DNA Building Blocks Research Articles

DNTP pool levels modulate mutator phenotypes of error-prone DNA polymerase ε variants

Mutator phenotypes create genetic diversity that fuels tumor evolution. DNA polymerase (Pol) ε mediates leading strand DNA replication. Proofreading defects in this enzyme drive a number of human malignancies. Here, using budding yeast, we show that mutator variants of Pol ε depend on damage uninducible (Dun)1, an S-phase checkpoint kinase that maintains dNTP levels during a normal cell cycle and up-regulates dNTP synthesis upon checkpoint activation. Deletion of DUN1 (dun1Δ) suppresses the mutator phenotype of pol2-4 (encoding Pol ε proofreading deficiency) and is synthetically lethal with pol2-M644G (encoding altered Pol ε base selectivity). Although pol2-4 cells cycle normally, pol2-M644G cells progress slowly through S-phase. The pol2-M644G cells tolerate deletions of mediator of the replication checkpoint (MRC) 1 (mrc1Δ) and radiation sensitive (Rad) 9 (rad9Δ), which encode mediators of checkpoint responses to replication stress and DNA damage, respectively. The pol2-M644G mutator phenotype is partially suppressed by mrc1Δ but not rad9Δ; neither deletion suppresses the pol2-4 mutator phenotype. Thus, checkpoint activation augments the Dun1 effect on replication fidelity but is not required for it. Deletions of genes encoding key Dun1 targets that negatively regulate dNTP synthesis, suppress the dun1Δ pol2-M644G synthetic lethality and restore the mutator phenotype of pol2-4 in dun1Δ cells. DUN1 pol2-M644G cells have constitutively high dNTP levels, consistent with checkpoint activation. In contrast, pol2-4 and POL2 cells have similar dNTP levels, which decline in the absence of Dun1 and rise in the absence of the negative regulators of dNTP synthesis. Thus, dNTP pool levels correlate with Pol ε mutator severity, suggesting that treatments targeting dNTP pools could modulate mutator phenotypes for therapy.

Development of a wavelength-shifting fluorescent module for the adenosine aptamer using photostable cyanine dyes.

DNA-based aptamers are commonly used recognition elements in biosensors for a range of target molecules. Here, the development of a wavelength-shifting optical module for a DNA-based adenosine-binding aptamer is described. It applies the combination of two photostable cyanine-styryl dyes as covalent modifications. This energy-transfer pair is postsynthetically attached to oligonucleotides via a copper(I)-catalyzed azide–alkyne cycloaddition by two structurally different approaches: 1) as nucleotide modifications at the 2′-position of uridines and 2) as nucleotide substitutions using (S)-amino-1,2-propanediol as acyclic linker between the phosphodiester bridges. Both dyes exhibit a remarkable photostability. A library of DNA aptamers consisting of different combinations of the two dyes in diagonal orientations were evaluated by their emission color contrast as readout. Further optimization led to aptasensors with improved fluorescent readout as compared with previously reported aptasensors. This approach described is synthetically facile using simple propargylated phosphoramidites as DNA building blocks. As such, this approach could be applied for other dyes and other chemical/biological applications.

Targeting the de novo biosynthesis of thymidylate for the development of a PET probe for pancreatic cancer imaging.

The development of cancer-specific probes for imaging by positron emission tomography (PET) is gaining impetus in cancer research and clinical oncology. One of the hallmarks of most cancer cells is incessant DNA replication, which requires the continuous synthesis of nucleotides. Thymidylate synthase (TSase) is unique in this context because it is the only enzyme in humans that is responsible for the de novo biosynthesis of the DNA building block 2'-deoxy-thymidylate (dTMP). TSase catalyzes the reductive methylation of 2'-deoxy-uridylate (dUMP) to dTMP using (R)-N(5),N(10)-methylene-5,6,7,8-tetrahydrofolate (MTHF) as a cofactor. Not surprisingly, several human cancers overexpress TSase, which makes it a common target for chemotherapy (e.g., 5-fluorouracil). We envisioned that [(11)C]-MTHF might be a PET probe that could specifically label cancerous cells. Using stable radiotracer [(14)C]-MTHF, we had initially found increased uptake by breast and colon cancer cell lines. In the current study, we examined the uptake of this radiotracer in human pancreatic cancer cell lines MIAPaCa-2 and PANC-1 and found predominant radiolabeling of cancerous versus normal pancreatic cells. Furthermore, uptake of the radiotracer is dependent on the intracellular level of the folate pool, cell cycle phase, expression of folate receptors on the cell membrane, and cotreatment with the common chemotherapeutic drug methotrexate (MTX, which blocks the biosynthesis of endogenous MTHF). These results point toward [(11)C]-MTHF being used as PET probe with broad specificity and being able to control its signal through MTX co-administration.

The Goldilocks Theory

The Goldilocks Theory

The general base in the thymidylate synthase catalyzed proton abstraction.

The enzyme thymidylate synthase (TSase), an important chemotherapeutic drug target, catalyzes the formation of 2'-deoxythymidine-5'-monophosphate (dTMP), a precursor of one of the DNA building blocks. TSase catalyzes a multi-step mechanism that includes the abstraction of a proton from the C5 of the substrate 2'-deoxyuridine-5'-monophosphate (dUMP). Previous studies on ecTSase proposed that an active-site residue, Y94 serves the role of the general base abstracting this proton. However, since Y94 is neither very basic, nor connected to basic residues, nor located close enough to the pyrimidine proton to be abstracted, the actual identity of this base remains enigmatic. Based on crystal structures, an alternative hypothesis is that the nearest potential proton-acceptor of C5 of dUMP is a water molecule that is part of a hydrogen bond (H-bond) network comprised of several water molecules and several protein residues including H147, E58, N177, and Y94. Here, we examine the role of the residue Y94 in the proton abstraction step by removing its hydroxyl group (Y94F mutant). We investigated the effect of the mutation on the temperature dependence of intrinsic kinetic isotope effects (KIEs) and found that these KIEs are more temperature dependent than those of the wild-type enzyme (WT). These results suggest that the phenolic -OH of Y94 is a component of the transition state for the proton abstraction step. The findings further support the hypothesis that no single functional group is the general base, but a network of bases and hydroxyls (from water molecules and tyrosine) sharing H-bonds across the active site can serve the role of the general base to remove the pyrimidine proton.

The Role of Organizational DNA in Improving Organizational Performance: A Study on the Industrial Companies in Egypt

Purpose: This paper attempts to highlight the significant role of organizational DNA in improving Organizational Performance (OP). Research Design/Methodology: Using Booz Allen Hamilton, 2002; Neilson, et al., 2003; 2004; Booz, 2004; Neilson, et al., 2005; Holoday, 2005; Remecker & Bowdin, 2005; Neilson, 2006; Vanmullem & Hondeghem, 2007; Soroush, et al., 2013 of organizational DNA, the study develops a number of hypotheses and tests them. This research is an applied form in terms of its goals and descriptive in terms of the method of data collection. Three groups of employees at industrial companies were examined. Of the 372 questionnaires that were distributed, 300 usable questionnaires were returned, a response rate of 81%. Findings: This study reveals that the four building blocks of organizational DNA (organizational structure, decision rights, motivators, and information) have a significantly direct effect on OP. Practical implications: The study suggests that the industrial companies can improve OP by influencing its organizational DNA, specifically, by developing the organizational structure, decision rights, motivators, and information. The study provided a set of recommendations including the necessity to pay more attention to the dimensions of organizational DNA as of a key source for organizations to enhance the competitive advantage which is of prime significance for OP. Originality/value: The study observes that there is a critical shortage of studying organizational DNA in Egypt and that a greater understanding of the factors that influence the OP, including organizational structure, decision rights, motivators, and information, is of great importance. Therefore, this study is to examine the relationship between organizational DNA and OP among employees in industrial companies in Egypt.

Perspectives actuelles dans la microdélétion 22q11.2 : prise en charge du phénotype neurocomportemental

The 22q11.2 deletion syndrome (22q11.2DS) is caused by hemizygous microdeletions on chromosome 22. 22q11.2DS has several presentations including Di George's syndrome, velo-cardio-facial syndrome or Shprintzen's syndrome and it is the most frequent microdeletion syndrome in the general population (prevalence estimated at 1/4000 births, de novo: 90%). The inheritance of the syndrome (10%) is autosomal dominant. Most people with 22q11.2DS are missing a sequence of about 3 million DNA building blocks (base pairs) on one copy of chromosome 22 in each cell. A small percentage of affected individuals have shorter deletions in the same region (contiguous gene deletion syndrome). The general features of 22q11.2DS vary widely (more than 180 phenotypic presentations) and the syndrome is under diagnosed. Characteristic symptoms may include congenital heart disease, defects in the palate, neuromuscular problems, velo-pharyngeal insufficiency, hypoparathyroidism, craniofacial features and problems with the immune system T-cell mediated response (caused by hypoplasia of the thymus). The neurocognitive phenotype of the 22q11.2DS is complex. Cognitive deficits are seen in the majority (80-100%) of individuals with 22q11DS with impairments in sustained attention, executive function, memory and visual-spatial perception. Borderline intellectual function (IQ: 70-75) is most common, mild intellectual disability (IQ: 55-75) is slightly less frequent and a small percentage of children fall into the low average intelligence range. Most children with 22q11.2DS achieve higher scores in verbal tasks than in non-verbal tasks, although this pattern of dysfunction being not universal. Brain MRI studies have shown volumetric changes in multiple cortical and subcortical regions in individuals with 22q11DS that could be related to both cognition and psychoses. General psychiatric features included anxiety disorders, attention deficit disorder and poor social skills (40-50%). An elevated risk of bipolar disorder and major depression occurs in adolescence and young adulthood. A strong and specific relationship exists between the presence of the 22q11.2 microdeletion and schizophrenia (30-40%). This risk is not associated with any other neurogenetic syndrome. Social cognition is impaired in 22q11.2 DS and this observation is correlated with psychotic features. So, long-term medical care is increasingly being directed towards the treatment and recognition of these symptoms. Required pharmacological treatment strategies have to be adapted to the syndrome. Moreover, cognitive remediation is a promising tool for treating neuro- and social cognitive deficits in 22q11.2DS. However, these new therapeutic strategies have to be developed to improve quality of life.

Absolute differential cross sections for elastic electron scattering from small biomolecules

The results of an experimental investigation of electrons colliding with a set of biomolecules that are assumed to be analogues of the building blocks of DNA (furan, 3- hydroxytetrahydrofuran and pyrimidine) and proteins (formamide, N-methylformamide) are presented. Absolute differential cross sections at medium incident electron energies 40 eV- 300 eV are presented and compared for these different targets. The experimental results are also compared with available calculations, based on the corrected form of independent atom model and show good agreement over the energy range studied.

Nucleic acid chemistry.

Since the discovery of the structure of the DNA double helix in 1953 by Watson and Crick [1], we know that DNA is of critical importance, carrying the genetic information for all living organisms. Only a few years later appeared the first reports on the chemical synthesis of oligonucleotides with a natural 3'-5' phosphodiester linker by Michelsen and Todd [2]. It took another two decades until the first step towards building block chemistry (the so-called phosphoramidte chemistry) was published by Letsinger and Lunsford, enabling efficient and fully synthetic access to oligonucleotides [3]. These authors discovered that DNA building blocks based on phosphorous(III) were significantly more reactive than phosphordiesters or -triesters. Finally, this approach using phosphoramidites as nucleoside building blocks was significantly further developed in 1981 by Beaucage and Caruthers [4]. Since then, oligonucleotides of up to 50-mers in length have become available by an extremely efficient solid-phase methodology that runs automatically on machines, so-called DNA synthesizers. With special care during each synthesis step, even longer oligonucleotides can be prepared in a similar, almost routine fashion. The field of nucleic acid chemistry has exploded: natural and artificial functionalities, as well as probes, markers or other biologically active molecules, can be synthetically introduced into DNA (as well as RNA) by preparing the corresponding artificial DNA and RNA building blocks. More recently, the chemistry was further developed for building blocks that are otherwise synthetically not obtainable. In such cases, postsynthetic strategies can allow for the desired oligonucleotides modification. This becomes an even more important issue for functionalities, probes or biologically relevant molecules that are incompatible with routine phosphoramidite chemistry. Although nucleic acid chemistry appears to be a mature part of organic and bioorganic chemistry, the many questions that are still being raised by research in biology and chemical biology give sufficient motivation to continue to synthesize new nucleic acid probes and thereby further develop nucleic acid chemistry. Moreover, in addition to their biological functionality, DNA and RNA are considered as increasingly important architectures and scaffolds for two- and three-dimensional objects, networks and materials for nanosciences. In conclusion, nucleic acid chemistry continues to maintain its appeal and affords good reason to focus on in this Thematic Series of the Beilstein Journal of Organic Chemistry. Hans-Achim Wagenknecht Karlsruhe, November 2014

Marine nucleosides: structure, bioactivity, synthesis and biosynthesis.

Nucleosides are glycosylamines that structurally form part of nucleotide molecules, the building block of DNA and RNA. Both nucleosides and nucleotides are vital components of all living cells and involved in several key biological processes. Some of these nucleosides have been obtained from a variety of marine resources. Because of the biological importance of these compounds, this review covers 68 marine originated nucleosides and their synthetic analogs published up to June 2014. The review will focus on the structures, bioactivities, synthesis and biosynthetic processes of these compounds.

The case for an early biological origin of DNA.

All life generates deoxyribonucleotides, the building blocks of DNA, via ribonucleotide reductases (RNRs). The complexity of this reaction suggests it did not evolve until well after the advent of templated protein synthesis, which in turn suggests DNA evolved later than both RNA and templated protein synthesis. However, deoxyribonucleotides may have first been synthesised via an alternative, chemically simpler route—the reversal of the deoxyriboaldolase (DERA) step in deoxyribonucleotide salvage. In light of recent work demonstrating that this reaction can drive synthesis of deoxyribonucleosides, we consider what pressures early adoption of this pathway would have placed on cell metabolism. This in turn provides a rationale for the replacement of DERA-dependent DNA production by RNR-dependent production.Electronic supplementary materialThe online version of this article (doi:10.1007/s00239-014-9656-6) contains supplementary material, which is available to authorized users.

Semiquinone-induced Maturation of Bacillus anthracis Ribonucleotide Reductase by a Superoxide Intermediate

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides, and represent the only de novo pathway to provide DNA building blocks. Three different classes of RNR are known, denoted I-III. Class I RNRs are heteromeric proteins built up by α and β subunits and are further divided into different subclasses, partly based on the metal content of the β-subunit. In subclass Ib RNR the β-subunit is denoted NrdF, and harbors a manganese-tyrosyl radical cofactor. The generation of this cofactor is dependent on a flavodoxin-like maturase denoted NrdI, responsible for the formation of an active oxygen species suggested to be either a superoxide or a hydroperoxide. Herein we report on the magnetic properties of the manganese-tyrosyl radical cofactor of Bacillus anthracis NrdF and the redox properties of B. anthracis NrdI. The tyrosyl radical in NrdF is stabilized through its interaction with a ferromagnetically coupled manganese dimer. Moreover, we show through a combination of redox titration and protein electrochemistry that in contrast to hitherto characterized NrdIs, the B. anthracis NrdI is stable in its semiquinone form (NrdIsq) with a difference in electrochemical potential of ∼110 mV between the hydroquinone and semiquinone state. The under anaerobic conditions stable NrdIsq is fully capable of generating the oxidized, tyrosyl radical-containing form of Mn-NrdF when exposed to oxygen. This latter observation strongly supports that a superoxide radical is involved in the maturation mechanism, and contradicts the participation of a peroxide species. Additionally, EPR spectra on whole cells revealed that a significant fraction of NrdI resides in its semiquinone form in vivo, underscoring that NrdIsq is catalytically relevant.

"Photoclick" postsynthetic modification of DNA.

A new DNA building block bearing a push-pull-substituted diaryltetrazole linked to the 5-position of 2'-deoxyuridine through an aminopropynyl group was synthesized. The accordingly modified oligonucleotide allows postsynthetic labeling with a maleimide-modified sulfo-Cy3 dye, N-methylmaleimide, and methylmethacrylate as dipolarophiles by irradiation at 365 nm (LED). The determined rate constant of (23±7) M(-1) s(-1) is remarkably high with respect to other copper-free bioorthogonal reactions and comparable with the copper-catalyzed cycloaddition between azides and acetylenes.

Regulating DNA building blocks

DNA Biochemistry The bulding blocks of DNA, deoxynucleotide triphosphates, are synthesized by the enzyme ribonucleotide reductase (RNR). Its central role in DNA synthesis and its aberrant expression in tumors have made it an important anticancer target, but nucleoside analog inhibitors of RNR can have adverse side effects. Arnaoutov and Dasso identify IRBIT (IP3-receptor-binding protein released with inositol 1,4,5-trisphosphate) as an RNR-binding protein. IRBIT regulates the activity of RNR by stabilizing the inactive form bound to deoxyadenosine triphosphate in the low-affinity A site. In turn, phosophorylation of IRBIT regulates its RNR-binding activity. IRBIT provides a new target for RNR inhibition that might avoid some of the side effects of current drugs. Science , this issue p. [1512][1] [1]: /lookup/doi/10.1126/science.1251550

THE PHOTOCHEMISTRY OF PYRIMIDINE IN REALISTIC ASTROPHYSICAL ICES AND THE PRODUCTION OF NUCLEOBASES

Nucleobases, together with deoxyribose/ribose and phosphoric acid, are the building blocks of DNA and RNA for all known life. The presence of nucleobase-like compounds in carbonaceous chondrites delivered to the Earth raises the question of an extraterrestrial origin for the molecules that triggered life on our planet. Whether these molecules are formed in interstellar/protostellar environments, in small parent bodies in the solar system, or both, is currently unclear. Recent experiments show that the UV irradiation of pyrimidine (C4H4N2) in H2O-rich ice mixtures that contain NH3, CH3OH, or CH4 leads to the formation of the pyrimidine-based nucleobases uracil, cytosine, and thymine. In this work, we discuss the low-temperature UV irradiation of pyrimidine in realistic astrophysical ice mixtures containing H2O, CH3OH, and NH3, with or without CH4, to search for the production of nucleobases and other prebiotic compounds. These experiments show the presence of uracil, urea, glycerol, hexamethylenetetramine, small amino acids, and small carboxylic acids in all samples. Cytosine was only found in one sample produced from ices irradiated with a higher UV dose, while thymine was not found in any sample, even after irradiation with a higher UV dose. Results are discussed to evaluate the role of the photochemistry of pyrimidine in the inventory of organic molecules detected in meteorites and their astrophysical/astrobiological implications.

2'-Deoxyuridine conjugated with a reactive monobenzocyclooctyne as a DNA building block for copper-free click-type postsynthetic modification of DNA.

The carboxymethylmonobenzocyclooctyne group attached to the 5-position of a 2'-deoxyuridine in DNA allows rapid and efficient copper-free postsynthetic modification as demonstrated with a far-red emitting fluorescent azide probe. Upon labeling strong fluorescence intensity enhancement is observed.

Concerted versus stepwise mechanism in thymidylate synthase.

Thymidylate synthase (TSase) catalyzes the intracellular de novo formation of thymidylate (a DNA building block) in most living organisms, making it a common target for chemotherapeutic and antibiotic drugs. Two mechanisms have been proposed for the rate-limiting hydride transfer step in TSase catalysis: a stepwise mechanism in which the hydride transfer precedes the cleavage of the covalent bond between the enzymatic cysteine and the product and a mechanism where both happen concertedly. Striking similarities between the enzyme-bound enolate intermediates formed in the initial and final step of the reaction supported the first mechanism, while QM/MM calculations favored the concerted mechanism. Here, we experimentally test these two possibilities using secondary kinetic isotope effect (KIE), mutagenesis study, and primary KIEs. The findings support the concerted mechanism and demonstrate the critical role of an active site arginine in substrate binding, activation of enzymatic nucleophile, and the hydride transfer studied here. The elucidation of this reduction/substitution sheds light on the critical catalytic step in TSase and may aid future drug or biomimetic catalyst design.

Ribonucleotide reductase and cancer: biological mechanisms and targeted therapies

Accurate DNA replication and repair is essential for proper development, growth and tumor-free survival in all multicellular organisms. A key requirement for the maintenance of genomic integrity is the availability of adequate and balanced pools of deoxyribonucleoside triphosphates (dNTPs), the building blocks of DNA. Notably, dNTP pool alterations lead to genomic instability and have been linked to multiple human diseases, including mitochondrial disorders, susceptibility to viral infection and cancer. In this review, we discuss how a key regulator of dNTP biosynthesis in mammals, the enzyme ribonucleotide reductase (RNR), impacts cancer susceptibility and serves as a target for anti-cancer therapies. Because RNR-regulated dNTP production can influence DNA replication fidelity while also supporting genome-protecting DNA repair, RNR has complex and stage-specific roles in carcinogenesis. Nevertheless, cancer cells are dependent on RNR for de novo dNTP biosynthesis. Therefore, elevated RNR expression is a characteristic of many cancers, and an array of mechanistically distinct RNR inhibitors serve as effective agents for cancer treatment. The dNTP metabolism machinery, including RNR, has been exploited for therapeutic benefit for decades and remains an important target for cancer drug development.

DGTP starvation in Escherichia coli provides new insights into the thymineless-death phenomenon.

Starvation of cells for the DNA building block dTTP is strikingly lethal (thymineless death, TLD), and this effect is observed in all organisms. The phenomenon, discovered some 60 years ago, is widely used to kill cells in anticancer therapies, but many questions regarding the precise underlying mechanisms have remained. Here, we show for the first time that starvation for the DNA precursor dGTP can kill E. coli cells in a manner sharing many features with TLD. dGTP starvation is accomplished by combining up-regulation of a cellular dGTPase with a deficiency of the guanine salvage enzyme guanine-(hypoxanthine)-phosphoribosyltransferase. These cells, when grown in medium without an exogenous purine source like hypoxanthine or adenine, display a specific collapse of the dGTP pool, slow-down of chromosomal replication, the generation of multi-branched nucleoids, induction of the SOS system, and cell death. We conclude that starvation for a single DNA building block is sufficient to bring about cell death.

Building blocks: the child health theme issue.

Blocks may be the ideal toy to represent childhood—alphabet blocks to spell words, wooden blocks to stack into tall towers, or plastic pieces to create grand imaginary structures. As evidence, in 2003, alphabet blocks were inducted into the National Toy Hall of Fame.1 Blocks have a literal role in medicine as part of the kit for the Denver Developmental Screening Test used by pediatricians, but they also have a prominent metaphorical role—amino acids as the building blocks of proteins, fatty acids as the building blocks of lipids, nucleotides as the building blocks of DNA.