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Dipankar Sen - One of the best experts on this subject based on the ideXlab platform.
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DNA repair by DNA the uv1c DNAzyme catalyzes photoreactivation of cyclobutane thymine dimers in DNA more effectively than their de novo formation
2016Co-Authors: Adam Barlev, Andrew J. Bennet, Gurpreet Sekhon, Dipankar SenAbstract:UV1C, a 42-nt DNA oligonucleotide, is a deoxyribozyme (DNAzyme) that optimally uses 305 nm wavelength light to catalyze photoreactivation of a cyclobutane thymine dimer placed within a gapped, unnatural DNA substrate, TDP. Herein we show that UV1C is also capable of photoreactivating thymine dimers within an authentic single-stranded DNA substrate, LDP. This bona fide UV1C substrate enables, for the first time, investigation of whether UV1C catalyzes only photoreactivation or also the de novo formation of thymine dimers. Single-turnover experiments carried out with LDP and UV1C, relative to control experiments with LDP alone in single-stranded and double-stranded contexts, show that while UV1C does modestly promote thymine dimer formation, its major activity is indeed photoreactivation. Distinct photostationary states are reached for LDP in its three contexts: as a single strand, as a constituent of a double-helix, and as a 1:1 complex with UV1C. The above results on the cofactor-independent photoreactivation capabilities of a Catalytic DNA reinforce a series of recent, unexpected reports that purely nucleotide-based photoreactivation is also operational within conventional double-helical DNA.
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DNA Repair by DNA: The UV1C DNAzyme Catalyzes Photoreactivation of Cyclobutane Thymine Dimers in DNA More Effectively than Their de Novo Formation
2016Co-Authors: Adam Barlev, Gurpreet S. Sekhon, Andrew J. Bennet, Dipankar SenAbstract:UV1C, a 42-nt DNA oligonucleotide, is a deoxyribozyme (DNAzyme) that optimally uses 305 nm wavelength light to catalyze photoreactivation of a cyclobutane thymine dimer placed within a gapped, unnatural DNA substrate, TDP. Herein we show that UV1C is also capable of photoreactivating thymine dimers within an authentic single-stranded DNA substrate, LDP. This bona fide UV1C substrate enables, for the first time, investigation of whether UV1C catalyzes only photoreactivation or also the de novo formation of thymine dimers. Single-turnover experiments carried out with LDP and UV1C, relative to control experiments with LDP alone in single-stranded and double-stranded contexts, show that while UV1C does modestly promote thymine dimer formation, its major activity is indeed photoreactivation. Distinct photostationary states are reached for LDP in its three contexts: as a single strand, as a constituent of a double-helix, and as a 1:1 complex with UV1C. The above results on the cofactor-independent photoreactivation capabilities of a Catalytic DNA reinforce a series of recent, unexpected reports that purely nucleotide-based photoreactivation is also operational within conventional double-helical DNA
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a ribozyme and a Catalytic DNA with peroxidase activity active sites versus cofactor binding sites
1999Co-Authors: Paola Travascio, Andrew J. Bennet, Dennis Y Wang, Dipankar SenAbstract:Abstract Background: An 18-nucleotide DNA oligomer, PS2.M , derived using an in vitro selection method was previously reported to bind hemin (Fe(III)-protoporphyrinIX) with submicromolar affinity. The DNA-hemin complex exhibited DNA-enhanced peroxidative activity. PS2.M is guanine-rich and requires potassium ions to fold to its active conformation, consistent with its forming a guanine-guaduplex. In investigating the specific Catalytic features of PS2.M we tested the peroxidative properties of its RNA version ( rPS2.M ) as well as that of an unrelated DNA guanine-quadruplex, OXY4 . Results: The hemin-binding affinity of rPS2.M was found to be 30-fold weaker than that of PS2.M . The UV-visible spectra and kinetics of enzymatic peroxidation of the RNA-hemin complex, however, were nearly identical to those of its DNA counterpart. Both displayed peroxidase activity substantially greater than those of heme proteins such as catalase and Fe(III)-myoglobin. Kinetic analysis suggested that PS2.M and rPS2.M catalyzed the breakdown of the hemin-hydrogen peroxide covalent complex to products. The hemin complex of folded OXY4 (which bound hemin as strongly as did rPS2.M ) had a distinct absorption spectrum and only a minor peroxidase activity above the background level. Conclusions: The results indicated that it is possible for RNA and DNA of the same sequence to fold to form comparable cofactor-binding sites, and to show comparable Catalytic behavior. The results further suggest that only a subset of cofactor-binding sites formed within folded nucleic acids might be able to function as active sites, by providing the appropriate chemical environments for catalysis.
Jack C Yalowich - One of the best experts on this subject based on the ideXlab platform.
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the Catalytic DNA topoisomerase ii inhibitor dexrazoxane icrf 187 induces differentiation and apoptosis in human leukemia k562 cells
2001Co-Authors: Michael E Abram, Norman Barnabe, William P Allan, Tayeb Khelifa, Jack C YalowichAbstract:The bisdioxopiperazines ICRF-187 (dexrazoxane), ICRF-193, and ICRF-154 are Catalytic noncleavable complex-forming inhibitors of DNA topoisomerase II that do not produce protein-linked DNA strand breaks. In this study, we showed that bisdioxopiperazines induced erythroid differentiation, inhibited human leukemia K562 cell growth, and caused a slow induction of apoptosis. Dexrazoxane treatment caused DNA endoreduplication resulting in large highly polyploid cells. This result suggested the lack of a DNA topoisomerase II activity-based cell cycle checkpoint. The percentage of K562 cells that became apoptotic was much larger than the percentage of cells that stained for hemoglobin, suggesting that prior differentiation was not required for induction of apoptosis. Use of the Bcr-Abl tyrosine kinase inhibitor STI-571 resulted in a reduction in Bcl-xL levels and potentiation of dexrazoxane-induced apoptosis related to an earlier onset and more extensive cleavage of caspase-3. These results indicated that dexrazoxane-induced apoptosis is associated with a caspase-3 activation/cleavage pathway. In addition, these results were consistent with the antiapoptotic signaling function of Bcr-Abl to regulate expression of Bcl-xL. The ability of dexrazoxane to induce differentiation and apoptosis suggests that bisdioxopiperazines may be useful in treating some types of leukemia.
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the Catalytic DNA topoisomerase ii inhibitor dexrazoxane icrf 187 induces endopolyploidy in chinese hamster ovary cells
2000Co-Authors: Michael E Abram, Victor J Ferrans, Gaiklean Chee, Erwin Huebner, Edward H Byard, Norman Barnabe, Zuxi Yu, Jack C YalowichAbstract:The bisdioxopiperazines, including dexrazoxane (ICRF-187), are Catalytic or noncleavable complex-forming inhibitors of DNA topoisomerase II that do not produce DNA strand breaks. In this study we show that dexrazoxane inhibits the division of Chinese hamster ovary (CHO) cells resulting in marked increases in cell size (up to 80 μm in diameter), volume (up to 150-fold greater), and ploidy (as high as 32N). This last result indicates that the dexrazoxane-induced DNA reduplication was restricted to once per cell cycle. Kinetic analysis of the flow cytometry data indicated that the conversion between successively higher ploidy levels was progressively slowed at longer times of exposure to dexrazoxane. Both the protein and DNA content of dexrazoxane-treated CHO cells increased linearly over time in the same proportion. Light and electron microscopic studies of dexrazoxane-treated cells showed ring-like multilobulated nuclei. Immunohistochemical staining of dexrazoxane-treated cells showed that F-actin and acetylated α-tubulin were present in large, highly organized networks. Immunohistochemical staining of the dexrazoxane-treated CHO cells also showed that the topoisomerase IIα colocalized with the DNA of the multilobulated nuclei. Staining of γ-tubulin revealed that the dexrazoxane-treated cells contained multiple centrosomes, indicating that dexrazoxane prevents cytokinesis but not centrosome reduplication. It is concluded that dexrazoxane inhibits CHO cytokinesis in cells by virtue of its ability to inhibit topoisomerase II.
Andrew J. Bennet - One of the best experts on this subject based on the ideXlab platform.
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DNA repair by DNA the uv1c DNAzyme catalyzes photoreactivation of cyclobutane thymine dimers in DNA more effectively than their de novo formation
2016Co-Authors: Adam Barlev, Andrew J. Bennet, Gurpreet Sekhon, Dipankar SenAbstract:UV1C, a 42-nt DNA oligonucleotide, is a deoxyribozyme (DNAzyme) that optimally uses 305 nm wavelength light to catalyze photoreactivation of a cyclobutane thymine dimer placed within a gapped, unnatural DNA substrate, TDP. Herein we show that UV1C is also capable of photoreactivating thymine dimers within an authentic single-stranded DNA substrate, LDP. This bona fide UV1C substrate enables, for the first time, investigation of whether UV1C catalyzes only photoreactivation or also the de novo formation of thymine dimers. Single-turnover experiments carried out with LDP and UV1C, relative to control experiments with LDP alone in single-stranded and double-stranded contexts, show that while UV1C does modestly promote thymine dimer formation, its major activity is indeed photoreactivation. Distinct photostationary states are reached for LDP in its three contexts: as a single strand, as a constituent of a double-helix, and as a 1:1 complex with UV1C. The above results on the cofactor-independent photoreactivation capabilities of a Catalytic DNA reinforce a series of recent, unexpected reports that purely nucleotide-based photoreactivation is also operational within conventional double-helical DNA.
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DNA Repair by DNA: The UV1C DNAzyme Catalyzes Photoreactivation of Cyclobutane Thymine Dimers in DNA More Effectively than Their de Novo Formation
2016Co-Authors: Adam Barlev, Gurpreet S. Sekhon, Andrew J. Bennet, Dipankar SenAbstract:UV1C, a 42-nt DNA oligonucleotide, is a deoxyribozyme (DNAzyme) that optimally uses 305 nm wavelength light to catalyze photoreactivation of a cyclobutane thymine dimer placed within a gapped, unnatural DNA substrate, TDP. Herein we show that UV1C is also capable of photoreactivating thymine dimers within an authentic single-stranded DNA substrate, LDP. This bona fide UV1C substrate enables, for the first time, investigation of whether UV1C catalyzes only photoreactivation or also the de novo formation of thymine dimers. Single-turnover experiments carried out with LDP and UV1C, relative to control experiments with LDP alone in single-stranded and double-stranded contexts, show that while UV1C does modestly promote thymine dimer formation, its major activity is indeed photoreactivation. Distinct photostationary states are reached for LDP in its three contexts: as a single strand, as a constituent of a double-helix, and as a 1:1 complex with UV1C. The above results on the cofactor-independent photoreactivation capabilities of a Catalytic DNA reinforce a series of recent, unexpected reports that purely nucleotide-based photoreactivation is also operational within conventional double-helical DNA
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a ribozyme and a Catalytic DNA with peroxidase activity active sites versus cofactor binding sites
1999Co-Authors: Paola Travascio, Andrew J. Bennet, Dennis Y Wang, Dipankar SenAbstract:Abstract Background: An 18-nucleotide DNA oligomer, PS2.M , derived using an in vitro selection method was previously reported to bind hemin (Fe(III)-protoporphyrinIX) with submicromolar affinity. The DNA-hemin complex exhibited DNA-enhanced peroxidative activity. PS2.M is guanine-rich and requires potassium ions to fold to its active conformation, consistent with its forming a guanine-guaduplex. In investigating the specific Catalytic features of PS2.M we tested the peroxidative properties of its RNA version ( rPS2.M ) as well as that of an unrelated DNA guanine-quadruplex, OXY4 . Results: The hemin-binding affinity of rPS2.M was found to be 30-fold weaker than that of PS2.M . The UV-visible spectra and kinetics of enzymatic peroxidation of the RNA-hemin complex, however, were nearly identical to those of its DNA counterpart. Both displayed peroxidase activity substantially greater than those of heme proteins such as catalase and Fe(III)-myoglobin. Kinetic analysis suggested that PS2.M and rPS2.M catalyzed the breakdown of the hemin-hydrogen peroxide covalent complex to products. The hemin complex of folded OXY4 (which bound hemin as strongly as did rPS2.M ) had a distinct absorption spectrum and only a minor peroxidase activity above the background level. Conclusions: The results indicated that it is possible for RNA and DNA of the same sequence to fold to form comparable cofactor-binding sites, and to show comparable Catalytic behavior. The results further suggest that only a subset of cofactor-binding sites formed within folded nucleic acids might be able to function as active sites, by providing the appropriate chemical environments for catalysis.
Frank Lyko - One of the best experts on this subject based on the ideXlab platform.
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rna methylation by dnmt2 protects transfer rnas against stress induced cleavage
2010Co-Authors: Matthias Schaefer, Katharina Hanna, Tim Pollex, Madeleine Meusburger, Francesca Tuorto, Mark Helm, Frank LykoAbstract:The covalent modification of nucleic acids plays an important role in regulating the functions of DNA and RNA. DNA modifications have been analyzed in considerable detail, and the characterization of (cytosine-5) DNA methylation has been crucial for understanding the molecular basis of epigenetic gene regulation (Klose and Bird 2006). (Cytosine-5) methylation has also been documented in various RNA species, including tRNA, but the function of RNA methylation has not been firmly established yet (Motorin et al. 2010). Dnmt2 proteins were originally assigned to the DNA methyltransferase family, because of their strong sequence conservation of Catalytic DNA methyltransferase motifs (Okano et al. 1998; Yoder and Bestor 1998). A recent study has suggested that Dnmt2-mediated DNA methylation is important for transposon silencing in Drosophila (Phalke et al. 2009). However, only a weak and distributive DNA methylation activity has been reported in various systems (Jeltsch et al. 2006). The ambiguities associated with the DNA methyltransferase activity of Dnmt2 have also prompted the search for alternative enzyme substrates, and resulted in the discovery of a tRNA methyltransferase activity of Dnmt2 (Goll et al. 2006). Purified recombinant human Dnmt2 methylated RNA preparations from Dnmt2 mutant mice, flies, and plants. Further experiments identified C38 in the anti-codon loop of tRNAAsp as the methylation target site of Dnmt2 (Goll et al. 2006). However, the functional relevance of the tRNA methyltransferase activity of Dnmt2 remains to be established. Dnmt2 mutant mice, flies, and plants were reported to be viable and fertile (Goll et al. 2006) under standard laboratory conditions. A distinct Dnmt2 mutant phenotype, caused by morpholino knockdown experiments, has so far been reported only in zebrafish, leading to lethal differentiation defects in the retina, liver, and brain (Rai et al. 2007). In addition, two studies have indicated increased stress tolerance in Dnmt2-overexpressing flies and amoebas (Lin et al. 2005; Fisher et al. 2006). However, the underlying molecular mechanisms have not been investigated yet.
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Establishment and functional validation of a structural homology model for human DNA methyltransferase 1.
2003Co-Authors: Pawel Siedlecki, Regine Garcia Boy, S. Comagic, Ralf Schirrmacher, Manfred Wiessler, Piotr Zielenkiewicz, Sándor Suhai, Frank LykoAbstract:Changes in DNA methylation patterns play an important role in tumorigenesis. The DNA methyltransferase 1 (DNMT1) protein represents a major DNA methyltransferase activity in human cells and is therefore a prominent target for experimental cancer therapies. However, there are only few available inhibitors and their high toxicity and low specificity have so far precluded their broad use in chemotherapy. Based on the strong conservation of Catalytic DNA methyltransferase domains we have used a homology modeling approach to determine the three-dimensional structure of the DNMT1 Catalytic domain. Our results suggest an overall structural conservation with other DNA methyltransferases but also indicate local conformational differences. To prove the validity of our model we used it as a template to design a novel derivative of the known DNA methyltransferase inhibitor 5-azacytidine. The resulting compound (N4-fluoroacetyl-5-azacytidine) functioned as an efficient inhibitor of DNA methylation in human tumor cell lines and also provides novel opportunities for pharmacological applications.
Michael E Abram - One of the best experts on this subject based on the ideXlab platform.
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the Catalytic DNA topoisomerase ii inhibitor dexrazoxane icrf 187 induces differentiation and apoptosis in human leukemia k562 cells
2001Co-Authors: Michael E Abram, Norman Barnabe, William P Allan, Tayeb Khelifa, Jack C YalowichAbstract:The bisdioxopiperazines ICRF-187 (dexrazoxane), ICRF-193, and ICRF-154 are Catalytic noncleavable complex-forming inhibitors of DNA topoisomerase II that do not produce protein-linked DNA strand breaks. In this study, we showed that bisdioxopiperazines induced erythroid differentiation, inhibited human leukemia K562 cell growth, and caused a slow induction of apoptosis. Dexrazoxane treatment caused DNA endoreduplication resulting in large highly polyploid cells. This result suggested the lack of a DNA topoisomerase II activity-based cell cycle checkpoint. The percentage of K562 cells that became apoptotic was much larger than the percentage of cells that stained for hemoglobin, suggesting that prior differentiation was not required for induction of apoptosis. Use of the Bcr-Abl tyrosine kinase inhibitor STI-571 resulted in a reduction in Bcl-xL levels and potentiation of dexrazoxane-induced apoptosis related to an earlier onset and more extensive cleavage of caspase-3. These results indicated that dexrazoxane-induced apoptosis is associated with a caspase-3 activation/cleavage pathway. In addition, these results were consistent with the antiapoptotic signaling function of Bcr-Abl to regulate expression of Bcl-xL. The ability of dexrazoxane to induce differentiation and apoptosis suggests that bisdioxopiperazines may be useful in treating some types of leukemia.
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the Catalytic DNA topoisomerase ii inhibitor dexrazoxane icrf 187 induces endopolyploidy in chinese hamster ovary cells
2000Co-Authors: Michael E Abram, Victor J Ferrans, Gaiklean Chee, Erwin Huebner, Edward H Byard, Norman Barnabe, Zuxi Yu, Jack C YalowichAbstract:The bisdioxopiperazines, including dexrazoxane (ICRF-187), are Catalytic or noncleavable complex-forming inhibitors of DNA topoisomerase II that do not produce DNA strand breaks. In this study we show that dexrazoxane inhibits the division of Chinese hamster ovary (CHO) cells resulting in marked increases in cell size (up to 80 μm in diameter), volume (up to 150-fold greater), and ploidy (as high as 32N). This last result indicates that the dexrazoxane-induced DNA reduplication was restricted to once per cell cycle. Kinetic analysis of the flow cytometry data indicated that the conversion between successively higher ploidy levels was progressively slowed at longer times of exposure to dexrazoxane. Both the protein and DNA content of dexrazoxane-treated CHO cells increased linearly over time in the same proportion. Light and electron microscopic studies of dexrazoxane-treated cells showed ring-like multilobulated nuclei. Immunohistochemical staining of dexrazoxane-treated cells showed that F-actin and acetylated α-tubulin were present in large, highly organized networks. Immunohistochemical staining of the dexrazoxane-treated CHO cells also showed that the topoisomerase IIα colocalized with the DNA of the multilobulated nuclei. Staining of γ-tubulin revealed that the dexrazoxane-treated cells contained multiple centrosomes, indicating that dexrazoxane prevents cytokinesis but not centrosome reduplication. It is concluded that dexrazoxane inhibits CHO cytokinesis in cells by virtue of its ability to inhibit topoisomerase II.