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Ukrains'kyi Biokhimichnyi Zhurnal (1999... 2011Quantitative characteristics of structural flexibility of the DNA elementary monomer units -5'-deoxycytidylic, 5'-thymidylic, 5'-deoxyadenylic and 5'-deoxyguanylic acid...
Quantitative characteristics of structural flexibility of the DNA elementary monomer units -5'-deoxycytidylic, 5'-thymidylic, 5'-deoxyadenylic and 5'-deoxyguanylic acid molecules--have been calculated with original methods. Root-mean-square deviations from equilibrium for all conformational parameters, caused by nuclei thermal or quantum zero-point vibrations, have been found to lie within 4 degrees divided by 25 degrees at 0 K and 7 degrees divided by 50 degrees at 298 K and corresponding relaxed force constants--within 1 divided by 35 kcal/mol x rad(-2). Their values have been found to be sensitive to the molecule's conformation. It has been proven, that the torsion angle gamma is the most rigid one whereas relaxed force constants for all other conformational variables are lower and comparable to each other. The data obtained could serve for development of structural-dynamical models of the DNA.
Topics: Chemistry, Physical; DNA; Deoxyribonucleotides; Models, Molecular; Nucleic Acid Conformation; Pliability; Quantum Theory; Temperature; Thermodynamics
PubMed: 22276428
DOI: No ID Found -
Analytical Chemistry Nov 2019Accurate, traceable quantification of ribonucleotide or deoxyribonucleotide oligomers is achievable using acid hydrolysis and isotope dilution mass spectrometry (ID-MS)....
Accurate, traceable quantification of ribonucleotide or deoxyribonucleotide oligomers is achievable using acid hydrolysis and isotope dilution mass spectrometry (ID-MS). In this work, formic acid hydrolysis is demonstrated to generate stoichiometric release of nucleobases from intact oligonucleotides, which then can be measured by ID-MS, facilitating true and precise absolute quantification of RNA, short linearized DNA, or genomic DNA. Surrogate nucleobases are quantified with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow, using multiple reaction monitoring (MRM). Nucleobases were chromatographically resolved using a novel cation-exchange separation, incorporating a pH gradient. Trueness of this quantitative assay is estimated from agreement among the surrogate nucleobases and by comparison to concentrations provided for commercial materials or Standard Reference Materials (SRMs) from the National Institute of Standards and Technology (NIST). Comparable concentration estimates using NanoDrop spectrophotometry or established from droplet-digital polymerase chain reaction (ddPCR) techniques agree well with the results. Acid hydrolysis-ID-LC-MS/MS provides excellent quantitative selectivity and accuracy while enabling traceability to mass unit. Additionally, this approach can be uniquely useful for quantifying modified nucleobases or mixtures.
Topics: BK Virus; Chromatography, Liquid; DNA, Viral; Deoxyribonucleotides; Formates; Humans; Hydrolysis; RNA; Ribonucleotides; Tandem Mass Spectrometry
PubMed: 31638773
DOI: 10.1021/acs.analchem.9b03625 -
Nucleic Acids Research Oct 1994Eight 3'-modified-dNTPs were synthesized and tested in two different DNA template assays for incorporation activity. From this enzymatic screen, two 3'-O-methyl-dNTPs...
Eight 3'-modified-dNTPs were synthesized and tested in two different DNA template assays for incorporation activity. From this enzymatic screen, two 3'-O-methyl-dNTPs were shown to terminate DNA syntheses mediated by a number of polymerases and may be used as alternative terminators in Sanger sequencing. 3'-O-(2-Nitrobenzyl)-dATP is a UV sensitive nucleotide and was shown to be incorporated by several thermostable DNA polymerases. Base specific termination and efficient photolytic removal of the 3'-protecting group was demonstrated. Following deprotection, DNA synthesis was reinitiated by the incorporation of natural nucleotides into DNA. The identification of this labile terminator and the demonstration of a one cycle stop-start DNA synthesis are initial steps in the development of a novel sequencing strategy.
Topics: Base Sequence; Chromatography, High Pressure Liquid; DNA; DNA-Directed DNA Polymerase; Deoxyribonucleotides; Molecular Sequence Data; Molecular Structure; Templates, Genetic
PubMed: 7937154
DOI: 10.1093/nar/22.20.4259 -
Journal of Molecular Biology Sep 1987The 14-mer oligodeoxynucleotide d(C3GC3GC3GC2) acts as a template to facilitate the cooligomerization of guanosine 5'-phospho-2-methylimidazolide and cytidine...
The 14-mer oligodeoxynucleotide d(C3GC3GC3GC2) acts as a template to facilitate the cooligomerization of guanosine 5'-phospho-2-methylimidazolide and cytidine 5'-phospho-2-methylimidazolide. The predominant products are a series of 3'-5'-linked oligonucleotides, complementary to the template, ranging in length from GGC to GGCGGGCGGGCGGG. Thus simple, non-enzymatic template-directed reactions can result in the accurate transfer of substantial amounts of information from template to products. The 15-mer oligodeoxynucleotide d(C3GC3GC3GC3) is also an efficient template, but directs the synthesis of the same family of products that are formed on the 14-mer template. This unexpected finding is explained by the preferential conversion of the dimer GG to GGC rather than to GGG. These results are interesting in the context of molecular evolution. They suggest that the detailed kinetics of template-directed synthesis could form the basis for the selection of one replicating oligonucleotide from a family of closely related oligonucleotides.
Topics: Base Sequence; Chromatography, High Pressure Liquid; Deoxyribonucleotides; Kinetics; Models, Genetic; Templates, Genetic; Transcription, Genetic
PubMed: 3681994
DOI: 10.1016/0022-2836(87)90117-3 -
Nucleic Acids Research Sep 2017While most DNA polymerases discriminate against ribonucleotide triphosphate (rNTP) incorporation very effectively, the Family X member DNA polymerase μ (Pol μ)...
While most DNA polymerases discriminate against ribonucleotide triphosphate (rNTP) incorporation very effectively, the Family X member DNA polymerase μ (Pol μ) incorporates rNTPs almost as efficiently as deoxyribonucleotides. To gain insight into how this occurs, here we have used X-ray crystallography to describe the structures of pre- and post-catalytic complexes of Pol μ with a ribonucleotide bound at the active site. These structures reveal that Pol μ binds and incorporates a rNTP with normal active site geometry and no distortion of the DNA substrate or nucleotide. Moreover, a comparison of rNTP incorporation kinetics by wildtype and mutant Pol μ indicates that rNTP accommodation involves synergistic interactions with multiple active site residues not found in polymerases with greater discrimination. Together, the results are consistent with the hypothesis that rNTP incorporation by Pol μ is advantageous in gap-filling synthesis during DNA double strand break repair by nonhomologous end joining, particularly in nonreplicating cells containing very low deoxyribonucleotide concentrations.
Topics: Amino Acid Motifs; Base Sequence; Catalytic Domain; Cloning, Molecular; Crystallography, X-Ray; DNA; DNA End-Joining Repair; DNA-Directed DNA Polymerase; Deoxyribonucleotides; Escherichia coli; Gene Expression; Humans; Kinetics; Models, Molecular; Nucleic Acid Conformation; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Recombinant Proteins; Ribonucleotides; Substrate Specificity; Thermodynamics
PubMed: 28911097
DOI: 10.1093/nar/gkx527 -
Chembiochem : a European Journal of... Oct 2015Previous studies of polymerase synthesis of base-modified DNAs and their cleavage by restriction enzymes have mostly related only to 5-substituted pyrimidine and...
Previous studies of polymerase synthesis of base-modified DNAs and their cleavage by restriction enzymes have mostly related only to 5-substituted pyrimidine and 7-substituted 7-deazaadenine nucleotides. Here we report the synthesis of a series of 7-substituted 7-deazaguanine 2'-deoxyribonucleoside 5'-O-triphosphates (dG(R) TPs), their use as substrates for polymerase synthesis of modified DNA and the influence of the modification on their cleavage by type II restriction endonucleases (REs). The dG(R) TPs were generally good substrates for polymerases but the PCR products could not be visualised on agarose gels by intercalator staining, due to fluorescence quenching. The presence of 7-substituted 7-deazaguanine residues in recognition sequences of REs in most cases completely blocked the cleavage.
Topics: DNA; DNA-Directed DNA Polymerase; Deoxyribonucleotides; Guanine
PubMed: 26382079
DOI: 10.1002/cbic.201500315 -
ELife Apr 2023Life requires ribonucleotide reduction for de novo synthesis of deoxyribonucleotides. As ribonucleotide reduction has on occasion been lost in parasites and...
Life requires ribonucleotide reduction for de novo synthesis of deoxyribonucleotides. As ribonucleotide reduction has on occasion been lost in parasites and endosymbionts, which are instead dependent on their host for deoxyribonucleotide synthesis, it should in principle be possible to knock this process out if growth media are supplemented with deoxyribonucleosides. We report the creation of a strain of where all three ribonucleotide reductase operons have been deleted following introduction of a broad spectrum deoxyribonucleoside kinase from Our strain shows slowed but substantial growth in the presence of deoxyribonucleosides. Under limiting deoxyribonucleoside levels, we observe a distinctive filamentous cell morphology, where cells grow but do not appear to divide regularly. Finally, we examined whether our lines can adapt to limited supplies of deoxyribonucleosides, as might occur in the switch from de novo synthesis to dependence on host production during the evolution of parasitism or endosymbiosis. Over the course of an evolution experiment, we observe a 25-fold reduction in the minimum concentration of exogenous deoxyribonucleosides necessary for growth. Genome analysis reveals that several replicate lines carry mutations in and . deoB codes for phosphopentomutase, a key part of the deoxyriboaldolase pathway, which has been hypothesised as an alternative to ribonucleotide reduction for deoxyribonucleotide synthesis. Rather than complementing the loss of ribonucleotide reduction, our experiments reveal that mutations appear that reduce or eliminate the capacity for this pathway to catabolise deoxyribonucleotides, thus preventing their loss via central metabolism. Mutational inactivation of both and is also observed in a number of obligate intracellular bacteria that have lost ribonucleotide reduction. We conclude that our experiments recapitulate key evolutionary steps in the adaptation to life without ribonucleotide reduction.
Topics: Ribonucleotides; Escherichia coli; Symbiosis; Ribonucleotide Reductases; Deoxyribonucleotides; Deoxyribonucleosides
PubMed: 37022136
DOI: 10.7554/eLife.83845 -
Nucleic Acids Research Jun 2014The transmission of genetic information relies on Watson-Crick base pairing between nucleoside phosphates and template bases in template-primer complexes. Enzyme-free...
The transmission of genetic information relies on Watson-Crick base pairing between nucleoside phosphates and template bases in template-primer complexes. Enzyme-free primer extension is the purest form of the transmission process, without any chaperon-like effect of polymerases. This simple form of copying of sequences is intimately linked to the origin of life and provides new opportunities for reading genetic information. Here, we report the dissociation constants for complexes between (deoxy)nucleotides and template-primer complexes, as determined by nuclear magnetic resonance and the inhibitory effect of unactivated nucleotides on enzyme-free primer extension. Depending on the sequence context, Kd's range from 280 mM for thymidine monophosphate binding to a terminal adenine of a hairpin to 2 mM for a deoxyguanosine monophosphate binding in the interior of a sequence with a neighboring strand. Combined with rate constants for the chemical step of extension and hydrolytic inactivation, our quantitative theory explains why some enzyme-free copying reactions are incomplete while others are not. For example, for GMP binding to ribonucleic acid, inhibition is a significant factor in low-yielding reactions, whereas for amino-terminal DNA hydrolysis of monomers is critical. Our results thus provide a quantitative basis for enzyme-free copying.
Topics: DNA; DNA Primers; Deoxyribonucleotides; Templates, Genetic
PubMed: 24875480
DOI: 10.1093/nar/gku314 -
The FEBS Journal Jun 2010A series of RNA-cleaving or DNA-cleaving DNAzymes have been obtained by in vitro selection. However, engineering an allosteric DNAzyme with dual RNA-cleaving and...
A series of RNA-cleaving or DNA-cleaving DNAzymes have been obtained by in vitro selection. However, engineering an allosteric DNAzyme with dual RNA-cleaving and DNA-cleaving activities is very challenging. We used an in vitro-selected pistol-like (PL) DNAzyme as a DNA scaffold for designing a DNAzyme with dual catalytic activities. We prepared the 46-nucleotide DNAzyme with DNA-cleaving activity (PL DNAzyme), and then grafted the deoxyribonucleotide residues from an 8-17 variant DNAzyme into the region of stem-loop I and the catalytic core of the PL DNAzyme scaffold. This deoxyribonucleotide residue grafting resulted in a DNAzyme with dual RNA-cleaving and DNA-cleaving activities (DRc DNAzyme). Drc DNAzyme has properties different from those of the original PL DNAzyme, including DNA cleavage sites and the required metal ion concentration. Interestingly, the RNA substrate and RNase A can act as effectors to mediate the DNA cleavage. Our results show that RNA-cleaving and DNA-cleaving activities simultaneously coexist in DRc DNAzyme, and the DNA cleavage activity can be reversibly regulated by a conformational transition.
Topics: Allosteric Regulation; Base Sequence; Catalytic Domain; DNA; DNA, Catalytic; Deoxyribonucleases; Deoxyribonucleotides; Genetic Variation; Kinetics; Molecular Sequence Data; Nucleic Acid Conformation; RNA; Ribonuclease, Pancreatic
PubMed: 20553490
DOI: 10.1111/j.1742-4658.2010.07669.x -
The Journal of Biological Chemistry Sep 2004Nucleic acid polymerases have evolved elaborate mechanisms that prevent incorporation of the non-cognate substrates, which are distinguished by both the base and the...
Nucleic acid polymerases have evolved elaborate mechanisms that prevent incorporation of the non-cognate substrates, which are distinguished by both the base and the sugar moieties. While the mechanisms of substrate selection have been studied in single-subunit DNA and RNA polymerases (DNAPs and RNAPs, respectively), the determinants of substrate binding in the multisubunit RNAPs are not yet known. Molecular modeling of Thermus thermophilus RNAP-substrate NTP complex identified a conserved beta' subunit Asn(737) residue in the active site that could play an essential role in selection of the substrate ribose. We utilized the Escherichia coli RNAP model system to assess this prediction. Functional in vitro analysis demonstrates that the substitutions of the corresponding beta' Asn(458) residue lead to the loss of discrimination between ribo- and deoxyribonucleotide substrates as well as to defects in RNA chain extension. Thus, in contrast to the mechanism utilized by the single-subunit T7 RNAP where substrate selection commences in the inactive pre-insertion site prior to its delivery to the catalytic center, the bacterial RNAPs likely recognize the sugar moiety in the active (insertion) site.
Topics: Asparagine; Binding Sites; Carbohydrates; Catalytic Domain; DNA-Directed RNA Polymerases; Deoxyribonucleotides; Escherichia coli; Models, Molecular; Plasmids; RNA, Messenger; Substrate Specificity; Thermus thermophilus; Transcription, Genetic; Viral Proteins
PubMed: 15262972
DOI: 10.1074/jbc.C400316200