis that an increase in DNA MeTase activity is critical for tumorigenesis and is reversible by pharmacological inhibition of DNA MeTase. PMID:9012845

  15. The Polymerase Activity of Mammalian DNA Pol ζ Is Specifically Required for Cell and Embryonic Viability

    PubMed Central

    Lange, Sabine S.; Tomida, Junya; Boulware, Karen S.; Bhetawal, Sarita; Wood, Richard D.

    2016-01-01

    DNA polymerase ζ (pol ζ) is exceptionally important for maintaining genome stability. Inactivation of the Rev3l gene encoding the polymerase catalytic subunit causes a high frequency of chromosomal breaks, followed by lethality in mouse embryos and in primary cells. Yet it is not known whether the DNA polymerase activity of pol ζ is specifically essential, as the large REV3L protein also serves as a multiprotein scaffold for translesion DNA synthesis via multiple conserved structural domains. We report that Rev3l cDNA rescues the genomic instability and DNA damage sensitivity of Rev3l-null immortalized mouse fibroblast cell lines. A cDNA harboring mutations of conserved catalytic aspartate residues in the polymerase domain of REV3L could not rescue these phenotypes. To investigate the role of REV3L DNA polymerase activity in vivo, a Rev3l knock-in mouse was constructed with this polymerase-inactivating alteration. No homozygous mutant mice were produced, with lethality occurring during embryogenesis. Primary fibroblasts from mutant embryos showed growth defects, elevated DNA double-strand breaks and cisplatin sensitivity similar to Rev3l-null fibroblasts. We tested whether the severe Rev3l-/- phenotypes could be rescued by deletion of DNA polymerase η, as has been reported with chicken DT40 cells. However, Rev3l-/- Polh-/- mice were inviable, and derived primary fibroblasts were as sensitive to DNA damage as Rev3l-/- Polh+/+ fibroblasts. Therefore, the functions of REV3L in maintaining cell viability, embryonic viability and genomic stability are directly dependent on its polymerase activity, and cannot be ameliorated by an additional deletion of pol η. These results validate and encourage the approach of targeting the DNA polymerase activity of pol ζ to sensitize tumors to DNA damaging agents. PMID:26727495

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

    PubMed

    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. PMID:27496999

  17. Unraveling the Fanconi anaemia-DNA repair connection through DNA helicase and translocase activities

    SciTech Connect

    Thompson, L H

    2005-08-16

    How the Fanconi anaemia (FA) chromosome stability pathway functions to cope with interstrand crosslinks and other DNA lesions has been elusive, even after FANCD1 proved to be BRCA2, a partner of Rad51 in homologous recombination. The identification and characterization of two new Fanconi proteins having helicase motifs, FANCM and FANCJ/BRIP1/BACH1, implicates the FANC nuclear core complex as a participant in recognizing or processing damaged DNA, and the BRIP1 helicase as acting independently of this complex.

  18. Adenovirus preterminal protein synthesized in COS cells from cloned DNA is active in DNA replication in vitro.

    PubMed Central

    Pettit, S C; Horwitz, M S; Engler, J A

    1988-01-01

    Replication of the DNA genome of human adenovirus serotype 2 requires three virus-encoded proteins. Two of these proteins, the preterminal protein (pTP) and the adenovirus DNA polymerase, are transcribed from a single promoter at early times after virus infection. The mRNAs for these proteins share several exons, including one encoded near adenovirus genome coordinate 39. By using plasmids containing DNA fragments postulated to encode the various exons of pTP mRNA, the contributions of each exon to the synthesis of an active pTP have been measured. Only plasmids that contain both the open reading frame for pTP (genome coordinates 29.4 to 23.9) and the HindIII J fragment that contains the exon at genome coordinate 39 can express functional pTP. Images PMID:3336069

  19. Hypomethylation of the Treg-Specific Demethylated Region in FOXP3 Is a Hallmark of the Regulatory T-cell Subtype in Adult T-cell Leukemia.

    PubMed

    Shimazu, Yayoi; Shimazu, Yutaka; Hishizawa, Masakatsu; Hamaguchi, Masahide; Nagai, Yuya; Sugino, Noriko; Fujii, Sumie; Kawahara, Masahiro; Kadowaki, Norimitsu; Nishikawa, Hiroyoshi; Sakaguchi, Shimon; Takaori-Kondo, Akifumi

    2016-02-01

    Adult T-cell leukemia (ATL) is an aggressive T-cell malignancy caused by human T-cell leukemia virus type 1. Because of its immunosuppressive property and resistance to treatment, patients with ATL have poor prognoses. ATL cells possess the regulatory T cell (Treg) phenotype, such as CD4 and CD25, and usually express forkhead box P3 (FOXP3). However, the mechanisms of FOXP3 expression and its association with Treg-like characteristics in ATL remain unclear. Selective demethylation of the Treg-specific demethylated region (TSDR) in the FOXP3 gene leads to stable FOXP3 expression and defines natural Tregs. Here, we focus on the functional and clinical relationship between the epigenetic pattern of the TSDR and ATL. Analysis of DNA methylation in specimens from 26 patients with ATL showed that 15 patients (58%) hypomethylated the TSDR. The FOXP3(+) cells were mainly observed in the TSDR-hypomethylated cases. The TSDR-hypomethylated ATL cells exerted more suppressive function than the TSDR-methylated ATL cells. Thus, the epigenetic analysis of the FOXP3 gene identified a distinct subtype with Treg properties in heterogeneous ATL. Furthermore, we observed that the hypomethylation of TSDR was associated with poor outcomes in ATL. These results suggest that the DNA methylation status of the TSDR is an important hallmark to define this heterogeneous disease and to predict ATL patient prognosis. PMID:26681759

  20. DNA binding, nuclease activity, DNA photocleavage and cytotoxic properties of Cu(II) complexes of N-substituted sulfonamides.

    PubMed

    García-Giménez, José Luis; Hernández-Gil, Javier; Martínez-Ruíz, Aloma; Castiñeiras, Alfonso; Liu-González, Malva; Pallardó, Federico V; Borrás, Joaquín; Alzuet Piña, Gloria

    2013-04-01

    Ternary copper(II) complexes [Cu(NST)2(phen)] (1) and [Cu(NST)2(NH3)2]·H2O (2) [HNST=N-(4,5-dimethylthiazol-2-yl)naphthalene-1-sulfonamide] were prepared and characterized by physico-chemical techniques. Both 1 and 2 were structurally characterized by X-ray crystallography. The crystal structures show the presence of a distorted square planar CuN4 geometry in which the deprotonated sulfonamide, acting as monodentate ligand, binds to the metal ion through the thiazole N atom. Both complexes present intermolecular π-π stacking interactions between phenanthroline rings (compound 1) and between naphthalene rings (compound 2). The interaction of the complexes with CT DNA was studied by means of thermal denaturation, viscosity measurements and fluorescence spectroscopy. The complexes display good binding propensity to the calf thymus DNA giving the order: 1>2. Complex 1, which has a higher capability for binding to DNA, showed better nuclease activity than 2 in the presence of ascorbate/H2O2. Both the kinetics and the mechanism of the DNA cleavage reaction were investigated. Furthermore, complex 1 showed efficient photo-induced DNA cleavage activity on irradiation with UV light in the absence of any external reagent. The UV light induced DNA cleavage follows a photo-redox pathway with generation of hydroxyl radicals as reactive species. In addition, the cytotoxic properties of both complexes (1 and 2) were evaluated in human cancer cells (HeLa, Caco-2 and MDA-468). The low IC50 values, in particular those against Caco-2, have indicated that the compounds can be considered as promising chemotherapeutic agents. PMID:23384854

  1. Os2 -Os4 Switch Controls DNA Knotting and Anticancer Activity.

    PubMed

    Fu, Ying; Romero, María J; Salassa, Luca; Cheng, Xi; Habtemariam, Abraha; Clarkson, Guy J; Prokes, Ivan; Rodger, Alison; Costantini, Giovanni; Sadler, Peter J

    2016-07-25

    Dinuclear trihydroxido-bridged osmium-arene complexes are inert and biologically inactive, but we show here that linking dihydroxido-bridged Os(II) -arene fragments by a bridging di-imine to form a metallacycle framework results in strong antiproliferative activity towards cancer cells and distinctive knotting of DNA. The shortened spacer length reduces biological activity and stability in solution towards decomposition to biologically inactive dimers. Significant differences in behavior toward plasmid DNA condensation are correlated with biological activity. PMID:27240103

  2. Human cytochrome P-450PA (P-450IA2), the phenacetin O-deethylase, is primarily responsible for the hepatic 3-demethylation of caffeine and N-oxidation of carcinogenic arylamines.

    PubMed Central

    Butler, M A; Iwasaki, M; Guengerich, F P; Kadlubar, F F

    1989-01-01

    Aromatic amines are well known as occupational carcinogens and are found in cooked foods, tobacco smoke, synthetic fuels, and agricultural chemicals. For the primary arylamines, metabolic N-oxidation by hepatic cytochromes P-450 is generally regarded as an initial activation step leading to carcinogenesis. The metabolic activation of 4-aminobiphenyl, 2-naphthylamine, and several heterocyclic amines has been shown recently to be catalyzed by rat cytochrome P-450ISF-G and by its human ortholog, cytochrome P-450PA. We now report that human hepatic microsomal caffeine 3-demethylation, the initial major step in caffeine biotransformation in humans, is selectively catalyzed by cytochrome P-450PA. Caffeine 3-demethylation was highly correlated with 4-aminobiphenyl N-oxidation (r = 0.99; P less than 0.0005) in hepatic microsomal preparations obtained from 22 human organ donors, and both activities were similarly decreased by the selective inhibitor, 7,8-benzoflavone. The rates of microsomal caffeine 3-demethylation, 4-aminobiphenyl N-oxidation, and phenacetin O-deethylation were also significantly correlated with each other and with the levels of immunoreactive human cytochrome P-450PA. Moreover, a rabbit polyclonal antibody raised to human cytochrome P-450PA was shown to inhibit strongly all three of these activities and to inhibit the N-oxidation of the carcinogen 2-naphthylamine and the heterocyclic amines, 2-amino-6-methyldipyrido-[1,2-a:3',2'-d]imidazole and 2-amino-3-methylimidazo[4,5-f]-quinoline. Human liver cytochrome P-450PA was also shown to catalyze caffeine 3-demethylation, 4-aminobiphenyl N-oxidation, and phenacetin O-deethylation. Thus, estimation of caffeine 3-demethylation activity in humans may be useful in the characterization of arylamine N-oxidation phenotypes and in the assessment of whether or not the hepatic levels of cytochrome P-450PA, as affected by environmental or genetic factors, contribute to interindividual differences in susceptibility to

  3. DNA binding activities of the Caenorhabditis elegans Tc3 transposase.

    PubMed Central

    Colloms, S D; van Luenen, H G; Plasterk, R H

    1994-01-01

    Tc3 is a member of the Tc1/mariner family of transposable elements. All these elements have terminal inverted repeats, encode related transposases and insert exclusively into TA dinucleotides. We have studied the DNA binding properties of Tc3 transposase and found that an N-terminal domain of 65 amino acids binds specifically to two regions within the 462 bp Tc3 inverted repeat; one region is located at the end of the inverted repeat, the other is located approximately 180 bp from the end. Methylation interference experiments indicate that this N-terminal DNA binding domain of the Tc3 transposase interacts with nucleotides on one face of the DNA helix over adjacent major and minor grooves. Images PMID:7838706

  4. Phosphorus(V) corrole: DNA binding, photonuclease activity and cytotoxicity toward tumor cells.

    PubMed

    Wang, Yi-Guang; Zhang, Zhao; Wang, Hui; Liu, Hai-Yang

    2016-08-01

    A new phosphorus(V) corrole, 10-(4-hydroxylphenyl)-5,15-bis(pentafluorophenyl)corrolato (trans-dihydroxo)phosphorus(V) 1-P, was synthesized and characterized. The interaction of 1-P with calf thymus DNA (CT-DNA) was studied by multi-spectroscopic methods. The photonuclease activity of this complex was examined by agarose gel electrophoresis. 1-P may bind to CT-DNA via an outside binding mode and display good photonuclease activity. 1-P displayed low dark toxicity but high photocytotoxic activity against H460 and A549 tumor cell lines. PMID:27281437

  5. 75 FR 31795 - Office of Biotechnology Activities; Recombinant DNA Research: Amended Notice of Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-04

    ..., 2010 (75 FR 28811) is withdrawn. The discussion that was to be held at the June 16-17, 2010 meeting of... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA... ] under Section III-A-1 of the NIH Guidelines for Research Involving Recombinant DNA Molecules...

  6. DNA

    ERIC Educational Resources Information Center

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  7. Sensing Enzymatic Activity by Exposure and Selection of DNA-Encoded Probes.

    PubMed

    Jetson, Rachael R; Krusemark, Casey J

    2016-08-01

    A sensing approach is applied to encode quantitative enzymatic activity information into DNA sequence populations. The method utilizes DNA-linked peptide substrates as activity probes. Signal detection involves chemical manipulation of a probe population downstream of sample exposure and application of purifying, selective pressure for enzyme products. Selection-induced changes in DNA abundance indicate sample activity. The detection of protein kinase, protease, and farnesyltransferase activities is demonstrated. The assays were employed to measure enzyme inhibition by small molecules and activity in cell lysates using parallel DNA sequencing or quantitative PCR. This strategy will allow the extensive infrastructure for genetic analysis to be applied to proteomic assays, which has a number of advantages in throughput, sensitivity, and sample multiplexing. PMID:27355201

  8. Low concentration of arsenite exacerbates UVR-induced DNA strand breaks by inhibiting PARP-1 activity

    SciTech Connect

    Qin Xujun; Hudson, Laurie G.; Liu Wenlan; Timmins, Graham S.; Liu Kejian

    2008-10-01

    Epidemiological studies have associated arsenic exposure with many types of human cancers. Arsenic has also been shown to act as a co-carcinogen even at low concentrations. However, the precise mechanism of its co-carcinogenic action is unknown. Recent studies indicate that arsenic can interfere with DNA-repair processes. Poly(ADP-ribose) polymerase (PARP)-1 is a zinc-finger DNA-repair protein, which can promptly sense DNA strand breaks and initiate DNA-repair pathways. In the present study, we tested the hypothesis that low concentrations of arsenic could inhibit PAPR-1 activity and so exacerbate levels of ultraviolet radiation (UVR)-induced DNA strand breaks. HaCat cells were treated with arsenite and/or UVR, and then DNA strand breaks were assessed by comet assay. Low concentrations of arsenite ({<=} 2 {mu}M) alone did not induce significant DNA strand breaks, but greatly enhanced the DNA strand breaks induced by UVR. Further studies showed that 2 {mu}M arsenite effectively inhibited PARP-1 activity. Zinc supplementation of arsenite-treated cells restored PARP-1 activity and significantly diminished the exacerbating effect of arsenite on UVR-induced DNA strand breaks. Importantly, neither arsenite treatment, nor zinc supplementation changed UVR-triggered reactive oxygen species (ROS) formation, suggesting that their effects upon UVR-induced DNA strand breaks are not through a direct free radical mechanism. Combination treatments of arsenite with PARP-1 inhibitor 3-aminobenzamide or PARP-1 siRNA demonstrate that PARP-1 is the target of arsenite. Together, these findings show that arsenite at low concentration exacerbates UVR-induced DNA strand breaks by inhibiting PARP-1 activity, which may represent an important mechanism underlying the co-carcinogenicity of arsenic.

  9. Characterization of two nuclear mammalian homologous DNA-pairing activities that do not require associated exonuclease activity.

    PubMed Central

    Akhmedov, A T; Bertrand, P; Corteggiani, E; Lopez, B S

    1995-01-01

    We have developed an assay to study homologous DNA-pairing activities in mammalian nuclear extracts. This assay is derived from the POM blot assay, described earlier, which was specific for RecA activity in bacterial crude extracts. In the present work, proteins from mammalian nuclear extracts were resolved by electrophoresis on SDS/polyacrylamide gels and then electrotransferred onto a nitrocellulose membrane coated with circular single-stranded DNA (ssDNA). The blot obtained was incubated with a labeled homologous double-stranded DNA (dsDNA). Homologous pairing between the ssDNA and the labeled dsDNA was detected by autoradiography as a radioactive spot on the membrane. In nuclear extracts from mammalian cells, we found two major polypeptides of 100 and 75 kDa, able to promote the formation of stable plectonemic joints. Joint molecule formation required at least one homologous end on the dsDNA, but either end of the dsDNA could be recruited to initiate the reaction. For each polypeptide, the reaction required divalent cations such as Mg2+, Ca2+, or Mn2+. Although ATP was not necessary, ADP was inhibitory in each case. Unlike most of the known eukaryotic DNA-pairing proteins, both activities identified here were able to promote the formation of joint molecules without requiring an associated exonuclease activity. In addition, these two proteins were detected in cell lines from different tissues and from different mammalian species (human, mouse, and hamster). Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7878049

  10. Differential modulation of base excision repair activities during brain ontogeny: implications for repair of transcribed DNA.

    PubMed

    Englander, Ella W; Ma, Huaxian

    2006-01-01

    DNA repair sustains fidelity of genomic replication in proliferating cells and integrity of transcribed sequences in postmitotic tissues. The repair process is critical in the brain, because high oxygen consumption exacerbates the risk for accumulation of oxidative DNA lesions in postmitotic neurons. Most oxidative DNA damage is repaired by the base excision repair (BER) pathway, which is initiated by specialized DNA glycosylases. Because the newly discovered Nei-like mammalian DNA glycosylases (NEIL1/2) proficiently excise oxidized bases from bubble structured DNA, it was suggested that NEILs favor repair of transcribed or replicated DNA. In addition, since NEILs generate 3'-phosphate termini, which are poor targets for AP endonuclease (APE1), it was proposed that APE1-dependent and independent BER sub-pathways exist in mammalian cells. We measured expression and activities of BER enzymes during brain ontogeny, i.e., during a physiologic transition from proliferative to postmitotic differentiated state. While a subset of BER enzymes, exhibited declining expression and excision activities, expression of NEIL1 and NEIL2 glycosylases increased during brain development. Furthermore, the capacity for excision of 5-hydroxyuracil from bubble structured DNA was retained in the mature rat brain suggesting a role for NEIL glycosylases in maintaining the integrity of transcribed DNA in postmitotic brain. PMID:16257035

  11. Evaluation of DNA extraction methods for the analysis of microbial community in biological activated carbon.

    PubMed

    Zheng, Lu; Gao, Naiyun; Deng, Yang

    2012-01-01

    It is difficult to isolate DNA from biological activated carbon (BAC) samples used in water treatment plants, owing to the scarcity of microorganisms in BAC samples. The aim of this study was to identify DNA extraction methods suitable for a long-term, comprehensive ecological analysis of BAC microbial communities. To identify a procedure that can produce high molecular weight DNA, maximizes detectable diversity and is relatively free from contaminants, the microwave extraction method, the cetyltrimethylammonium bromide (CTAB) extraction method, a commercial DNA extraction kit, and the ultrasonic extraction method were used for the extraction of DNA from BAC samples. Spectrophotometry, agarose gel electrophoresis and polymerase chain reaction (PCR)-restriction fragment length polymorphisms (RFLP) analysis were conducted to compare the yield and quality of DNA obtained using these methods. The results showed that the CTAB method produce the highest yield and genetic diversity of DNA from BAC samples, but DNA purity was slightly less than that obtained with the DNA extraction-kit method. This study provides a theoretical basis for establishing and selecting DNA extraction methods for BAC samples. PMID:22629615

  12. Interleukin-4 enhances PARP-dependent DNA repair activity in vitro.

    PubMed

    Ciszewski, Wojciech Michał; Wagner, Waldemar; Kania, Katarzyna Dominika; Dastych, Jarosław

    2014-09-01

    Eukaryotic cells possess several DNA repair mechanisms, including homologous recombination and the non-homologous end-joining (NHEJ) system. There are two known NHEJ systems. The major mechanism depends on the catalytic unit of DNA-dependent protein kinase (DNA-PKcs) and DNA ligase IV, and an alternative mechanism (B-NHEJ) depends on poly(ADP-ribose) polymerase (PARP). These systems are upregulated by genotoxic agents. Interleukin 4 (IL-4) is an immunoregulatory cytokine that is secreted by immune cells upon contact with certain genotoxic compounds and is known to regulate several genes encoding components of DNA repair systems in human monocytes. We have investigated the possible effects of IL-4 on the DNA repair process within murine and human cells exposed to selected genotoxic compounds. In a series of experiments, including the comet assay, cell surface annexin V staining, analysis of histone H2AX phosphorylation, and a DNA end-joining assay, we observed that IL-4 decreased DNA damage in murine fibroblasts and human glioblastoma cells exposed to genotoxic agents and increased DNA ligation activity in the nuclei of these cells in a process that depended on PARP. These observations suggest that IL-4 is capable of upregulating the alternative NHEJ DNA repair mechanism in murine and human cells. PMID:24724620

  13. Immune cell activation from multivalent interactions with liquid-crystalline polycation-DNA complexes

    NASA Astrophysics Data System (ADS)

    Schmidt, Nathan; Jin, Fan; Lande, Roberto; Curk, Tine; Xian, Wujing; Frasca, Loredana; Dobnikar, Jure; Frenkel, Daan; Gilliet, Michel; Wong, Gerard

    2014-03-01

    Microbial DNA can trigger type I interferon (IFN) production in plasmacytoid cells (pDCs) by binding to endosomal toll-like receptor 9 (TLR9). TLR9 in pDCs do not normally respond to self-DNA, but in certain autoimmune diseases self-DNA can complex with the polycationic antimicrobial peptide LL37 into condensed structures which allow DNA to access endosomal compartments and stimulate TLR9 in pDCs. We use x-ray studies and cell measurements of IFN secretion by pDCs to show that a broad range of polycation-DNA complexes stimulate pDCs and elucidate the criterion for high IFN production. Furthermore, we show via experiments and computer simulations that the distinguishing factor for why certain complexes activate pDCs while others do not is the self-assembled structure of the liquid-crystalline polycation-DNA complex.

  14. Food mutagens: Mutagenic activity, DNA mechanisms, and cancer risk

    SciTech Connect

    1995-09-01

    Heterocyclic amines are potent mutagens, and they are carcinogenic in laboratory animals. We have shown that typical Western diets rich in well-done meta cooked at high temperatures have significant levels of at least five different heterocyclic amines. We are beginning to understand the process by which food mutagens become adducted to DNA. This molecular binding to DNA is an important step that can lead to cancer. The binding of mutagens depends on the formation of intermediate, biologically reactive molecules. The intermediates appear to link preferentially to the DNA base guanine. The extent of DNA adduction and the subsequent occurrence of tumors vary considerably in different types of tissues, such as the liver and pancreas, and in different animal species. A comparison of fried meats shows that beef and chicken are the most mutagenic types of food in a typical Western diet. When account is taken of the relative amounts of different meats consumed by Americans and of the potency of mutagens in them, ground beef may be the most important source of food mutagens in the U.S. diet. The overall, average, upper-bound estimate of cancer risk in the U.S. from eating heterocyclic amines in cooked foods is about one in ten thousand. The consumption of meat and chicken contributes most to the total risk. Some individuals eating large amounts of well-done muscle foods may be at much greater risk.

  15. Checkpoint Activation of an Unconventional DNA Replication Program in Tetrahymena

    PubMed Central

    Sandoval, Pamela Y.; Lee, Po-Hsuen; Meng, Xiangzhou; Kapler, Geoffrey M.

    2015-01-01

    The intra-S phase checkpoint kinase of metazoa and yeast, ATR/MEC1, protects chromosomes from DNA damage and replication stress by phosphorylating subunits of the replicative helicase, MCM2-7. Here we describe an unprecedented ATR-dependent pathway in Tetrahymena thermophila in which the essential pre-replicative complex proteins, Orc1p, Orc2p and Mcm6p are degraded in hydroxyurea-treated S phase cells. Chromosomes undergo global changes during HU-arrest, including phosphorylation of histone H2A.X, deacetylation of histone H3, and an apparent diminution in DNA content that can be blocked by the deacetylase inhibitor sodium butyrate. Most remarkably, the cell cycle rapidly resumes upon hydroxyurea removal, and the entire genome is replicated prior to replenishment of ORC and MCMs. While stalled replication forks are elongated under these conditions, DNA fiber imaging revealed that most replicating molecules are produced by new initiation events. Furthermore, the sole origin in the ribosomal DNA minichromosome is inactive and replication appears to initiate near the rRNA promoter. The collective data raise the possibility that replication initiation occurs by an ORC-independent mechanism during the recovery from HU-induced replication stress. PMID:26218270

  16. Fluorescent Single-Stranded DNA Binding Protein as a Probe for Sensitive, Real-Time Assays of Helicase Activity

    PubMed Central

    Dillingham, Mark S.; Tibbles, Katherine L.; Hunter, Jackie L.; Bell, Jason C.; Kowalczykowski, Stephen C.; Webb, Martin R.

    2008-01-01

    The formation and maintenance of single-stranded DNA (ssDNA) are essential parts of many processes involving DNA. For example, strand separation of double-stranded DNA (dsDNA) is catalyzed by helicases, and this exposure of the bases on the DNA allows further processing, such as replication, recombination, or repair. Assays of helicase activity and probes for their mechanism are essential for understanding related biological processes. Here we describe the development and use of a fluorescent probe to measure ssDNA formation specifically and in real time, with high sensitivity and time resolution. The reagentless biosensor is based on the ssDNA binding protein (SSB) from Escherichia coli, labeled at a specific site with a coumarin fluorophore. Its use in the study of DNA manipulations involving ssDNA intermediates is demonstrated in assays for DNA unwinding, catalyzed by DNA helicases. PMID:18599625

  17. DNA binding induces active site conformational change in the human TREX2 3'-exonuclease.

    PubMed

    de Silva, Udesh; Perrino, Fred W; Hollis, Thomas

    2009-04-01

    The TREX enzymes process DNA as the major 3'-->5' exonuclease activity in mammalian cells. TREX2 and TREX1 are members of the DnaQ family of exonucleases and utilize a two metal ion catalytic mechanism of hydrolysis. The structure of the dimeric TREX2 enzyme in complex with single-stranded DNA has revealed binding properties that are distinct from the TREX1 protein. The TREX2 protein undergoes a conformational change in the active site upon DNA binding including ordering of active site residues and a shift of an active site helix. Surprisingly, even when a single monomer binds DNA, both monomers in the dimer undergo the structural rearrangement. From this we have proposed a model for DNA binding and 3' hydrolysis for the TREX2 dimer. The structure also shows how TREX proteins potentially interact with double-stranded DNA and suggest features that might be involved in strand denaturation to provide a single-stranded substrate for the active site. PMID:19321497

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

    PubMed Central

    2012-01-01

    Background 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. Methods 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. Results 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. Conclusions 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. PMID:22429326

  19. Effects of 5-azacytidine on natural killer cell activating receptor expression in patients with refractory anemia with excess of blasts.

    PubMed

    Costello, Régis T; Leclercq, Amélie; Treut, Thérèse Le; Sanchez, Carole; Mercier, Delphine; Sébahoun, Gérard

    2015-01-01

    Epigenetic drugs modify DNA methylation and are used in refractory anemia with excess of blasts (RAEB). These drugs may reactivate anti-oncogene expression and restore a normal phenotype instead of inducing antitumor toxicity, although they also have immunosuppressive effects on T-lymphocytes [1] In RAEB and acute myeloid leukemia, a defect in natural killer (NK) cell cytotoxicity has been shown, which relies on abnormal expression of activating receptors. Previous study has shown that 5-azacytidine impaired mRNA synthesis and induced apoptosis in NK cells [2]. In this study we investigated the effect of the demethylating drug 5-azacytidine (Vidaza(®)) on NK receptors with the hypothesis that demethylation of the promoters of activating NK receptor genes induces gene reactivation and thus may increase their expression. PMID:25709892

  20. Effects of 5-azacytidine on natural killer cell activating receptor expression in patients with refractory anemia with excess of blasts

    PubMed Central

    Costello, Régis T.; Leclercq, Amélie; Treut, Thérèse Le; Sanchez, Carole; Mercier, Delphine; Sébahoun, Gérard

    2015-01-01

    Epigenetic drugs modify DNA methylation and are used in refractory anemia with excess of blasts (RAEB). These drugs may reactivate anti-oncogene expression and restore a normal phenotype instead of inducing antitumor toxicity, although they also have immunosuppressive effects on T-lymphocytes [1] In RAEB and acute myeloid leukemia, a defect in natural killer (NK) cell cytotoxicity has been shown, which relies on abnormal expression of activating receptors. Previous study has shown that 5-azacytidine impaired mRNA synthesis and induced apoptosis in NK cells [2]. In this study we investigated the effect of the demethylating drug 5-azacytidine (Vidaza®) on NK receptors with the hypothesis that demethylation of the promoters of activating NK receptor genes induces gene reactivation and thus may increase their expression. PMID:25709892

  1. DNA repair activity in fish and interest in ecotoxicology: a review.

    PubMed

    Kienzler, Aude; Bony, Sylvie; Devaux, Alain

    2013-06-15

    The knowledge of DNA repair in a target species is of first importance as it is the primary line of defense against genotoxicants, and a better knowledge of DNA repair capacity in fish could help to interpret genotoxicity data and/or assist in the choice of target species, developmental stage and tissues to focus on, both for environmental biomonitoring studies and DNA repair testing. This review focuses in a first part on what is presently known on a mechanistic basis, about the various DNA repair systems in fish, in vivo and in established cell lines. Data on base excision repair (BER), direct reversal with O⁶-alkylguanine transferase and double strand breaks repair, although rather scarce, are being reviewed, as well as nucleotide excision repair (NER) and photoreactivation repair (PER), which are by far the most studied repair mechanisms in fish. Most of these repair mechanisms seem to be strongly species and tissue dependent; they also depend on the developmental stage of the organisms. BER is efficient in vivo, although no data has been found on in vitro models. NER activity is quite low or even inexistent depending on the studies; however this lack is partly compensated by a strong PER activity, especially in early developmental stage. In a second part, a survey of the ecotoxicological studies integrating DNA repair as a parameter responding to single or mixture of contaminant is realized. Three main approaches are being used: the measurement of DNA repair gene expression after exposure, although it has not yet been clearly established whether gene expression is indicative of repair capacity; the monitoring of DNA damage removal by following DNA repair kinetics; and the modulation of DNA repair activity following exposure in situ, in order to assess the impact of exposure history on DNA repair capacity. Since all DNA repair processes are possible targets for environmental pollutants, we can also wonder at which extent such a modulation of repair capacities

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

    PubMed

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

    2012-04-01

    Neocarzinostatin (NCS), a potent mutagen and carcinogen, consists of an enediyne prodrug and a protein carrier. It has a unique double role in that it intercalates into DNA and imposes radical-mediated damage after thiol activation. Here we employed NCS as a probe to examine the DNA-protection capability of caffeine, one of common dietary phytochemicals with potential cancer-chemopreventive activity. NCS at the nanomolar concentration range could induce significant single- and double-strand lesions in DNA, but up to 75 ± 5% of such lesions were found to be efficiently inhibited by caffeine. The percentage of inhibition was caffeine-concentration dependent, but was not sensitive to the DNA-lesion types. The well-characterized activation reactions of NCS allowed us to explore the effect of caffeine on the enediyne-generated radicals. Postactivation analyses by chromatographic and mass spectroscopic methods identified a caffeine-quenched enediyne-radical adduct, but the yield was too small to fully account for the large inhibition effect on DNA lesions. The affinity between NCS chromophore and DNA was characterized by a fluorescence-based kinetic method. The drug-DNA intercalation was hampered by caffeine, and the caffeine-induced increases in DNA-drug dissociation constant was caffeine-concentration dependent, suggesting importance of binding affinity in the protection mechanism. Caffeine has been shown to be both an effective free radical scavenger and an intercalation inhibitor. Our results demonstrated that caffeine ingeniously protected DNA against the enediyne-induced damages mainly by inhibiting DNA intercalation beforehand. The direct scavenging of the DNA-bound NCS free radicals by caffeine played only a minor role. PMID:21538576

  3. Polymerase/DNA interactions and enzymatic activity: multi-parameter analysis with electro-switchable biosurfaces

    NASA Astrophysics Data System (ADS)

    Langer, Andreas; Schräml, Michael; Strasser, Ralf; Daub, Herwin; Myers, Thomas; Heindl, Dieter; Rant, Ulrich

    2015-07-01

    The engineering of high-performance enzymes for future sequencing and PCR technologies as well as the development of many anticancer drugs requires a detailed analysis of DNA/RNA synthesis processes. However, due to the complex molecular interplay involved, real-time methodologies have not been available to obtain comprehensive information on both binding parameters and enzymatic activities. Here we introduce a chip-based method to investigate polymerases and their interactions with nucleic acids, which employs an electrical actuation of DNA templates on microelectrodes. Two measurement modes track both the dynamics of the induced switching process and the DNA extension simultaneously to quantitate binding kinetics, dissociation constants and thermodynamic energies. The high sensitivity of the method reveals previously unidentified tight binding states for Taq and Pol I (KF) DNA polymerases. Furthermore, the incorporation of label-free nucleotides can be followed in real-time and changes in the DNA polymerase conformation (finger closing) during enzymatic activity are observable.

  4. Depletion of RNase HI activity in Escherichia coli lacking DNA topoisomerase I leads to defects in DNA supercoiling and segregation

    PubMed Central

    Usongo, Valentine; Nolent, Flora; Sanscartier, Patrick; Tanguay, Cynthia; Broccoli, Sonia; Baaklini, Imad; Drlica, Karl; Drolet, Marc

    2010-01-01

    Gyrase-mediated hypernegative supercoiling is one manifestation of R-loop formation, a phenomenon that is normally suppressed by topoisomerase I (topA) in Escherichia coli. Overproduction of RNase HI (rnhA), an enzyme that removes the RNA moiety of R-loops, prevents hypernegative supercoiling and allows growth of topA null mutants. We previously showed that topA and rnhA null mutations are incompatible. We now report that such mutants were viable when RNase HI or topoisomerase III was expressed from a plasmid-borne gene. Surprisingly, DNA of topA null mutants became relaxed rather than hypernegatively supercoiled following depletion of RNase HI activity. This result failed to correlate with the cellular concentration of gyrase or topoisomerase IV (the other relaxing enzyme in the cell) or with transcription-induced supercoiling. Rather, intracellular DNA relaxation in the absence of RNase HI was related to inhibition of gyrase activity both in vivo and in extracts. Cells lacking topA and rnhA also exhibited properties consistent with segregation defects. Overproduction of topoisomerase III, an enzyme that can carry out DNA decatenation, corrected the segregation defects without restoring supercoiling activity. Collectively these data reveal 1) the existence of a cellular response to loss of RNase HI that counters the supercoiling activity of gyrase and 2) supercoiling-independent segregation defects due to loss of RNase HI from topA null mutants. Thus RNase HI plays a more central role in DNA topology than previously thought. PMID:18554330

  5. Depletion of RNase HI activity in Escherichia coli lacking DNA topoisomerase I leads to defects in DNA supercoiling and segregation.

    PubMed

    Usongo, Valentine; Nolent, Flora; Sanscartier, Patrick; Tanguay, Cynthia; Broccoli, Sonia; Baaklini, Imad; Drlica, Karl; Drolet, Marc

    2008-08-01

    Gyrase-mediated hypernegative supercoiling is one manifestation of R-loop formation, a phenomenon that is normally suppressed by topoisomerase I (topA) in Escherichia coli. Overproduction of RNase HI (rnhA), an enzyme that removes the RNA moiety of R-loops, prevents hypernegative supercoiling and allows growth of topA null mutants. We previously showed that topA and rnhA null mutations are incompatible. We now report that such mutants were viable when RNase HI or topoisomerase III was expressed from a plasmid-borne gene. Surprisingly, DNA of topA null mutants became relaxed rather than hypernegatively supercoiled following depletion of RNase HI activity. This result failed to correlate with the cellular concentration of gyrase or topoisomerase IV (the other relaxing enzyme in the cell) or with transcription-induced supercoiling. Rather, intracellular DNA relaxation in the absence of RNase HI was related to inhibition of gyrase activity both in vivo and in extracts. Cells lacking topA and rnhA also exhibited properties consistent with segregation defects. Overproduction of topoisomerase III, an enzyme that can carry out DNA decatenation, corrected the segregation defects without restoring supercoiling activity. Collectively these data reveal (i) the existence of a cellular response to loss of RNase HI that counters the supercoiling activity of gyrase, and (ii) supercoiling-independent segregation defects due to loss of RNase HI from topA null mutants. Thus RNase HI plays a more central role in DNA topology than previously thought. PMID:18554330

  6. Antimalarial, antimicrobial, cytotoxic, DNA interaction and SOD like activities of tetrahedral copper(II) complexes.

    PubMed

    Mehta, Jugal V; Gajera, Sanjay B; Patel, Mohan N

    2014-11-01

    The mononuclear copper(II) complexes with P, O-donor ligand and different fluoroquinolones have been synthesized and characterized by elemental analysis, electronic spectra, TGA, EPR, FT-IR and LC-MS spectroscopy. An antimicrobial efficiency of the complexes has been tested against five different microorganisms in terms of minimum inhibitory concentration (MIC) and displays very good antimicrobial activity. The binding strength and binding mode of the complexes with Herring Sperm DNA (HS DNA) have been investigated by absorption titration and viscosity measurement studies. The studies suggest the classical intercalative mode of DNA binding. Gel electrophoresis assay determines the ability of the complexes to cleave the supercoiled form of pUC19 DNA. Synthesized complexes have been tested for their SOD mimic activity using nonenzymatic NBT/NADH/PMS system and found to have good antioxidant activity. All the complexes show good cytotoxic and in vitro antimalarial activities. PMID:25467683

  7. Antimalarial, antimicrobial, cytotoxic, DNA interaction and SOD like activities of tetrahedral copper(II) complexes

    NASA Astrophysics Data System (ADS)

    Mehta, Jugal V.; Gajera, Sanjay B.; Patel, Mohan N.

    2015-02-01

    The mononuclear copper(II) complexes with P, O-donor ligand and different fluoroquinolones have been synthesized and characterized by elemental analysis, electronic spectra, TGA, EPR, FT-IR and LC-MS spectroscopy. An antimicrobial efficiency of the complexes has been tested against five different microorganisms in terms of minimum inhibitory concentration (MIC) and displays very good antimicrobial activity. The binding strength and binding mode of the complexes with Herring Sperm DNA (HS DNA) have been investigated by absorption titration and viscosity measurement studies. The studies suggest the classical intercalative mode of DNA binding. Gel electrophoresis assay determines the ability of the complexes to cleave the supercoiled form of pUC19 DNA. Synthesized complexes have been tested for their SOD mimic activity using nonenzymatic NBT/NADH/PMS system and found to have good antioxidant activity. All the complexes show good cytotoxic and in vitro antimalarial activities.

  8. Dda helicase tightly couples translocation on single-stranded DNA to unwinding of duplex DNA: Dda is an optimally active helicase

    PubMed Central

    Byrd, Alicia K.; Matlock, Dennis L.; Bagchi, Debjani; Aarattuthodiyil, Suja; Harrison, David; Croquette, Vincent; Raney, Kevin D.

    2012-01-01

    Helicases utilize the energy of ATP hydrolysis to unwind double-stranded DNA while translocating on the DNA. Mechanisms for melting the duplex have been characterized as active or passive, depending on whether the enzyme actively separates the base pairs or simply sequesters single-stranded DNA that forms due to thermal fraying. Here we show that Dda translocates unidirectionally on single-stranded DNA at the same rate at which it unwinds double-stranded DNA in both ensemble and single molecule experiments. Further, the unwinding rate is largely insensitive to the duplex stability and to applied force. Thus, Dda transduces all of its translocase activity into DNA unwinding activity so that the rate of unwinding is limited by the rate of translocation and the enzyme actively separates the duplex. Active and passive helicases have been characterized by dividing the velocity of DNA unwinding in base pairs per second (Vun) by the velocity of translocation on single-stranded DNA in nucleotides per second (Vtrans). If the resulting fraction is 0.25, then a helicase is considered to be at the lower end of the “active” range. In the case of Dda, the average DNA unwinding velocity was 257 ± 42 bp/s and the average translocation velocity was 267 ± 15 nucleotides/s. The Vun/Vtrans value of 0.96 places Dda in a unique category of being an essentially “perfectly” active helicase. PMID:22504228

  9. The Roles of a Flavone-6-Hydroxylase and 7-O-Demethylation in the Flavone Biosynthetic Network of Sweet Basil*

    PubMed Central

    Berim, Anna; Gang, David R.

    2013-01-01

    Lipophilic flavonoids found in the Lamiaceae exhibit unusual 6- and 8-hydroxylations whose enzymatic basis is unknown. We show that crude protein extracts from peltate trichomes of sweet basil (Ocimum basilicum L.) cultivars readily hydroxylate position 6 of 7-O-methylated apigenin but not apigenin itself. The responsible protein was identified as a P450 monooxygenase from the CYP82 family, a family not previously reported to be involved in flavonoid metabolism. This enzyme prefers flavones but also accepts flavanones in vitro and requires a 5-hydroxyl in addition to a 7-methoxyl residue on the substrate. A peppermint (Mentha × piperita L.) homolog displayed identical substrate requirements, suggesting that early 7-O-methylation of flavones might be common in the Lamiaceae. This hypothesis is further substantiated by the pioneering discovery of 2-oxoglutarate-dependent flavone demethylase activity in basil, which explains the accumulation of 7-O-demethylated flavone nevadensin. PMID:23184958

  10. The role of human equilibrative nucleoside transporter 1 on the cellular transport of the DNA methyltransferase inhibitors 5-azacytidine and CP-4200 in human leukemia cells.

    PubMed

    Hummel-Eisenbeiss, Johanna; Hascher, Antje; Hals, Petter-Arnt; Sandvold, Marit Liland; Müller-Tidow, Carsten; Lyko, Frank; Rius, Maria

    2013-09-01

    The nucleoside analog 5-azacytidine is an archetypical drug for epigenetic cancer therapy, and its clinical effectiveness has been demonstrated in the treatment of myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML). However, therapy resistance in patients with MDS/AML remains a challenging issue. Membrane proteins that are involved in drug uptake are potential mediators of drug resistance. The responsible proteins for the transport of 5-azacytidine into MDS/AML cells are unknown. We have now systematically analyzed the expression and activity of various nucleoside transporters. We identified the human equilibrative nucleoside transporter 1 (hENT1) as the most abundant nucleoside transporter in leukemia cell lines and in AML patient samples. Transport assays using [¹⁴C]5-azacytidine demonstrated Na⁺-independent uptake of the drug into the cells, which was inhibited by S-(4-nitrobenzyl)-6-thioinosine (NBTI), a hENT1 inhibitor. The cellular toxicity of 5-azacytidine and its DNA demethylating activity were strongly reduced after hENT1 inhibition. In contrast, the cellular activity of the 5-azacytidine derivative 5-azacytidine-5'-elaidate (CP-4200), a nucleoside transporter-independent drug, persisted after hENT1 inhibition. A strong dependence of 5-azacytidine-induced DNA demethylation on hENT1 activity was also confirmed by array-based DNA methylation profiling, which uncovered hundreds of loci that became demethylated only when hENT1-mediated transport was active. Our data establish hENT1 as a key transporter for the cellular uptake of 5-azacytidine in leukemia cells and raise the possibility that hENT1 expression might be a useful biomarker to predict the efficiency of 5-azacytidine treatments. Furthermore, our data suggest that CP-4200 may represent a valuable compound for the modulation of transporter-related 5-azacytidine resistances. PMID:23814180

  11. De novo DNA methylation drives 5hmC accumulation in mouse zygotes

    PubMed Central

    Amouroux, Rachel; Hill, Peter WS; D’Souza, Zelpha; Nakayama, Manabu; Matsuda, Masashi; Turp, Aleksandra; Ndjetehe, Elodie; Encheva, Vesela; Kudo, Nobuaki R; Koseki, Haruhiko; Sasaki, Hiroyuki; Hajkova, Petra

    2016-01-01

    Zygotic epigenetic reprogramming entails genome-wide DNA demethylation that is accompanied by Ten-Eleven Translocation 3 (Tet3)-driven oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC)1-4. Here we demonstrate using detailed immunofluorescence analysis and ultra-sensitive LC/MS based quantitative measurements that the initial loss of paternal 5mC does not require 5hmC formation. Small molecule inhibition of Tet3 activity as well as genetic ablation impedes 5hmC accumulation in zygotes without affecting the early loss of paternal 5mC. Instead, 5hmC accumulation is dependent on the activity of zygotic Dnmt3a and Dnmt1, documenting a role for Tet3 driven hydroxylation in targeting de novo methylation activities present in the early embryo. Our data thus provide further insights into the dynamics of zygotic reprogramming revealing intricate interplay between DNA demethylation, de novo methylation and Tet3 driven hydroxylation. PMID:26751286

  12. De novo DNA methylation drives 5hmC accumulation in mouse zygotes.

    PubMed

    Amouroux, Rachel; Nashun, Buhe; Shirane, Kenjiro; Nakagawa, Shoma; Hill, Peter W S; D'Souza, Zelpha; Nakayama, Manabu; Matsuda, Masashi; Turp, Aleksandra; Ndjetehe, Elodie; Encheva, Vesela; Kudo, Nobuaki R; Koseki, Haruhiko; Sasaki, Hiroyuki; Hajkova, Petra

    2016-02-01

    Zygotic epigenetic reprogramming entails genome-wide DNA demethylation that is accompanied by Tet methylcytosine dioxygenase 3 (Tet3)-driven oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC; refs 1-4). Here we demonstrate using detailed immunofluorescence analysis and ultrasensitive LC-MS-based quantitative measurements that the initial loss of paternal 5mC does not require 5hmC formation. Small-molecule inhibition of Tet3 activity, as well as genetic ablation, impedes 5hmC accumulation in zygotes without affecting the early loss of paternal 5mC. Instead, 5hmC accumulation is dependent on the activity of zygotic Dnmt3a and Dnmt1, documenting a role for Tet3-driven hydroxylation in targeting de novo methylation activities present in the early embryo. Our data thus provide further insights into the dynamics of zygotic reprogramming, revealing an intricate interplay between DNA demethylation, de novo methylation and Tet3-driven hydroxylation. PMID:26751286

  13. Genome-wide localization of Rrm3 and Pif1 DNA helicases at stalled active and inactive DNA replication forks of Saccharomyces cerevisiae.

    PubMed

    Rossi, Silvia Emma; Carotenuto, Walter; Giannattasio, Michele

    2016-03-01

    The genome of the budding yeast Saccharomyces cerevisiae is sequenced and the location and dynamic of activation of DNA replication origins are known. G1 synchronized yeast cells can be released into S-phase in the presence of hydroxyurea (HU) (1), which slows down DNA replication and retains replication forks in proximity of DNA replication origins. In this condition, the Chromatin Immuno-Precipitation on chip (ChIP on chip) (2-4) of replisome components allows the precise localization of all active DNA replication forks. This analysis can be coupled with the ssDNA-BromodeoxyUridine (ssDNA-BrdU) Immuno-Precipitation on chip (ssDNA-BrdU IP on chip) technique (5-7), which detects the location of newly synthesized DNA. Comparison of binding and BrdU incorporation profiles allows to locate a factor of interest at DNA replication forks genome wide. We present datasets deposited in the gene expression omnibus (GEO) database under accession number GSE68214, which show how the DNA helicases Rrm3 and Pif1 (8) associate to active and inactive DNA replication forks. PMID:26981397

  14. Hormone stimulation of androgen receptor mediates dynamic changes in DNA methylation patterns at regulatory elements

    PubMed Central

    Dhiman, Vineet K.; Attwood, Kristopher; Campbell, Moray J.; Smiraglia, Dominic J.

    2015-01-01

    DNA methylation is an epigenetic modification that contributes to stable gene silencing by interfering with the ability of transcriptional regulators to bind to DNA. Recent findings have revealed that hormone stimulation of certain nuclear receptors induces rapid, dynamic changes in DNA methylation patterns alongside transcriptional responses at a subset of target loci, over time. However, the ability of androgen receptor (AR) to dynamically regulate gene transcription is relatively under-studied and its role in the regulation of DNA methylation patterns remains to be elucidated. Here we demonstrate in normal prostate cells that hormone stimulated AR activity results in dynamic changes in the transcription rate and DNA methylation patterns at the AR target genes, TIPARP and SGK1. Time-resolved chromatin immunoprecipitation experiments on the SGK1 locus reveals dynamic recruitment of AR and RNA Polymerase II, as well as the recruitment of proteins involved in the DNA demethylation process, TET1 and TDG. Furthermore, the presence of DNA methylation at dynamic regions inhibits protein binding and transcriptional activity of SGK1. These findings establish AR activity as a contributing factor to the dynamic regulation of DNA methylation patterns at target genes in prostate biology and infer further complexity involved in nuclear receptor mediation of transcriptional regulation. PMID:26646795

  15. Ubiquitin ligase activity of TFIIH and the transcriptional response to DNA damage.

    PubMed

    Takagi, Yuichiro; Masuda, Claudio A; Chang, Wei-Hau; Komori, Hirofumi; Wang, Dong; Hunter, Tony; Joazeiro, Claudio A P; Kornberg, Roger D

    2005-04-15

    Core transcription factor (TF) IIH purified from yeast possesses an E3 ubiquitin (Ub) ligase activity, which resides, at least in part, in a RING finger (RNF) domain of the Ssl1 subunit. Yeast strains mutated in the Ssl1 RNF domain are sensitive to ultraviolet (UV) light and to methyl methanesulfonate (MMS). This increased sensitivity to DNA-damaging agents does not reflect a deficiency in nucleotide excision repair. Rather, it correlates with reduced transcriptional induction of genes involved in DNA repair, suggesting that the E3 Ub ligase activity of TFIIH mediates the transcriptional response to DNA damage. PMID:15837426

  16. Binding Potency of Heparin Immobilized on Activated Charcoal for DNA Antibodies.

    PubMed

    Snezhkova, E A; Tridon, A; Evrard, B; Nikolaev, V G; Uvarov, V Yu; Tsimbalyuk, R S; Ivanuk, A A; Komov, V V; Sakhno, L A

    2016-02-01

    In vitro experiments showed that heparin adsorbed on activated charcoal can bind antibodies raised against native and single-stranded DNA in a diluted sera pool with a high level of these DNA. Thus, heparin used as anticoagulant during hemosorption procedure can demonstrate supplementary therapeutic activity resulting from its interaction with various agents involved in acute and chronic inflammatory reactions such as DNA- and RNA-binding substances, proinflammatory cytokines, complement components, growth factors, etc. Research and development of heparin-containing carbonic adsorbents for the therapy of numerous inflammatory and autoimmune diseases seems to be a promising avenue in hematology. PMID:26902353

  17. HMGB1-DNA Complex-induced Autophagy Limits AIM2 Inflammasome Activation through RAGE

    PubMed Central

    Liu, Liying; Yang, Minghua; Kang, Rui; Yu, Yan; Dai, Yunpen; Gao, Fei; Wang, Hongmei; Sun, Xiaojun; Li, Xiuli; Li, Jianhua; Wang, Haichao; Cao, Lizhi; Tang, Daolin

    2014-01-01

    High mobility group box 1 (HMGB1) is a prototype damage-associated molecular pattern (DAMP) that can induce inflammatory and immune responses alone as well as in combination with other molecules such as DNA. However, the intricate molecular mechanisms underlying HMGB1-DNA complex-mediated innate immune response remains largely elusive. In this study, we demonstrated that HMGB1-DNA complex initially induced absent in melanoma 2 (AIM2)-dependent inflammasome activation, and promoted rapid release of inflammasome-dependent early proinflammatory cytokines such as interleukin 1β (IL-1β). Subsequently, HMGB1-DNA complex stimulated an ATG5-dependent cellular degradation process, autophagy, which was paralleled by a cessation of AIM2 inflammasome activation and IL-1β release. These HMGB1-DNA complex-induced inflammasome activation and autophagy were both dependent on the receptor for advanced glycation endproducts (RAGE) that recognizes a wide array of ligands (including HMGB1 and DNA). Thus, autophagy may function as a negative counter-regulatory mechanism for HMGB1-DNA complex-induced inflammasome activation, and provide a checkpoint to limit the development of inflammation. PMID:24971542

  18. DNA-recognition by a σ54 transcriptional activator from Aquifex aeolicus

    PubMed Central

    Vidangos, Natasha K.; Heideker, Johanna; Lyubimov, Artem; Lamers, Meindert; Huo, Yixin; Pelton, Jeffrey G.; Ton, Jimmy; Gralla, Jay; Berger, James; Wemmer, David E.

    2014-01-01

    Transcription initiation by bacterial σ54-polymerase requires the action of a transcriptional activator protein. Activators bind sequence-specifically upstream of the transcription initiation site via a DNA-binding domain. The structurally characterized DNA-binding domains from activators all belong to the Factor for Inversion Stimulation (Fis) family of helix-turn-helix DNA-binding proteins. We report here structures of the free and DNA-bound forms of the DNA-binding domain of NtrC4 (4DBD) from Aquifex aeolicus, a member of the NtrC family of σ54 activators. Two NtrC4 binding sites were identified upstream (−145 and −85 base pairs) from the start of the lpxC gene, which is responsible for the first committed step in Lipid A biosynthesis. This is the first experimental evidence for σ54 regulation in lpxC expression. 4DBD was crystallized both without DNA and in complex with the −145 binding site. The structures, together with biochemical data, indicate that NtrC4 binds to DNA in a manner that is similar to that of its close homologue, Fis. The greater sequence specificity for the binding of 4DBD relative to Fis seems to arise from a larger number of base specific contacts contributing to affinity than for Fis. PMID:25158097

  19. DNA Recognition by a σ54 Transcriptional Activator from Aquifex aeolicus

    SciTech Connect

    Vidangos, Natasha K.; Heideker, Johanna; Lyubimov, Artem; Lamers, Meindert; Huo, Yixin; Pelton, Jeffrey G.; Ton, Jimmy; Gralla, Jay; Berger, James; Wemmer, David E.

    2014-08-23

    Transcription initiation by bacterial σ54-polymerase requires the action of a transcriptional activator protein. Activators bind sequence-specifically upstream of the transcription initiation site via a DNA-binding domain. The structurally characterized DNA-binding domains from activators all belong to the Factor for Inversion Stimulation (Fis) family of helix-turn-helix DNA-binding proteins. We report here structures of the free and DNA-bound forms of the DNA-binding domain of NtrC4 (4DBD) from Aquifex aeolicus, a member of the NtrC family of σ54 activators. Two NtrC4 binding sites were identified upstream (-145 and -85 base pairs) from the start of the lpxC gene, which is responsible for the first committed step in Lipid A biosynthesis. This is the first experimental evidence for σ54 regulation in lpxC expression. 4DBD was crystallized both without DNA and in complex with the -145 binding site. The structures, together with biochemical data, indicate that NtrC4 binds to DNA in a manner that is similar to that of its close homologue, Fis. Ultimately, the greater sequence specificity for the binding of 4DBD relative to Fis seems to arise from a larger number of base specific contacts contributing to affinity than for Fis.

  20. DNA Recognition by a σ54 Transcriptional Activator from Aquifex aeolicus

    DOE PAGESBeta

    Vidangos, Natasha K.; Heideker, Johanna; Lyubimov, Artem; Lamers, Meindert; Huo, Yixin; Pelton, Jeffrey G.; Ton, Jimmy; Gralla, Jay; Berger, James; Wemmer, David E.

    2014-08-23

    Transcription initiation by bacterial σ54-polymerase requires the action of a transcriptional activator protein. Activators bind sequence-specifically upstream of the transcription initiation site via a DNA-binding domain. The structurally characterized DNA-binding domains from activators all belong to the Factor for Inversion Stimulation (Fis) family of helix-turn-helix DNA-binding proteins. We report here structures of the free and DNA-bound forms of the DNA-binding domain of NtrC4 (4DBD) from Aquifex aeolicus, a member of the NtrC family of σ54 activators. Two NtrC4 binding sites were identified upstream (-145 and -85 base pairs) from the start of the lpxC gene, which is responsible for themore » first committed step in Lipid A biosynthesis. This is the first experimental evidence for σ54 regulation in lpxC expression. 4DBD was crystallized both without DNA and in complex with the -145 binding site. The structures, together with biochemical data, indicate that NtrC4 binds to DNA in a manner that is similar to that of its close homologue, Fis. Ultimately, the greater sequence specificity for the binding of 4DBD relative to Fis seems to arise from a larger number of base specific contacts contributing to affinity than for Fis.« less

  1. Importance of Endosomal Cathelicidin Degradation To Enhance DNA-Induced Chicken Macrophage Activation.

    PubMed

    Coorens, Maarten; van Dijk, Albert; Bikker, Floris; Veldhuizen, Edwin J A; Haagsman, Henk P

    2015-10-15

    Cathelicidins are essential in the protection against invading pathogens through both their direct antimicrobial activity and their immunomodulatory functions. Although cathelicidins are known to modulate activation by several TLR ligands, little is known about their influence on DNA-induced macrophage activation. In this study, we explored the effects of cathelicidins on DNA-induced activation of chicken macrophages and elucidated the intracellular processes underlying these effects. Our results show that chicken cathelicidin (CATH)-2 strongly enhances DNA-induced activation of both chicken and mammalian macrophages because of enhanced endocytosis of DNA-CATH-2 complexes. After endocytosis, DNA is liberated from the complex because of proteolytic breakdown of CATH-2, after which TLR21 is activated. This leads to increased cytokine expression and NO production. Through the interaction with DNA, CATH-2 can play an important role in modulating the immune response at sites of infection. These observations underline the importance of cathelicidins in sensing bacterial products and regulating immune responses. PMID:26378074

  2. Raman spectroscopic characterisations and analytical discrimination between caffeine and demethylated analogues of pharmaceutical relevance

    NASA Astrophysics Data System (ADS)

    Edwards, H. G. M.; Munshi, T.; Anstis, M.

    2005-05-01

    The FT Raman spectrum of caffeine was analysed along with that of its demethylated analogues, theobromine and theophylline. The similar but not identical structures of these three compounds allowed a more detailed assignment of the Raman bands. Noticeable differences in the Raman spectra of these compounds were apparent and key marker bands have been identified for the spectroscopic identification of these three compounds.

  3. Developmental Heterogeneity in DNA Packaging Patterns Influences T-Cell Activation and Transmigration

    PubMed Central

    Garg, Megha; R., Indulaxmi; Perumalsamy, Lakshmi R.; Sarin, Apurva; Shivashankar, G. V.

    2012-01-01

    Cellular differentiation programs are accompanied by large-scale changes in nuclear organization and gene expression. In this context, accompanying transitions in chromatin assembly that facilitates changes in gene expression and cell behavior in a developmental system are poorly understood. Here, we address this gap and map structural changes in chromatin organization during murine T-cell development, to describe an unusual heterogeneity in chromatin organization and associated functional correlates in T-cell lineage. Confocal imaging of DNA assembly in cells isolated from bone marrow, thymus and spleen reveal the emergence of heterogeneous patterns in DNA organization in mature T-cells following their exit from the thymus. The central DNA pattern dominated in immature precursor cells in the thymus whereas both central and peripheral DNA patterns were observed in naïve and memory cells in circulation. Naïve T-cells with central DNA patterns exhibited higher mechanical pliability in response to compressive loads in vitro and transmigration assays in vivo, and demonstrated accelerated expression of activation-induced marker CD69. T-cell activation was characterized by marked redistribution of DNA assembly to a central DNA pattern and increased nuclear size. Notably, heterogeneity in DNA patterns recovered in cells induced into quiescence in culture, suggesting an internal regulatory mechanism for chromatin reorganization. Taken together, our results uncover an important component of plasticity in nuclear organization, reflected in chromatin assembly, during T-cell development, differentiation and transmigration. PMID:22957031

  4. Multispectral studies of DNA binding, antioxidant and cytotoxic activities of a new pyranochromene derivative

    NASA Astrophysics Data System (ADS)

    Dehkordi, Mahvash Farajzadeh; Dehghan, Gholamreza; Mahdavi, Majid; Hosseinpour Feizi, Mohammad Ali

    2015-06-01

    The binding properties of a new pyranochromene derivative, 2-amino-4-(3-hydroxyphenyl)-5-oxo-4H, 5H-pyrano-[3, 2-c] chromene-3-carbonitrile (3-HC) with calf thymus DNA (ctDNA) have been investigated by UV-vis absorption, circular dichroism, fluorescence spectroscopy and viscosity measurement. These results indicated that 3-HC can interact with DNA through non-intercalative mode and the intrinsic binding constant (Kb) for 3-HC with DNA was estimated to be 3.6 × 103 M-1. The antioxidant activity experiments show that 3-HC also exhibit good antioxidant activity in DPPH free radical scavenging and ferric reducing ability methods. Moreover, 3-HC exhibited cytotoxic activity against K562, human chronic myelogenous leukemia cells, with IC50 value of 146 μM and the cells responded to the treatment with mostly through apoptosis.

  5. DNA intercalation, topoisomerase II inhibition and cytotoxic activity of the plant alkaloid neocryptolepine.

    PubMed

    Bailly, C; Laine, W; Baldeyrou, B; De Pauw-Gillet, M C; Colson, P; Houssier, C; Cimanga, K; Van Miert, S; Vlietinck, A J; Pieters, L

    2000-06-01

    Cryptolepine and neocryptolepine are two indoloquinoline alkaloids isolated from the roots of the African plant Cryptolepis sanguinolenta. Both drugs have revealed antibacterial and antiparasitic activities and are strongly cytotoxic to tumour cells. We have recently shown that cryptolepine can intercalate into DNA and stimulates DNA cleavage by human topoisomerase II. In this study, we have investigated the mechanism of action and cytotoxicity of neocryptolepine, which differs from the parent isomer only by the orientation of the indole unit with respect to the quinoline moiety. The biochemical and physicochemical results presented here indicate that neocryptolepine also intercalates into DNA, preferentially at GC-rich sequences, but exhibits a reduced affinity for DNA compared with cryptolepine. The two alkaloids interfere with the catalytic activity of human topoisomerase II but the poisoning activity is slightly more pronounced with cryptolepine than with its isomer. The data provide a molecular basis to account for the reduced cytotoxicity of neocryptolepine compared with the parent drug. PMID:11049087

  6. DNA-Binding, Photocleavage, and Photodynamic Anti-cancer Activities of Pyridyl Corroles.

    PubMed

    Liang, Zhen-Hua; Liu, Hai-Yang; Zhou, Rong; Zhang, Zao; Ali, Atif; Han, Bing-Jie; Liu, Yun-Jun; Xiao, Xin-Yan

    2016-08-01

    The DNA-binding, photocleavage, and antitumor activity of three free base pyridyl corroles 1, 2, and 3 have been investigated. The binding affinity toward CT-DNA decreases with increasing number of pentafluorophenyl, whereas the photocleavage activity toward pBR322 DNA becomes more efficient. Singlet oxygen was demonstrated as active species responsible for DNA cleavage. These corroles exhibited high cytotoxicity against three tested cancer cells (Hela, HapG2, and A549) and the cytotoxicity could be further enhanced under irradiation. Intracellular reactive oxygen species level was also monitored using HeLa Cells upon the combined treatment of corroles and light. These corroles could be absorbed by HeLa cells at low concentration. They can induce the decrease of mitochondrial membrane potential and apoptosis of tumor cells under irradiation. PMID:26895317

  7. Probing the Run-On Oligomer of Activated SgrAI Bound to DNA

    PubMed Central

    Shah, Santosh; Sanchez, Jonathan; Stewart, Andrew; Piperakis, Michael M.; Cosstick, Richard; Nichols, Claire; Park, Chad K.; Ma, Xin; Wysocki, Vicki; Bitinaite, Jurate; Horton, Nancy C.

    2015-01-01

    SgrAI is a type II restriction endonuclease with an unusual mechanism of activation involving run-on oligomerization. The run-on oligomer is formed from complexes of SgrAI bound to DNA containing its 8 bp primary recognition sequence (uncleaved or cleaved), and also binds (and thereby activates for DNA cleavage) complexes of SgrAI bound to secondary site DNA sequences which contain a single base substitution in either the 1st/8th or the 2nd/7th position of the primary recognition sequence. This modulation of enzyme activity via run-on oligomerization is a newly appreciated phenomenon that has been shown for a small but increasing number of enzymes. One outstanding question regarding the mechanistic model for SgrAI is whether or not the activating primary site DNA must be cleaved by SgrAI prior to inducing activation. Herein we show that an uncleavable primary site DNA containing a 3’-S-phosphorothiolate is in fact able to induce activation. In addition, we now show that cleavage of secondary site DNA can be activated to nearly the same degree as primary, provided a sufficient number of flanking base pairs are present. We also show differences in activation and cleavage of the two types of secondary site, and that effects of selected single site substitutions in SgrAI, as well as measured collisional cross-sections from previous work, are consistent with the cryo-electron microscopy model for the run-on activated oligomer of SgrAI bound to DNA. PMID:25880668

  8. Binding of 2,7-diaminomitosene to DNA: model for the precovalent recognition of DNA by activated mitomycin C.

    PubMed

    Kumar, G S; He, Q Y; Behr-Ventura, D; Tomasz, M

    1995-02-28

    Mitomycin C (MC), mitomycin A, porfiromycin, BMY-25067, and BMY-25287, antitumor antibiotics collectively termed "mitosanes", were found to have no appreciable binding affinity to various natural and synthetic DNAs, as tested by UV spectrophotometry and equilibrium dialysis. Further tests of DNA binding applied to MC including thermal melting measurements, displacement of ethidium fluorescence, and unwinding of closed circular DNA were similarly negative. In contrast, 2,7-diaminomitosene (2,7-DAM), a major end product of the reductive activation of MC, binds to the same series of DNAs by all of these criteria. In the presence of DNA its UV absorbance at the 313 nm maximum decreased and underwent a slight red shift. This effect was used for determining DNA binding constants (Kb) by the spectrophotometric titration method. At pH 6.0 the Kbs of three natural DNAs with varying GC content, as well as poly(dA-dT).poly(dA-dT), and poly(dG-dC).poly(dG-dC), were all in the range of (1.2-5.3) x 10(4) (M nucleotide)-1, with no apparent specificity of binding. Poly(dG-m5dC).poly(dG-m5dC) displayed a slightly higher Kb ((7.5-8.4) x 10(4)). Binding of other, closely related mitosenes was tested to calf thymus DNA by equilibrium dialysis. Neither the presence of a 1-OH substituent, removal of the 10-carbamoyl group, nor methylation of the 2-amino group modifies the binding affinity of the mitosenes significantly. The 1-phosphate substituent abolishes binding. The binding of 2,7-DAM to DNA increased with decreasing pH and decreasing ionic strength. It was determined that 2,7-DAM is protonated at the 2-amino group with a pKa = 7.55, and this correlated well with the observed pH dependence of the binding, indicating that the binding affinity has a strong electrostatic component. This was confirmed by the finding that the extrapolated Kb to 1 M Na+ concentration diminishes to only 10% of the value of Kb at 0.01 M Na+ concentration. Viscosity tests showed conclusively that 2,7-DAM

  9. Deoxyribonucleic acid of Cancer pagurus. II. Tempiate activity for a DNA-dependent DNA polymerase of eukaryotic cells

    PubMed Central

    De Recondo, Anne-Marie; Londos-Gagliardi, Danielle; Aubel-Sadron, Geneviève

    1974-01-01

    The template activity of Cancer pagurus DNA and its two components (poly d(A-T) and main component) in response to a DNA polymerase purified from regenerating rat liver has been studied and compared to the results previously obtained with synthetic templates. In the double-stranded native state, whole crab DNA and the main component were poor templates. Their replication was increased by thermal denaturation and inhibited by actinomycin. Like the synthetic copolymer poly[d(A-T)·d(T-A)], native crab poly d(A-T) could be copied and its duplication was not inhibited by actinomycin. The structural difference between native poly d(A-T) Form I, isolated on a density gradient, and partially renatured poly d(A-T) Form II, isolated on hydroxylapatite, resulted in a modification of their template activity. The kinetic studies of [3H] dGMP and [3H] dAMP incorporation confirmed the importance of single-stranded regions (particulary dC regions) in the initiation of the in vitro duplication. PMID:10793685

  10. Lamin A Is an Endogenous SIRT6 Activator and Promotes SIRT6-Mediated DNA Repair.

    PubMed

    Ghosh, Shrestha; Liu, Baohua; Wang, Yi; Hao, Quan; Zhou, Zhongjun

    2015-11-17

    The nuclear lamins are essential for various molecular events in the nucleus, such as chromatin organization, DNA replication, and provision of mechanical support. A specific point mutation in the LMNA gene creates a truncated prelamin A termed progerin, causing Hutchinson-Gilford progeria syndrome (HGPS). SIRT6 deficiency leads to defective genomic maintenance and accelerated aging similar to HGPS, suggesting a potential link between lamin A and SIRT6. Here, we report that lamin A is an endogenous activator of SIRT6 and facilitates chromatin localization of SIRT6 upon DNA damage. Lamin A promotes SIRT6-dependent DNA-PKcs (DNA-PK catalytic subunit) recruitment to chromatin, CtIP deacetylation, and PARP1 mono-ADP ribosylation in response to DNA damage. The presence of progerin jeopardizes SIRT6 activation and compromises SIRT6-mediated molecular events in response to DNA damage. These data reveal a critical role for lamin A in regulating SIRT6 activities, suggesting that defects in SIRT6 functions contribute to impaired DNA repair and accelerated aging in HGPS. PMID:26549451

  11. Differential regulation of DNA damage response activation between somatic and germline cells in Caenorhabditis elegans

    PubMed Central

    Vermezovic, J; Stergiou, L; Hengartner, M O; d'Adda di Fagagna, F

    2012-01-01

    The germline of Caenorhabditis elegans is a well-established model for DNA damage response (DDR) studies. However, the molecular basis of the observed cell death resistance in the soma of these animals remains unknown. We established a set of techniques to study ionizing radiation-induced DNA damage generation and DDR activation in a whole intact worm. Our single-cell analyses reveal that, although germline and somatic cells show similar levels of inflicted DNA damage, somatic cells, differently from germline cells, do not activate the crucial apical DDR kinase ataxia-telengiectasia mutated (ATM). We also show that DDR signaling proteins are undetectable in all somatic cells and this is due to transcriptional repression. However, DNA repair genes are expressed and somatic cells retain the ability to efficiently repair DNA damage. Finally, we demonstrate that germline cells, when induced to transdifferentiate into somatic cells within the gonad, lose the ability to activate ATM. Overall, these observations provide a molecular mechanism for the known, but hitherto unexplained, resistance to DNA damage-induced cell death in C. elegans somatic cells. We propose that the observed lack of signaling and cell death but retention of DNA repair functions in the soma is a Caenorhabditis-specific evolutionary-selected strategy to cope with its lack of adult somatic stem cell pools and regenerative capacity. PMID:22705849

  12. The reversible DNA-alkylating activity of duocarmycin and its analogues.

    PubMed Central

    Asai, A; Nagamura, S; Saito, H; Takahashi, I; Nakano, H

    1994-01-01

    Intact drugs with spirocyclopropylhexadienone moieties can be regenerated from the covalent DNA adducts induced by antitumor antibiotics duocarmycin (DUM) A, SA and some DUMA analogues in neutral aqueous solution. We detected the reversible nature of DUMs by determination of the antimicrobial activity and cytotoxicity of DUM-DNA adducts. All of the adducts selectively inhibited the growth of a sensitive strain of Bacillus but not that of the wild type strain, a property of parent DUM and its analogues. Most of the DNA adducts were also cytotoxic to HeLa S3. These results suggested that active drugs can be released from their covalent DNA adducts under these biological assay conditions. Regeneration of intact drugs was quantitatively analyzed by HPLC and the amount of free drug released from DNA adducts revealed that the rate and efficiency of this reversal were dependent on structural variables among the drugs. The differences in rates of reversibility were correlated with the biological activity of DUMs. The effect of pH, temperature and salt concentration on the regeneration of drugs from their DNA adducts suggest a catalytic role of double-helical DNA on the reversal pathway. Images PMID:8127659

  13. Oxidative DNA adducts after Cu(2+)-mediated activation of dihydroxy PCBs: role of reactive oxygen species.

    PubMed

    Spencer, Wendy A; Lehmler, Hans-Joachim; Robertson, Larry W; Gupta, Ramesh C

    2009-05-15

    Polychlorinated biphenyls (PCBs) are toxic industrial chemicals, complete carcinogens, and efficacious tumor promoters. However, the mechanism(s) of PCB-mediated carcinogenicity remains largely undefined. One likely pathway by which these agents may play a role in carcinogenesis is the generation of oxidative DNA damage by redox cycling of dihydroxylated PCB metabolites. We have now employed a new (32)P-postlabeling system to examine novel oxidative DNA lesions induced by Cu(2+)-mediated activation of PCB metabolites. (32)P postlabeling of DNA incubated with various PCB metabolites resulted in over a dozen novel polar oxidative DNA adducts that were chromatographically similar for all active agents. The most potent metabolites tested were the hydroquinones (hydroxyl groups arranged para to each other), yielding polar oxidative adduct levels ranging from 55 to 142 adducts/10(6) nucleotides. PCB catechols, or ortho-dihydroxy metabolites, were up to 40% less active than their corresponding hydroquinone congeners, whereas monohydroxylated and quinone metabolites did not produce detectable oxidative damage over that of vehicle. With the exception of 2,4,5-Cl-2',5'-dihydroxybiphenyl, this oxidative DNA damage seemed to be inversely related to chlorine content: no chlorine approximately mono->di->trichlorinated metabolites. Importantly, copper, but not iron, was essential for activation of the PCB metabolites to these polar oxidative DNA adducts, because in its absence or in the presence of the Cu(+)-specific scavenger bathocuproine, no adducts were detected. Intervention studies with known reactive oxygen species (ROS) modifiers suggested that H(2)O(2), singlet oxygen, hydroxyl radical, and superoxide may also be involved in this PCB-mediated oxidative DNA damage. These data indicate a mechanistic role for several ROS, in addition to copper, in PCB-induced DNA damage and provide further support for oxidative DNA damage in PCB-mediated carcinogenesis. PMID:19233261

  14. PDIP46 (DNA polymerase δ interacting protein 46) is an activating factor for human DNA polymerase δ

    PubMed Central

    Zheng, Rong; Yue, Fu; Lin, Szu Hua Sharon; Rahmeh, Amal A.; Lee, Ernest Y. C.; Zhang, Zhongtao; Lee, Marietta Y. W. T.

    2016-01-01

    PDIP46 (SKAR, POLDIP3) was discovered through its interaction with the p50 subunit of human DNA polymerase δ (Pol δ). Its functions in DNA replication are unknown. PDIP46 associates with Pol δ in cell extracts both by immunochemical and protein separation methods, as well as by ChIP analyses. PDIP46 also interacts with PCNA via multiple copies of a novel PCNA binding motif, the APIMs (AlkB homologue-2 PCNA-Interacting Motif). Sites for both p50 and PCNA binding were mapped to the N-terminal region containing the APIMs. Functional assays for the effects of PDIP46 on Pol δ activity on singly primed ssM13 DNA templates revealed that it is a novel and potent activator of Pol δ. The effects of PDIP46 on Pol δ in primer extension, strand displacement and synthesis through simple hairpin structures reveal a mechanism where PDIP46 facilitates Pol δ4 synthesis through regions of secondary structure on complex templates. In addition, evidence was obtained that PDIP46 is also capable of exerting its effects by a direct interaction with Pol δ, independent of PCNA. Mutation of the Pol δ and PCNA binding region resulted in a loss of PDIP46 functions. These studies support the view that PDIP46 is a novel accessory protein for Pol δ that is involved in cellular DNA replication. This raises the possibility that altered expression of PDIP46 or its mutation may affect Pol δ functions in vivo, and thereby be a nexus for altered genomic stability. PMID:26819372

  15. Human placental DNA polymerase delta: identification of a 170-kilodalton polypeptide by activity staining and immunoblotting

    SciTech Connect

    Lee, M.Y.W.T.; Toomey, N.L.

    1987-02-24

    DNA polymerase delta was isolated from human placenta and identified as such on the basis of its association with a 3'- to 5'-exonuclease activity. The association of the polymerase and exonuclease activities was maintained throughout purification and attempted separations by physical or electrophoretic methods. Moreover, ratios of the two activities remained constant during the purification steps, and both activities were inhibited by aphidicolin, oxidized glutathione, and n-ethylmaleimide. The purified enzyme had an estimated molecular weight of 172,000, on the basis of a Stokes radius of 53.6 A and a sedimentation coefficient of 7.8 S. On sodium dodecyl sulfate (SDS) gel electrophoresis, polymerase delta preparations contained a band of ca. 170 kilodaltons (kDa) as well as several smaller polypeptides. The 170-kDa polypeptide was identified as the largest polypeptides component in the preparation possessing DNA polymerase activity by an activity staining procedure following gel electrophoresis in the presence of SDS. Western blotting of DNA polymerase delta with polyclonal antisera also revealed a single 170-kDa immunoreactive polypeptide. Monoclonal antibodies to KB cell polymerase ..cap alpha.. inhibited placental polymerase ..cap alpha.. but did not inhibit DNA polymerase delta, while the murine polyclonal antisera to polymerase delta inhibited delta but not ..cap alpha... These findings establish the existence of DNA polymerase delta in a human tissue and support the view that both its polymerase and its exonuclease activities may be associated with a single protein.

  16. Engineering molecularly-active nanoplasmonic surfaces for DNA detection via colorimetry and Raman scattering

    NASA Astrophysics Data System (ADS)

    Heydari, Esmaeil; Mabbott, Samuel; Thompson, David; Graham, Duncan; Cooper, Jonathan M.; Clark, Alasdair W.

    2016-03-01

    We report a novel nanophotonic biosensor surface capable of both colorimetric detection and Raman-scattered detection of DNA infection markers at extreme sensitivities. Combining direct-write lithography, dip-pen nanolithography based DNA patterning, and molecular self-assembly, we create molecularly-active plasmonic nanostructures onto which metallic nanoparticles are located via DNA-hybridization. Arraying these structures enables optical surfaces that change state when contacted by specific DNA sequences; shifting the surface color while simultaneously generating strong Raman-scattering signals. Patterning the DNA markers onto the plasmonic surface as micro-scale symbols results in easily identifiable color shifts, making this technique applicable to multiplexed lab-on-a-chip and point-of-care diagnostic applications.

  17. Tissue Inhibitor of Metalloproteinase 1 Expression Associated with Gene Demethylation Confers Anoikis Resistance in Early Phases of Melanocyte Malignant Transformation1

    PubMed Central

    Ricca, Tatiana I; Liang, Gangning; Suenaga, Ana Paula M; Han, Sang W; Jones, Peter A; Jasiulionis, Miriam G

    2009-01-01

    Although anoikis resistance has been considered a hallmark of malignant phenotype, the causal relation between neoplastic transformation and anchorage-independent growth remains undefined. We developed an experimental model of murine melanocyte malignant transformation, where a melanocyte lineage (melan-a) was submitted to sequential cycles of anchorage blockade, resulting in progressive morphologic alterations, and malignant transformation. Throughout this process, cells corresponding to premalignant melanocytes and melanoma cell lines were established and show progressive anoikis resistance and increased expression of Timp1. In melan-a melanocytes, Timp1 expression is suppressed by DNA methylation as indicated by its reexpression after 5-aza-2′-deoxycytidine treatment. Methylation-sensitive single-nucleotide primer extension analysis showed increased demethylation in Timp1 in parallel with its expression along malignant transformation. Interestingly, TIMP1 expression has already been related with negative prognosis in some human cancers. Although described as a MMP inhibitor, this protein has been associated with apoptosis resistance in different cell types. Melan-a cells overexpressing Timp1 showed increased survival in suspension but were unable to form tumors in vivo, whereas Timp1-overexpressing melanoma cells showed reduced latency time for tumor appearance and increased metastatic potential. Here, we demonstrated for the first time an increment in Timp1 expression since the early phases of melanocyte malignant transformation, associated to a progressive gene demethylation, which confers anoikis resistance. In this way, Timp1 might be considered as a valued marker for melanocyte malignant transformation. PMID:19956395

  18. Novel FOXC2 Mutation in Hereditary Distichiasis Impairs DNA-Binding Activity and Transcriptional Activation.

    PubMed

    Zhang, Leilei; He, Jie; Han, Bing; Lu, Linna; Fan, Jiayan; Zhang, He; Ge, Shengfang; Zhou, Yixiong; Jia, Renbing; Fan, Xianqun

    2016-01-01

    Distichiasis presents as double rows of eyelashes arising from aberrant differentiation of the meibomian glands of the eyelids, and it may be sporadic or hereditary. FOXC2 gene mutations in hereditary distichiasis are rarely reported. Here, we examined two generations of a Chinese family with hereditary distichiasis but without lymphedema or other features of LD syndrome. The FOXC2 gene was amplified and sequenced in all family members. Subcellular localization and luciferase assays were performed to assess the activity of the mutant FOXC2 protein. Clinical examinations showed distichiasis, lower eyelid ectropion, congenital ptosis and photophobia in all affected individuals. Sequence analysis revealed a novel frameshift mutation, c.964_965insG, in the coding region of the FOXC2 gene. This mutation caused protein truncation due to the presence of a premature stop codon. A fluorescence assay showed that this mutation did not change the nuclear localization of the protein. However, it impaired DNA-binding activity and decreased transcriptional activation. This is the first report of a FOXC2 mutation in hereditary distichiasis in the Chinese population. The findings of our study expand the FOXC2 mutation spectrum and contribute to the understanding of the genotype-phenotype correlation of this disease. PMID:27570485

  19. Novel FOXC2 Mutation in Hereditary Distichiasis Impairs DNA-Binding Activity and Transcriptional Activation

    PubMed Central

    Zhang, Leilei; He, Jie; Han, Bing; Lu, Linna; Fan, Jiayan; Zhang, He; Ge, Shengfang; Zhou, Yixiong; Jia, Renbing; Fan, Xianqun

    2016-01-01

    Distichiasis presents as double rows of eyelashes arising from aberrant differentiation of the meibomian glands of the eyelids, and it may be sporadic or hereditary. FOXC2 gene mutations in hereditary distichiasis are rarely reported. Here, we examined two generations of a Chinese family with hereditary distichiasis but without lymphedema or other features of LD syndrome. The FOXC2 gene was amplified and sequenced in all family members. Subcellular localization and luciferase assays were performed to assess the activity of the mutant FOXC2 protein. Clinical examinations showed distichiasis, lower eyelid ectropion, congenital ptosis and photophobia in all affected individuals. Sequence analysis revealed a novel frameshift mutation, c.964_965insG, in the coding region of the FOXC2 gene. This mutation caused protein truncation due to the presence of a premature stop codon. A fluorescence assay showed that this mutation did not change the nuclear localization of the protein. However, it impaired DNA-binding activity and decreased transcriptional activation. This is the first report of a FOXC2 mutation in hereditary distichiasis in the Chinese population. The findings of our study expand the FOXC2 mutation spectrum and contribute to the understanding of the genotype-phenotype correlation of this disease. PMID:27570485

  20. The metabolic activator FOXO1 binds hepatitis B virus DNA and activates its transcription

    SciTech Connect

    Shlomai, Amir; Shaul, Yosef

    2009-04-17

    Hepatitis B virus (HBV) is a small DNA virus that targets the liver and infects humans worldwide. Recently we have shown that the metabolic regulator PGC-1{alpha} coactivates HBV transcription thereby rendering the virus susceptible to fluctuations in the nutritional status of the liver. PGC-1{alpha} coactivation of HBV is mediated through the liver-enriched nuclear receptor HNF4{alpha} and through another yet unknown transcription factor(s). Here we show that the forkhead transcription factor FOXO1, a known target for PGC-1{alpha} coactivation and a central mediator of glucose metabolism in the liver, binds HBV core promoter and activates its transcription. This activation is further enhanced in the presence of PGC-1{alpha}, implying that FOXO1 is a target for PGC-1{alpha} coactivation of HBV transcription. Thus, our results identify another key metabolic regulator as an activator of HBV transcription, thereby supporting the principle that HBV gene expression is regulated in a similar way to key hepatic metabolic genes.

  1. Circulating Differentially Methylated Amylin DNA as a Biomarker of β-Cell Loss in Type 1 Diabetes

    PubMed Central

    Olsen, John A.; Kenna, Lauren A.; Spelios, Michael G.; Hessner, Martin J.; Akirav, Eitan M.

    2016-01-01

    In type 1 diabetes (T1D), β-cell loss is silent during disease progression. Methylation-sensitive quantitative real-time PCR (qPCR) of β-cell-derived DNA in the blood can serve as a biomarker of β-cell death in T1D. Amylin is highly expressed by β-cells in the islet. Here we examined whether demethylated circulating free amylin DNA (cfDNA) may serve as a biomarker of β-cell death in T1D. β cells showed unique methylation patterns within the amylin coding region that were not observed with other tissues. The design and use of methylation-specific primers yielded a strong signal for demethylated amylin in purified DNA from murine islets when compared with other tissues. Similarly, methylation-specific primers detected high levels of demethylated amylin DNA in human islets and enriched human β-cells. In vivo testing of the primers revealed an increase in demethylated amylin cfDNA in sera of non-obese diabetic (NOD) mice during T1D progression and following the development of hyperglycemia. This increase in amylin cfDNA did not mirror the increase in insulin cfDNA, suggesting that amylin cfDNA may detect β-cell loss in serum samples where insulin cfDNA is undetected. Finally, purified cfDNA from recent onset T1D patients yielded a high signal for demethylated amylin cfDNA when compared with matched healthy controls. These findings support the use of demethylated amylin cfDNA for detection of β-cell-derived DNA. When utilized in conjunction with insulin, this latest assay provides a comprehensive multi-gene approach for the detection of β-cell loss. PMID:27111653

  2. Autophosphorylation and Pin1 binding coordinate DNA damage-induced HIPK2 activation and cell death.

    PubMed

    Bitomsky, Nadja; Conrad, Elisa; Moritz, Christian; Polonio-Vallon, Tilman; Sombroek, Dirk; Schultheiss, Kathrin; Glas, Carolina; Greiner, Vera; Herbel, Christoph; Mantovani, Fiamma; del Sal, Giannino; Peri, Francesca; Hofmann, Thomas G

    2013-11-01

    Excessive genome damage activates the apoptosis response. Protein kinase HIPK2 is a key regulator of DNA damage-induced apoptosis. Here, we deciphered the molecular mechanism of HIPK2 activation and show its relevance for DNA damage-induced apoptosis in cellulo and in vivo. HIPK2 autointeracts and site-specifically autophosphorylates upon DNA damage at Thr880/Ser882. Autophosphorylation regulates HIPK2 activity and mutation of the phosphorylation-acceptor sites deregulates p53 Ser46 phosphorylation and apoptosis in cellulo. Moreover, HIPK2 autophosphorylation is conserved between human and zebrafish and is important for DNA damage-induced apoptosis in vivo. Mechanistically, autophosphorylation creates a binding signal for the phospho-specific isomerase Pin1. Pin1 links HIPK2 activation to its stabilization by inhibiting HIPK2 polyubiquitination and modulating Siah-1-HIPK2 interaction. Concordantly, Pin1 is required for DNA damage-induced HIPK2 stabilization and p53 Ser46 phosphorylation and is essential for induction of apotosis both in cellulo and in zebrafish. Our results identify an evolutionary conserved mechanism regulating DNA damage-induced apoptosis. PMID:24145406

  3. Distinct mechanisms for DNA cleavage by myoglobin with a designed heme active center.

    PubMed

    Zhao, Yuan; Du, Ke-Jie; Gao, Shu-Qin; He, Bo; Wen, Ge-Bo; Tan, Xiangshi; Lin, Ying-Wu

    2016-03-01

    Heme proteins perform diverse biological functions, of which myoglobin (Mb) is a representative protein. In this study, the O2 carrier Mb was shown to cleave double stranded DNA upon aerobic dithiothreitol-induced reduction, which is fine-tuned by an additional distal histidine, His29 or His43, engineered in the heme active center. Spectroscopic (UV-vis and EPR) and inhibition studies suggested that free radicals including singlet oxygen and hydroxyl radical are responsible for efficient DNA cleavage via an oxidative cleavage mechanism. On the other hand, L29E Mb, with a distinct heme active center involving three water molecules in the met form, was found to exhibit an excellent DNA cleavage activity that was not depending on O2. Inhibition and ligation studies demonstrated for the first time that L29E Mb cleaves double stranded DNA into both the nicked circular and linear forms via a hydrolytic cleavage mechanism, which resembles native endonucleases. This study provides valuable insights into the distinct mechanisms for DNA cleavage by heme proteins, and lays down a base for creating artificial DNA endonucleases by rational design of heme proteins. Moreover, this study suggests that the diverse functions of heme proteins can be fine-tuned by rational design of the heme active center with a hydrogen-bonding network. PMID:26775281

  4. Label-free molecular beacon for real-time monitoring of DNA polymerase activity.

    PubMed

    Ma, Changbei; Liu, Haisheng; Wang, Jun; Jin, Shunxin; Wang, Kemin

    2016-05-01

    Traditional methods for assaying DNA polymerase activity are discontinuous, time consuming, and laborious. Here, we report a new approach for label-free and real-time monitoring of DNA polymerase activity using a Thioflavin T (ThT) probe. In the presence of DNA polymerase, the DNA primer could be elongated through polymerase reaction to open MB1, leading to the release of the G-quartets. These then bind to ThT to form ThT/G-quadruplexes with an obvious fluorescence generation. It exhibits a satisfying detection result for the activity of DNA polymerase with a low detection limit of 0.05 unit/ml. In addition, no labeling with a fluorophore or a fluorophore-quencher pair is required; this method is fairly simple, fast, and low cost. Furthermore, the proposed method was also applied to assay the inhibition of DNA polymerase activity. This approach may offer potential applications in drug screening, clinical diagnostics, and some other related biomedical research. PMID:26894757

  5. Structure-function relationships governing activity and stability of a DNA alkylation damage repair thermostable protein.

    PubMed

    Perugino, Giuseppe; Miggiano, Riccardo; Serpe, Mario; Vettone, Antonella; Valenti, Anna; Lahiri, Samarpita; Rossi, Franca; Rossi, Mosè; Rizzi, Menico; Ciaramella, Maria

    2015-10-15

    Alkylated DNA-protein alkyltransferases repair alkylated DNA bases, which are among the most common DNA lesions, and are evolutionary conserved, from prokaryotes to higher eukaryotes. The human ortholog, hAGT, is involved in resistance to alkylating chemotherapy drugs. We report here on the alkylated DNA-protein alkyltransferase, SsOGT, from an archaeal species living at high temperature, a condition that enhances the harmful effect of DNA alkylation. The exceptionally high stability of SsOGT gave us the unique opportunity to perform structural and biochemical analysis of a protein of this class in its post-reaction form. This analysis, along with those performed on SsOGT in its ligand-free and DNA-bound forms, provides insights in the structure-function relationships of the protein before, during and after DNA repair, suggesting a molecular basis for DNA recognition, catalytic activity and protein post-reaction fate, and giving hints on the mechanism of alkylation-induced inactivation of this class of proteins. PMID:26227971

  6. Structure-function relationships governing activity and stability of a DNA alkylation damage repair thermostable protein

    PubMed Central

    Perugino, Giuseppe; Miggiano, Riccardo; Serpe, Mario; Vettone, Antonella; Valenti, Anna; Lahiri, Samarpita; Rossi, Franca; Rossi, Mosè; Rizzi, Menico; Ciaramella, Maria

    2015-01-01

    Alkylated DNA-protein alkyltransferases repair alkylated DNA bases, which are among the most common DNA lesions, and are evolutionary conserved, from prokaryotes to higher eukaryotes. The human ortholog, hAGT, is involved in resistance to alkylating chemotherapy drugs. We report here on the alkylated DNA-protein alkyltransferase, SsOGT, from an archaeal species living at high temperature, a condition that enhances the harmful effect of DNA alkylation. The exceptionally high stability of SsOGT gave us the unique opportunity to perform structural and biochemical analysis of a protein of this class in its post-reaction form. This analysis, along with those performed on SsOGT in its ligand-free and DNA-bound forms, provides insights in the structure-function relationships of the protein before, during and after DNA repair, suggesting a molecular basis for DNA recognition, catalytic activity and protein post-reaction fate, and giving hints on the mechanism of alkylation-induced inactivation of this class of proteins. PMID:26227971

  7. New metal based drugs: Spectral, electrochemical, DNA-binding, surface morphology and anticancer activity properties

    NASA Astrophysics Data System (ADS)

    Çeşme, Mustafa; Gölcü, Aysegul; Demirtaş, Ibrahim

    2015-01-01

    The NSAID piroxicam (PRX) drug was used for complex formation reactions with Cu(II), Zn(II) and Pt(II) metal salts have been synthesized. Then, these complexes have been characterized by spectroscopic and analytical techniques. Thermal behavior of the complexes were also investigated. The electrochemical properties of all complexes have been investigated by cyclic voltammetry (CV) using glassy carbon electrode. The biological activity of the complexes has been evaluated by examining their ability to bind to fish sperm double strand DNA (FSFSdsDNA) with UV spectroscopy. UV studies of the interaction of the PRX and its complexes with FSdsDNA have shown that these compounds can bind to FSdsDNA. The binding constants of the compounds with FSdsDNA have also been calculated. The morphology of the FSdsDNA, PRX, metal ions and metal complexes has been investigated by scanning electron microscopy (SEM). To get the SEM images, the interaction of compounds with FSdsDNA has been studied by means of differential pulse voltammetry (DPV) at FSdsDNA modified pencil graphite electrode (PGE). The decrease in intensity of the guanine oxidation signals has been used as an indicator for the interaction mechanism. The effect of proliferation PRX and complexes were examined on the HeLA and C6 cells using real-time cell analyzer with four different concentrations.

  8. Purification of transcriptionally active multimeric plasmid DNA using zwitterionic detergent and carbonate apatite nano-particles.

    PubMed

    Tee, Lau Khye; Ling, Chong Siew; Chua, Ming Jang; Abdullah, Syahril; Rosli, Rozita; Chowdhury, Ezharul Hoque

    2011-10-01

    Plasmid DNA is one of the indispensable components in molecular biology research and a potential biomaterial for gene therapy and DNA vaccination. Both quality and quantity of extracted plasmid DNA are of the great interests in cloning and subsequent expression of genes in vitro and in vivo for basic research and therapeutic interventions. Bacteria with extremely short generation times are the valuable source of plasmid DNA that can be isolated through a number of existing techniques. However, the current methods have some limitations in isolating high quality plasmid DNA since the multimeric plasmid which is believed to be more efficiently transcribed by RNA polymerase than the monomeric form, is almost lost during the extraction process. Recently, we developed a rapid isolation technique for multimeric plasmid based on generation of a 'protein aggregate' using a zwitterionic detergent and alkali. Here we have investigated the roles of different parameters in the whole extraction process to optimise the production of high quality multimeric plasmid DNA. Moreover, we have showed the advantageous effects of nanoparticles to effectively sediment the 'protein aggregate' for smooth elution of multimeric plasmid DNA from it. Finally, quality assessment study has revealed that the isolated multimeric DNA is at least 10 times more transcriptionally active than the monomeric form isolated by the commercially available Qiaget kit. PMID:21419794

  9. Evidence for the kinetic partitioning of polymerase activity on G-quadruplex DNA

    PubMed Central

    Eddy, Sarah; Maddukuri, Leena; Ketkar, Amit; Zafar, Maroof K.; Henninger, Erin E.; Pursell, Zachary F.; Eoff, Robert L.

    2015-01-01

    We have investigated the action of the human DNA polymerase epsilon (hpol ε) and eta (hpol η) catalytic cores on G-quadruplex (G4) DNA substrates derived from the promoter of the c-MYC proto-oncogene. The translesion enzyme hpol η exhibits a 6.2-fold preference for binding to G4 DNA relative to non-G4 DNA, while hpol ε binds both G4 and non-G4 substrates with near equal affinity. Kinetic analysis of single-nucleotide insertion by hpol η reveals that it is able to maintain greater than 25% activity on G4 substrates compared to non-G4 DNA substrates, even when the primer template junction is positioned directly adjacent to G22 (the first tetrad-associated guanine in the c-MYC G4 motif). Surprisingly, hpol η fidelity increases ~15-fold when copying G22. By way of comparison, hpol ε retains ~4% activity and has a 33-fold decrease in fidelity when copying G22. The fidelity of hpol η is ~100-fold more accurate than hpol ε when comparing the mis-insertion frequencies of the two enzymes opposite a tetrad-associated guanine. The kinetic differences observed for the B- and Y-family pols on G4 DNA support a model where a simple kinetic switch between replicative and TLS pols could help govern fork progress during G4 DNA replication. PMID:25903680

  10. [p53 activation by PI-3K family kinases after DNA double-strand breaks].

    PubMed

    Pernin, D; Uhrhammer, N; Verrelle, P; Bignon, Y J; Bay, J O

    2000-09-01

    p53 plays a central role in the cellular response to DNA double-strand breaks (DSBs), and to DNA damage in general. The protein kinases ATM, ATR and DNA-PK detect DSBs and transmit this information to p53 by phosphorylation. This phosphorylation dissociates p53 from its negative regulator, mdm2. p53 then undergoes further modification and activates transcription of the genes responsible for cell cycle arrest. In certain circumstances, p53 also activates transcription of the genes responsible for apoptosis. The dysfunction of this cascade of events is oncogenic, with P53 itself being the most commonly mutated gene in malignant cells, although mutations in both the DNA damage sensors and cell cycle checkpoint and apoptosis effectors are frequent. A more complete understanding of p53 and the proteins it interacts with may allow the development of new cancer treatments. PMID:11038413

  11. Effect of pH on DNA alkylation by enzyme-activated mitomycin C and porfiromycin.

    PubMed

    Yu, F; Pan, S S

    1993-06-01

    DNA adduct formation by enzyme-activated antibiotics, mitomycin C (MMC) or porfiromycin (PFM), at pH 7.6 or pH 6.0 under anaerobic conditions was analyzed by a 32P-postlabeling method. Antibiotic activation by rat liver NADPH-cytochrome P-450 reductase (EC 1.6.2.4) and bovine milk xanthine oxidase (EC 1.2.3.2) produced similar results. Five 32P-labeled MMC adducts were separated by thin layer chromatography and high performance liquid chromatography from DNA alkylated at either pH. Four of the radioactive spots separated by thin layer chromatography were identified as two monofunctional monoadducts [1" alpha and 1" beta forms of N2-(2" beta,7"-diaminomitosen-1"-yl)-2'-deoxyguanylic acid], one bifunctional monoadduct [N2-(10"-decarbamoyl-2",7"-diaminomitosen-1" alpha-yl)-2'-deoxyguanylic acid], and one cross-linked adduct [N2-(2" beta,7"-diamino-10"-deoxyguanyl-N2-yl-mitosen- 1" alpha-yl)-2'-deoxyguanylic acid]. One minor radioactive spot was not identified. By comparing DNA alkylated at the two pH values, based on equal amounts of 32P radioactivity, similar amounts of cross-links were detected. However, the DNA showed different ratios of the alpha and beta isomers of the monofunctional monoadduct. Furthermore, the DNA alkylated at pH 6.0 showed more bifunctional monoadducts than did the DNA alkylated at pH 7.6. Analysis of alkylated DNA by enzyme-activated PFM showed a similar spectrum of DNA adduct formation. The effect of pH on the distribution of the five PFM-DNA adducts was similar to that observed for the five MMC-DNA adducts. The distribution of adducts in DNA alkylated at the same pH was similar irrespective of which enzyme activated MMC or PFM. The pH of the reaction during DNA and MMC interaction was the determining factor for the quantitative distribution of the adducts. This pH effect may be important for the cytotoxicity of MMC and PFM in tumor cells that have high levels of reductive enzymes with low optimal pH values. PMID:8391116

  12. Light-activated DNA binding in a designed allosteric protein

    SciTech Connect

    Strickland, Devin; Moffat, Keith; Sosnick, Tobin R.

    2008-09-03

    An understanding of how allostery, the conformational coupling of distant functional sites, arises in highly evolvable systems is of considerable interest in areas ranging from cell biology to protein design and signaling networks. We reasoned that the rigidity and defined geometry of an {alpha}-helical domain linker would make it effective as a conduit for allosteric signals. To test this idea, we rationally designed 12 fusions between the naturally photoactive LOV2 domain from Avena sativa phototropin 1 and the Escherichia coli trp repressor. When illuminated, one of the fusions selectively binds operator DNA and protects it from nuclease digestion. The ready success of our rational design strategy suggests that the helical 'allosteric lever arm' is a general scheme for coupling the function of two proteins.

  13. DNA-PK mediates AKT activation and apoptosis inhibition in clinically acquired platinum resistance.

    PubMed

    Stronach, Euan A; Chen, Michelle; Maginn, Elaina N; Agarwal, Roshan; Mills, Gordon B; Wasan, Harpreet; Gabra, Hani

    2011-11-01

    Clinical resistance to chemotherapy is a frequent event in cancer treatment and is closely linked to poor outcome. High-grade serous (HGS) ovarian cancer is characterized by p53 mutation and high levels of genomic instability. Treatment includes platinum-based chemotherapy and initial response rates are high; however, resistance is frequently acquired, at which point treatment options are largely palliative. Recent data indicate that platinum-resistant clones exist within the sensitive primary tumor at presentation, implying resistant cell selection after treatment with platinum chemotherapy. The AKT pathway is central to cell survival and has been implicated in platinum resistance. Here, we show that platinum exposure induces an AKT-dependent, prosurvival, DNA damage response in clinically platinum-resistant but not platinum-sensitive cells. AKT relocates to the nucleus of resistant cells where it is phosphorylated specifically on S473 by DNA-dependent protein kinase (DNA-PK), and this activation inhibits cisplatin-mediated apoptosis. Inhibition of DNA-PK or AKT, but not mTORC2, restores platinum sensitivity in a panel of clinically resistant HGS ovarian cancer cell lines: we also demonstrate these effects in other tumor types. Resensitization is associated with prevention of AKT-mediated BAD phosphorylation. Strikingly, in patient-matched sensitive cells, we do not see enhanced apoptosis on combining cisplatin with AKT or DNA-PK inhibition. Insulin-mediated activation of AKT is unaffected by DNA-PK inhibitor treatment, suggesting that this effect is restricted to DNA damage-mediated activation of AKT and that, clinically, DNA-PK inhibition might prevent platinum-induced AKT activation without interfering with normal glucose homeostasis, an unwanted toxicity of direct AKT inhibitors. PMID:22131882

  14. DNA-PK Mediates AKT Activation and Apoptosis Inhibition in Clinically Acquired Platinum Resistance12

    PubMed Central

    Stronach, Euan A; Chen, Michelle; Maginn, Elaina N; Agarwal, Roshan; Mills, Gordon B; Wasan, Harpreet; Gabra, Hani

    2011-01-01

    Clinical resistance to chemotherapy is a frequent event in cancer treatment and is closely linked to poor outcome. High-grade serous (HGS) ovarian cancer is characterized by p53 mutation and high levels of genomic instability. Treatment includes platinum-based chemotherapy and initial response rates are high; however, resistance is frequently acquired, at which point treatment options are largely palliative. Recent data indicate that platinum-resistant clones exist within the sensitive primary tumor at presentation, implying resistant cell selection after treatment with platinum chemotherapy. The AKT pathway is central to cell survival and has been implicated in platinum resistance. Here, we show that platinum exposure induces an AKT-dependent, prosurvival, DNA damage response in clinically platinum-resistant but not platinum-sensitive cells. AKT relocates to the nucleus of resistant cells where it is phosphorylated specifically on S473 by DNA-dependent protein kinase (DNA-PK), and this activation inhibits cisplatin-mediated apoptosis. Inhibition of DNA-PK or AKT, but not mTORC2, restores platinum sensitivity in a panel of clinically resistant HGS ovarian cancer cell lines: we also demonstrate these effects in other tumor types. Resensitization is associated with prevention of AKT-mediated BAD phosphorylation. Strikingly, in patient-matched sensitive cells, we do not see enhanced apoptosis on combining cisplatin with AKT or DNA-PK inhibition. Insulin-mediated activation of AKT is unaffected by DNA-PK inhibitor treatment, suggesting that this effect is restricted to DNA damage-mediated activation of AKT and that, clinically, DNA-PK inhibition might prevent platinum-induced AKT activation without interfering with normal glucose homeostasis, an unwanted toxicity of direct AKT inhibitors. PMID:22131882

  15. TALEored Epigenetics: A DNA-Binding Scaffold for Programmable Epigenome Editing and Analysis.

    PubMed

    Kubik, Grzegorz; Summerer, Daniel

    2016-06-01

    Epigenetic modification of the cytosine 5-position is an important regulator of gene expression with essential roles in genome stability, development, and disease. In addition to 5-methylcytosine (mC), the oxidized mC derivatives 5-hydroxymethyl-, 5-formyl-, and 5-carboxylcytosine (hmC, fC, and caC) have recently been discovered. These are intermediates of an active demethylation pathway but might also represent new epigenetic marks with individual biological roles. This increase in chemical complexity of DNA-encoded information has created a pressing need for new approaches that allow reading and editing of this information. Transcription-activator-like effectors (TALEs) are DNA-binding domains with programmable sequence selectivity that enable the direct reading of epigenetic cytosine modifications but can also guide enzymatic editing domains to genomic loci of choice. Here, we review recent advances in employing TALEs for these applications. PMID:26972580

  16. Dynamic DNA methylation regulates neuronal intrinsic membrane excitability.

    PubMed

    Meadows, Jarrod P; Guzman-Karlsson, Mikael C; Phillips, Scott; Brown, Jordan A; Strange, Sarah K; Sweatt, J David; Hablitz, John J

    2016-01-01

    Epigenetic modifications, such as DNA cytosine methylation, contribute to the mechanisms underlying learning and memory by coordinating adaptive gene expression and neuronal plasticity. Transcription-dependent plasticity regulated by DNA methylation includes synaptic plasticity and homeostatic synaptic scaling. Memory-related plasticity also includes alterations in intrinsic membrane excitability mediated by changes in the abundance or activity of ion channels in the plasma membrane, which sets the threshold for action potential generation. We found that prolonged inhibition of DNA methyltransferase (DNMT) activity increased intrinsic membrane excitability of cultured cortical pyramidal neurons. Knockdown of the cytosine demethylase TET1 or inhibition of RNA polymerase blocked the increased membrane excitability caused by DNMT inhibition, suggesting that this effect was mediated by subsequent cytosine demethylation and de novo transcription. Prolonged DNMT inhibition blunted the medium component of the after-hyperpolarization potential, an effect that would increase neuronal excitability, and was associated with reduced expression of the genes encoding small-conductance Ca(2+)-activated K(+) (SK) channels. Furthermore, the specific SK channel blocker apamin increased neuronal excitability but was ineffective after DNMT inhibition. Our results suggested that DNMT inhibition enables transcriptional changes that culminate in decreased expression of SK channel-encoding genes and decreased activity of SK channels, thus providing a mechanism for the regulation of neuronal intrinsic membrane excitability by dynamic DNA cytosine methylation. This study has implications for human neurological and psychiatric diseases associated with dysregulated intrinsic excitability. PMID:27555660

  17. Electrochemical Assay for the Signal-on Detection of Human DNA Methyltransferase Activity

    PubMed Central

    Muren, Natalie B.; Barton, Jacqueline K.

    2013-01-01

    Strategies to detect human DNA methyltransferases are needed, given that aberrant methylation by these enzymes is associated with cancer initiation and progression. Here we describe a non-radioactive, antibody-free, electrochemical assay in which methyltransferase activity on DNA-modified electrodes confers protection from restriction for signal-on detection. We implement this assay with a multiplexed chip platform and show robust detection of both bacterial (SssI) and human (Dnmt1) methyltransferase activity. Essential to work with human methyltransferases, our unique assay design allows activity measurements on both unmethylated and hemimethylated DNA substrates. We validate this assay by comparison with a conventional radioactive method. The advantages of electrochemistry over radioactivity and fluorescence make this assay an accessible and promising new approach for the sensitive, label-free detection of human methyltransferase activity. PMID:24164112

  18. DNA methylation/demethylation programming during peach flower bud dormancy release, development and blooming

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Peach flower bud development undergoes a long, complex and temperature-dependent regulation process with cessation of growth in response to cool temperatures in late fall, a slow but gradual development during the chilling period in winter, and eventually blooming in early spring. It has been demon...

  19. Os2–Os4 Switch Controls DNA Knotting and Anticancer Activity

    PubMed Central

    Fu, Ying; Romero, María J.; Salassa, Luca; Cheng, Xi; Habtemariam, Abraha; Clarkson, Guy J.; Prokes, Ivan; Rodger, Alison; Costantini, Giovanni

    2016-01-01

    Abstract Dinuclear trihydroxido‐bridged osmium–arene complexes are inert and biologically inactive, but we show here that linking dihydroxido‐bridged OsII–arene fragments by a bridging di‐imine to form a metallacycle framework results in strong antiproliferative activity towards cancer cells and distinctive knotting of DNA. The shortened spacer length reduces biological activity and stability in solution towards decomposition to biologically inactive dimers. Significant differences in behavior toward plasmid DNA condensation are correlated with biological activity. PMID:27240103

  20. 5mC oxidation by Tet2 modulates enhancer activity and timing of transcriptome reprogramming during differentiation

    PubMed Central

    Hon, Gary C.; Song, Chun-Xiao; Du, Tingting; Jin, Fulai; Selvaraj, Siddarth; Lee, Ah Young; Yen, Chia-an; Ye, Zhen; Mao, Shi-Qing; Wang, Bang-An; Kuan, Samantha; Edsall, Lee E.; Zhao, Boxuan Simen; Xu, Guo-Liang; He, Chuan; Ren, Bing

    2014-01-01

    Summary In mammals, cytosine methylation (5mC) is widely distributed throughout the genome, but is notably depleted from active promoters and enhancers. While the role of DNA methylation in promoter silencing has been well documented, the function of this epigenetic mark at enhancers remains unclear. Recent experiments have demonstrated that enhancers are enriched for 5-hydroxymethylcytosine (5hmC), an oxidization product of the Tet family of 5mC dioxygenases and an intermediate of DNA demethylation. These results support the involvement of Tet proteins in regulation of dynamic DNA methylation at enhancers. By mapping DNA methylation and hydroxymethylation at base resolution, we find that deletion of Tet2 causes extensive loss of 5hmC at enhancers, accompanied by enhancer hypermethylation, reduction of enhancer activity, and delayed gene induction in the early steps of differentiation. Our results reveal that DNA demethylation modulates enhancer activity, and its disruption influences the timing of transcriptome reprogramming during cellular differentiation. PMID:25263596

  1. Highly sensitive detection of M.SssI DNA methyltransferase activity using a personal glucose meter.

    PubMed

    Deng, Huimin; Peng, Si Ying; Gao, Zhiqiang

    2016-08-01

    A simple method for highly sensitive and selective detection of M.SssI CpG methyltransferase (M.SssI MTase) activity is developed, leveraging on the portability and ease of use of a personal glucose meter (PGM). Briefly, DNA-invertase conjugates are hybridized with their complementary DNA strands pre-immobilized on magnetic beads. The 5'-CCGG-3' sequence present in the DNA duplexes serves as the recognition site for both Hpa II restriction enzyme and M.SssI MTase (5'-CG-3'). Hpa II restriction enzyme specifically cleaves at unmethylated 5'-CCGG-3' sequence, and the invertase that remains on the methylated DNA catalyzes the hydrolysis of sucrose to glucose and fructose. It is found that the amount of glucose is proportional to the M.SssI MTase methylation activity in the range of 0.5 to 80 U/mL with a detection limit of 0.37 U/mL. Due to the specific recognition sequence present in the DNA strands, this method also shows high selectivity for M.SssI MTase. In addition, inhibition studies with 5'-azacytidine demonstrate the capability of inhibition screening using this method. Graphical abstract Deteciton of M.SssI DNA methyltransferase activity by a personal glucose meter. PMID:27311957

  2. Liquid-crystalline ordering of antimicrobial peptide-DNA complexes controls TLR9 activation.

    PubMed

    Schmidt, Nathan W; Jin, Fan; Lande, Roberto; Curk, Tine; Xian, Wujing; Lee, Calvin; Frasca, Loredana; Frenkel, Daan; Dobnikar, Jure; Gilliet, Michel; Wong, Gerard C L

    2015-07-01

    Double-stranded DNA (dsDNA) can trigger the production of type I interferon (IFN) in plasmacytoid dendritic cells (pDCs) by binding to endosomal Toll-like receptor-9 (TLR9; refs 1-5). It is also known that the formation of DNA-antimicrobial peptide complexes can lead to autoimmune diseases via amplification of pDC activation. Here, by combining X-ray scattering, computer simulations, microscopy and measurements of pDC IFN production, we demonstrate that a broad range of antimicrobial peptides and other cationic molecules cause similar effects, and elucidate the criteria for amplification. TLR9 activation depends on both the inter-DNA spacing and the multiplicity of parallel DNA ligands in the self-assembled liquid-crystalline complex. Complexes with a grill-like arrangement of DNA at the optimum spacing can interlock with multiple TLR9 like a zipper, leading to multivalent electrostatic interactions that drastically amplify binding and thereby the immune response. Our results suggest that TLR9 activation and thus TLR9-mediated immune responses can be modulated deterministically. PMID:26053762

  3. Liquid-crystalline ordering of antimicrobial peptide-DNA complexes controls TLR9 activation

    NASA Astrophysics Data System (ADS)

    Schmidt, Nathan W.; Jin, Fan; Lande, Roberto; Curk, Tine; Xian, Wujing; Lee, Calvin; Frasca, Loredana; Frenkel, Daan; Dobnikar, Jure; Gilliet, Michel; Wong, Gerard C. L.

    2015-07-01

    Double-stranded DNA (dsDNA) can trigger the production of type I interferon (IFN) in plasmacytoid dendritic cells (pDCs) by binding to endosomal Toll-like receptor-9 (TLR9; refs , , , , ). It is also known that the formation of DNA-antimicrobial peptide complexes can lead to autoimmune diseases via amplification of pDC activation. Here, by combining X-ray scattering, computer simulations, microscopy and measurements of pDC IFN production, we demonstrate that a broad range of antimicrobial peptides and other cationic molecules cause similar effects, and elucidate the criteria for amplification. TLR9 activation depends on both the inter-DNA spacing and the multiplicity of parallel DNA ligands in the self-assembled liquid-crystalline complex. Complexes with a grill-like arrangement of DNA at the optimum spacing can interlock with multiple TLR9 like a zipper, leading to multivalent electrostatic interactions that drastically amplify binding and thereby the immune response. Our results suggest that TLR9 activation and thus TLR9-mediated immune responses can be modulated deterministically.

  4. Novel DNA mismatch repair activity involving YB-1 in human mitochondria

    PubMed Central

    de Souza-Pinto, Nadja C.; Mason, Penelope A.; Hashiguchi, Kazunari; Weissman, Lior; Tian, Jingyan; Guay, David; Lebel, Michel; Stevnsner, Tinna V.; Rasmussen, Lene Juel; Bohr, Vilhelm A.

    2009-01-01

    Maintenance of the mitochondrial genome (mtDNA) is essential for proper cellular function. The accumulation of damage and mutations in the mtDNA leads to diseases, cancer, and aging. Mammalian mitochondria have proficient base excision repair, but the existence of other DNA repair pathways is still unclear. Deficiencies in DNA mismatch repair (MMR), which corrects base mismatches and small loops, are associated with DNA microsatellite instability, accumulation of mutations, and cancer. MMR proteins have been identified in yeast and coral mitochondria; however, MMR proteins and function have not yet been detected in human mitochondria. Here we show that human mitochondria have a robust mismatch-repair activity, which is distinct from nuclear MMR. Key nuclear MMR factors were not detected in mitochondria, and similar mismatch-binding activity was observed in mitochondrial extracts from cells lacking MSH2, suggesting distinctive pathways for nuclear and mitochondrial MMR. We identified the repair factor YB-1 as a key candidate for a mitochondrial mismatch-binding protein. This protein localizes to mitochondria in human cells, and contributes significantly to the mismatch-binding and mismatch-repair activity detected in HeLa mitochondrial extracts, which are significantly decreased when the intracellular levels of YB-1 are diminished. Moreover, YB-1 depletion in cells increases mitochondrial DNA mutagenesis. Our results show that human mitochondria contain a functional MMR repair pathway in which YB-1 participates, likely in the mismatch binding and recognition steps. PMID:19272840

  5. Role of DNA methylation and the DNA methyltransferases in learning and memory

    PubMed Central

    Morris, Michael J.; Monteggia, Lisa M.

    2014-01-01

    Dynamic regulation of chromatin structure in postmitotic neurons plays an important role in learning and memory. Methylation of cytosine nucleotides has historically been considered the strongest and least modifiable of epigenetic marks. Accumulating recent data suggest that rapid and dynamic methylation and demethylation of specific genes in the brain may play a fundamental role in learning, memory formation, and behavioral plasticity. The current review focuses on the emergence of data that support the role of DNA methylation and demethylation, and its molecular mediators in memory formation. PMID:25364286

  6. Sensitization for death receptor- or drug-induced apoptosis by re-expression of caspase-8 through demethylation or gene transfer.

    PubMed

    Fulda, S; Küfer, M U; Meyer, E; van Valen, F; Dockhorn-Dworniczak, B; Debatin, K M

    2001-09-13

    Resistance of tumors to treatment with cytotoxic drugs, irradiation or immunotherapy may be due to disrupted apoptosis programs. Here, we report in a variety of different tumor cells including Ewing tumor, neuroblastoma, malignant brain tumors and melanoma that caspase-8 expression acts as a key determinant of sensitivity for apoptosis induced by death-inducing ligands or cytotoxic drugs. In tumor cell lines resistant to TRAIL, anti-CD95 or TNFalpha, caspase-8 protein and mRNA expression was decreased or absent without caspase-8 gene loss. Methylation-specific PCR revealed hypermethylation of caspase-8 regulatory sequences in cells with impaired caspase-8 expression. Treatment with the demethylation agent 5-Aza-2'-deoxycytidine (5-dAzaC) reversed hypermethylation of caspase-8 resulting in restoration of caspase-8 expression and recruitment and activation of caspase-8 at the CD95 DISC upon receptor cross-linking thereby sensitizing for death receptor-, and importantly, also for drug-induced apoptosis. Inhibition of caspase-8 activity also inhibited apoptosis sensitization by 5-dAzaC. Similar to demethylation, introduction of caspase-8 by gene transfer sensitized for apoptosis induction. Hypermethylation of caspase-8 was linked to reduced caspase-8 expression in different tumor cell lines in vitro and, most importantly, also in primary tumor samples. Thus, these findings indicate that re-expression of caspase-8, e.g. by demethylation or caspase-8 gene transfer, might be an effective strategy to restore sensitivity for chemotherapy- or death receptor-induced apoptosis in various tumors in vivo. PMID:11593392

  7. A caspase active site probe reveals high fractional inhibition needed to block DNA fragmentation.

    PubMed

    Méthot, Nathalie; Vaillancourt, John P; Huang, JingQi; Colucci, John; Han, Yongxin; Ménard, Stéphane; Zamboni, Robert; Toulmond, Sylvie; Nicholson, Donald W; Roy, Sophie

    2004-07-01

    Apoptotic markers consist of either caspase substrate cleavage products or phenotypic changes that manifest themselves as a consequence of caspase-mediated substrate cleavage. We have shown recently that pharmacological inhibitors of caspase activity prevent the appearance of two such apoptotic manifestations, alphaII-spectrin cleavage and DNA fragmentation, but that blockade of the latter required a significantly higher concentration of inhibitor. We investigated this phenomenon through the use of a novel radiolabeled caspase inhibitor, [(125)I]M808, which acts as a caspase active site probe. [(125)I]M808 bound to active caspases irreversibly and with high sensitivity in apoptotic cell extracts, in tissue extracts from several commonly used animal models of cellular injury, and in living cells. Moreover, [(125)I]M808 detected active caspases in septic mice when injected intravenously. Using this caspase probe, an active site occupancy assay was developed and used to measure the fractional inhibition required to block apoptosis-induced DNA fragmentation. In thymocytes, occupancy of up to 40% of caspase active sites had no effect on DNA fragmentation, whereas inhibition of half of the DNA cleaving activity required between 65 and 75% of active site occupancy. These results suggest that a high and persistent fractional inhibition will be required for successful caspase inhibition-based therapies. PMID:15067000

  8. Purification of a 24-kD protease from apoptotic tumor cells that activates DNA fragmentation.

    PubMed

    Wright, S C; Wei, Q S; Zhong, J; Zheng, H; Kinder, D H; Larrick, J W

    1994-12-01

    We report the purification of a protease from tumor cells undergoing apoptosis that is involved in activating DNA fragmentation. Initial studies revealed that two inhibitors of serine proteases, N-1-tosylamide-2-phenylethylchloromethyl ketone and carbobenzoxy-Ala-Ala-borophe (DK120), suppressed tumor necrosis factor or ultraviolet (UV) light-induced DNA fragmentation in the U937 histiocytic lymphoma as well as UV light-induced DNA fragmentation in the BT-20 breast carcinoma, HL-60 myelocytic leukemia, and 3T3 fibroblasts. The protease was purified by affinity chromatography with DK120 as ligand and showed high activity on a synthetic substrate preferred by elastase-like enzymes (Ala-Ala-Pro-Val p-nitroanilide), but was inactive on the trypsin substrate, N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester, or the chymotrypsin substrate, Ala-Ala-Pro-Phe p-nitroanilide. The activity of the DK120-binding protease purified from U937 cells undergoing apoptosis was increased approximately 10-fold over that recovered from normal cells. Further purification to homogeneity by heparin-Sepharose affinity chromatography followed by reverse phase high-performance liquid chromatography revealed a single band of 24 kD on a silver-stained sodium dodecyl sulfate gel. In addition to protease activity, the purified enzyme induced DNA fragmentation into multiples of 180 basepairs in isolated U937 nuclei. These findings suggest the 24-kD protease is a novel enzyme that activates DNA fragmentation in U937 cells undergoing apoptosis. PMID:7964487

  9. DNA-binding activity of TNF-{alpha} inducing protein from Helicobacter pylori

    SciTech Connect

    Kuzuhara, T. Suganuma, M.; Oka, K.; Fujiki, H.

    2007-11-03

    Tumor necrosis factor-{alpha} (TNF-{alpha}) inducing protein (Tip{alpha}) is a carcinogenic factor secreted from Helicobacter pylori (H. pylori), mediated through both enhanced expression of TNF-{alpha} and chemokine genes and activation of nuclear factor-{kappa}B. Since Tip{alpha} enters gastric cancer cells, the Tip{alpha} binding molecules in the cells should be investigated. The direct DNA-binding activity of Tip{alpha} was observed by pull down assay using single- and double-stranded genomic DNA cellulose. The surface plasmon resonance assay, indicating an association between Tip{alpha} and DNA, revealed that the affinity of Tip{alpha} for (dGdC)10 is 2400 times stronger than that of del-Tip{alpha}, an inactive Tip{alpha}. This suggests a strong correlation between DNA-binding activity and carcinogenic activity of Tip{alpha}. And the DNA-binding activity of Tip{alpha} was first demonstrated with a molecule secreted from H. pylori.

  10. Actin and DNA Protect Histones from Degradation by Bacterial Proteases but Inhibit Their Antimicrobial Activity

    PubMed Central

    Sol, Asaf; Skvirsky, Yaniv; Blotnick, Edna; Bachrach, Gilad; Muhlrad, Andras

    2016-01-01

    Histones are small polycationic proteins located in the cell nucleus. Together, DNA and histones are integral constituents of the nucleosomes. Upon apoptosis, necrosis, and infection – induced cell death, histones are released from the cell. The extracellular histones have strong antimicrobial activity but are also cytotoxic and thought as mediators of cell death in sepsis. The antimicrobial activity of the cationic extracellular histones is inhibited by the polyanionic DNA and F-actin, which also become extracellular upon cell death. DNA and F-actin protect histones from degradation by the proteases of Pseudomonas aeruginosa and Porphyromonas gingivalis. However, though the integrity of the histones is protected, the activity of histones as antibacterial agents is lost. The inhibition of the histone’s antibacterial activity and their protection from proteolysis by DNA and F-actin indicate a tight electrostatic interaction between the positively charged histones and negatively charged DNA and F-actin, which may have physiological significance in maintaining the equilibrium between the beneficial antimicrobial activity of extracellular histones and their cytotoxic effects. PMID:27555840

  11. AGE-induced keratinocyte MMP-9 expression is linked to TET2-mediated CpG demethylation.

    PubMed

    Zhang, Jinglu; Yang, Chuan; Wang, Chuan; Liu, Dan; Lao, Guojuan; Liang, Ying; Sun, Kan; Luo, Hengcong; Tan, Qin; Ren, Meng; Yan, Li

    2016-05-01

    Studies have documented that unusually high expression of matrix metalloproteinase-9 (MMP-9) suppresses wound healing during the late stages of diabetic foot ulcers. Recently, it has been reported that the presence of advanced glycation end products-bovine serum albumin (AGE-BSA) resulted in a higher expression of MMP-9 in skin primary keratinocytes. The aim of the present study was to elucidate the molecular machinery that is responsible for the inappropriately high AGE-BSA-induced expression of MMP-9. It has been demonstrated that site-specific DNA demethylation played an important role in MMP-9 expression in AGE-BSA-stimulated keratinocytes. Ten-eleven translocation-2 (TET2) was up-regulated, whereas the percentage of methylation in the MMP-9 promoter was reduced. Furthermore, TET2 directly bound to a fragment surrounding the transcriptional start site in the MMP-9 promoter region, contributing to the regulation of MMP-9 expression. In addition, evidence indicated that TET2 affected the migration and proliferation in vitro of cultured skin primary keratinocytes. These findings indicated that TET2 directly interacted with the promoter region of MMP-9 in diabetic tissues and may be a novel master regulator of wound healing. PMID:26913994

  12. A Distinct Triplex DNA Unwinding Activity of ChlR1 Helicase*

    PubMed Central

    Guo, Manhong; Hundseth, Kristian; Ding, Hao; Vidhyasagar, Venkatasubramanian; Inoue, Akira; Nguyen, Chi-Hung; Zain, Rula; Lee, Jeremy S.; Wu, Yuliang

    2015-01-01

    Mutations in the human ChlR1 (DDX11) gene are associated with a unique genetic disorder known as Warsaw breakage syndrome characterized by cellular defects in genome maintenance. The DNA triplex helix structures that form by Hoogsteen or reverse Hoogsteen hydrogen bonding are examples of alternate DNA structures that can be a source of genomic instability. In this study, we have examined the ability of human ChlR1 helicase to destabilize DNA triplexes. Biochemical studies demonstrated that ChlR1 efficiently melted both intermolecular and intramolecular DNA triplex substrates in an ATP-dependent manner. Compared with other substrates such as replication fork and G-quadruplex DNA, triplex DNA was a preferred substrate for ChlR1. Also, compared with FANCJ, a helicase of the same family, the triplex resolving activity of ChlR1 is unique. On the other hand, the mutant protein from a Warsaw breakage syndrome patient failed to unwind these triplexes. A previously characterized triplex DNA-specific antibody (Jel 466) bound triplex DNA structures and inhibited ChlR1 unwinding activity. Moreover, cellular assays demonstrated that there were increased triplex DNA content and double-stranded breaks in ChlR1-depleted cells, but not in FANCJ−/− cells, when cells were treated with a triplex stabilizing compound benzoquinoquinoxaline, suggesting that ChlR1 melting of triple-helix structures is distinctive and physiologically important to defend genome integrity. On the basis of our results, we conclude that the abundance of ChlR1 known to exist in vivo is likely to be a strong deterrent to the stability of triplexes that can potentially form in the human genome. PMID:25561740

  13. Absence of superoxide dismutase activity causes nuclear DNA fragmentation during the aging process

    SciTech Connect

    Muid, Khandaker Ashfaqul; Karakaya, Hüseyin Çaglar; Koc, Ahmet

    2014-02-07

    Highlights: • Aging process increases ROS accumulation. • Aging process increases DNA damage levels. • Absence of SOD activity does not cause DNA damage in young cells. • Absence of SOD activity accelerate aging and increase oxidative DNA damages during the aging process. - Abstract: Superoxide dismutases (SOD) serve as an important antioxidant defense mechanism in aerobic organisms, and deletion of these genes shortens the replicative life span in the budding yeast Saccharomyces cerevisiae. Even though involvement of superoxide dismutase enzymes in ROS scavenging and the aging process has been studied extensively in different organisms, analyses of DNA damages has not been performed for replicatively old superoxide dismutase deficient cells. In this study, we investigated the roles of SOD1, SOD2 and CCS1 genes in preserving genomic integrity in replicatively old yeast cells using the single cell comet assay. We observed that extend of DNA damage was not significantly different among the young cells of wild type, sod1Δ and sod2Δ strains. However, ccs1Δ mutants showed a 60% higher amount of DNA damage in the young stage compared to that of the wild type cells. The aging process increased the DNA damage rates 3-fold in the wild type and more than 5-fold in sod1Δ, sod2Δ, and ccs1Δ mutant cells. Furthermore, ROS levels of these strains showed a similar pattern to their DNA damage contents. Thus, our results confirm that cells accumulate DNA damages during the aging process and reveal that superoxide dismutase enzymes play a substantial role in preserving the genomic integrity in this process.

  14. Water-dependent optical activity inversion of chiral DNA-silica assemblies.

    PubMed

    Liu, Ben; Cao, Yuanyuan; Duan, Yingying; Che, Shunai

    2013-11-25

    Chirality is widely found in nature and is expressed hierarchically in many organic-inorganic hybrid materials. Optical activity (OA) is the most fundamental attribute of these chiral materials. In this study, we found that the OA of impeller-like chiral DNA-silica assemblies (CDSAs) was inverted with the addition of water. The state of DNA under dry and wet conditions, and the dual chirality of chiral DNA layers and twisted helical arrays of opposite handedness in CDSAs were considered to exert predominant effects on the OAs. The circular dichroism (CD) responses for the dry CDSAs were mostly attributed to the chiral arrangement of DNA layers, whereas the opposite CD responses for the wet CDSAs primarily originated from twisted helical arrays of DNA molecules. The observed CD signals were a super-position of the two opposing OA responses. The increase in the longitudinal relation of DNA molecules due to the recovery of a double-helical structure of DNA in the presence of water was considered to be the reason for the increase in intensity of the CD signals that originated from the twisted helical array, which led to the inversion of OA of the CDSAs. The inversion of the plasmon-resonance-based OAs for the chiral-arranged achiral Ag nanoparticles (NPs) located in the channels of the CDSAs in dry and wet states further confirmed the dual chirality of DNA packing. Such research on DNA assemblies and metal NPs with dual, opposite chirality assists in the understanding of DNA hierarchical chirality in living systems and the creation of macroscopic ordered helical materials and biosensors. PMID:24136856

  15. Thymine DNA glycosylase exhibits negligible affinity for nucleobases that it removes from DNA

    PubMed Central

    Malik, Shuja S.; Coey, Christopher T.; Varney, Kristen M.; Pozharski, Edwin; Drohat, Alexander C.

    2015-01-01

    Thymine DNA Glycosylase (TDG) performs essential functions in maintaining genetic integrity and epigenetic regulation. Initiating base excision repair, TDG removes thymine from mutagenic G·T mispairs caused by 5-methylcytosine (mC) deamination and other lesions including uracil (U) and 5-hydroxymethyluracil (hmU). In DNA demethylation, TDG excises 5-formylcytosine (fC) and 5-carboxylcytosine (caC), which are generated from mC by Tet (ten–eleven translocation) enzymes. Using improved crystallization conditions, we solved high-resolution (up to 1.45 Å) structures of TDG enzyme–product complexes generated from substrates including G·U, G·T, G·hmU, G·fC and G·caC. The structures reveal many new features, including key water-mediated enzyme–substrate interactions. Together with nuclear magnetic resonance experiments, the structures demonstrate that TDG releases the excised base from its tight product complex with abasic DNA, contrary to previous reports. Moreover, DNA-free TDG exhibits no significant binding to free nucleobases (U, T, hmU), indicating a Kd >> 10 mM. The structures reveal a solvent-filled channel to the active site, which might facilitate dissociation of the excised base and enable caC excision, which involves solvent-mediated acid catalysis. Dissociation of the excised base allows TDG to bind the beta rather than the alpha anomer of the abasic sugar, which might stabilize the enzyme–product complex. PMID:26358812

  16. LOX-1, mtDNA damage, and NLRP3 inflammasome activation in macrophages: implications in atherogenesis

    PubMed Central

    Ding, Zufeng; Liu, Shijie; Wang, Xianwei; Dai, Yao; Khaidakov, Magomed; Deng, Xiaoyan; Fan, Yubo; Xiang, David; Mehta, Jawahar L.

    2014-01-01

    Aims Lectin-like ox-LDL scavenger receptor-1 (LOX-1) and mitochondrial DNA (mtDNA) damage play a key role in a variety of cardiovascular diseases, including atherosclerosis, hypertension, and inflammation. We posited that damaged mtDNA could trigger autophagy and NLRP3 inflammasome activation, and LOX-1 may play a critical role in this process. Methods and results In order to examine this hypothesis, cultured human THP-1 macrophages exposed to lipopolysaccharide (LPS) were applied to study the link between LOX-1, mtDNA damage, autophagy, and NLRP3 inflammasome expression. Our data showed that LPS markedly induced LOX-1 expression, reactive oxygen species (ROS) generation, autophagy, mtDNA damage, and NLRP3 inflammasome. LOX-1 inhibition with a binding antibody or siRNA inhibited ROS generation, autophagy and mtDNA damage, and a decreased expression of NLRP3 inflammasome. To study the LOX-1–NLRP3 inflammasome signalling, we performed studies using ROS inhibitors and an autophagy inducer, and found that both decreased the expression of NLRP3. On the other hand, autophagy inhibitor enhanced the expression of NLRP3 inflammasome. Knockdown of DNase II inhibited autophagy and NLRP3 inflammasome, providing further support for our hypothesis. Finally, we confirmed the relationship between LOX-1, ROS, mtDNA damage, autophagy, and NLRP3 inflammasome activation in primary macrophages. Conclusions This study based on THP-1 macrophages and primary macrophages indicates that LOX-1-mediated autophagy and mtDNA damage play an essential role in NLRP3 inflammasome activation in inflammatory disease states. PMID:24776598

  17. Novel DNA Motif Binding Activity Observed In Vivo With an Estrogen Receptor α Mutant Mouse

    PubMed Central

    Li, Leping; Grimm, Sara A.; Winuthayanon, Wipawee; Hamilton, Katherine J.; Pockette, Brianna; Rubel, Cory A.; Pedersen, Lars C.; Fargo, David; Lanz, Rainer B.; DeMayo, Francesco J.; Schütz, Günther; Korach, Kenneth S.

    2014-01-01

    Estrogen receptor α (ERα) interacts with DNA directly or indirectly via other transcription factors, referred to as “tethering.” Evidence for tethering is based on in vitro studies and a widely used “KIKO” mouse model containing mutations that prevent direct estrogen response element DNA- binding. KIKO mice are infertile, due in part to the inability of estradiol (E2) to induce uterine epithelial proliferation. To elucidate the molecular events that prevent KIKO uterine growth, regulation of the pro-proliferative E2 target gene Klf4 and of Klf15, a progesterone (P4) target gene that opposes the pro-proliferative activity of KLF4, was evaluated. Klf4 induction was impaired in KIKO uteri; however, Klf15 was induced by E2 rather than by P4. Whole uterine chromatin immunoprecipitation-sequencing revealed enrichment of KIKO ERα binding to hormone response elements (HREs) motifs. KIKO binding to HRE motifs was verified using reporter gene and DNA-binding assays. Because the KIKO ERα has HRE DNA-binding activity, we evaluated the “EAAE” ERα, which has more severe DNA-binding domain mutations, and demonstrated a lack of estrogen response element or HRE reporter gene induction or DNA-binding. The EAAE mouse has an ERα null–like phenotype, with impaired uterine growth and transcriptional activity. Our findings demonstrate that the KIKO mouse model, which has been used by numerous investigators, cannot be used to establish biological functions for ERα tethering, because KIKO ERα effectively stimulates transcription using HRE motifs. The EAAE-ERα DNA-binding domain mutant mouse demonstrates that ERα DNA-binding is crucial for biological and transcriptional processes in reproductive tissues and that ERα tethering may not contribute to estrogen responsiveness in vivo. PMID:24713037

  18. Novel DNA motif binding activity observed in vivo with an estrogen receptor α mutant mouse.

    PubMed

    Hewitt, Sylvia C; Li, Leping; Grimm, Sara A; Winuthayanon, Wipawee; Hamilton, Katherine J; Pockette, Brianna; Rubel, Cory A; Pedersen, Lars C; Fargo, David; Lanz, Rainer B; DeMayo, Francesco J; Schütz, Günther; Korach, Kenneth S

    2014-06-01

    Estrogen receptor α (ERα) interacts with DNA directly or indirectly via other transcription factors, referred to as "tethering." Evidence for tethering is based on in vitro studies and a widely used "KIKO" mouse model containing mutations that prevent direct estrogen response element DNA- binding. KIKO mice are infertile, due in part to the inability of estradiol (E2) to induce uterine epithelial proliferation. To elucidate the molecular events that prevent KIKO uterine growth, regulation of the pro-proliferative E2 target gene Klf4 and of Klf15, a progesterone (P4) target gene that opposes the pro-proliferative activity of KLF4, was evaluated. Klf4 induction was impaired in KIKO uteri; however, Klf15 was induced by E2 rather than by P4. Whole uterine chromatin immunoprecipitation-sequencing revealed enrichment of KIKO ERα binding to hormone response elements (HREs) motifs. KIKO binding to HRE motifs was verified using reporter gene and DNA-binding assays. Because the KIKO ERα has HRE DNA-binding activity, we evaluated the "EAAE" ERα, which has more severe DNA-binding domain mutations, and demonstrated a lack of estrogen response element or HRE reporter gene induction or DNA-binding. The EAAE mouse has an ERα null-like phenotype, with impaired uterine growth and transcriptional activity. Our findings demonstrate that the KIKO mouse model, which has been used by numerous investigators, cannot be used to establish biological functions for ERα tethering, because KIKO ERα effectively stimulates transcription using HRE motifs. The EAAE-ERα DNA-binding domain mutant mouse demonstrates that ERα DNA-binding is crucial for biological and transcriptional processes in reproductive tissues and that ERα tethering may not contribute to estrogen responsiveness in vivo. PMID:24713037

  19. Mutation of Gly721 Alters DNA Topoisomerase I Active Site Architecture and Sensitivity to Camptothecin*

    PubMed Central

    van der Merwe, Marie; Bjornsti, Mary-Ann

    2015-01-01

    DNA topoisomerase I (Top1p) catalyzes the relaxation of supercoiled DNA via a concerted mechanism of DNA strand cleavage and religation. Top1p is the cellular target of the anticancer drug camptothecin (CPT), which reversibly stabilizes a covalent enzyme-DNA intermediate. Top1p clamps around duplex DNA, wherein the core and C-terminal domains are connected by extended α-helices (linker domain), which position the active site Tyr of the C-terminal domain within the catalytic pocket. The physical connection of the linker with the Top1p clamp as well as linker flexibility affect enzyme sensitivity to CPT. Crystallographic data reveal that a conserved Gly residue (located at the juncture between the linker and C-terminal domains) is at one end of a short α-helix, which extends to the active site Tyr covalently linked to the DNA. In the presence of drug, the linker is rigid and this α-helix extends to include Gly and the preceding Leu. We report that mutation of this conserved Gly in yeast Top1p alters enzyme sensitivity to CPT. Mutating Gly to Asp, Glu, Asn, Gln, Leu, or Ala enhanced enzyme CPT sensitivity, with the acidic residues inducing the greatest increase in drug sensitivity in vivo and in vitro. By contrast, Val or Phe substituents rendered the enzyme CPT-resistant. Mutation-induced alterations in enzyme architecture preceding the active site Tyr suggest these structural transitions modulate enzyme sensitivity to CPT, while enhancing the rate of DNA cleavage. We postulate that this conserved Gly residue provides a flexible hinge within the Top1p catalytic pocket to facilitate linker dynamics and the structural alterations that accompany drug binding of the covalent enzyme-DNA intermediate. PMID:18056711

  20. CpGB DNA activates dermal macrophages and specifically recruits inflammatory monocytes into the skin.

    PubMed

    Mathes, Allison L; Rice, Lisa; Affandi, Alsya J; DiMarzio, Michael; Rifkin, Ian R; Stifano, Giuseppina; Christmann, Romy B; Lafyatis, Robert

    2015-02-01

    Toll-like receptor 9 (TLR9) drives innate immune responses after recognition of foreign or endogenous DNA containing unmethylated CpG motifs. DNA-mediated TLR9 activation is highly implicated in the pathogenesis of several autoimmune skin diseases, yet its contribution to the inflammation seen in these diseases remains unclear. In this study, TLR9 ligand, CpGB DNA, was administered to mice via a subcutaneous osmotic pump with treatment lasting 1 or 4 weeks. Gene expression and immunofluorescence analyses were used to determine chemokine expression and cell recruitment in the skin surrounding the pump outlet. CpGB DNA skin treatment dramatically induced a marked influx of CD11b+ F4/80+ macrophages, increasing over 4 weeks of treatment, and induction of IFNγ and TNFα expression. Chemokines, CCL2, CCL4, CCL5, CXCL9 and CXCL10, were highly induced in CpGB DNA-treated skin, although abrogation of these signalling pathways individually did not alter macrophage accumulation. Flow cytometry analysis showed that TLR9 activation in the skin increased circulating CD11b+ CD115+ Ly6C(hi) inflammatory monocytes following 1 week of CpGB DNA treatment. Additionally, skin-resident CD11b+ cells were found to initially take up subcutaneous CpGB DNA and propagate the subsequent immune response. Using diphtheria toxin-induced monocyte depletion mouse model, gene expression analysis demonstrated that CD11b+ cells are responsible for the CpGB DNA-induced cytokine and chemokine response. Overall, these data demonstrate that chronic TLR9 activation induces a specific inflammatory response, ultimately leading to a striking and selective accumulation of macrophages in the skin. PMID:25425469