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Journal of Biomaterials Applications Jul 2017The encapsulation mechanism of DNA in sol-gel derived silica has been explored in order to elucidate the effect of DNA conformation on encapsulation and to identify the...
The encapsulation mechanism of DNA in sol-gel derived silica has been explored in order to elucidate the effect of DNA conformation on encapsulation and to identify the nature of chemical/physical interaction of DNA with silica during and after sol-gel transition. In this respect, double stranded DNA and dAMP (2'-deoxyadenosine 5'-monophosphate) were encapsulated in silica using an alkoxide-based sol-gel route. Biomolecule-encapsulating gels have been characterized using UV-Vis, Si NMR, FTIR spectroscopy and gas adsorption (BET) to investigate chemical interactions of biomolecules with the porous silica network and to examine the extent of sol-gel reactions upon encapsulation. Ethidium bromide intercalation and leach out tests showed that helix conformation of DNA was preserved after encapsulation. For both biomolecules, high water-to-alkoxide ratio promoted water-producing condensation and prevented alcoholic denaturation. NMR and FTIR analyses confirmed high hydraulic reactivity (water adsorption) for more silanol groups-containing DNA and dAMP encapsulated gels than plain silica gel. No chemical binding/interaction occurred between biomolecules and silica network. DNA and dAMP encapsulated silica gelled faster than plain silica due to basic nature of DNA or dAMP containing buffer solutions. DNA was not released from silica gels to aqueous environment up to 9 days. The chemical association between DNA/dAMP and silica host was through phosphate groups and molecular water attached to silanols, acting as a barrier around biomolecules. The helix morphology was found not to be essential for such interaction. BET analyses showed that interconnected, inkbottle-shaped mesoporous silica network was condensed around DNA and dAMP molecules.
Topics: DNA; Deoxyadenine Nucleotides; Drug Carriers; Nucleic Acid Conformation; Phase Transition; Porosity; Silanes; Silica Gel; Silicon Dioxide
PubMed: 28566001
DOI: 10.1177/0885328217713104 -
Nature Communications Apr 2021Oxidized dGTP (8-oxo-7,8-dihydro-2´-deoxyguanosine triphosphate, 8-oxodGTP) insertion by DNA polymerases strongly promotes cancer and human disease. How DNA polymerases...
Oxidized dGTP (8-oxo-7,8-dihydro-2´-deoxyguanosine triphosphate, 8-oxodGTP) insertion by DNA polymerases strongly promotes cancer and human disease. How DNA polymerases discriminate against oxidized and undamaged nucleotides, especially in error-prone double strand break (DSB) repair, is poorly understood. High-resolution time-lapse X-ray crystallography snapshots of DSB repair polymerase μ undergoing DNA synthesis reveal that a third active site metal promotes insertion of oxidized and undamaged dGTP in the canonical anti-conformation opposite template cytosine. The product metal bridged O8 with product oxygens, and was not observed in the syn-conformation opposite template adenine (A). Rotation of A into the syn-conformation enabled undamaged dGTP misinsertion. Exploiting metal and substrate dynamics in a rigid active site allows 8-oxodGTP to circumvent polymerase fidelity safeguards to promote pro-mutagenic double strand break repair.
Topics: Adenine; Base Pairing; Biocatalysis; Catalytic Domain; Cytosine; DNA Breaks, Double-Stranded; DNA Repair; DNA-Directed DNA Polymerase; Deoxyguanine Nucleotides; Humans; Models, Molecular; Mutagenesis; Mutagenesis, Insertional; Nucleotides; Oxidation-Reduction
PubMed: 33824325
DOI: 10.1038/s41467-021-21354-6 -
Life Science Alliance Apr 2022Eukaryotic cells have evolved a replication stress response that helps to overcome stalled/collapsed replication forks and ensure proper DNA replication. The replication...
Eukaryotic cells have evolved a replication stress response that helps to overcome stalled/collapsed replication forks and ensure proper DNA replication. The replication checkpoint protein Mrc1 plays important roles in these processes, although its functional interactions are not fully understood. Here, we show that negatively interacts with , which encodes the helicase protein Chl1, suggesting distinct roles for these factors during the replication stress response. Indeed, whereas Mrc1 is known to facilitate the restart of stalled replication forks, we uncovered that Chl1 controls replication fork rate under replication stress conditions. Chl1 loss leads to increased gene expression and dNTP levels at the onset of S phase likely without activating the DNA damage response. This in turn impairs the formation of RPA-coated ssDNA and subsequent checkpoint activation. Thus, the Chl1 helicase affects RPA-dependent checkpoint activation in response to replication fork arrest by ensuring proper intracellular dNTP levels, thereby controlling replication fork progression under replication stress conditions.
Topics: Cell Cycle Proteins; Cells, Cultured; Chromosomal Proteins, Non-Histone; DEAD-box RNA Helicases; DNA Helicases; DNA Replication; Deoxyribonucleotides; Humans; Saccharomyces cerevisiae Proteins
PubMed: 35017203
DOI: 10.26508/lsa.202101153 -
Langmuir : the ACS Journal of Surfaces... Dec 2022The fate of biomolecules in the environment depends in part on understanding the surface chemistry occurring at the biological-geochemical (bio-geo) interface. Little is...
The fate of biomolecules in the environment depends in part on understanding the surface chemistry occurring at the biological-geochemical (bio-geo) interface. Little is known about how environmental DNA (eDNA) or smaller components, like nucleotides and oligonucleotides, persist in aquatic environments and the role of surface interactions. This study aims to probe surface interactions and adsorption behavior of nucleotides on oxide surfaces. We have investigated the interactions of individual nucleotides (dGMP, dCMP, dAMP, and dTMP) on TiO particle surfaces as a function of pH and in the presence of complementary and noncomplementary base pairs. Using attenuated total reflectance-Fourier transform infrared spectroscopy, there is an increased number of adsorbed nucleotides at lower pH with a preferential interaction of the phosphate group with the oxide surface. Additionally, differential adsorption behavior is seen where purine nucleotides are preferentially adsorbed, with higher surface saturation coverage, over their pyrimidine derivatives. These differences may be a result of intermolecular interactions between coadsorbed nucleotides. When the TiO surface was exposed to two-component solutions of nucleotides, there was preferential adsorption of dGMP compared to dCMP and dTMP, and dAMP compared to dTMP and dCMP. Complementary nucleotide base pairs showed hydrogen-bond interactions between a strongly adsorbed purine nucleotide layer and a weaker interacting hydrogen-bonded pyrimidine second layer. Noncomplementary base pairs did not form a second layer. These results highlight several important findings: (i) there is differential adsorption of nucleotides; (ii) complementary coadsorbed nucleotides show base pairing with a second layer, and the stability depends on the strength of the hydrogen bonding interactions and; (iii) the first layer coverage strongly depends on pH. Overall, the importance of surface interactions in the adsorption of nucleotides and the templating of specific interactions between nucleotides are discussed.
Topics: Thymidine Monophosphate; Deoxycytidine Monophosphate; Oxides; Hydrogen Bonding; Hydrogen
PubMed: 36445255
DOI: 10.1021/acs.langmuir.2c01604 -
Proceedings of the National Academy of... Jun 2021Bacteriophages (phages) have evolved efficient means to take over the machinery of the bacterial host. The molecular tools at their disposal may be applied to manipulate...
Bacteriophages (phages) have evolved efficient means to take over the machinery of the bacterial host. The molecular tools at their disposal may be applied to manipulate bacteria and to divert molecular pathways at will. Here, we describe a bacterial growth inhibitor, gene product T5.015, encoded by the T5 phage. High-throughput sequencing of genomic DNA of bacterial mutants, resistant to this inhibitor, revealed disruptive mutations in the gene, suggesting that growth inhibition mediated by T5.015 depends on the uracil-excision activity of Ung. We validated that growth inhibition is abrogated in the absence of and confirmed physical binding of Ung by T5.015. In addition, biochemical assays with T5.015 and Ung indicated that T5.015 mediates endonucleolytic activity at abasic sites generated by the base-excision activity of Ung. Importantly, the growth inhibition resulting from the endonucleolytic activity is manifested by DNA replication and cell division arrest. We speculate that the phage uses this protein to selectively cause cleavage of the host DNA, which possesses more misincorporated uracils than that of the phage. This protein may also enhance phage utilization of the available resources in the infected cell, since halting replication saves nucleotides, and stopping cell division maintains both daughters of a dividing cell.
Topics: Bacteriophages; Cell Cycle Checkpoints; Cell Division; DNA; Deoxyuracil Nucleotides; Endonucleases; Escherichia coli; High-Throughput Nucleotide Sequencing; Mutation; Uracil
PubMed: 34074772
DOI: 10.1073/pnas.2026354118 -
The Journal of Biological Chemistry Nov 2019Ribonucleotide reductase (RNR) catalyzes the first committed reaction in DNA synthesis. Most of what we know about RNR regulation comes from studies with cultured cells...
Ribonucleotide reductase (RNR) catalyzes the first committed reaction in DNA synthesis. Most of what we know about RNR regulation comes from studies with cultured cells and with purified proteins. In this study, Tran use technology to inactivate RNR large subunit expression in heart and skeletal muscle of mouse embryos. Analysis of these mutants paints a picture of dNTP regulation in whole animals quite different from that seen in studies of purified proteins and cultured cells.
Topics: Animals; DNA Replication; Deoxyribonucleotides; Heart; Mice; Ribonucleotide Reductases
PubMed: 31676554
DOI: 10.1074/jbc.H119.011335 -
Langmuir : the ACS Journal of Surfaces... 2018Calcium phosphate (CaP) has long been used for DNA delivery, although its fundamental interaction with DNA, especially with single-stranded DNA oligonucleotides, remains...
Calcium phosphate (CaP) has long been used for DNA delivery, although its fundamental interaction with DNA, especially with single-stranded DNA oligonucleotides, remains to be fully understood. Using fluorescently labeled oligonucleotides, we herein studied DNA adsorption isotherm and the effect of DNA length and sequence. Longer DNAs are adsorbed more strongly, and at neutral pH, poly-C DNAs are adsorbed more than the other three DNA homopolymers. However, at near pH 11, the pH of CaP synthesis, T DNA is adsorbed more strongly than C or A. This can explain why T and G can fully inhibit the growth of CaP, while A and C only retarded its growth kinetics. DNA adsorption also reduces aggregation of CaP. DNA desorption experiments were carried out using concentrated urea, thymidine, or inorganic phosphate as competitors, and desorption was observed only in the presence of phosphate, suggesting that DNA uses its phosphate backbone to interact with the CaP surface. Desorption was also promoted by raising the NaCl concentration suggesting the electrostatic nature of interaction. Finally, ten different metal phosphate materials were synthesized by co-precipitating each metal ion (Ce, Fe, Ca, Ni, Zn, Mn, Ba, Cu, Sr, Co), and DNA adsorption by these phosphate precipitants was found to be related to their surface charge and metal chemistry. This work has revealed fundamental surface science of DNA adsorption by CaP and other metal phosphate salts, and this knowledge might be useful for gene delivery, biomineralization, and DNA-directed assembly of metal phosphate materials.
Topics: Adsorption; Calcium Phosphates; Crystallization; Deoxyribonucleotides; Fluorescence; Hydrogen-Ion Concentration; Metals, Heavy; Molecular Structure; Poly G; Poly T
PubMed: 29228772
DOI: 10.1021/acs.langmuir.7b03204 -
Biochemistry Jul 2017The causative agent of tuberculosis, Mycobacterium tuberculosis, is a bacterium with a complex cell wall and a complicated life cycle. The genome of M. tuberculosis...
The causative agent of tuberculosis, Mycobacterium tuberculosis, is a bacterium with a complex cell wall and a complicated life cycle. The genome of M. tuberculosis contains well over 4000 genes thought to encode proteins. One of these codes for a putative enzyme referred to as Rv3404c, which has attracted research attention as a potential virulence factor for over 12 years. Here we demonstrate that Rv3404c functions as a sugar N-formyltransferase that converts dTDP-4-amino-4,6-dideoxyglucose into dTDP-4-formamido-4,6-dideoxyglucose using N-formyltetrahydrofolate as the carbon source. Kinetic analyses demonstrate that Rv3404c displays a significant catalytic efficiency of 1.1 × 10 M s. In addition, we report the X-ray structure of a ternary complex of Rv3404c solved in the presence of N-formyltetrahydrofolate and dTDP-4-amino-4,6-dideoxyglucose. The final model of Rv3404c was refined to an overall R-factor of 16.8% at 1.6 Å resolution. The results described herein are especially intriguing given that there have been no published reports of N-formylated sugars associated with M. tuberculosis. The data thus provide a new avenue of research into this fascinating, yet deadly, organism that apparently has been associated with human infection since ancient times.
Topics: Bacterial Proteins; Catalysis; Crystallography, X-Ray; Deoxy Sugars; Formyltetrahydrofolates; Hydroxymethyl and Formyl Transferases; Kinetics; Models, Molecular; Mycobacterium tuberculosis; Thymine Nucleotides; Virulence Factors
PubMed: 28665588
DOI: 10.1021/acs.biochem.7b00506 -
The Plant Cell Sep 2022Thymidylates are generated by several partially overlapping metabolic pathways in different subcellular locations. This interconnectedness complicates an understanding...
Thymidylates are generated by several partially overlapping metabolic pathways in different subcellular locations. This interconnectedness complicates an understanding of how thymidylates are formed in vivo. Analyzing a comprehensive collection of mutants and double mutants on the phenotypic and metabolic level, we report the effect of de novo thymidylate synthesis, salvage of thymidine, and conversion of cytidylates to thymidylates on thymidylate homeostasis during seed germination and seedling establishment in Arabidopsis (Arabidopsis thaliana). During germination, the salvage of thymidine in organelles contributes predominantly to the thymidylate pools and a mutant lacking organellar (mitochondrial and plastidic) thymidine kinase has severely altered deoxyribonucleotide levels, less chloroplast DNA, and chlorotic cotyledons. This phenotype is aggravated when mitochondrial thymidylate de novo synthesis is additionally compromised. We also discovered an organellar deoxyuridine-triphosphate pyrophosphatase and show that its main function is not thymidylate synthesis but probably the removal of noncanonical nucleotide triphosphates. Interestingly, cytosolic thymidylate synthesis can only compensate defective organellar thymidine salvage in seedlings but not during germination. This study provides a comprehensive insight into the nucleotide metabolome of germinating seeds and demonstrates the unique role of enzymes that seem redundant at first glance.
Topics: Arabidopsis; Arabidopsis Proteins; Chloroplasts; DNA, Chloroplast; Deoxyribonucleotides; Deoxyuridine; Germination; Metabolome; Nucleotides; Phosphorylation; Pyrophosphatases; Seedlings; Seeds; Thymidine; Thymidine Kinase
PubMed: 35861422
DOI: 10.1093/plcell/koac207 -
Veterinary Journal (London, England :... Mar 2016In the equine large intestine, the knowledge of the basic mechanisms underlying motility function is crucial to properly treat motility disorders. P2Y1 receptors are...
In the equine large intestine, the knowledge of the basic mechanisms underlying motility function is crucial to properly treat motility disorders. P2Y1 receptors are responsible for mediating purinergic colonic relaxation in several species. In vitro experimental studies of the circular muscle from the equine pelvic flexure (n = 6) were performed to characterize inhibitory and excitatory neuromuscular transmission. Electrophysiological studies showed that electrical field stimulation (EFS) evoked biphasic inhibitory junction potentials (IJPs) in smooth muscle cells: a fast IJP (IJPf) followed by a sustained IJP (IJPs). IJPs was sensitive to L-NNA 1 mM (a nitric oxide synthase inhibitor) (P <0.01), while IJPf was abolished by MRS2500 1 µM (a P2Y1 receptor antagonist) (P <0.001). EFS (5 Hz for 2 min) in the organ bath inhibited rhythmic contractions to 3.0 ± 2.5% of basal area under the curve (P <0.0001). EFS under MRS2500 1 µM or L-NNA 1 mM incubation inhibited contractions to 6.0 ± 2.8% (P <0.05) and 24.4 ± 11.3% respectively (P <0.05). Combination of MRS2500 1 µM and L-NNA 1 mM completely reversed the EFS-induced inhibition of colonic motility. Non-nitrergic, non-purinergic conditions were used to reveal voltage-dependent EFS-induced contractions sensitive to atropine 1 µM (P <0.001) and, therefore, cholinergic. In conclusion, nerve-mediated relaxation and contraction in the equine pelvic flexure involve the same mechanisms as those observed in the human colon. P2Y1 receptors mediate purinergic relaxations and are potential targets for the treatment of equine colonic motor disorders.
Topics: Animals; Colon; Deoxyadenine Nucleotides; Enzyme Inhibitors; Gastrointestinal Motility; Horses; Muscle Contraction; Muscle Relaxation; Nitroarginine; Purinergic P2Y Receptor Antagonists
PubMed: 26831180
DOI: 10.1016/j.tvjl.2016.01.002