Canonical Nucleotides Research Articles

Synthesis of barbituric acid containing nucleotides and their implications for the origin of primitive informational polymers.

Given that all processes in modern biology are encoded and orchestrated by polymers, the origin of informational molecules had to be a crucial and significant step in the origin of life on Earth. An important molecule in this context is RNA that is thought to have allowed the transition from chemistry to biology. However, the RNA molecule is comprised of intramolecular bonds which are prone to hydrolysis, especially so under the harsh conditions of the early Earth. Furthermore, the formation of nucleotides with extant bases and their subsequent polymerization have both been problematic, to say the least. Alternate heterocycles, in contrast, have resulted in nucleosides in higher yields, suggesting a viable and prebiotically relevant solution to the longstanding "nucleoside problem". In the present study, we have synthesized a nucleotide using ribose 5'-monophosphate (rMP) and barbituric acid (BA), as the base analog, using dry-heating conditions that are thought to be prevalent in several regimes of the early Earth. Polymerization of the resultant monomers, i.e. BA-nucleotides, was also observed when dehydration-rehydration cycles were carried out at low pH and high temperature. The resulting RNA-like oligomers have intact bases unlike in reactions that were carried out with canonical nucleotides, which resulted in abasic sites under acidic conditions due to cleavage of the N-glycosidic linkages. Furthermore, the incorporation of BA directly into preformed sugar-phosphate backbones was also observed when rMP oligomers were subjected to heating with BA. The results from our aforementioned experiments provide preliminary evidence that BA could have been a putative precursor of modern nucleobases, which could have been incorporated into primitive informational polymers that predated the molecules of an RNA world. Moreover, they also highlight that the prebiotic soup, which would have been replete with alternate heterocycles, could have allowed the sampling of other such heterocycles, which would have had a selective advantage under pertinent selection pressures. Importantly, these kinds of processes have implications for shaping the prebiotic landscape that allowed for the emergence of primitive informational polymers of the pre-RNA world(s), prior to the emergence of a putative RNA world.

Exogenous adenosine 5′-phosphoramidate behaves as a signal molecule in plants; it augments metabolism of phenylpropanoids and salicylic acid in Arabidopsis thaliana seedlings

Cells contain various congeners of the canonical nucleotides. Some of these accumulate in cells under stress and may function as signal molecules. Their cellular levels are enzymatically controlled. Previously, we demonstrated a signaling function for diadenosine polyphosphates and cyclic nucleotides in Arabidopsis thaliana and grape, Vitis vinifera. These compounds increased the expression of genes for and the specific activity of enzymes of phenylpropanoid pathways resulting in the accumulation of certain products of these pathways. Here, we show that adenosine 5′-phosphoramidate, whose level can be controlled by HIT-family proteins, induced similar effects. This natural nucleotide, when added to A. thaliana seedlings, activated the genes for phenylalanine:ammonia lyase, 4-coumarate:coenzyme A ligase, cinnamate-4-hydroxylase, chalcone synthase, cinnamoyl-coenzyme A:NADP oxidoreductase and isochorismate synthase, which encode proteins catalyzing key reactions of phenylpropanoid pathways, and caused accumulation of lignins, anthocyanins and salicylic acid. Adenosine 5′-phosphofluoridate, a synthetic congener of adenosine 5′-phosphoramidate, behaved similarly. The results allow us to postulate that adenosine 5′-phosphoramidate should be considered as a novel signaling molecule.

Nanopore Sequencing of “Alien” DNA Bases

Recently, it was shown for the first time that Escherichia coli can efficiently propagate a genetic alphabet expanded from the four canonical DNA nucleotides to include the synthetic “alien” bases d5SICS and dNaM. Traditional sequencing platforms cannot detect these alien bases. We tested whether nanopore sequencing with the protein pore Mycobacterium smegmatis porin A (MspA) is sensitive to d5SICS and dNaM. In nanopore sequencing, a nanometer scale pore provides the only electrical connection between two electrolyte solutions. An applied voltage causes a current to flow through the pore. Negatively-charged single stranded DNA is drawn through the pore, causing a nucleotide specific reduction in the measured current. Thus-far, MspA is the only nanopore shown to demonstrate single-nucleotide sensitivity to standard nucleotides as well as methyl cytosine and hydroxymethyl cytosine. We use the φ29 DNA polymerase to regulate DNA motion to single-nucleotide steps. Here, we demonstrate the direct detection d5SICS and dNaM with MspA-enabled nanopore sequencing.

From canonical to non-canonical cyclic nucleotides as second messengers: pharmacological implications.

This review summarizes our knowledge on the non-canonical cyclic nucleotides cCMP, cUMP, cIMP, cXMP and cTMP. We place the field into a historic context and discuss unresolved questions and future directions of research. We discuss the implications of non-canonical cyclic nucleotides for experimental and clinical pharmacology, focusing on bacterial infections, cardiovascular and neuropsychiatric disorders and reproduction medicine. The canonical cyclic purine nucleotides cAMP and cGMP fulfill the criteria of second messengers. (i) cAMP and cGMP are synthesized by specific generators, i.e. adenylyl and guanylyl cyclases, respectively. (ii) cAMP and cGMP activate specific effector proteins, e.g. protein kinases. (iii) cAMP and cGMP exert specific biological effects. (iv) The biological effects of cAMP and cGMP are terminated by phosphodiesterases and export. The effects of cAMP and cGMP are mimicked by (v) membrane-permeable cyclic nucleotide analogs and (vi) bacterial toxins. For decades, the existence and relevance of cCMP and cUMP have been controversial. Modern mass-spectrometric methods have unequivocally demonstrated the existence of cCMP and cUMP in mammalian cells. For both, cCMP and cUMP, the criteria for second messenger molecules are now fulfilled as well. There are specific patterns by which nucleotidyl cyclases generate cNMPs and how they are degraded and exported, resulting in unique cNMP signatures in biological systems. cNMP signaling systems, specifically at the level of soluble guanylyl cyclase, soluble adenylyl cyclase and ExoY from Pseudomonas aeruginosa are more promiscuous than previously appreciated. cUMP and cCMP are evolutionary new molecules, probably reflecting an adaption to signaling requirements in higher organisms.

Post-transcriptional modifications are very important after all

In the beginning there was RNA and our lives as molecular biologists and biochemists proceeded simply. All one needed to know, to explain all things nucleic acid, were 4 canonical nucleotides: G, A...

Mechanisms of targeted frameshift mutations: Insertions arising during error-prone or SOS synthesis of DNA containing cis-syn cyclobutane thymine dimers

It is still unclear how frameshift mutations arise at cyclobutane pyrimidine dimers. The polymerase model is commonly used to explain the mechanisms of various mutations. An alternative polymerase-tautomer model was developed for UV-induced mutagenesis. A mechanism was proposed for targeted insertions caused by cis-syn cyclobutane thymine dimers. Targeted insertions are frameshift mutations due to addition of one or more nucleotides in a DNA sequence opposite to a lesion capable of stopping DNA synthesis. Among other factors, cyclobutane pyrimidine dimers can cause targeted insertions. UV irradiation can change the tautomeric form of DNA bases. Five rare tautomeric forms are possible for thymine, and they are stable when the thymine is a component of a cyclobutane dimer. A structural analysis showed that none of the canonical nucleotides can be added opposite to a specific rare thymine tautomer so that hydrogen bonds form between the two bases. A single nucleotide gap is consequently left in the corresponding site of the nascent strand when a specialized or modified DNA polymerase drives SOS or error-prone DNA synthesis on a template containing cis-syn cyclobutane thymine dimers with a base occurring in the rare tautomeric form. If the DNA composition is homogenous within the region, the end of the growing DNA strand may slip to form a complementary pair with the nucleotide adjacent to the dimer according to the Streisinger model, thus producing a loop. A targeted insertion is thereby generated to make the daughter strand longer. Targeted insertions were for the first time assumed to result from the cis-syn cyclobutane thymine dimers wherein one or both of the bases occur in the specific tautomeric form that does not allow the addition and hydrogen bonding of any canonical nucleotide in the opposite position. A model was developed to explain how targeted insertions of one or more nucleotides are caused by cis-syn cyclobutane thymine dimers. Thus, the polymerase-tautomer model can explain the nature and formation of targeted frameshift mutations in addition to hot and cold spots or targeted or untargeted nucleotide substitutions.

Biochemical and Structural Studies of Conserved Maf Proteins Revealed Nucleotide Pyrophosphatases with a Preference for Modified Nucleotides

SummaryMaf (for multicopy associated filamentation) proteins represent a large family of conserved proteins implicated in cell division arrest but whose biochemical activity remains unknown. Here, we show that the prokaryotic and eukaryotic Maf proteins exhibit nucleotide pyrophosphatase activity against 5-methyl-UTP, pseudo-UTP, 5-methyl-CTP, and 7-methyl-GTP, which represent the most abundant modified bases in all organisms, as well as against canonical nucleotides dTTP, UTP, and CTP. Overexpression of the Maf protein YhdE in E. coli cells increased intracellular levels of dTMP and UMP, confirming that dTTP and UTP are the in vivo substrates of this protein. Crystal structures and site-directed mutagenesis of Maf proteins revealed the determinants of their activity and substrate specificity. Thus, pyrophosphatase activity of Maf proteins toward canonical and modified nucleotides might provide the molecular mechanism for a dual role of these proteins in cell division arrest and house cleaning.

The Role of Soluble 5'.-Nucleotidases in the Conversion of Nucleotide Analogs: Metabolic and Therapeutic Aspects

A variety of anti-proliferative drugs is based on the structure of purine or pyrimidine nucleosides. These compounds, after phosphorylation, act as analogs of natural nucleotides. In vivo they are recognized by enzymes that transform them either into anti-metabolites targeted to the synthesis of DNA or RNA, or to inactive products of detoxification. 5'-Nucleotidases of different specificity and cellular localization can either remove the phosphate residue from the 5'- position of (deoxy) nucleotide or transfer it from nucleoside monophosphate onto other nucleosides. Such a nucleoside phosphotransferase activity also works with analogs of canonical nucleotides and nucleosides. The majority of nucleoside analogs is metabolized by intracellular cytoplasmic or mitochondrial 5'-nucleotidases and only few reactions proceed on the cell surface. This review summarizes our knowledge of cytoplasmic and mitochondrial forms of 5'-nucleotidases and focuses on their ability to dephosphorylate different analogs of canonical nucleoside 5'-monophosphates. The involvement of 5'-nucleotidases in the phosphotransfer reaction with some nucleoside analogs has been also presented. The importance of the reactions catalyzed by 5'-nucleotidases in clinical resistance to nucleoside-based drugs used in the treatment of cancer or viral diseases, as well as in activation of pro-drugs has been highlighted.

Electrical Current Signatures of DNA Base Modifications in Single Molecules Immobilized in the α-Hemolysin Ion Channel.

Nanopore technology holds high potential for next-generation DNA sequencing. This method operates by drawing an individual single-stranded DNA molecule through a nanoscale pore while monitoring the current deflections that occur as the DNA passes through. Individual current levels for the four DNA nucleotides have been established by immobilization of an end biotinylated strand in the pore in which the nucleotide of interest is suspended at the most sensitive region of the ion channel. Due to the inherent reactivity of the DNA bases, many modified nucleotides in the genome exist resulting from oxidative and UV insults, among others. Herein, the current levels for the common DNA damages 8-oxo-7,8-dihydroguanine (OG), spiroiminodihydantoin (Sp), guanidinohydantoin (Gh), uridine (U), abasic sites (AP), thymine dimers (T=T), thymine glycol (Tg) and 5-iodocytosine (5-I-C) were assessed via immobilization experiments. In some cases, the current difference between the damaged and canonical nucleotides was not well resolved; therefore, we took advantage of the chemical reactivity of the new functional groups present to make amine adducts that shifted the current levels outside the range of the native nucleotides. Among adducts studied, only the 2-aminomethyl-18-crown-6 adduct was able to give a large current shift in the immobilization experiment, as well as to be observed in a translocation experiment. The results show potential in providing current level modulators for identification of some types of DNA damage. In principle, any DNA base modification that can be converted chemically or enzymatically to an abasic site could be identified in this way.

Mutations in C8orf37, Encoding a Ciliary Protein, are Associated with Autosomal-Recessive Retinal Dystrophies with Early Macular Involvement

Cone-rod dystrophy (CRD) and retinitis pigmentosa (RP) are clinically and genetically overlapping heterogeneous retinal dystrophies. By using homozygosity mapping in an individual with autosomal-recessive (ar) RP from a consanguineous family, we identified three sizeable homozygous regions, together encompassing 46 Mb. Next-generation sequencing of all exons, flanking intron sequences, microRNAs, and other highly conserved genomic elements in these three regions revealed a homozygous nonsense mutation (c.497T>A [p.Leu166(∗)]) in C8orf37, located on chromosome 8q22.1. This mutation was not present in 150 ethnically matched control individuals, single-nucleotide polymorphism databases, or the 1000 Genomes database. Immunohistochemical studies revealed C8orf37 localization at the base of the primary cilium of human retinal pigment epithelium cells and at the base of connecting cilia of mouse photoreceptors. C8orf37 sequence analysis of individuals who had retinal dystrophy and carried conspicuously large homozygous regions encompassing C8orf37 revealed a homozygous splice-site mutation (c.156-2A>G) in two siblings of a consanguineous family and homozygous missense mutations (c.529C>T [p.Arg177Trp]; c.545A>G [p.Gln182Arg]) in siblings of two other consanguineous families. The missense mutations affect highly conserved amino acids, and in silico analyses predicted that both variants are probably pathogenic. Clinical assessment revealed CRD in four individuals and RP with early macular involvement in two individuals. The two CRD siblings with the c.156-2A>G mutation also showed unilateral postaxial polydactyly. These results underline the importance of disrupted ciliary processes in the pathogenesis of retinal dystrophies.

Crystal Structure of Escherichia coli MazG, the Regulator of Nutritional Stress Response

MazG is a nucleoside triphosphate pyrophosphohydrolase that hydrolyzes all canonical nucleoside triphosphates. The mazG gene located downstream from the chromosomal mazEF "addiction module," that mediated programmed cell death in Escherichia coli. MazG activity is inhibited by the MazEF complex both in vivo and in vitro. Enzymatic activity of MazG in vivo affects the cellular level of guanosine 3',5'-bispyrophosphate (ppGpp), synthesized by RelA under amino acid starvation. The reduction of ppGpp, caused by MazG, may extend the period of cell survival under nutritional stress. Here we describe the first crystal structure of active MazG from E. coli, which is composed of two similarly folded globular domains in tandem. Among the two putative catalytic domains, only the C-terminal domain has well ordered active sites and exhibits an NTPase activity. The MazG-ATP complex structure and subsequent mutagenesis studies explain the peculiar active site environment accommodating all eight canonical NTPs as substrates. In vivo nutrient starvation experiments show that the C terminus NTPase activity is responsible for the regulation of bacterial cell survival under nutritional stress.

Crystal Structure of Human Inosine Triphosphatase: SUBSTRATE BINDING AND IMPLICATION OF THE INOSINE TRIPHOSPHATASE DEFICIENCY MUTATION P32T

Inosine triphosphatase (ITPA) is a ubiquitous key regulator of cellular non-canonical nucleotide levels. It breaks down inosine and xanthine nucleotides generated by deamination of purine bases. Its enzymatic action prevents accumulation of ITP and reduces the risk of incorporation of potentially mutagenic inosine nucleotides into nucleic acids. Here we describe the crystal structure of human ITPA in complex with its prime substrate ITP, as well as the apoenzyme at 2.8 and 1.1A, respectively. These structures show for the first time the site of substrate and Mg2+ coordination as well as the conformational changes accompanying substrate binding in this class of enzymes. Enzyme substrate interactions induce an extensive closure of the nucleotide binding grove, resulting in tight interactions with the base that explain the high substrate specificity of ITPA for inosine and xanthine over the canonical nucleotides. One of the dimer contact sites is made up by a loop that is involved in coordinating the metal ion in the active site. We predict that the ITPA deficiency mutation P32T leads to a shift of this loop that results in a disturbed affinity for nucleotides and/or a reduced catalytic activity in both monomers of the physiological dimer.

Tuning the Specificity of a Synthetic Receptor Using a Selected Nucleic Acid Receptor

Because of their relative simplicity, synthetic receptors often lack the selectivity observed for biopolymer receptors, such as aptamers. However, aptamer recognition of ligands is limited by the chemistries inherent in the four canonical nucleotides. Here, we report the design and selection of a ternary complex in which the specificity of a bis-boronic acid synthetic host (1) that binds to various carboxylic acids is tuned by a surrounding aptamer. Although, the synthetic receptor alone has higher selectivity for citrate over DL-tartrate, the formation of the aptamer:receptor complex reversed the organic host selectivity to preferentially bind tartrate. The RNA conformation changed upon the introduction of the synthetic host, consistent with an induced-fit mechanism for binding.

Variations of the Mononucleotide and Short Oligonucleotide Distributions in the Genomes of Various Organisms

We calculated the variation coefficients of the mononucleotide and short oligonucleotide distributions in over 1700 long genomic sequences originating from six organisms to demonstrate that the human and Escherichia coli genomic sequences were the least and the most uniform, respectively. The most non-random genomic distributions were exhibited by the four canonical nucleotides, followed by the strong and weak nucleotides, while the distributions of purine or pyrimidine nucleotides and especially the distributions of (A+C) and (G+T) were significantly more uniform even in the human genome. In the human and mouse genomes, the highest coefficients of variation were further observed with the oligonucleotides where CG was combined with the strong nucleotides while its combination with the weak nucleotides significantly decreased the variation which, however, was still very high. High variation was also exhibited by the remaining oligonucleotides composed exclusively of the strong nucleotides or those containing only weak nucleotides. On the other hand, the distributions of oligonucleotides containing similar and especially the same numbers of the strong and weak nucleotides, but no CG or TA dinucleotide, were the most uniform. The information following from the present analysis will be useful not only in the identification of important genomic regions but also in computer simulations of the genomic nucleotide sequences in order to trace and reproduce the pathways of genome evolution.

Major identity determinants for enzymatic formation of ribothymidine and pseudouridine in the TΨ-loop of yeast tRNAs

Almost all transfer RNA molecules sequenced so far contain two universal modified nucleosides at positions 54 and 55, respectively: ribothymidine (T 54) and pseudouridine (Ψ 55). To identify the tRNA elements recognized by tRNA:m 5uridine-54 methyltransferase and tRNA:pseudouridine-55 synthase from the yeast Saccharomyces cerevisiae, a set of 43 yeast tRNA Aspmutants were used. Some variants contained point mutations, while the others included progressive reductions in size down to a tRNA minisubstrate consisting of the TΨ-loop with only one G·C base-pair as stem (9-mer). All substrates (full-sized tRNA Aspand various minihelices) were produced in vitro by T7 transcription and tested using yeast extract (S100) as a source of enzymatic activities and S-adenosyl- l-methionine as a methyl donor. The results indicate that the minimal substrate for enzymatic formation of Ψ 55is a stem/loop structure with only four G·C base-pairs in the stem, while a longer stem is required for efficient T 54formation. None of the conserved nucleotides (G 53, C 56, A 58and C 61) and U 54for Ψ 55 or U 55 for T 54 formation can be replaced by any of the other three canonical nucleotides. Yeast tRNA:m 5uridine-54 methyltransferase additionally requires the presence of a pyrimidine-60 in the loop. Interestingly, in a tRNA Aspvariant in which the TΨ-loop was permuted with the anticodon-loop, the new U 32 and U 33 residues derived from the TΨ-loop were quantitatively converted to T 32and Ψ 33, respectively. Structural mapping of this variant with ethylnitrosourea confirmed that the intrinsic characteristic structure of the TΨ-loop was conserved upon permutation and that the displaced anticodon-loop did not acquire a TΨ-loop structure. These results demonstrate that a local conformation rather than the exact location of the U-U sequence within the tRNA architecture is the important identity determinant for recognition by yeast tRNA:m 5uridine-54 methyltransferase and tRNA:pseudouridine-55 synthase.

Crystal structure of bacteriophage T4 deoxynucleotide kinase with its substrates dGMP and ATP.

NMP kinases catalyse the phosphorylation of the canonical nucleotides to the corresponding diphosphates using ATP as a phosphate donor. Bacteriophage T4 deoxynucleotide kinase (DNK) is the only member of this family of enzymes that recognizes three structurally dissimilar nucleotides: dGMP, dTMP and 5-hydroxymethyl-dCMP while excluding dCMP and dAMP. The crystal structure of DNK with its substrate dGMP has been determined at 2.0 A resolution by single isomorphous replacement. The structure of the ternary complex with dGMP and ATP has been determined at 2.2 A resolution. The polypeptide chain of DNK is folded into two domains of equal size, one of which resembles the mononucleotide binding motif with the glycine-rich P-loop. The second domain, consisting of five alpha-helices, forms the NMP binding pocket. A hinge connection between the domains allows for large movements upon substrate binding which are not restricted by dimerization of the enzyme. The mechanism of active centre formation via domain closure is described. Comparison with other P-loop-containing proteins indicates an induced-fit mode of NTP binding. Protein-substrate interactions observed at the NMP and NTP sites provide the basis for understanding the principles of nucleotide discrimination.

A rapid and efficient method for targeted random mutagenesis

We describe a new rapid method for random introduction of single-nucleotide (nt) substitutions into a small segment of cloned DNA. A DNA fragment containing a sequence to be mutagenized is inserted into a multiple cloning site sequence of a vector plasmid. The plasmid is linearized with two adjacent cuts (generating 5 ' and 3 ' protruding ends) and then synchronously and unidirectionally digested with exonuclease III (Exo III) so that the 3' termini generated are localized within the target region. A non-complementary α-thiophosphate nucleotide is misincorporated into the 3' terminus generated by Exo III. Since the nucleotide analogue is resistant to the 3'–5' exonuclease activity of DNA polymerase I, its misincorporation into the 3' termini is irreversible. Then, the single-stranded region is filled-in with four canonical nucleotides, and the plasmid is recircularized. This procedure was used to mutagenize a specific region of the mpB gene of E. coli. By sequencing 72 randomly selected clones, we found that 27 clones (37.5%) had nucleotide substitutions distributed within the desired region of a 55-nt-long segment of the gene. The procedure is simple and is applicable to any DNA molecule.