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Applied and Environmental Microbiology May 2015Analysis of the genome of Bacillus halodurans strain C125 indicated that two pathways leading from a cytosine deoxyribonucleotide to dUMP, used for dTMP synthesis, were...
Analysis of the genome of Bacillus halodurans strain C125 indicated that two pathways leading from a cytosine deoxyribonucleotide to dUMP, used for dTMP synthesis, were encoded by the genome of the bacterium. The genes that were responsible, the comEB gene and the dcdB gene, encoding dCMP deaminase and the bifunctional dCTP deaminase:dUTPase (DCD:DUT), respectively, were both shown to be expressed in B. halodurans, and both genes were subject to repression by the nucleosides thymidine and deoxycytidine. The latter nucleoside presumably exerts its repression after deamination by cytidine deaminase. Both comEB and dcdB were cloned, overexpressed in Escherichia coli, and purified to homogeneity. Both enzymes were active and displayed the expected regulatory properties: activation by dCTP for dCMP deaminase and dTTP inhibition for both enzymes. Structurally, the B. halodurans enzyme resembled the Mycobacterium tuberculosis enzyme the most. An investigation of sequenced genomes from other species of the genus Bacillus revealed that not only the genome of B. halodurans but also the genomes of Bacillus pseudofirmus, Bacillus thuringiensis, Bacillus hemicellulosilyticus, Bacillus marmarensis, Bacillus cereus, and Bacillus megaterium encode both the dCMP deaminase and the DCD:DUT enzymes. In addition, eight dcdB homologs from Bacillus species within the genus for which the whole genome has not yet been sequenced were registered in the NCBI Entrez database.
Topics: Amino Acid Sequence; Bacillus; Bacterial Proteins; Biosynthetic Pathways; Crystallography, X-Ray; Cytosine; DCMP Deaminase; Deoxyribonucleotides; Deoxyuracil Nucleotides; Kinetics; Molecular Sequence Data; Nucleotide Deaminases; Substrate Specificity
PubMed: 25746996
DOI: 10.1128/AEM.00268-15 -
Structure (London, England : 1993) Sep 2012In this issue of Structure, Rymer and colleagues present the first crystal structures of a bacterial DnaG primase with bound substrate NTPs and alarmone inhibitors. A...
In this issue of Structure, Rymer and colleagues present the first crystal structures of a bacterial DnaG primase with bound substrate NTPs and alarmone inhibitors. A thoughtful comparative structural analysis provides important insights into the chemical mechanism of primase.
Topics: Bacterial Proteins; DNA Primase; Deoxyribonucleotides; Guanosine Pentaphosphate; Guanosine Tetraphosphate; Staphylococcus aureus
PubMed: 22958638
DOI: 10.1016/j.str.2012.08.013 -
Biochemistry Dec 2014Ribonucleoside triphosphates (rNTPs) are frequently incorporated during DNA synthesis by replicative DNA polymerases (DNAPs), and once incorporated are not efficiently...
Ribonucleoside triphosphates (rNTPs) are frequently incorporated during DNA synthesis by replicative DNA polymerases (DNAPs), and once incorporated are not efficiently edited by the DNAP exonucleolytic function. We examined the kinetic mechanisms that govern selection of complementary deoxyribonucleoside triphosphates (dNTPs) over complementary rNTPs and that govern the probability of a complementary ribonucleotide at the primer terminus escaping exonucleolytic editing and becoming stably incorporated. We studied the quantitative responses of individual Φ29 DNAP complexes to ribonucleotides using a kinetic framework, based on our prior work, in which transfer of the primer strand from the polymerase to exonuclease site occurs prior to translocation, and translocation precedes dNTP binding. We determined transition rates between the pre-translocation and post-translocation states, between the polymerase and exonuclease sites, and for dNTP or rNTP binding, with single-nucleotide spatial precision and submillisecond temporal resolution, from ionic current time traces recorded when individual DNAP complexes are held atop a nanopore in an electric field. The predominant response to the presence of a ribonucleotide in Φ29 DNAP complexes before and after covalent incorporation is significant destabilization, relative to the presence of a deoxyribonucleotide. This destabilization is manifested in the post-translocation state prior to incorporation as a substantially higher rNTP dissociation rate and manifested in the pre-translocation state after incorporation as rate increases for both primer strand transfer to the exonuclease site and the forward translocation, with the probability of editing not directly increased. In the post-translocation state, the primer terminal 2'-OH group also destabilizes dNTP binding.
Topics: Bacillus Phages; DNA Primers; DNA Replication; DNA-Directed DNA Polymerase; Deoxyribonucleotides; Kinetics; Models, Biological; Models, Molecular; Mutagenesis, Site-Directed; Nanopores; Recombinant Proteins; Viral Proteins
PubMed: 25478721
DOI: 10.1021/bi501216a -
Nucleic Acids Research Aug 2015The substitution of 2'-fluoro for 2'-hydroxyl moieties in RNA substantially improves the stability of RNA. RNA stability is a major issue in RNA research and...
The substitution of 2'-fluoro for 2'-hydroxyl moieties in RNA substantially improves the stability of RNA. RNA stability is a major issue in RNA research and applications involving RNA. We report that the RNA polymerase from the marine cyanophage Syn5 has an intrinsic low discrimination against the incorporation of 2'-fluoro dNMPs during transcription elongation. The presence of both magnesium and manganese ions at high concentrations further reduce this discrimination without decreasing the efficiency of incorporation. We have constructed a Syn5 RNA polymerase in which tyrosine 564 is replaced with phenylalanine (Y564F) that further decreases the discrimination against 2'-fluoro-dNTPs during RNA synthesis. Sequence elements in DNA templates that affect the yield of RNA and incorporation of 2'-fluoro-dNMPs by Syn5 RNA polymerase have been identified.
Topics: DNA-Directed RNA Polymerases; Deoxyribonucleotides; Fluorine; Manganese; Mutation; Podoviridae; RNA; RNA Stability; Transcription Initiation Site; Transcription, Genetic
PubMed: 25897116
DOI: 10.1093/nar/gkv367 -
Biochemistry Nov 2012We carried out free-energy calculations and transient kinetic experiments for the insertion of the right (dC) and wrong (dA) nucleotides by wild-type (WT) and six mutant...
We carried out free-energy calculations and transient kinetic experiments for the insertion of the right (dC) and wrong (dA) nucleotides by wild-type (WT) and six mutant variants of human DNA polymerase β (Pol β). Since the mutated residues in the point mutants, I174S, I260Q, M282L, H285D, E288K, and K289M, were not located in the Pol β catalytic site, we assumed that the WT and its point mutants share the same dianionic phosphorane transition-state structure of the triphosphate moiety of deoxyribonucleotide 5'-triphosphate (dNTP) substrate. On the basis of this assumption, we have formulated a thermodynamic cycle for calculating relative dNTP insertion efficiencies, Ω = (k(pol)/K(D))(mut)/(k(pol)/K(D))(WT) using free-energy perturbation (FEP) and linear interaction energy (LIE) methods. Kinetic studies on five of the mutants have been published previously using different experimental conditions, e.g., primer-template sequences. We have performed a presteady kinetic analysis for the six mutants for comparison with wild-type Pol β using the same conditions, including the same primer/template DNA sequence proximal to the dNTP insertion site used for X-ray crystallographic studies. This consistent set of kinetic and structural data allowed us to eliminate the DNA sequence from the list of factors that can adversely affect calculated Ω values. The calculations using the FEP free energies scaled by 0.5 yielded 0.9 and 1.1 standard deviations from the experimental log Ω values for the insertion of the right and wrong dNTP, respectively. We examined a hybrid FEP/LIE method in which the FEP van der Waals term for the interaction of the mutated amino acid residue with its surrounding environment was replaced by the corresponding van der Waals term calculated using the LIE method, resulting in improved 0.4 and 1.0 standard deviations from the experimental log Ω values. These scaled FEP and FEP/LIE methods were also used to predict log Ω for R283A and R283L Pol β mutants.
Topics: Amino Acid Sequence; Amino Acid Substitution; Base Sequence; Catalysis; DNA Polymerase beta; Deoxyribonucleotides; Humans; Kinetics; Molecular Dynamics Simulation; Point Mutation; Thermodynamics
PubMed: 23013478
DOI: 10.1021/bi300783t -
Nucleic Acids Research Feb 2016The absolute and relative concentrations of the four dNTPs are key determinants of DNA replication fidelity, yet the consequences of altered dNTP pools on replication...
The absolute and relative concentrations of the four dNTPs are key determinants of DNA replication fidelity, yet the consequences of altered dNTP pools on replication fidelity have not previously been investigated on a genome-wide scale. Here, we use deep sequencing to determine the types, rates and locations of uncorrected replication errors that accumulate in the nuclear genome of a mismatch repair-deficient diploid yeast strain with elevated dCTP and dTTP concentrations. These imbalanced dNTP pools promote replication errors in specific DNA sequence motifs suggesting increased misinsertion and increased mismatch extension at the expense of proofreading. Interestingly, substitution rates are similar for leading and lagging strand replication, but are higher in regions replicated late in S phase. Remarkably, the rate of single base deletions is preferentially increased in coding sequences and in short rather than long mononucleotides runs. Based on DNA sequence motifs, we propose two distinct mechanisms for generating single base deletions in vivo. Collectively, the results indicate that elevated dCTP and dTTP pools increase mismatch formation and decrease error correction across the nuclear genome, and most strongly increases mutation rates in coding and late replicating sequences.
Topics: Base Sequence; Cell Cycle; DNA Mismatch Repair; DNA Replication; Deoxyribonucleotides; Genome, Fungal; High-Throughput Nucleotide Sequencing; Mutagenesis; Mutation; Mutation Rate; Saccharomyces cerevisiae
PubMed: 26609135
DOI: 10.1093/nar/gkv1298 -
The Journal of Biological Chemistry Aug 2012Gene 1.7 protein is the only known nucleotide kinase encoded by bacteriophage T7. The enzyme phosphorylates dTMP and dGMP to dTDP and dGDP, respectively, in the presence...
Gene 1.7 protein is the only known nucleotide kinase encoded by bacteriophage T7. The enzyme phosphorylates dTMP and dGMP to dTDP and dGDP, respectively, in the presence of a phosphate donor. The phosphate donors are dTTP, dGTP, and ribo-GTP as well as the thymidine and guanosine triphosphate analogs ddTTP, ddGTP, and dITP. The nucleotide kinase is found in solution as a 256-kDa complex consisting of ~12 monomers of the gene 1.7 protein. The two molecular weight forms co-purify as a complex, but each form has nearly identical kinase activity. Although gene 1.7 protein does not require a metal ion for its kinase activity, the presence of Mg(2+) in the reaction mixture results in either inhibition or stimulation of the rate of kinase reactions depending on the substrates used. Both the dTMP and dGMP kinase reactions are reversible. Neither dTDP nor dGDP is a phosphate acceptor of nucleoside triphosphate donors. Gene 1.7 protein exhibits two different equilibrium patterns toward deoxyguanosine and thymidine substrates. The K(m) of 4.4 × 10(-4) M obtained with dTTP for dTMP kinase is ~3-fold higher than that obtained with dGTP for dGMP kinase (1.3 × 10(-4) M), indicating that a higher concentration of dTTP is required to saturate the enzyme. Inhibition studies indicate a competitive relationship between dGDP and both dGTP, dGMP, whereas dTDP appears to have a mixed type of inhibition of dTMP kinase. Studies suggest two functions of dTTP, as a phosphate donor and a positive effector of the dTMP kinase reaction.
Topics: Bacteriophage T7; Deoxyribonucleotides; Magnesium; Phosphotransferases (Alcohol Group Acceptor); Substrate Specificity; Viral Proteins
PubMed: 22761426
DOI: 10.1074/jbc.M112.389619 -
Nucleic Acids Research May 2010Synthetic biology projects aim to produce physical DNA that matches a designed target sequence. Chemically synthesized oligomers are generally used as the starting point...
Synthetic biology projects aim to produce physical DNA that matches a designed target sequence. Chemically synthesized oligomers are generally used as the starting point for building larger and larger sequences. Due to the error rate of chemical synthesis, these oligomers can have many differences from the target sequence. As oligomers are joined together to make larger and larger synthetic intermediates, it becomes essential to perform quality control to eliminate intermediates with errors and retain only those DNA molecules that are error free with respect to the target. This step is often performed by transforming bacteria with synthetic DNA and sequencing colonies until a clone with a perfect sequence is identified. Here we present CloneQC, a lightweight software pipeline available as a free web server and as source code that performs quality control on sequenced clones. Input to the server is a list of desired sequences and forward and reverse reads for each clone. The server generates summary statistics (error rates and success rates target-by-target) and a detailed report of perfect clones. This software will be useful to laboratories conducting in-house DNA synthesis and is available at http://cloneqc.thruhere.net/ and as Berkeley Software Distribution (BSD) licensed source.
Topics: Base Sequence; DNA; Deoxyribonucleotides; Quality Control; Sequence Analysis, DNA; Software
PubMed: 20211841
DOI: 10.1093/nar/gkq093 -
Journal of Bacteriology Oct 1993
Review
Topics: Bacteriophage T4; DNA, Viral; Deoxyribonucleotides; Enzymes; Kinetics; Macromolecular Substances
PubMed: 8407814
DOI: 10.1128/jb.175.20.6377-6381.1993 -
Nucleic Acids Research May 2020Cells maintain a fine-tuned, dynamic concentration balance in the pool of deoxyribonucleoside 5'-triphosphates (dNTPs). This balance is essential for physiological...
Cells maintain a fine-tuned, dynamic concentration balance in the pool of deoxyribonucleoside 5'-triphosphates (dNTPs). This balance is essential for physiological processes including cell cycle control or antiviral defense. Its perturbation results in increased mutation frequencies, replication arrest and may promote cancer development. An easily accessible and relatively high-throughput method would greatly accelerate the exploration of the diversified consequences of dNTP imbalances. The dNTP incorporation based, fluorescent TaqMan-like assay published by Wilson et al. has the aforementioned advantages over mass spectrometry, radioactive or chromatography based dNTP quantification methods. Nevertheless, the assay failed to produce reliable data in several biological samples. Therefore, we applied enzyme kinetics analysis on the fluorescent dNTP incorporation curves and found that the Taq polymerase exhibits a dNTP independent exonuclease activity that decouples signal generation from dNTP incorporation. Furthermore, we found that both polymerization and exonuclease activities are unpredictably inhibited by the sample matrix. To resolve these issues, we established a kinetics based data analysis method which identifies the signal generated by dNTP incorporation. We automated the analysis process in the nucleoTIDY software which enables even the inexperienced user to calculate the final and accurate dNTP amounts in a 96-well-plate setup within minutes.
Topics: Deoxyribonucleotides; Exodeoxyribonucleases; Fluorescence; Kinetics; Software; Taq Polymerase
PubMed: 32103262
DOI: 10.1093/nar/gkaa116