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Analytical Chemistry Jan 2019Investigation into intracellular ribonucleotides (RNs) and deoxyribonucleotides (dRNs) is important for studies of the mechanism of many biological processes, such as...
Method for Quantification of Ribonucleotides and Deoxyribonucleotides in Human Cells Using (Trimethylsilyl)diazomethane Derivatization Followed by Liquid Chromatography-Tandem Mass Spectrometry.
Investigation into intracellular ribonucleotides (RNs) and deoxyribonucleotides (dRNs) is important for studies of the mechanism of many biological processes, such as RNA and DNA synthesis and DNA repair, as well as metabolic and therapeutic efficacy of nucleoside analogues. However, current methods are still unsatisfactory for determination of nucleotides in complex matrixes. Here we describe a novel method for the determination of RN and dRN pools in cells based on fast derivatization with (trimethylsilyl)diazomethane (TMSD) followed by quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Derivatization was accomplished in 3 min, and each derivatized nucleotide not only had a sufficient retention on reversed-phase column by introduction of methyl groups but also exhibited a unique ion transition which consequently eliminated mutual interference in LC-MS/MS. Chromatographic separation was performed on a C column with a simple acetonitrile-water gradient elution system, which avoided contamination and ion suppression caused by ion-pairing reagents. The developed method was fully validated and applied to the analysis of RNs and dRNs in cell samples. Moreover, results demonstrated that the applicability of this method could be extended to nucleoside analogues and their metabolites and could facilitate many applications in future studies.
Topics: A549 Cells; Chromatography, Liquid; Deoxyribonucleotides; Diazomethane; HCT116 Cells; Humans; Ribonucleotides; Tandem Mass Spectrometry; Tumor Cells, Cultured
PubMed: 30525455
DOI: 10.1021/acs.analchem.8b04281 -
Acta Crystallographica. Section D,... Oct 2020Mycobacterium smegmatis MutT1 (MsMutT1) is a sanitation enzyme made up of an N-terminal Nudix hydrolase domain and a C-terminal domain resembling a histidine...
Mycobacterium smegmatis MutT1 (MsMutT1) is a sanitation enzyme made up of an N-terminal Nudix hydrolase domain and a C-terminal domain resembling a histidine phosphatase. It has been established that the action of MutT1 on 8-oxo-dGTP, 8-oxo-GTP and diadenosine polyphosphates is modulated by intermolecular interactions. In order to further explore this and to elucidate the structural basis of its differential action on 8-oxo-NTPs and unsubstituted NTPs, the crystal structures of complexes of MsMutT1 with 8-oxo-dGTP, GMPPNP and GMPPCP have been determined. Replacement soaking was used in order to ensure that the complexes were isomorphous to one another. Analysis of the structural data led to the elucidation of a relationship between the arrangements of molecules observed in the crystals, molecular plasticity and the action of the enzyme on nucleotides. The dominant mode of arrangement involving a head-to-tail sequence predominantly leads to the generation of NDPs. The other mode of packing arrangement appears to preferentially generate NMPs. This work also provides interesting insights into the dependence of enzyme action on the conformation of the ligand. The possibility of modulating the enzyme action through differences in intermolecular interactions and ligand conformations makes MsMutT1 a versatile enzyme.
Topics: Bacterial Proteins; Crystallography, X-Ray; Deoxyguanine Nucleotides; Ligands; Models, Molecular; Mycobacterium smegmatis; Protein Domains; Pyrophosphatases; Substrate Specificity; Nudix Hydrolases
PubMed: 33021500
DOI: 10.1107/S2059798320010992 -
Nature Structural & Molecular Biology Dec 2016Changes in telomere length are associated with degenerative diseases and cancer. Oxidative stress and DNA damage have been linked to both positive and negative...
Changes in telomere length are associated with degenerative diseases and cancer. Oxidative stress and DNA damage have been linked to both positive and negative alterations in telomere length and integrity. Here we examined how the common oxidative lesion 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxoG) regulates telomere elongation by human telomerase. When 8-oxoG is present in the dNTP pool as 8-oxodGTP, telomerase utilization of the oxidized nucleotide during telomere extension is mutagenic and terminates further elongation. Depletion of MTH1, the enzyme that removes oxidized dNTPs, increases telomere dysfunction and cell death in telomerase-positive cancer cells with shortened telomeres. In contrast, a preexisting 8-oxoG within the telomeric DNA sequence promotes telomerase activity by destabilizing the G-quadruplex DNA structure. We show that the mechanism by which 8-oxoG arises in telomeres, either by insertion of oxidized nucleotides or by direct reaction with free radicals, dictates whether telomerase is inhibited or stimulated and thereby mediates the biological outcome.
Topics: Base Sequence; Cell Death; Cell Line; Cell Line, Tumor; DNA; DNA Adducts; DNA Damage; Deoxyguanine Nucleotides; Enzyme Activation; G-Quadruplexes; Humans; Mutagens; Oxidation-Reduction; Oxidative Stress; Telomerase; Telomere; Telomere Shortening
PubMed: 27820808
DOI: 10.1038/nsmb.3319 -
Biochemistry Feb 2021DNA polymerases play vital roles in the maintenance and replication of genomic DNA by synthesizing new nucleotide polymers using nucleoside triphosphates as substrates....
DNA polymerases play vital roles in the maintenance and replication of genomic DNA by synthesizing new nucleotide polymers using nucleoside triphosphates as substrates. Deoxynucleoside triphosphates (dNTPs) are the canonical substrates for DNA polymerases; however, some bacterial polymerases have been demonstrated to insert deoxynucleoside diphosphates (dNDPs), which lack a third phosphate group, the γ-phosphate. Whether eukaryotic polymerases can efficiently incorporate dNDPs has not been investigated, and much about the chemical or structural role played by the γ-phosphate of dNTPs remains unknown. Using the model mammalian polymerase (Pol) β, we examine how Pol β incorporates a substrate lacking a γ-phosphate [deoxyguanosine diphosphate (dGDP)] utilizing kinetic and crystallographic approaches. Using single-turnover kinetics, we determined dGDP insertion across a templating dC by Pol β to be drastically impaired when compared to dGTP insertion. We found the most significant impairment in the apparent insertion rate (), which was reduced 32000-fold compared to that of dGTP insertion. X-ray crystal structures revealed similar enzyme-substrate contacts for both dGDP and dGTP. These findings suggest the insertion efficiency of dGDP is greatly decreased due to impairments in polymerase chemistry. This work is the first instance of a mammalian polymerase inserting a diphosphate nucleotide and provides insight into the nature of polymerase mechanisms by highlighting how these enzymes have evolved to use triphosphate nucleotide substrates.
Topics: DNA; DNA Polymerase beta; DNA-Directed DNA Polymerase; Deoxyguanine Nucleotides; Deoxyguanosine; Diphosphates; Humans; Kinetics; Substrate Specificity
PubMed: 33475337
DOI: 10.1021/acs.biochem.0c00847 -
Chemistry (Weinheim An Der Bergstrasse,... May 2017The base-pair sequences are the foundation for the biological processes of DNA or RNA, and base-pair mismatch is very important to reveal genetic diseases and DNA...
The base-pair sequences are the foundation for the biological processes of DNA or RNA, and base-pair mismatch is very important to reveal genetic diseases and DNA rearrangements. However, the lack of well-defined structural information about base-pair mismatch is obstructing the investigation of this issue. The challenge is to crystallize the materials containing the base-pair mismatch. Engineering the small-molecule mimics or model is an effective strategy to solve this issue. Here, six cytidine-5'-monophosphate (CMP) and 2'-deoxycytidine-5'-monophosphate (dCMP) coordination polymers were reported containing cytosine-cytosine base-pair mismatch (i-motif), and their single-crystal structures and chiralities were studied. The precise control over the formation of the i-motif was demonstrated, in which the regulating of supramolecular interactions was achieved based on molecular design. In addition, the chiralities of these coordination polymers were investigated according to their crystal structures and solution- and solid-state circular dichroism spectroscopy.
Topics: Base Pair Mismatch; Base Pairing; Coordination Complexes; Crystallography, X-Ray; Cytidine Monophosphate; Cytosine; DNA; Deoxycytidine Monophosphate; Hydrogen Bonding; Models, Molecular; Nucleotides; Stereoisomerism
PubMed: 28370519
DOI: 10.1002/chem.201700930 -
The Journal of Biological Chemistry Nov 2019The building blocks of DNA, dNTPs, can be produced or can be salvaged from deoxyribonucleosides. However, to what extent the absence of dNTP production can be...
The building blocks of DNA, dNTPs, can be produced or can be salvaged from deoxyribonucleosides. However, to what extent the absence of dNTP production can be compensated for by the salvage pathway is unknown. Here, we eliminated dNTP synthesis in the mouse heart and skeletal muscle by inactivating ribonucleotide reductase (RNR), a key enzyme for the production of dNTPs, at embryonic day 13. All other tissues had normal dNTP synthesis and theoretically could supply heart and skeletal muscle with deoxyribonucleosides needed for dNTP production by salvage. We observed that the dNTP and NTP pools in WT postnatal hearts are unexpectedly asymmetric, with unusually high dGTP and GTP levels compared with those in whole mouse embryos or murine cell cultures. We found that RNR inactivation in heart led to strongly decreased dGTP and increased dCTP, dTTP, and dATP pools; aberrant DNA replication; defective expression of muscle-specific proteins; progressive heart abnormalities; disturbance of the cardiac conduction system; and lethality between the second and fourth weeks after birth. We conclude that dNTP salvage cannot substitute for dNTP synthesis in the heart and that cardiomyocytes and myocytes initiate DNA replication despite an inadequate dNTP supply. We discuss the possible reasons for the observed asymmetry in dNTP and NTP pools in WT hearts.
Topics: Animals; DNA Replication; Deoxyribonucleotides; Heart; Mice; Mice, Inbred C57BL; Muscle Proteins; Myocytes, Cardiac; Ribonucleotide Reductases
PubMed: 31300555
DOI: 10.1074/jbc.RA119.009492 -
Molecular Microbiology Apr 2019Biosynthesis of the nucleotide sugar precursor dTDP-L-rhamnose is critical for the viability and virulence of many human pathogenic bacteria, including Streptococcus...
Biosynthesis of the nucleotide sugar precursor dTDP-L-rhamnose is critical for the viability and virulence of many human pathogenic bacteria, including Streptococcus pyogenes (Group A Streptococcus; GAS), Streptococcus mutans and Mycobacterium tuberculosis. Streptococcal pathogens require dTDP-L-rhamnose for the production of structurally similar rhamnose polysaccharides in their cell wall. Via heterologous expression in S. mutans, we confirmed that GAS RmlB and RmlC are critical for dTDP-L-rhamnose biosynthesis through their action as dTDP-glucose-4,6-dehydratase and dTDP-4-keto-6-deoxyglucose-3,5-epimerase enzymes respectively. Complementation with GAS RmlB and RmlC containing specific point mutations corroborated the conservation of previous identified catalytic residues. Bio-layer interferometry was used to identify and confirm inhibitory lead compounds that bind to GAS dTDP-rhamnose biosynthesis enzymes RmlB, RmlC and GacA. One of the identified compounds, Ri03, inhibited growth of GAS, other rhamnose-dependent streptococcal pathogens as well as M. tuberculosis with an IC of 120-410 µM. Importantly, we confirmed that Ri03 inhibited dTDP-L-rhamnose formation in a concentration-dependent manner through a biochemical assay with recombinant rhamnose biosynthesis enzymes. We therefore conclude that inhibitors of dTDP-L-rhamnose biosynthesis, such as Ri03, affect streptococcal and mycobacterial viability and can serve as lead compounds for the development of a new class of antibiotics that targets dTDP-rhamnose biosynthesis in pathogenic bacteria.
Topics: Anti-Bacterial Agents; Biosynthetic Pathways; Hydro-Lyases; Inhibitory Concentration 50; Nucleoside Diphosphate Sugars; Racemases and Epimerases; Streptococcus; Thymine Nucleotides
PubMed: 30600561
DOI: 10.1111/mmi.14197 -
The Journal of Biological Chemistry Apr 2020MutT homologue 1 (MTH1) removes oxidized nucleotides from the nucleotide pool and thereby prevents their incorporation into the genome and thereby reduces genotoxicity....
MutT homologue 1 (MTH1) removes oxidized nucleotides from the nucleotide pool and thereby prevents their incorporation into the genome and thereby reduces genotoxicity. We previously reported that MTH1 is an efficient catalyst of O6-methyl-dGTP hydrolysis suggesting that MTH1 may also sanitize the nucleotide pool from other methylated nucleotides. We here show that MTH1 efficiently catalyzes the hydrolysis of N6-methyl-dATP to N6-methyl-dAMP and further report that N6-methylation of dATP drastically increases the MTH1 activity. We also observed MTH1 activity with N6-methyl-ATP, albeit at a lower level. We show that N6-methyl-dATP is incorporated into DNA , as indicated by increased N6-methyl-dA DNA levels in embryos developed from MTH1 knock-out zebrafish eggs microinjected with N6-methyl-dATP compared with noninjected embryos. N6-methyl-dATP activity is present in MTH1 homologues from distantly related vertebrates, suggesting evolutionary conservation and indicating that this activity is important. Of note, N6-methyl-dATP activity is unique to MTH1 among related NUDIX hydrolases. Moreover, we present the structure of N6-methyl-dAMP-bound human MTH1, revealing that the N6-methyl group is accommodated within a hydrophobic active-site subpocket explaining why N6-methyl-dATP is a good MTH1 substrate. N6-methylation of DNA and RNA has been reported to have epigenetic roles and to affect mRNA metabolism. We propose that MTH1 acts in concert with adenosine deaminase-like protein isoform 1 (ADAL1) to prevent incorporation of N6-methyl-(d)ATP into DNA and RNA. This would hinder potential dysregulation of epigenetic control and RNA metabolism via conversion of N6-methyl-(d)ATP to N6-methyl-(d)AMP, followed by ADAL1-catalyzed deamination producing (d)IMP that can enter the nucleotide salvage pathway.
Topics: Animals; Catalytic Domain; DNA Repair Enzymes; Deoxyadenine Nucleotides; Deoxyribonucleotides; Embryo, Nonmammalian; Evolution, Molecular; Humans; Hydrolysis; Kinetics; Phosphoric Monoester Hydrolases; Pyrophosphatases; Substrate Specificity; Zebrafish; Nudix Hydrolases
PubMed: 32144205
DOI: 10.1074/jbc.RA120.012636 -
Chembiochem : a European Journal of... May 2021The observables associated with protein intrinsic fluorescence - spectra, time decays, anisotropies - offer opportunities to monitor in real time and non-invasively a...
The observables associated with protein intrinsic fluorescence - spectra, time decays, anisotropies - offer opportunities to monitor in real time and non-invasively a protein's functional form and its interchange with other forms with different functions. We employed these observables to sketch the fluorometric profiles of two functional forms of human thymidylate synthase (hTS), a homodimeric enzyme crucial for cell proliferation and thus targeted by anticancer drugs. The protein takes an active and an inactive form. Stabilization of the latter by peptides that, unlike classical hTS inhibitors, bind it at the monomer/monomer interface offers an alternative inhibition mechanism that promises to avoid the onset of drug resistance in anticancer therapy. The fluorescence features depicted herein can be used as tools to identify and quantify each of the two protein forms in solution, thus making it possible to investigate the kinetic and thermodynamic aspects of the active/inactive conformational interchange. Two examples of fluorometrically monitored interconversion kinetics are provided.
Topics: Deoxyuracil Nucleotides; Fluorescence Polarization; Humans; Kinetics; Molecular Dynamics Simulation; Mutagenesis, Site-Directed; Protein Structure, Quaternary; Thymidylate Synthase
PubMed: 33554411
DOI: 10.1002/cbic.202000722 -
Molecular Therapy : the Journal of the... Oct 2022Existing evidence indicates that gut fungal dysbiosis might play a key role in the pathogenesis of colorectal cancer (CRC). We sought to explore whether reversing the...
Existing evidence indicates that gut fungal dysbiosis might play a key role in the pathogenesis of colorectal cancer (CRC). We sought to explore whether reversing the fungal dysbiosis by terbinafine, an approved antifungal drug, might inhibit the development of CRC. A population-based study from Sweden identified a total of 185 patients who received terbinafine after their CRC diagnosis and found that they had a decreased risk of death (hazard ratio = 0.50) and metastasis (hazard ratio = 0.44) compared with patients without terbinafine administration. In multiple mouse models of CRC, administration of terbinafine decreased the fungal load, the fungus-induced myeloid-derived suppressor cell (MDSC) expansion, and the tumor burden. Fecal microbiota transplantation from mice without terbinafine treatment reversed MDSC infiltration and partially restored tumor proliferation. Mechanistically, terbinafine directly impaired tumor cell proliferation by reducing the ratio of nicotinamide adenine dinucleotide phosphate (NADP) to reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), suppressing the activity of glucose-6-phosphate dehydrogenase (G6PD), resulting in nucleotide synthesis disruption, deoxyribonucleotide (dNTP) starvation, and cell-cycle arrest. Collectively, terbinafine can inhibit CRC by reversing fungal dysbiosis, suppressing tumor cell proliferation, inhibiting fungus-induced MDSC infiltration, and restoring antitumor immune response.
Topics: Animals; Antifungal Agents; Colorectal Neoplasms; Deoxyribonucleotides; Dysbiosis; Glucosephosphate Dehydrogenase; Mice; NADP; Terbinafine
PubMed: 35765243
DOI: 10.1016/j.ymthe.2022.06.015