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While alarmone nucleotides guanosine-3′,5′-bisdiphosphate (ppGpp) and guanosine-5′-triphosphate-3′-diphosphate (pppGpp) are archetypical bacterial second messengers, their adenosine analogues ppApp (adenosine-3′,5′-bisdiphosphate) and pppApp (adenosine-5′-triphosphate-3′-diphosphate) are toxic effectors that abrogate bacterial growth. The alarmones are both synthesized and degraded by the members of the RelA-SpoT Homologue (RSH) enzyme family. Because of the chemical and enzymatic liability of (p)ppGpp and (p)ppApp, these alarmones are prone to degradation during structural biology experiments. To overcome this limitation, we have established an efficient and straightforward procedure for synthesizing nonhydrolysable (p)ppNuNpp analogues starting from 3′-azido-3′-deoxyribonucleotides as key intermediates. To demonstrate the utility of (p)ppGNpp as a molecular tool, we show that (i) as an HD substrate mimic, ppGNpp competes with ppGpp to inhibit the enzymatic activity of human MESH1 Small Alarmone Hyrolase, SAH; and (ii) mimicking the allosteric effects of (p)ppGpp, (p)ppGNpp acts as a positive regulator of the synthetase activity of long ribosome-associated RSHs Rel and RelA. Finally, by solving the structure of the N-terminal domain region (NTD) of T. thermophilus Rel complexed with pppGNpp, we show that as an HD substrate mimic, the analogue serves as a bona fide orthosteric regulator that promotes the same intra-NTD structural rearrangements as the native substrate., SCOPUS: ar.j, info:eu-repo/semantics/published

SAMHD1 is a fundamental regulator of cellular dNTPs that catalyzes their hydrolysis into 2'-deoxynucleoside and triphosphate, restricting the replication of viruses, including HIV-1, in CD4+ myeloid lineage and resting T-cells. SAMHD1 mutations are associated with the autoimmune disease Aicardi-Goutieres syndrome (AGS) and certain cancers. More recently, SAMHD1 has been linked to anticancer drug resistance and the suppression of the interferon response to cytosolic nucleic acids after DNA damage. Here, we probe dNTP hydrolysis and inhibition of SAMHD1 using the Rp and Sp diastereomers of dNTPαS nucleotides. Our biochemical and enzymological data show that the α-phosphorothioate substitution in Sp-dNTPαS but not Rp-dNTPαS diastereomers prevents Mg2+ ion coordination at both the allosteric and catalytic sites, rendering SAMHD1 unable to form stable, catalytically active homotetramers or hydrolyze substrate dNTPs at the catalytic site. Furthermore, we find that Sp-dNTPαS diastereomers competitively inhibit dNTP hydrolysis, while Rp-dNTPαS nucleotides stabilize tetramerization and are hydrolyzed with similar kinetic parameters to cognate dNTPs. For the first time, we present a cocrystal structure of SAMHD1 with a substrate, Rp-dGTPαS, in which an Fe-Mg-bridging water species is poised for nucleophilic attack on the Pα. We conclude that it is the incompatibility of Mg2+, a hard Lewis acid, and the α-phosphorothioate thiol, a soft Lewis base, that prevents the Sp-dNTPαS nucleotides coordinating in a catalytically productive conformation. On the basis of these data, we present a model for SAMHD1 stereospecific hydrolysis of Rp-dNTPαS nucleotides and for a mode of competitive inhibition by Sp-dNTPαS nucleotides that competes with formation of the enzyme-substrate complex.

Information about the cellular concentrations of deoxyribonucleoside triphosphates (dNTPs) is instrumental for mechanistic studies of DNA replication and for understanding diseases caused by defects in dNTP metabolism. The dNTPs are measured by methods based on either HPLC or DNA polymerization. An advantage with the HPLC-based techniques is that the parallel analysis of ribonucleoside triphosphates (rNTPs) can serve as an internal quality control of nucleotide integrity and extraction efficiency. We have developed a Freon-free trichloroacetic acid-based method to extract cellular nucleotides and an isocratic reverse phase HPLC-based technique that is able to separate dNTPs, rNTPs and ADP in a single run. The ability to measure the ADP levels improves the control of nucleotide integrity, and the use of an isocratic elution overcomes the shifting baseline problems in previously developed gradient-based reversed phase protocols for simultaneously measuring dNTPs and rNTPs. An optional DNA-polymerase-dependent step is used for confirmation that the dNTP peaks do not overlap with other components of the extracts, further increasing the reliability of the analysis. The method is compatible with a wide range of biological samples and has a sensitivity better than other UV-based HPLC protocols, closely matching that of mass spectrometry-based detection.

Nucleotide synthesis is a metabolically demanding process essential for cell division. Several anti-cancer drugs that inhibit nucleotide metabolism induce apoptosis. How inhibition of nucleotide metabolism impacts non-apoptotic cell death is less clear. Here, we report that inhibition of nucleotide metabolism by the p53 pathway is sufficient to suppress the non-apoptotic cell death process of ferroptosis. Mechanistically, stabilization of wild-type p53 and induction of the p53 target gene CDKN1A (p21) leads to decreased expression of the ribonucleotide reductase (RNR) subunits RRM1 and RRM2. RNR is the rate-limiting enzyme of de novo nucleotide synthesis that reduces ribonucleotides to deoxyribonucleotides in a glutathione-dependent manner. Direct inhibition of RNR conserves glutathione which can then be used to limit the accumulation of toxic lipid peroxides, preventing the onset of ferroptosis. These results support a mechanism linking p53-dependent regulation of nucleotide metabolism to non-apoptotic cell death.

Sterile α-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) is an intrinsic antiviral restriction factor known to play a vital role in preventing multiple viral infections and in the control of the cellular deoxynucleoside triphosphate (dNTP) pool. Human and mouse SAMHD1 have both been extensively studied; however, our knowledge on porcine SAMHD1 is limited. Here, we report our findings from comprehensive structural and functional studies on porcine SAMHD1. We determined the crystal structure of porcine SAMHD1 and showed that it forms a symmetric tetramer. Moreover, we modified the deoxynucleotide triphosphohydrolase (dNTPase) activity of SAMHD1 by site-directed mutagenesis based on the crystal structure, and obtained an artificial dimeric enzyme possessing high dNTPase activity. Taken together, our results define the mechanism underlying dNTP regulation and provide a deeper understanding of the regulation of porcine SAMHD1 functions. Directed modification of key residues based on the protein structure enhances the activity of the enzyme, which will be beneficial in the search for new antiviral strategies and for future translational applications.

Previously, we reported the creation of a semi-synthetic organism (SSO) that stores and retrieves increased information by virtue of stably maintaining an unnatural base pair (UBP) in its DNA, transcribing the corresponding unnatural nucleotides into the codons and anticodons of mRNAs and tRNAs, and then using them to produce proteins containing noncanonical amino acids (ncAAs). Here we report a systematic extension of the effort to optimize the SSO by exploring a variety of deoxy- and ribonucleotide analogues. Importantly, this includes the first in vivo structure-activity relationship (SAR) analysis of unnatural ribonucleoside triphosphates. Similarities and differences between how DNA and RNA polymerases recognize the unnatural nucleotides were observed, and remarkably, we found that a wide variety of unnatural ribonucleotides can be efficiently transcribed into RNA and then productively and selectively paired at the ribosome to mediate the synthesis of proteins with ncAAs. The results extend previous studies, demonstrating that nucleotides bearing no significant structural or functional homology to the natural nucleotides can be efficiently and selectively paired during replication, to include each step of the entire process of information storage and retrieval. From a practical perspective, the results identify the most optimal UBP for replication and transcription, as well as the most optimal unnatural ribonucleoside triphosphates for transcription and translation. The optimized SSO is now, for the first time, able to efficiently produce proteins containing multiple, proximal ncAAs.

Bacteriophage possess a variety of auxiliary metabolic genes (AMGs) of bacterial origin. These proteins enable them to maximise infection efficiency, subverting bacterial metabolic processes for the purpose of viral genome replication and synthesis of the next generation of virion progeny. Here, we examined the enzymatic activity of a cyanophage MazG protein – a putative pyrophosphohydrolase previously implicated in regulation of the stringent response via reducing levels of the central alarmone molecule (p)ppGpp. We demonstrate however, that the purified viral MazG shows no binding or hydrolysis activity against (p)ppGpp. Instead, dGTP and dCTP appear to be the preferred substrates of this protein, consistent with a role preferentially hydrolysing deoxyribonucleotides from the high GC content host Synechococcus genome. This showcases a new example of the fine‐tuned nature of viral metabolic processes.\ud

For oligonucleotide therapeutics, chemical modifications of the sugar-phosphate backbone are frequently used to confer drug-like properties. Because 2′-deoxy-2′-fluoro (2′-F) nucleotides are not known to occur naturally, their safety profile was assessed when used in revusiran and ALN-TTRSC02, two short interfering RNAs (siRNAs), of the same sequence but different chemical modification pattern and metabolic stability, conjugated to an N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Exposure to 2′-F-monomer metabolites was low and transient in rats and humans. In vitro, 2′-F-nucleoside 5′-triphosphates were neither inhibitors nor preferred substrates for human polymerases, and no obligate or non-obligate chain termination was observed. Modest effects on cell viability and mitochondrial DNA were observed in vitro in a subset of cell types at high concentrations of 2′-F-nucleosides, typically not attained in vivo. No apparent functional impact on mitochondria and no significant accumulation of 2′-F-monomers were observed after weekly administration of two GalNAc–siRNA conjugates in rats for ∼2 years. Taken together, the results support the conclusion that 2′-F nucleotides can be safely applied for the design of metabolically stabilized therapeutic GalNAc–siRNAs with favorable potency and prolonged duration of activity allowing for low dose and infrequent dosing.

Site-specific modification of synthetic and cellular RNA such as with specific nucleobases, fluorophores and attachment chemistries is important for a variety of basic and applied research applications. However, simple and efficient methods to modify RNA such as at the 3′ terminus with specific nucleobases or nucleotide analogs conjugated to various chemical moieties are lacking. Here, we develop and characterize a one-step enzymatic method to modify RNA 3′ termini using recombinant human polymerase theta (Polθ). We demonstrate that Polθ efficiently adds 30–50 2′-deoxyribonucleotides to the 3′ terminus of RNA molecules of various lengths and sequences, and extends RNA 3′ termini with an assortment of 2′-deoxy and 2′,3′-dideoxy ribonucleotide analogs containing functional chemistries, such as high affinity attachment moieties and fluorophores. In contrast to Polθ, terminal deoxynucleotidyl transferase (TdT) is unable to use RNA as a substrate altogether. Overall, Polθ shows a strong preference for adding deoxyribonucleotides to RNA, but can also add ribonucleotides with relatively high efficiency in particular sequence contexts. We anticipate that this unique activity of Polθ will become invaluable for applications requiring 3′ terminal modification of RNA and potentially enzymatic synthesis of RNA.

Polθ reverse transcribes RNA by undergoing a significant conformational change and promotes RNA-templated DNA repair., Genome-embedded ribonucleotides arrest replicative DNA polymerases (Pols) and cause DNA breaks. Whether mammalian DNA repair Pols efficiently use template ribonucleotides and promote RNA-templated DNA repair synthesis remains unknown. We find that human Polθ reverse transcribes RNA, similar to retroviral reverse transcriptases (RTs). Polθ exhibits a significantly higher velocity and fidelity of deoxyribonucleotide incorporation on RNA versus DNA. The 3.2-Å crystal structure of Polθ on a DNA/RNA primer-template with bound deoxyribonucleotide reveals that the enzyme undergoes a major structural transformation within the thumb subdomain to accommodate A-form DNA/RNA and forms multiple hydrogen bonds with template ribose 2′-hydroxyl groups like retroviral RTs. Last, we find that Polθ promotes RNA-templated DNA repair in mammalian cells. These findings suggest that Polθ was selected to accommodate template ribonucleotides during DNA repair.

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides, the building blocks of DNA. RNRs are specific for either ribonucleoside diphosphates or triphosphates as substrates. As far as is known, oxygen-dependent class I RNRs (NrdAB) all reduce ribonucleoside diphosphates, and oxygen-sensitive class III RNRs (NrdD) are all ribonucleoside triphosphate reducers, whereas the adenosylcobalamin-dependent class II (NrdJ) contains both ribonucleoside diphosphate and triphosphate reducers. However, it is unknown how this specificity is conveyed by the active site of the enzymes and how this feature developed in RNR evolution. By structural comparison of the active sites in different RNRs, we identified the apical loop of the phosphate-binding site as a potential structural determinant of substrate specificity. Grafting two residues from this loop from a diphosphate- to a triphosphate-specific RNR caused a change in preference from ribonucleoside triphosphate to diphosphate substrates in a class II model enzyme, confirming them as the structural determinants of phosphate specificity. The investigation of the phylogenetic distribution of this motif in class II RNRs yielded a likely monophyletic clade with the diphosphate-defining motif. This indicates a single evolutionary-split event early in NrdJ evolution in which diphosphate specificity developed from the earlier triphosphate specificity. For those interesting cases where organisms contain more than one nrdJ gene, we observed a preference for encoding enzymes with diverse phosphate specificities, suggesting that this varying phosphate specificity confers a selective advantage.

The abiotic synthesis of ribonucleotides is thought to have been an essential step toward the emergence of the RNA world. However, it is likely that the prebiotic synthesis of ribonucleotides was accompanied by the simultaneous synthesis of arabinonucleotides, 2'-deoxyribonucleotides, and other variations on the canonical nucleotides. In order to understand how relatively homogeneous RNA could have emerged from such complex mixtures, we have examined the properties of arabinonucleotides and 2'-deoxyribonucleotides in nonenzymatic template-directed primer extension reactions. We show that nonenzymatic primer extension with activated arabinonucleotides is much less efficient than with activated ribonucleotides, and furthermore that once an arabinonucleotide is incorporated, continued primer extension is strongly inhibited. As previously shown, 2'-deoxyribonucleotides are also less efficiently incorporated in primer extension reactions, but the difference is more modest. Experiments with mixtures of nucleotides suggest that the coexistence of ribo- and arabinonucleotides does not impede the copying of RNA templates. Moreover, chimeric oligoribonucleotides containing 2'-deoxy- or arabinonucleotides are effective templates for RNA synthesis. We propose that the initial genetic polymers were random sequence chimeric oligonucleotides formed by untemplated polymerization, but that template copying chemistry favored RNA synthesis; multiple rounds of replication may have led to pools of oligomers composed mainly of RNA.

Eukaryotic ribonucleotide reductases are iron-dependent enzymes that catalyze the rate-limiting step in the de novo synthesis of deoxyribonucleotides. Multiple mechanisms regulate the activity of ribonucleotide reductases in response to genotoxic stresses and iron deficiency. Upon iron starvation, the Saccharomyces cerevisiae Aft1 transcription factor specifically binds to iron-responsive cis elements within the promoter of a group of genes, known as the iron regulon, activating their transcription. Members of the iron regulon participate in iron acquisition, mobilization and recycling, and trigger a genome-wide metabolic remodeling of iron-dependent pathways. Here, we describe a mechanism that optimizes the activity of yeast ribonucleotide reductase when iron is scarce. We demonstrate that Aft1 and the DNA-binding protein Ixr1 enhance the expression of the gene encoding for its catalytic subunit, RNR1, in response to iron limitation, leading to an increase in both mRNA and protein levels. By mutagenesis of the Aft1-binding sites within RNR1 promoter, we conclude that RNR1 activation by iron depletion is important for Rnr1 protein and deoxyribonucleotide synthesis. Remarkably, Aft1 also activates the expression of IXR1 upon iron scarcity through an iron-responsive element located within its promoter. These results provide a novel mechanism for the direct activation of ribonucleotide reductase function by the iron-regulated Aft1 transcription factor., This work was supported by predoctoral contracts from the Spanish Ministry of Economy, Industry and Competitiveness to LRA and AMR; and by the BIO2017-87828-C2-1-P grant from the Spanish Ministry of Science, Innovation and Universities, and FEDER (Fondo Europeo de Desarrollo Regional) funds to SP.

Retroviral reverse transcriptase catalyses the synthesis of an integration-competent dsDNA molecule, using as a substrate the viral RNA. Using optical tweezers, we follow the Murine Leukemia Virus reverse transcriptase as it performs strand-displacement polymerization on a template under mechanical force. Our results indicate that reverse transcriptase functions as a Brownian ratchet, with dNTP binding as the rectifying reaction of the ratchet. We also found that reverse transcriptase is a relatively passive enzyme, able to polymerize on structured templates by exploiting their thermal breathing. Finally, our results indicate that the enzyme enters the recently characterized backtracking state from the pre-translocation complex.

The hydrophobic hinge region of DNA polymerase β (pol β) is located between the fingers and palm subdomains. The hydrophobicity of the hinge region is important for maintaining the geometry of the binding pocket and for selectivity of the enzyme. Various cancer-associated pol β variants in the hinge region have reduced fidelity resulting from decreased discrimination at the level of dNTP binding. Specifically, I260M, a prostate cancer-associated variant of pol β, has been shown to have reduced discrimination during dNTP binding and also during nucleotidyl transfer. To test whether fidelity of the I260M variant is dependent on leaving group chemistry, we employed a tool-kit comprising dNTP bisphosphonate analogues modified at the β-γ bridging methylene to modulate leaving group (pCXYp mimicking PPi) basicity. Construction of LFER plots for the dependence of log(kpol) on the leaving group pKa4 revealed that I260M catalyzes dNMP incorporation with a marked negative dependence of leaving group basicity, consistent with a chemical transition state, during both correct and incorrect incorporation. Additionally, we provide evidence that the I260M fidelity is altered in the presence of some of the analogues, possibly resulting from a lack of coordination between the fingers and palm subdomains in the presence of the I260M mutation.

Deoxyribonucleoside triphosphates (dNTPs) are vital for the biosynthesis and repair of DNA. Their cellular concentration peaks during the S phase of the cell cycle. In non-proliferating cells, dNTP concentrations are low, making their reliable quantification from tissue samples of heterogeneous cellular composition challenging. Partly because of this, the current knowledge related to the regulation of and disturbances in cellular dNTP concentrations derive mostly from cell culture experiments with little corroboration at the tissue or organismal level. Here, we fill the methodological gap by presenting a simple non-radioactive microplate assay for the quantification of dNTPs with a minimum requirement of 4-12 mg of biopsy material. In contrast to published assays, this assay is based on long synthetic single-stranded DNA templates (50-200 nucleotides), an inhibitor-resistant high-fidelity DNA polymerase, and the double-stranded-DNA-binding EvaGreen dye. The assay quantified reliably less than 50 fmol of each of the four dNTPs and discriminated well against ribonucleotides. Additionally, thermostable RNAse HII-mediated nicking of the reaction products and a subsequent shift in their melting temperature allowed near-complete elimination of the interfering ribonucleotide signal, if present. Importantly, the assay allowed measurement of minute dNTP concentrations in mouse liver, heart and skeletal muscle.

The levels of the four deoxynucleoside triphosphates (dNTPs) are under strict control in the cell, as improper or imbalanced dNTP pools may lead to growth defects and oncogenesis. Upon treatment of cancer cells with therapeutic agents, changes in the canonical dNTPs levels may provide critical information for evaluating drug response and mode of action. The radioisotope-labeling enzymatic assay has been commonly used for quantitation of cellular dNTP levels. However, the disadvantage of this method is the handling of biohazard materials. Here, we described the use of click chemistry to replace radioisotope-labeling in template-dependent DNA polymerization for quantitation of the four canonical dNTPs. Specific oligomers were designed for dCTP, dTTP, dATP and dGTP measurement, and the incorporation of 5-ethynyl-dUTP or C8-alkyne-dCTP during the polymerization reaction allowed for fluorophore conjugation on immobilized oligonucleotides. The four reactions gave a linear correlation coefficient >0.99 in the range of the concentration of dNTPs present in 106 cells, with little interference of cellular rNTPs. We present evidence indicating that data generated by this methodology is comparable to radioisotope-labeling data. Furthermore, the design and utilization of a robust microplate assay based on this technology evidenced the modulation of dNTPs in response to different chemotherapeutic agents in cancer cells., We thank Hsin-Yen Wang for maintaining K562 cells. This study was financially supported by the “Center of Precision Medicine” from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan and by the grant, MOST 107-3017-F-002-002, MOST 107-2321-B-002-041, and MOST 106-2113-M-002-021-MY3 from the Ministry of Science and Technology, Taiwan, (R.O.C.).

A new approach to single-molecule DNA sequencing in which dNTPs, released by pyrophosphorolysis from the strand to be sequenced, are captured in microdroplets and read directly could have substantial advantages over current sequence-by-synthesis methods; however, there is no existing method sensitive enough to detect a single nucleotide in a microdroplet. We have developed a method for dNTP detection based on an enzymatic two-stage reaction which produces a robust fluorescent signal that is easy to detect and process. By taking advantage of the inherent specificity of DNA polymerases and ligases, coupled with volume restriction in microdroplets, this method allows us to simultaneously detect the presence of and distinguish between, the four natural dNTPs at the single-molecule level, with negligible cross-talk.

Oligonucleotides are key compounds widely used for research, diagnostics, and therapeutics. The rapid increase in oligonucleotide-based applications, together with the progress in nucleic acids research, has led to the design of nucleotide analogs that, when part of these oligomers, enhance their efficiency, bioavailability, or stability. One of the most useful nucleotide analogs is the first-generation bridged nucleic acids (BNA), also known as locked nucleic acids (LNA), which were used in combination with ribonucleotides, deoxyribonucleotides, or other analogs to construct oligomers with diverse applications. However, there is still room to improve their efficiency, bioavailability, stability, and, importantly, toxicity. A second-generation BNA, BNANC (20 -O,40 -aminoethylene bridged nucleic acid), has been recently made available. Oligomers containing these analogs not only showed less toxicity when compared to LNA-containing compounds but, in some cases, also exhibited higher specificity. Although there are still few applications where BNANC-containing compounds have been researched, the promising results warrant more effort in incorporating these analogs for other applications. Furthermore, newer BNA compounds will be introduced in the near future, offering great hope to oligonucleotide-based fields of research and applications. Fil: Soler Bistue, Alfonso J. C.. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Zorreguieta, Ángeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Tolmasky, Marcelo E.. California State University; Estados Unidos

Class I ribonucleotide reductases (RNRs) share a common mechanism of nucleotide reduction in a catalytic α subunit. All RNRs initiate catalysis with a thiyl radical, generated in class I enzymes by a metallocofactor in a separate β subunit. Class Id RNRs use a simple mechanism of cofactor activation involving oxidation of a Mn(II)(2) cluster by free superoxide to yield a metal-based Mn(III)Mn(IV) oxidant. This simple cofactor assembly pathway suggests that class Id RNRs may be representative of the evolutionary precursors to more complex class Ia-c enzymes. X-ray crystal structures of two class Id α proteins from Flavobacterium johnsoniae (Fj) and Actinobacillus ureae (Au) reveal that this subunit is distinctly small. The enzyme completely lacks common N-terminal ATP cone allosteric motifs that regulate overall activity, a process that normally occurs by dATP-induced formation of inhibitory quaternary structures to prevent productive β subunit association. Class Id RNR activity is insensitive to dATP in the Fj and Au enzymes evaluated here, as expected. However, the class Id α protein from Fj adopts higher order structures, detected crystallographically and in solution. The Au enzyme does not exhibit these quaternary forms. Our study reveals structural similarity between bacterial class Id and eukaryotic class Ia α subunits in conservation of an internal auxiliary domain. Our findings with the Fj enzyme illustrate that nucleotide-independent higher-order quaternary structures can form in simple RNRs with truncated or missing allosteric motifs.

The base-excision repair enzyme uracil-DNA glycosylase (UDG) plays a crucial role in the maintenance of genome integrity. The authors describe a fluorometric method for the detection of the activity of UDG. It is making use of (a) a 3’-FAM-labeled hairpin DNA probe with two uracil deoxyribonucleotides in the self-complementary duplex region of its hairpin structure, (b) exonuclease I (Exo I) that catalyzes the release of FAM from the UDG-induced stretched ssDNA probe, and (c) graphene oxide that quenches the green FAM fluorescence of the intact hairpin DNA probe in the absence of UDG. If Exo I causes the release of FAM from the hairpin DNA probe, the fluorescence peaking at 517 nm is turned off in the absence of UDG but turned on in its presence. The resulting assay has a wide linear range (0.008 to 1 U·mL−1) and a detection limit as low as 0.005 U·mL−1. It has good specificity for UDG over potentially interfering enzymes and gave satisfactory results when applied to biological samples. Conceivably, the method may be used in a wide range of applications such as in diagnosis, drug screening, and in studying the repair of DNA lesions.

Sterile alpha motif (SAM) and Histidine-Aspartate (HD)-domain containing protein 1 (SAMHD1) is a triphosphohydrolase that converts deoxyribonucleoside triphosphates (dNTPs) into deoxyribonucleosides and triphosphates. SAMHD1 exists in multiple states. The monomer and apo- or GTP-bound dimer are catalytically inactive. Binding of dNTP at allosteric site 2 (AS2), adjacent to the GTP-binding, allosteric site 1 (AS1), induces formation of tetramer, the catalytically active form. We have developed an enzyme kinetic assay, tailored to control specific dNTP binding at each site, allowing us to determine the kinetic binding parameters of individual dNTPs at both the AS2 and catalytic site for all possible combinations of dNTP binding at both sites. Here, we show that the apparent Km values of dNTPs at AS2 vary, in order of dCTP

Ribonucleotide Reductase (RNR) is the sole enzyme that catalyzes the reduction of ribonucleotides into deoxyribonucleotides. Even though RNR is a recognized target for antiproliferative molecules, and the main target of the approved drug hydroxyurea, few new leads targeted to this enzyme have been developed. We have evaluated a recently identified set of RNR inhibitors with respect to inhibition of the human enzyme and cellular toxicity. One compound, NSC73735, is particularly interesting; it is specific for leukemia cells and is the first identified compound that hinders oligomerization of the mammalian large RNR subunit. Similar to hydroxyurea, it caused a disruption of the cell cycle distribution of cultured HL‐60 cells. In contrast to hydroxyurea, the disruption was reversible, indicating higher specificity. NSC73735 thus defines a potential lead candidate for RNR‐targeted anticancer drugs, as well as a chemical probe with better selectivity for RNR inhibition than hydroxyurea.

Conjugation of single-strand DNA aptamers and enzymes has been of great significance in bioanalytical and biomedical applications because of the unlimited functions provided by DNA aptamer direction. Therefore, we developed efficient tailing of a DNA aptamer, with end-specific conjugation of multiple enzymes, through enzymatic catalysis. Terminal deoxynucleotidyl transferase (TdT) added multiple Z-Gln-Gly (Z-QG) moieties to the 3'-end of a DNA aptamer via the addition of Z-QG-modified deoxyuridine triphosphate (Z-QG-dUTP) and deoxynucleoside triphosphates (dNTPs). The resultant (Z-QG)m -(dN)l-aptamer, whose Z-QGs with dN spacers served as stickers for microbial transglutaminase (MTG), were crosslinked between the Z-QGs on the DNA and a substrate peptide sequence containing lysine (K), fused to a recombinant enzyme (i.e. bacterial alkaline phosphatase; BAP) by MTG. The incorporation efficiency of Z-QG moieties on the aptamer tail and the subsequent conjugation efficiency with multiple enzyme molecules were dramatically altered by the presence of dNTPs, revealing that a combination of Z-QG-dUTP/dTTP comprised the best labeling efficiency and corresponding properties during analytical performance. Thus, a novel optimized platform for designing (BAP)n -(dT)l-DNA aptamers was demonstrated for the first time in this article, offering unique opportunities for tailoring new types of covalent protein-nucleic acid conjugates in a controllable way.

One frontier in synthetic biology seeks to move artificially expanded genetic information systems (AEGIS) into natural living cells and to arrange the metabolism of those cells to allow them to replicate plasmids built from these unnatural genetic systems. In addition to requiring polymerases that replicate AEGIS oligonucleotides, such cells require metabolic pathways that biosynthesize the triphosphates of AEGIS nucleosides, the substrates for those polymerases. Such pathways generally require nucleoside and nucleotide kinases to phosphorylate AEGIS nucleosides and nucleotides on the path to these triphosphates. Thus, constructing such pathways focuses on engineering natural nucleoside and nucleotide kinases, which often do not accept the unnatural AEGIS biosynthetic intermediates. This, in turn, requires assays that allow the enzyme engineer to follow the kinase reaction, assays that are easily confused by ATPase and other spurious activities that might arise through "site-directed damage" of the natural kinases being engineered. This article introduces three assays that can detect the formation of both natural and unnatural deoxyribonucleoside triphosphates, assessing their value as polymerase substrates at the same time as monitoring the progress of kinase engineering. Here, we focus on two complementary AEGIS nucleoside diphosphates, 6-amino-5-nitro-3-(1'-β-D-2'-deoxyribofuranosyl)-2(1H)-pyridone and 2-amino-8-(1'-β-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one. These assays provide new ways to detect the formation of unnatural deoxyribonucleoside triphosphates in vitro and to confirm their incorporation into DNA. Thus, these assays can be used with other unnatural nucleotides.

Ribonucleotide reductases (RNRs) are responsible for all de novo biosynthesis of DNA precursors in nature by catalyzing the conversion of ribonucleotides to deoxyribonucleotides. Because of its essential role in cell division, human RNR is a target for a number of anticancer drugs in clinical use. Like other class Ia RNRs, human RNR requires both a radical-generation subunit (β) and nucleotide-binding subunit (α) for activity. Because of their complex dependence on allosteric effectors, however, the active and inactive quaternary forms of many class Ia RNRs have remained in question. Here, we present an X-ray crystal structure of the human α subunit in the presence of inhibiting levels of dATP, depicting a ring-shaped hexamer (α6) where the active sites line the inner hole. Surprisingly, our small-angle X-ray scattering (SAXS) results indicate that human α forms a similar hexamer in the presence of ATP, an activating effector. In both cases, α6 is assembled from dimers (α2) without a previously proposed tetramer intermediate (α4). However, we show with SAXS and electron microscopy that at millimolar ATP, the ATP-induced α6 can further interconvert with higher-order filaments. Differences in the dATP- and ATP-induced α6 were further examined by SAXS in the presence of the β subunit and by activity assays as a function of ATP or dATP. Together, these results suggest that dATP-induced α6 is more stable than the ATP-induced α6 and that stabilization of this ring-shaped configuration provides a mechanism to prevent access of the β subunit to the active site of α.

In this method, E. coli DNA Pol I binds to a nick or short gap in duplex DNA. The 5′ → 3′ exonuclease activity of Pol I then removes nucleotides from one strand of the DNA, creating a template for the synthesis of DNA by the 5′ → 3′ polymerase activity of Pol I. The simultaneous elimination of nucleotides from the 5′ side and the addition of nucleotides to the 3′ side result in movement of the nick (nick translation) along the DNA, which becomes labeled to high specific activity.

Ribonucleotide reductases (RNRs) use a conserved radical-based mechanism to catalyze the conversion of ribonucleotides to deoxyribonucleotides. Within the RNR family, class Ib RNRs are notable for being largely restricted to bacteria, including many pathogens, and for lacking an evolutionarily mobile ATP-cone domain that allosterically controls overall activity. In this study, we report the emergence of a distinct and unexpected mechanism of activity regulation in the sole RNR of the model organism Bacillus subtilis. Using a hypothesis-driven structural approach that combines the strengths of small-angle X-ray scattering (SAXS), crystallography, and cryo-electron microscopy (cryo-EM), we describe the reversible interconversion of six unique structures, including a flexible active tetramer and two inhibited helical filaments. These structures reveal the conformational gymnastics necessary for RNR activity and the molecular basis for its control via an evolutionarily convergent form of allostery., Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides, which is an essential step in DNA synthesis. Here the authors use small-angle X-ray scattering, X-ray crystallography, and cryo-electron microscopy to capture active and inactive forms of the Bacillus subtilis RNR and provide mechanistic insights into a convergent form of allosteric regulation.

Investigation into intracellular ribonucleotides (RNs) and deoxyribonucleotides (dRNs) is important for studies of the mechanism of many biological processes, such as RNA and DNA synthesis and DNA repair, as well as metabolic and therapeutic efficacy of nucleoside analogues. However, current methods are still unsatisfactory for determination of nucleotides in complex matrixes. Here we describe a novel method for the determination of RN and dRN pools in cells based on fast derivatization with (trimethylsilyl)diazomethane (TMSD) followed by quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Derivatization was accomplished in 3 min, and each derivatized nucleotide not only had a sufficient retention on reversed-phase column by introduction of methyl groups but also exhibited a unique ion transition which consequently eliminated mutual interference in LC-MS/MS. Chromatographic separation was performed on a C18 column with a simple acetonitrile-water gradient elution system, which avoided contamination and ion suppression caused by ion-pairing reagents. The developed method was fully validated and applied to the analysis of RNs and dRNs in cell samples. Moreover, results demonstrated that the applicability of this method could be extended to nucleoside analogues and their metabolites and could facilitate many applications in future studies.

Deoxyxylonucleic acid (dxNA) is a synthetic polymer that might have potential for heredity and evolution. Because of dxNA's unusual backbone geometry, sequence information stored in it is presumed to be inaccessible to natural nucleic acids or proteins. Despite a large structural similarity with natural nucleotides, incorporation of 2'-deoxyxylonucleotides (dxNTs) through the action of polymerases is limited. We present the identification of a mutant of the DNA polymerase Therminator with increased tolerance to deoxyxylose-induced backbone distortions. Whereas the original polymerase stops after incorporation of two consecutive dxNTs, the mutant is able to catalyse the extension of incorporated dxNTs with 2'-deoxyribonucleotides (dNTs) and the incorporation of up to four dxNTs alternates with dNTs, thereby translocating a highly distorted double helix throughout the entire polymerase. A single His-to-Arg substitution very close to the catalytic site residues is held to be responsible for interaction with the primer phosphate groups and for stabilizing nucleotide sugar-induced distortions during incorporation and translocation. ispartof: CHEMBIOCHEM vol:19 issue:22 pages:2410-2420 ispartof: location:Germany status: published

Ribonucleotide reductase (RNR) catalyses the only known de novo pathway for the production of all four deoxyribonucleotides that are required for DNA synthesis1,2. It is essential for all organisms that use DNA as their genetic material and is a current drug target3,4. Since the discovery that iron is required for function in the aerobic, class I RNR found in all eukaryotes and many bacteria, a dinuclear metal site has been viewed as necessary to generate and stabilize the catalytic radical that is essential for RNR activity5–7. Here we describe a group of RNR proteins in Mollicutes—including Mycoplasma pathogens—that possess a metal-independent stable radical residing on a modified tyrosyl residue. Structural, biochemical and spectroscopic characterization reveal a stable 3,4-dihydroxyphenylalanine (DOPA) radical species that directly supports ribonucleotide reduction in vitro and in vivo. This observation overturns the presumed requirement for a dinuclear metal site in aerobic ribonucleotide reductase. The metal-independent radical requires new mechanisms for radical generation and stabilization, processes that are targeted by RNR inhibitors. It is possible that this RNR variant provides an advantage under metal starvation induced by the immune system. Organisms that encode this type of RNR—some of which are developing resistance to antibiotics—are involved in diseases of the respiratory, urinary and genital tracts. Further characterization of this RNR family and its mechanism of cofactor generation will provide insight into new enzymatic chemistry and be of value in devising strategies to combat the pathogens that utilize it. We propose that this RNR subclass is denoted class Ie. A subclass of ribonucleotide reductase in Mycoplasma pathogens contains a stable radical formed from a modified tyrosine residue, overturning the presumed requirement for a dinuclear metal site in aerobic ribonucleotide reductase.

Understanding the mechanism of action (MOA) of new antimicrobial agents is a critical step in drug discovery but is notoriously difficult for compounds that appear to inhibit multiple cellular pathways. We recently described image-based approaches [bacterial cytological profiling and rapid inducible profiling (RIP)] for identifying the cellular pathways targeted by antibiotics. Here we have applied these methods to examine the effects of proteolytically degrading enzymes involved in pyrimidine nucleotide biosynthesis, a pathway that produces intermediates for transcription, DNA replication, and cell envelope synthesis. We show that rapid removal of enzymes directly involved in deoxyribonucleotide synthesis blocks DNA replication. However, degradation of cytidylate kinase (CMK), which catalyzes reactions involved in the synthesis of both ribonucleotides and deoxyribonucleotides, blocks both DNA replication and wall teichoic acid biosynthesis, producing cytological effects identical to those created by simultaneously inhibiting both processes with the antibiotics ciprofloxacin and tunicamycin. Our results suggest that RIP can be used to identify and characterize potential keystone enzymes like CMK whose inhibition dramatically affects multiple pathways, thereby revealing important metabolic connections. Identifying and understanding the role of keystone targets might also help to determine the MOAs of drugs that appear to inhibit multiple targets.

Class I ribonucleotide reductase (RNR) consists of a catalytic subunit (NrdA) and a radical-generating subunit (NrdB) that together catalyse reduction of the four ribonucleotides to their corresponding deoxyribonucleotides.Facklamia ignavaNrdB is an unprecedented fusion protein with N-terminal add-ons of a glutaredoxin (Grx) domain followed by an ATP-cone. Grx, which in general is encoded elsewhere in the genome than is the RNR operon, is a known physiological reductant of RNRs. Here we show that the fused Grx domain functions as an efficient reductant of theF. ignavaclass I RNR via the common dithiol mechanism and interestingly also via a monothiol mechanism, although less efficiently. A Grx that utilizes either or of these two reaction mechanisms has to our knowledge not been observed with a native substrate before. The ATP-cone, which is commonly found as an N-terminal domain of the catalytic subunit of RNRs, is an allosteric on/off switch that promotes dNDP reduction in presence of ATP and inhibits the enzyme activity in presence of dATP. Here we show that dATP bound to the ATP-cone ofF. ignavaNrdB promotes formation of tetramers that are unable to form enzymatically competent complexes withF. ignavaNrdA. The ATP-cone binds two molecules of dATP, but only one molecule of the activating nucleotide ATP.F. ignavaNrdB contains the recently identified radical factor Mn2III/IV. We show that NrdA from the firmicuteF. ignavacan form a catalytically competent RNR with the Mn2III/IV-containing NrdB from the flavobacteriumLeeuwenhoekiella blandensis.

The activity of DNA polymerase underlies numerous biotechnologies, cell division, and therapeutics, yet the enzyme remains incompletely understood. We demonstrate that both thermostable and mesophilic DNA polymerases readily utilize deoxyribonucleoside diphosphates (dNDPs) for DNA synthesis and inorganic phosphate for the reverse reaction, that is, phosphorolysis of DNA. For Taq DNA polymerase, the KMs of the dNDP and phosphate substrates are ∼20 and 200 times higher than for dNTP and pyrophosphate, respectively. DNA synthesis from dNDPs is about 17 times slower than from dNTPs, and DNA phosphorolysis about 200 times less efficient than pyrophosphorolysis. Such parameters allow DNA replication without requiring coupled metabolism to sequester the phosphate products, which consequently do not pose a threat to genome stability. This mechanism contrasts with DNA synthesis from dNTPs, which yield high-energy pyrophosphates that have to be hydrolyzed to phosphates to prevent the reverse reaction. Because the last common ancestor was likely a thermophile, dNDPs are plausible substrates for genome replication on early Earth and may represent metabolic intermediates later replaced by the higher-energy triphosphates.

Simultaneous, quantitative determination of intracellular nucleoside triphosphates and other polar metabolites using liquid chromatography with electrospray ionization tandem mass spectrometry (LC-MS/MS) represents a bioanalytic challenge because of charged, highly hydrophilic analytes presented at a large concentration range in a complex matrix. In this study, an ion pair LC-MS/MS method using triethylamine (TEA) – hexafluoroisopropanol (HFIP) ion-pair mobile phase was optimized and validated for simultaneous and unambiguous determination of 8 nucleoside triphosphates (including ATP, CTP, GTP, UTP, dATP, dCTP, dGTP, and dTTP) in cellular samples. Compared to the the less volatile ion-pair reagent, triethylammonium acetate (100 mM, pH 7.0), the combination of HFIP (100 mM) and TEA (8.6 mM) increased the MS signal intensity by about 50-fold, while retaining comparable chromatographic resolution. The isotope-labeled internal standard method was used for the quantitation. Lower limits of quantitation were determined at 0.5 nM for CTP, UTP, dATP, dCTP, and dTTP, at 1 nM for ATP, and at 5 nM for GTP and dGTP. The intra- and inter-day precision and accuracy were within the generally accepted criteria for bioanalytical method validation (< 15%). While the present method was validated for the quantitation of intracellular nucleoside triphosphates, it had a broad application potential for quantitative profiling of nucleoside mono- and bi-phosphates as well as other polar, ionic metabolic intermediates (including carbohydrate derivatives, carboxylic acid derivatives, co-acyl A derivatives, fatty acyls, and others) in biological samples.

The leadzyme refers to a small ribozyme that cleaves a RNA substrate in the presence of Pb(2+). In an optimized form, the enzyme strand contains only two unpaired nucleotides. Most RNA-cleaving DNAzymes are much longer. Two classical Pb(2+)-dependent DNAzymes, 8-17 and GR5, both contain around 15 nucleotides in the enzyme loop. This is also the size of most RNA-cleaving DNAzymes that use other metal ions for their activity. Such large enzyme loops make spectroscopic characterization difficult and so far no high-resolution structural information is available for active DNAzymes. The goal of this work is to search for DNAzymes with smaller enzyme loops. A simple replacement of the ribonucleotides in the leadzyme by deoxyribonucleotides failed to produce an active enzyme. A Pb(2+)-dependent in vitro selection combined with deep sequencing was then performed. After sequence alignment and DNA folding, a new DNAzyme named PbE22 was identified, which contains only 5 nucleotides in the enzyme catalytic loop. The biochemical characteristics of PbE22 were compared with those of the leadzyme and the two classical Pb(2+)-dependent DNAzymes. The rate of PbE22 rises with increase in Pb(2+) concentration, being 1.7 h(-1) in the presence of 100 μM Pb(2+) and reaching 3.5 h(-1) at 500 µM Pb(2+). The log of PbE22 rate rises linearly in a pH-dependent fashion (20 µM Pb(2+)) with a slope of 0.74. In addition, many other abundant sequences in the final library were studied. These sequences are quite varied in length and nucleotide composition, but some contain a few conserved nucleotides consistent with the GR5 structure. Interestingly, some sequences are active with Pb(2+) but none of them were active with even 50 mM Mg(2+), which is reminiscent of the difference between the GR5 and 8-17 DNAzymes.

Abasic sites (Apurinic/apyrimidinic (AP) sites), produced ∼50,000 times/cell/day, are very blocking and miscoding. To better understand miscoding mechanisms of abasic site for yeast DNA polymerase η, pre-steady-state nucleotide incorporation and LC–MS/MS sequence analysis of extension product were studied using pol ηcore (catalytic core, residues 1–513), which can completely eliminate the potential effects of the C-terminal C2H2 motif of pol η on dNTP incorporation. The extension beyond the abasic site was very inefficient. Compared with incorporation of dCTP opposite G, the incorporation efficiencies opposite abasic site were greatly reduced according to the order of dGTP > dATP >> dCTP and dTTP. Pol ηcore showed no fast burst phase for any incorporation opposite G or abasic site, suggesting that the catalytic step is not faster than the dissociation of polymerase from DNA. LC–MS/MS sequence analysis of extension products showed that 53% products were dGTP misincorporation, 33% were dATP and 14% were -1 frameshift, indicating that Pol ηcore bypasses abasic site by a combined G-rule, A-rule and -1 frameshift deletions. Compared with full-length pol η, pol ηcore relatively reduced the efficiency of incorporation of dCTP opposite G, increased the efficiencies of dNTP incorporation opposite abasic site and the exclusive incorporation of dGTP opposite abasic site, but inhibited the extension beyond abasic site, and increased the priority in extension of A: abasic site relative to G: abasic site. This study provides further understanding in the mutation mechanism of abasic sites for yeast DNA polymerase η.

Regulating gene expression programmes is a central facet of the DNA damage response. The Dun1 kinase protein controls expression of many DNA damage induced genes, including the ribonucleotide reductase genes, which regulate cellular dNTP pools. Using a combination of gene expression profiling and chromatin immunoprecipitation, we demonstrate that in the absence of DNA damage the yeast Rad4-Rad23 nucleotide excision repair complex binds to the promoters of certain DNA damage response genes including DUN1, inhibiting their expression. UV radiation promotes the loss of occupancy of the Rad4-Rad23 complex from the regulatory regions of these genes, enabling their induction and thereby controlling the production of dNTPs. We demonstrate that this regulatory mechanism, which is dependent on the ubiquitination of Rad4 by the GG-NER E3 ligase, promotes UV survival in yeast cells. These results support an unanticipated regulatory mechanism that integrates ubiquitination of NER DNA repair factors with the regulation of the transcriptional response controlling dNTP production and cellular survival after UV damage.

DNA polymerases maintain genomic integrity by copying DNA with high fidelity. A conformational change important for fidelity is the motion of the polymerase fingers subdomain from an open to a closed conformation upon binding of a complementary nucleotide. We previously employed intra-protein single-molecule FRET on diffusing molecules to observe fingers conformations in polymerase-DNA complexes. Here, we used the same FRET ruler on surface-immobilized complexes to observe fingers-opening and closing of individual polymerase molecules in real time. Our results revealed the presence of intrinsic dynamics in the binary complex, characterized by slow fingers-closing and fast fingers-opening. When binary complexes were incubated with increasing concentrations of complementary nucleotide, the fingers-closing rate increased, strongly supporting an induced-fit model for nucleotide recognition. Meanwhile, the opening rate in ternary complexes with complementary nucleotide was 6 s(-1), much slower than either fingers closing or the rate-limiting step in the forward direction; this rate balance ensures that, after nucleotide binding and fingers-closing, nucleotide incorporation is overwhelmingly likely to occur. Our results for ternary complexes with a non-complementary dNTP confirmed the presence of a state corresponding to partially closed fingers and suggested a radically different rate balance regarding fingers transitions, which allows polymerase to achieve high fidelity.

Model studies of prebiotic chemistry have re- vealed compelling routes for the formation of the building blocks of proteins and RNA, but not DNA. Today, deoxynu- cleotides required for the construction of DNA are produced by reduction of nucleotides catalysed by ribonucleotide re- ductases, which are radical enzymes. This study considers potential non-enzymatic routes via intermediate radicals for the ancient formation of deoxynucleotides. In this context, several mechanisms for ribonucleotide reduction, in a puta- tive H2S/HSC environment, are characterized using computa- tional chemistry. A bio-inspired mechanistic cycle involving a keto intermediate and HSSH production is found to be po- tentially viable. An alternative pathway, proceeding through an enol intermediate is found to exhibit similar energetic re- quirements. Non-cyclical pathways, in which HSSC is generat- ed in the final step instead of HSC, show a markedly in- creased thermodynamic driving force (ca. 70 kJ mol � 1 ) and thus warrant serious consideration in the context of the pre- biotic ribonucleotide reduction.

Fluorescent 2'-deoxynucleotides containing a protecting group at the 3'-O-position are reversible terminators enabling array-based DNA sequencing by synthesis (SBS) approaches. Herein, we describe the synthesis of a new family of 3'-OH unprotected cleavable fluorescent 2'-deoxynucleotides and their evaluation as reversible terminators for high-throughput DNA SBS strategies. In this first version, all four modified nucleotides bearing a cleavable disulfide Alexa Fluor(R) 594 dye were assayed for their ability to act as a reversible stop for the incorporation of the next labeled base. Their use in SBS leaded to a signal-no signal output after successive addition of each labeled nucleotide during the sequencing process (binary read-out). Solid-phase immobilized synthetic DNA target sequences were used to optimize the method that has been applied to DNA polymerized colonies or clusters obtained by in situ solid-phase amplification of fragments of genomic DNA templates.

The question of whether RNA is more stable or unstable compared to DNA or other nucleic acids has long been a subject of extensive scrutiny and public attention. Recently, thermodynamically stable and degradation-resistant RNA motifs have been utilized in RNA nanotechnology to build desired architectures and integrate multiple functional groups. Here we report the effects of phosphorothioate deoxyribonucleotides (PS-DNA), deoxyribonucleotides (DNA), ribonucleotides (RNA), 2′-F nucleotides (2′-F), and locked nucleic acids (LNA) on the thermal and in vivo stability of the three-way junction (3WJ) of bacteriophage phi29 motor packaging RNA. It was found that the thermal stability gradually increased following the order of PS-DNA/PS-DNA < DNA/DNA < DNA/RNA < RNA/RNA < RNA/2′-F RNA < 2’-F RNA/2′-F RNA < 2′-F RNA/LNA < LNA/LNA. This proposition is supported by studies on strand displacement and the melting of homogeneous and heterogeneous 3WJs. By simply mixing different chemically modified oligonucleotides, the thermal stability of phi29 pRNA 3WJ can be tuned to cover a wide range of melting temperatures from 21.2°C to over 95°C. The 3WJLNA was resistant to boiling temperature denaturation, urea denaturation, and 50% serum degradation. Intravenous injection of fluorescent LNA/2′-F hybrid 3WJs into mice revealed its exceptional in vivo stability and presence in urine. It is thus concluded that incorporation of LNA nucleotides, alone or in combination with 2′-F, into RNA nanoparticles derived from phi29 pRNA 3WJ can extend the half-life of the RNA nanoparticles in vivo and improve their pharmacokinetics profile.

How tumor cells adapt and survive under hypoxia significantly impacts patient prognosis. We recently demonstrated that the oxygen-requiring ribonucleotide reductase (RNR) enzyme, which provides cells with deoxyribonucleotides, responds to limited oxygen availability by switching small subunits from RRM2 to RRM2B. This property of RNR is essential for hypoxic cell viability and therefore contributes to the most aggressive and therapy-resistant fraction of tumors.

Sterile α motif and histidine-aspartate domain-containing protein 1 (SAMHD1) plays a critical role in inhibiting HIV infection, curtailing the pool of dNTPs available for reverse transcription of the viral genome. Recent structural data suggested a compelling mechanism for the regulation of SAMHD1 enzymatic activity and revealed dGTP-induced association of two inactive dimers into an active tetrameric enzyme. Here, we present the crystal structures of SAMHD1 catalytic core (residues 113-626) tetramers, complexed with mixtures of nucleotides, including dGTP/dATP, dGTP/dCTP, dGTP/dTTP, and dGTP/dUTP. The combined structural and biochemical data provide insight into dNTP promiscuity at the secondary allosteric site and how enzymatic activity is modulated. In addition, we present biochemical analyses of GTP-induced SAMHD1 full-length tetramerization and the structure of SAMHD1 catalytic core tetramer in complex with GTP/dATP, revealing the structural basis of GTP-mediated SAMHD1 activation. Altogether, the data presented here advance our understanding of SAMHD1 function during cellular homeostasis.

The human 5′(3′)-deoxyribonucleotidases catalyze the dephosphorylation of deoxyribonucleoside monophosphates to the corresponding deoxyribonucleosides and thus help to maintain the balance between pools of nucleosides and nucleotides. Here, the structures of human cytosolic deoxyribonucleotidase (cdN) at atomic resolution (1.08 Å) and mitochondrial deoxyribonucleotidase (mdN) at near-atomic resolution (1.4 Å) are reported. The attainment of an atomic resolution structure allowed interatomic distances to be used to assess the probable protonation state of the phosphate anion and the side chains in the enzyme active site. A detailed comparison of the cdN and mdN active sites allowed the design of a cdN-specific inhibitor.

Oligonucleotides labeled with a single fluorophore (fluorescein or tetramethylrhodamine) have been used previously as fluorogenic substrates for a number of DNA modifying enzymes. Here, it is shown that such molecules can be used as fluorogenic probes to detect the template-dependent binding of deoxynucleotide triphosphates by DNA polymerases. Two polymerases were used in this work: the Klenow fragment of the Escherichia coli DNA polymerase I and the Bacillus stearothermophilus polymerase, Bst. When complexes of these polymerases with dye-labeled hairpin-type oligonucleotides were mixed with various deoxynucleotide triphosphates in the presence of Sr2+ as the divalent metal cation, the formation of ternary DNA–polymerase–dNTP complexes was detected by concentration-dependent changes in the fluorescence intensities of the dyes. Fluorescein- and tetramethylrhodamine-labeled probes of identical sequences responded differently to the two polymerases. With Bst polymerase, the fluorescence intensities of all probes increased with the next correct dNTP; with Klenow polymerase, tetramethylrhodamine-labeled probes increased their fluorescence, but the intensity of fluorescein-labeled probes decreased on formation of ternary complexes with the correct incoming nucleotides. The use of Sr2+ as the divalent metal ion allowed the formation of catalytically inactive ternary complexes and obviated the need for using 2′,3′-dideoxy-terminated oligonucleotides as would have been needed in the case of Mg2+ as the metal ion.

Cytosolic dNT-1 nucleotidase plays a key role in the homeostasis of pyrimidine deoxyribonucleotides in mammalian cells. The enzyme is responsible for the dephosphorylation of physiological substrates as well as nucleoside analogues that are used in antiviral and anticancer therapies, therefore selective inhibition of the dNT-1 nucleotidase activity may lead to an increase in efficacy of this type of therapeutic compounds. Here, we report the backbone ¹H, ¹³C and ¹⁵N assignments for the 47 kDa dNT-1 dimer, which will be used for structural characterisation of dNT-1 complexes with small molecule inhibitors obtained through modification of pyrimidine nucleotide scaffolds or optimisation of successful binders obtained from the screening of fragment libraries.

An N-terminal-domain (NTD) and adjacent catalytic body (CB) make up subunit-α of ribonucleotide reductase (RNR), the rate-limiting enzyme for de novo dNTP biosynthesis. A strong linkage exists between ligand binding at the NTD and oligomerization-coupled RNR inhibition, inducible by both dATP and nucleotide chemotherapeutics. These observations have distinguished the NTD as an oligomeric regulation domain dictating the assembly of inactive RNR oligomers. Inactive states of RNR differ between eukaryotes and prokaryotes (α6 in human versus α4β4 in Escherichia coli , wherein β is RNR's other subunit); however, the NTD structurally interconnects individual α2 or α2 and β2 dimeric motifs within the respective α6 or α4β4 complexes. To elucidate the influence of NTD ligand binding on RNR allosteric and oligomeric regulation, we engineered a human- E. coli hybrid enzyme (HE) where human-NTD is fused to E. coli -CB. Both the NTD and the CB of the HE bind dATP. The HE specifically partners with E. coli -β to form an active holocomplex. However, although the NTD is the sole physical tether to support α2 and/or β2 associations in the dATP-bound α6 or α4β4 fully inhibited RNR complexes, the binding of dATP to the HE NTD only partially suppresses HE activity and fully precludes formation of higher-order HE oligomers. We postulate that oligomeric regulation is the ultimate mechanism for potent RNR inhibition, requiring species-specific NTD-CB interactions. Such interdomain cooperativity in RNR oligomerization is unexpected from structural studies alone or biochemical studies of point mutants.

Nucleoside analogs are an important class of anticancer agent that historically show better efficacy against hematological cancers versus solid tumors. This report describes the development and characterization of a new class of nucleoside analog that displays anticancer effects against both hematological and adherent cancer cell lines. These new analogs lack canonical hydrogen-bonding groups yet are effective nucleotide substrates for several high-fidelity DNA polymerases. Permutations in the position of the non-hydrogen-bonding functional group greatly influence the kinetic behavior of these nucleosides. One particular analog designated 4-nitroindolyl-2'-deoxynucleoside triphosphate (4-NITP) is unique as it is incorporated opposite C and T with high catalytic efficiencies. In addition, this analog functions as a nonobligate chain terminator of DNA synthesis, since it is poorly elongated. Consistent with this mechanism, the corresponding nucleoside, 4-nitroindolyl-2'-deoxynucleoside (4-NIdR), produces antiproliferative effects against leukemia cells. 4-NIdR also produces cytostatic and cytotoxic effects against several adherent cancer cell lines, especially those that are deficient in mismatch repair and p53. Cell death in this case appears to occur via mitotic catastrophe, a specialized form of apoptosis. Mass spectroscopy experiments performed on nucleic acid isolated from cells treated with 4-NIdR validate that the non-natural nucleoside is stably incorporated into DNA. Xenograft mouse studies demonstrate that administration of 4-NIdR delays tumor growth without producing adverse side effects such as anemia and thrombocytopenia. Collectively, the results of in vitro, cell-based, and animal studies provide evidence for the development of a novel nucleoside analog that shows enhanced effectiveness against solid tumors.

A large number of nucleoside analogues and 2′-deoxynucleoside triphosphates (dNTP) have been synthesized to interfere with DNA metabolism. However, in vivo the concentration and phosphorylation of these analogues are key limiting factors. In this context, we designed enzymes to switch nucleobases attached to a deoxyribose monophosphate. Active chimeras were made from two distantly related enzymes: a nucleoside deoxyribosyltransferase from lactobacilli and a 5′-monophosphate-2′-deoxyribonucleoside hydrolase from rat. Then their unprecedented activity was further extended to deoxyribose triphosphate, and in vitro biosyntheses could be successfully performed with several base analogues. These new enzymes provide new tools to synthesize dNTP analogues and to deliver them into cells. Background: The nucleoside deoxyribosyltransferase family contains hydrolases and transferases with different substrate specificities. Results: Chimeras exchange deoxyribose 5-(mono, di, and tri)-phosphate between natural bases and analogues. Conclusion: Comparison of the structures and catalytic mechanisms of members of the nucleoside deoxyribosyltransferase family allows the design of unprecedented enzymes. Significance: Phosphodeoxyribosyltransferases open the road to new deoxyribonucleotides synthetic pathways.