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International Journal of Molecular... Jan 2021MTH1 is an enzyme that hydrolyzes 8-oxo-dGTP, which is an oxidatively damaged nucleobase, into 8-oxo-dGMP in nucleotide pools to prevent its mis-incorporation into...
MTH1 is an enzyme that hydrolyzes 8-oxo-dGTP, which is an oxidatively damaged nucleobase, into 8-oxo-dGMP in nucleotide pools to prevent its mis-incorporation into genomic DNA. Selective and potent MTH1-binding molecules have potential as biological tools and drug candidates. We recently developed 8-halogenated 7-deaza-dGTP as an 8-oxo-dGTP mimic and found that it was not hydrolyzed, but inhibited enzyme activity. To further increase MTH1 binding, we herein designed and synthesized 7,8-dihalogenated 7-deaza-dG derivatives. We successfully synthesized multiple derivatives, including substituted nucleosides and nucleotides, using 7-deaza-dG as a starting material. Evaluations of the inhibition of MTH1 activity revealed the strong inhibitory effects on enzyme activity of the 7,8-dihalogenated 7-deaza-dG derivatives, particularly 7,8-dibromo 7-daza-dGTP. Based on the results obtained on kinetic parameters and from computational docking simulating studies, these nucleotide analogs interacted with the active site of MTH1 and competitively inhibited the substrate 8-oxodGTP. Therefore, novel properties of repair enzymes in cells may be elucidated using new compounds.
Topics: Binding Sites; DNA Damage; DNA Repair Enzymes; Deoxyguanine Nucleotides; Drug Design; Enzyme Assays; Halogenation; Humans; Hydrolysis; Kinetics; Molecular Docking Simulation; Molecular Mimicry; Oxidative Stress; Phosphoric Monoester Hydrolases; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Structure-Activity Relationship; Substrate Specificity
PubMed: 33525366
DOI: 10.3390/ijms22031274 -
ACS Chemical Biology Nov 2022Primase-DNA polymerase (PrimPol) is involved in reinitiating DNA synthesis at stalled replication forks. PrimPol also possesses DNA translesion (TLS) activity and...
Primase-DNA polymerase (PrimPol) is involved in reinitiating DNA synthesis at stalled replication forks. PrimPol also possesses DNA translesion (TLS) activity and bypasses several endogenous nonbulky DNA lesions in vitro. Little is known about the TLS activity of PrimPol across bulky carcinogenic adducts. We analyzed the DNA polymerase activity of human PrimPol on DNA templates with seven -dG lesions of different steric bulkiness. In the presence of Mg ions, bulky -isobutyl-dG, -benzyl-dG, -methyl(1-naphthyl)-dG, -methyl(9-anthracenyl)-dG, -methyl(1-pyrenyl)-dG, and -methyl(1,3-dimethoxyanthraquinone)-dG adducts fully blocked PrimPol activity. At the same time, PrimPol incorporated complementary deoxycytidine monophosphate (dCMP) opposite -ethyl-dG with moderate efficiency but did not extend DNA beyond the lesion. We also demonstrated that mutation of the Arg288 residue abrogated dCMP incorporation opposite the lesion in the presence of Mn ions. When Mn replaced Mg, PrimPol carried out DNA synthesis on all DNA templates with -dG adducts in standing start reactions with low efficiency and accuracy, possibly utilizing a lesion "skipping" mechanism. The TLS activity of PrimPol opposite -ethyl-dG but not bulkier adducts was stimulated by accessory proteins, polymerase delta-interacting protein 2 (PolDIP2), and replication protein A (RPA). Molecular dynamics studies demonstrated the absence of stable interactions with deoxycytidine triphosphate (dCTP), large reactions, and C1'-C1' distances for the -isobutyl-dG and -benzyl-dG PrimPol complexes, suggesting that the size of the adduct is a limiting factor for efficient TLS across minor groove adducts by PrimPol.
Topics: Humans; Deoxycytidine Monophosphate; DNA Damage; Deoxyguanosine; DNA Replication; DNA-Directed DNA Polymerase; DNA; DNA Adducts; Nuclear Proteins; DNA Primase; Multifunctional Enzymes
PubMed: 36318733
DOI: 10.1021/acschembio.2c00717 -
Nucleic Acids Research Jul 2018A huge diversity of modified nucleobases is used as a tool for studying DNA and RNA. Due to practical reasons, the most suitable positions for modifications are C5 of...
A huge diversity of modified nucleobases is used as a tool for studying DNA and RNA. Due to practical reasons, the most suitable positions for modifications are C5 of pyrimidines and C7 of purines. Unfortunately, by using these two positions only, one cannot expand a repertoire of modified nucleotides to a maximum. Here, we demonstrate the synthesis and enzymatic incorporation of novel N4-acylated 2'-deoxycytidine nucleotides (dCAcyl). We find that a variety of family A and B DNA polymerases efficiently use dCAcylTPs as substrates. In addition to the formation of complementary CAcyl•G pair, a strong base-pairing between N4-acyl-cytosine and adenine takes place when Taq, Klenow fragment (exo-), Bsm and KOD XL DNA polymerases are used for the primer extension reactions. In contrast, a proofreading phi29 DNA polymerase successfully utilizes dCAcylTPs but is prone to form CAcyl•A base pair under the same conditions. Moreover, we show that terminal deoxynucleotidyl transferase is able to incorporate as many as several hundred N4-acylated-deoxycytidine nucleotides. These data reveal novel N4-acylated deoxycytidine nucleotides as beneficial substrates for the enzymatic synthesis of modified DNA, which can be further applied for specific labelling of DNA fragments, selection of aptamers or photoimmobilization.
Topics: DNA; DNA-Directed DNA Polymerase; Deoxycytosine Nucleotides
PubMed: 29846697
DOI: 10.1093/nar/gky435 -
Angewandte Chemie (International Ed. in... Apr 2016The metabolic conversion of nucleoside analogues into their triphosphates often proceeds insufficiently. Rate-limitations can be at the mono-, but also at the di- and...
The metabolic conversion of nucleoside analogues into their triphosphates often proceeds insufficiently. Rate-limitations can be at the mono-, but also at the di- and triphosphorylation steps. We developed a nucleoside triphosphate (NTP) delivery system (TriPPPro-approach). In this approach, NTPs are masked by two bioreversible units at the γ-phosphate. Using a procedure involving H-phosphonate chemistry, a series of derivatives bearing approved, as well as potentially antivirally active, nucleoside analogues was synthesized. The enzyme-triggered delivery of NTPs was demonstrated by pig liver esterase, in human T-lymphocyte cell extracts and by a polymerase chain reaction using a prodrug of thymidine triphosphate. The TriPPPro-compounds of some HIV-inactive nucleoside analogues showed marked anti-HIV activity. For cellular uptake studies, a fluorescent TriPPPro-compound was prepared that delivered the triphosphorylated metabolite to intact CEM cells.
Topics: Animals; Anti-HIV Agents; CD4-Positive T-Lymphocytes; Cell Line; Cell Membrane Permeability; Esterases; HIV; HIV Infections; Humans; Nucleosides; Polyphosphates; Prodrugs; Swine; Thymine Nucleotides
PubMed: 27008042
DOI: 10.1002/anie.201511808 -
Journal of Molecular Biology Feb 2019PrimPol is the most recently discovered human DNA polymerase/primase and plays an emerging role in nuclear and mitochondrial genomic maintenance. As a member of...
PrimPol is the most recently discovered human DNA polymerase/primase and plays an emerging role in nuclear and mitochondrial genomic maintenance. As a member of archaeo-eukaryotic primase superfamily enzymes, PrimPol possesses DNA polymerase and primase activities that are important for replication fork progression in vitro and in cellulo. The enzymatic activities of PrimPol are critically dependent on the nucleotidyl-transfer reaction to incorporate deoxyribonucleotides successively; however, our knowledge concerning the kinetic mechanism of the reaction remains incomplete. Using enzyme kinetic analyses and computer simulations, we dissected the mechanism by which PrimPol transfers a nucleotide to a primer-template DNA, which comprises DNA binding, conformational transition, nucleotide binding, phosphoester bond formation, and dissociation steps. We obtained the rate constants of the steps by steady-state and pre-steady-state kinetic analyses and simulations. Our data demonstrate that the rate-limiting step of PrimPol-catalyzed DNA elongation depends on the metal cofactor involved. In the presence of Mn, a conformational transition step from non-productive to productive PrimPol:DNA complexes limits the enzymatic turnover, whereas in the presence of Mg, the chemical step becomes rate limiting. As evidenced from our kinetic and simulation data, PrimPol maintains the same kinetic mechanism under either millimolar or physiological micromolar Mn concentration. Our study revealed the underlying mechanism by which PrimPol catalyzes nucleotide incorporation with two common metal cofactors and provides a kinetic basis for further understanding the regulatory mechanism of this functionally diverse primase-polymerase.
Topics: Catalysis; Cations, Divalent; DNA Primase; DNA Primers; DNA Replication; DNA, Catalytic; DNA-Directed DNA Polymerase; Deoxyribonucleotides; Humans; Kinetics; Multifunctional Enzymes
PubMed: 30633872
DOI: 10.1016/j.jmb.2019.01.002 -
Nucleic Acids Research Oct 2019We document the preparation and properties of dimerized pentaphosphate-bridged deoxynucleotides (dicaptides) that contain reactive components of two different...
We document the preparation and properties of dimerized pentaphosphate-bridged deoxynucleotides (dicaptides) that contain reactive components of two different nucleotides simultaneously. Importantly, dicaptides are found to be considerably more stable to hydrolysis than standard dNTPs. Steady-state kinetics studies show that the dimers exhibit reasonably good efficiency with the Klenow fragment of DNA polymerase I, and we identify thermostable enzymes that process them efficiently at high temperature. Experiments show that the dAp5dT dimer successfully acts as a combination of dATP and dTTP in primer extension reactions, and the dGp5dC dimer as a combination of dGTP and dCTP. The two dimers in combination promote successful 4-base primer extension. The final byproduct of the reaction, triphosphate, is shown to be less inhibitory to primer extension than pyrophosphate, the canonical byproduct. Finally, we document PCR amplification of DNA with two dimeric nucleotides, and show that the dimers can promote amplification under extended conditions when PCR with normal dNTPs fails. These dimeric nucleotides represent a novel and simple approach for increasing stability of nucleotides and avoiding inhibition from pyrophosphate.
Topics: DNA; DNA Polymerase I; DNA Replication; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Kinetics; Nucleotides; Temperature
PubMed: 31504784
DOI: 10.1093/nar/gkz741 -
Nature Communications Aug 2021Reactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Non-homologous end-joining (NHEJ) attaches broken chromosomal ends...
Reactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Non-homologous end-joining (NHEJ) attaches broken chromosomal ends together in mammalian cells. Ribonucleotide insertion by DNA polymerase (pol) μ prepares breaks for end-joining and this is required for successful NHEJ in vivo. We previously showed that pol μ lacks discrimination against oxidized dGTP (8-oxo-dGTP), that can lead to mutagenesis, cancer, aging and human disease. Here we reveal the structural basis for proficient oxidized ribonucleotide (8-oxo-rGTP) incorporation during DSB repair by pol μ. Time-lapse crystallography snapshots of structural intermediates during nucleotide insertion along with computational simulations reveal substrate, metal and side chain dynamics, that allow oxidized ribonucleotides to escape polymerase discrimination checkpoints. Abundant nucleotide pools, combined with inefficient sanitization and repair, implicate pol μ mediated oxidized ribonucleotide insertion as an emerging source of widespread persistent mutagenesis and genomic instability.
Topics: Adenine; Calcium; Catalytic Domain; Cytosine; DNA Breaks, Double-Stranded; DNA Repair; DNA-Directed DNA Polymerase; Deoxyguanine Nucleotides; Humans; Kinetics; Manganese; Models, Molecular; Oxidation-Reduction; Ribonucleotides
PubMed: 34417448
DOI: 10.1038/s41467-021-24486-x -
ACS Chemical Biology Nov 2020Fluorescent nucleoside triphosphates are powerful probes of DNA synthesis, but their potential use in living animals has been previously underexplored. Here, we report...
Fluorescent nucleoside triphosphates are powerful probes of DNA synthesis, but their potential use in living animals has been previously underexplored. Here, we report the synthesis and characterization of 7-deaza-(1,2,3-triazole)-2'-deoxyadenosine-5'-triphosphate (dATP) derivatives of tetramethyl rhodamine ("TAMRA-dATP"), cyanine ("Cy3-dATP"), and boron-dipyrromethene ("BODIPY-dATP"). Upon microinjection into live zebrafish embryos, all three compounds were incorporated into the DNA of dividing cells; however, their impact on embryonic toxicity was highly variable, depending on the exact structure of the dye. TAMRA-EdATP exhibited superior characteristics in terms of its high brightness, low toxicity, and rapid incorporation and depletion kinetics in both a vertebrate (zebrafish) and a nematode (). TAMRA-EdATP allows for unprecedented, real-time visualization of DNA replication and chromosome segregation .
Topics: Animals; Boron Compounds; Caenorhabditis elegans; Carbocyanines; DNA; DNA Replication; Deoxyadenine Nucleotides; Fluorescent Dyes; Optical Imaging; Rhodamines; Zebrafish
PubMed: 33108866
DOI: 10.1021/acschembio.0c00654 -
Advances in Gerontology = Uspekhi... 2023In recent years, more and more attention of researchers has been paid to the study of dilated cardiomyopathy (DCMP). The prevalence of this disease in older age groups...
In recent years, more and more attention of researchers has been paid to the study of dilated cardiomyopathy (DCMP). The prevalence of this disease in older age groups is higher than previously thought, and the course of the disease is associated with a worse prognosis and treatment difficulties. Researchers are considering various signaling molecules whose expression changes are associated with myocardial damage and the development of DCMP; evaluation of changes in the expression of melatonin and its receptors in DCMP requires further study. The aim of the study was to study the age-related features of the expression of melatonin and its receptors (MT1, MT2) in the myocardium and their changes depending on the presence of dilated cardiomyopathy. Immunocytochemical and immunohistochemical methods were used to evaluate the expression of melatonin and its MT1, MT2 receptors in myocardial autopsy material and cardiomyocyte cultures of people of different ages with and without cardiovascular pathology. The study revealed age-associated changes in the form of a decrease in the expression of melatonin and its MT1 and MT2 receptors in the myocardium. In individuals with DCMP of all age groups, a more significant decrease in expression was noted: melatonin by 1,6-1,7 times in old age and 3,2 times in old age; MT1 by 1,8 and 2 times, respectively; MT2 by 1,4 and 4 times, respectively. The relationship between the decrease in the expression of melatonin and its receptors in myocardial tissues with age and the presence of DCMP was revealed. The data obtained allow us to clarify age-dependent changes in melatonin and its receptors, as well as to assume their important role in the development of DCMP, which requires further study.
Topics: Humans; Aged; Melatonin; Cardiomyopathy, Dilated; Deoxycytidine Monophosphate; Receptor, Melatonin, MT1; Myocardium; Receptor, Melatonin, MT2
PubMed: 38010187
DOI: No ID Found -
Chemistry (Weinheim An Der Bergstrasse,... Nov 2022Nanozymes have attracted wide attention for the unique advantages of low cost, high stability and designability. Molecularly imprinted polymers (MIPs) have demonstrated...
Nanozymes have attracted wide attention for the unique advantages of low cost, high stability and designability. Molecularly imprinted polymers (MIPs) have demonstrated great potential as a new type of nanozymes due to their excellent specificity and high affinity. However, effective approaches for creating molecularly imprinted nanozymes still remain limited. Herein, reverse microemulsion template docking surface imprinting (RMTD-SI) is reported as a new approach for the rational design and engineering of nanozymes with free substrate access for the ligation of ssDNA sequences. As a proof of the principle, octa-deoxyribonucleotide-imprinted nanoparticles were successfully prepared. Using tetradeoxyribonucleotides and octa-deoxyribonucleotide as substrates, the properties, catalytic activity and behavior of the imprinted nanoparticles were thoroughly investigated. The imprinted nanozyme exhibited an enhanced reaction speed (by up to 41-fold) and good sequence selectivity towards substrate tetra-deoxyribonucleotides. More interestingly, due to the open substrate access, the imprinted nanozyme also allowed the ligation of a ssDNA that fully matched with the imprinted cavity and other ssDNA substrates to form longer sequences, but at the price of substrate selectivity. Thus, this study provides not only a new avenue to the rational design and synthesis of molecularly imprinted nanozymes but also new insights of their catalytic behavior.
Topics: Molecular Imprinting; Polymers; Nanoparticles; Deoxyribonucleotides
PubMed: 35924666
DOI: 10.1002/chem.202202052