Disease-associated inosine misincorporation into RNA hinders translation.

Failure to prevent accumulation of the non-canonical nucleotide inosine triphosphate (ITP) by inosine triphosphate pyrophosphatase (ITPase) during nucleotide synthesis results in misincorporation of inosine into RNA and can cause severe and fatal developmental anomalies in humans. While the biochemical activity of ITPase is well understood, the pathogenic basis of ITPase deficiency and the molecular and cellular consequences of ITP misincorporation into RNA remain cryptic. Here, we demonstrate that excess ITP in the nucleotide pool during in vitro transcription results in T7 polymerase-mediated inosine misincorporation in luciferase RNA. In vitro translation of inosine-containing luciferase RNA reduces resulting luciferase activity, which is only partly explained by reduced abundance of the luciferase protein produced. Using Oxford Nanopore Direct RNA sequencing, we reveal inosine misincorporation to be stochastic but biased largely towards misincorporation in place of guanosine, with evidence for misincorporation also in place of cytidine, adenosine and uridine. Inosine misincorporation into RNA is also detected in Itpa-null mouse embryonic heart tissue as an increase in relative variants compared with the wild type using Illumina RNA sequencing. By generating CRISPR/Cas9 rat H9c2 Itpa-null cardiomyoblast cells, we validate a translation defect in cells that accumulate inosine within endogenous RNA. Furthermore, we observe hindered cellular translation of transfected luciferase RNA containing misincorporated inosine in both wild-type and Itpa-null cells. We therefore conclude that inosine misincorporation into RNA perturbs translation, thus providing mechanistic insight linking ITPase deficiency, inosine accumulation and pathogenesis.

Arginine-178 is an essential residue for ITPA function.

The inosine triphosphate pyrophosphatase (ITPA) enzyme plays a critical cellular role by removing noncanonical nucleoside triphosphates from nucleotide pools. One of the first pathological ITPA mutants identified is R178C (rs746930990), which causes a fatal infantile encephalopathy, termed developmental and epileptic encephalopathy 35 (DEE 35). The accumulation of noncanonical nucleotides such as inosine triphosphate (ITP), is suspected to affect RNA and/or interfere with normal nucleotide function, leading to development of DEE 35. Molecular dynamics simulations have shown that the very rare R178C mutation does not significantly perturb the overall structure of the protein, but results in a high level of structural flexibility and disrupts active-site hydrogen bond networks, while preliminary biochemical data indicate that ITP hydrolyzing activity is significantly reduced for the R178C mutant. Here we report Michaelis-Menten enzyme kinetics data for the R178C ITPA mutant and three other position 178 ITPA mutants. These data confirm that position 178 is essential for ITPA activity and even conservative mutation at this site (R178K) results in significantly reduced enzyme activity. Our data support that disruption of the active-site hydrogen bond network is a major cause of diminished ITP hydrolyzing activity for the R178C mutation. These results suggest an avenue for developing therapies to address DEE 35.

Finding Ways to Relax: A Revisionistic Analysis of the Chemistry of E. coli GTP Cyclohydrolase II.

Guanosine triphosphate (GTP) cyclohydrolase II (RibA) is one of three enzymes that hydrolytically cleave the C8-N9 bond of the GTP guanine. RibA also catalyzes a subsequent hydrolytic attack at the base liberating formate and in addition cleaves the α-ß phosphodiester bond of the triphosphate to form pyrophosphate (PPi). These hydrolytic reactions are promoted by tandem active-site metal ions, zinc and magnesium, that respectively function at the GTP guanine and triphosphate moieties. The RibA reaction is part of riboflavin biosynthesis and forms 2,5-diamino-6-ß-pyrimidinone 5'-phosphate, an exocyclic pyrimidine nucleotide that ultimately forms the pyrimidine ring of the isoalloxazine of riboflavin. The stoichiometry of the RibA reaction was defined in the study that first identified this activity in Escherichia coli (Foor, F., Brown, G. M. J. Biol. Chem., 1975, 250, 9, 3545-3551) and has not been quantitatively evaluated in subsequent works. Using primarily transient state approaches we examined the interaction of RibA from E. coli with the GTP, inosine triphosphate, and PPi. Our data indicate that PPi is a slow substrate for RibA that is cleaved to form two phosphate ions (Pi). A combination of real-time enzymatically coupled Pi reporter assays and end-point 31P NMR revealed that Pi is formed at a catalytically relevant rate in the native reaction of RibA with GTP, redefining the reaction stoichiometry. Furthermore, our data indicate that both PPi and GTP stimulate conformational changes prior to hydrolytic chemistry, and we conclude that the cleavage of PPi bound as a substrate or an intermediate state results in conformational relaxation.

Molecular mechanism of ATP versus GTP selectivity of adenylate kinase.

Enzymatic substrate selectivity is critical for the precise control of metabolic pathways. In cases where chemically related substrates are present inside cells, robust mechanisms of substrate selectivity are required. Here, we report the mechanism utilized for catalytic ATP versus GTP selectivity during adenylate kinase (Adk) -mediated phosphorylation of AMP. Using NMR spectroscopy we found that while Adk adopts a catalytically competent and closed structural state in complex with ATP, the enzyme is arrested in a catalytically inhibited and open state in complex with GTP. X-ray crystallography experiments revealed that the interaction interfaces supporting ATP and GTP recognition, in part, are mediated by coinciding residues. The mechanism provides an atomic view on how the cellular GTP pool is protected from Adk turnover, which is important because GTP has many specialized cellular functions. In further support of this mechanism, a structure-function analysis enabled by synthesis of ATP analogs suggests that a hydrogen bond between the adenine moiety and the backbone of the enzyme is vital for ATP selectivity. The importance of the hydrogen bond for substrate selectivity is likely general given the conservation of its location and orientation across the family of eukaryotic protein kinases.

2-Substituted 2'-deoxyinosine 5'-triphosphates as substrates for polymerase synthesis of minor-groove-modified DNA and effects on restriction endonuclease cleavage.

Five 2-substituted 2'-deoxyinosine triphosphates (dRITP) were synthesized and tested as substrates in enzymatic synthesis of minor-groove base-modified DNA. Only 2-methyl and 2-vinyl derivatives proved to be good substrates for Therminator DNA polymerase, whilst all other dRITPs and other tested DNA polymerases did not give full length products in primer extension. The DNA containing 2-vinylhypoxanthine was then further modified through thiol-ene reactions with thiols. Cross-linking reaction between cysteine-containing minor-groove binding dodecapeptide and DNA proceeded thanks to the proximity effect between thiol and vinyl groups inside the minor groove. 2-Substituted dIRTPs and also previously prepared 2-substituted 2'-deoxyadenosine triphosphates (dRATP) were then used for enzymatic synthesis of minor-groove modified DNA to study the effect of minor-groove modifications on cleavage of DNA by type II restriction endonucleases (REs). Although the REs should recognize the sequence through H-bonds in the major groove, some minor-groove modifications also had an inhibiting effect on the cleavage.

Transcription factors TFIIF and TFIIS promote transcript elongation by RNA polymerase II by synergistic and independent mechanisms.

Recent evidence suggests that transcript elongation by RNA polymerase II (RNAPII) is regulated by mechanical cues affecting the entry into, and exit from, transcriptionally inactive states, including pausing and arrest. We present a single-molecule optical-trapping study of the interactions of RNAPII with transcription elongation factors TFIIS and TFIIF, which affect these processes. By monitoring the response of elongation complexes containing RNAPII and combinations of TFIIF and TFIIS to controlled mechanical loads, we find that both transcription factors are independently capable of restoring arrested RNAPII to productive elongation. TFIIS, in addition to its established role in promoting transcript cleavage, is found to relieve arrest by a second, cleavage-independent mechanism. TFIIF synergistically enhances some, but not all, of the activities of TFIIS. These studies also uncovered unexpected insights into the mechanisms underlying transient pauses. The direct visualization of pauses at near-base-pair resolution, together with the load dependence of the pause-entry phase, suggests that two distinct mechanisms may be at play: backtracking under forces that hinder transcription and a backtrack-independent activity under assisting loads. The measured pause lifetime distributions are inconsistent with prevailing views of backtracking as a purely diffusive process, suggesting instead that the extent of backtracking may be modulated by mechanisms intrinsic to RNAPII. Pauses triggered by inosine triphosphate misincorporation led to backtracking, even under assisting loads, and their lifetimes were reduced by TFIIS, particularly when aided by TFIIF. Overall, these experiments provide additional insights into how obstacles to transcription may be overcome by the concerted actions of multiple accessory factors.

Structure and activity of the Saccharomyces cerevisiae dUTP pyrophosphatase DUT1, an essential housekeeping enzyme.

Genomes of all free-living organisms encode the enzyme dUTPase (dUTP pyrophosphatase), which plays a key role in preventing uracil incorporation into DNA. In the present paper, we describe the biochemical and structural characterization of DUT1 (Saccharomyces cerevisiae dUTPase). The hydrolysis of dUTP by DUT1 was strictly dependent on a bivalent metal cation with significant activity observed in the presence of Mg2+, Co2+, Mn2+, Ni2+ or Zn2+. In addition, DUT1 showed a significant activity against another potentially mutagenic nucleotide: dITP. With both substrates, DUT1 demonstrated a sigmoidal saturation curve, suggesting a positive co-operativity between the subunits. The crystal structure of DUT1 was solved at 2 Å resolution (1 Å=0.1 nm) in an apo state and in complex with the non-hydrolysable substrate α,ß-imido dUTP or dUMP product. Alanine-replacement mutagenesis of the active-site residues revealed seven residues important for activity including the conserved triad Asp87/Arg137/Asp85. The Y88A mutant protein was equally active against both dUTP and UTP, indicating that this conserved tyrosine residue is responsible for discrimination against ribonucleotides. The structure of DUT1 and site-directed mutagenesis support a role of the conserved Phe142 in the interaction with the uracil base. Our work provides further insight into the molecular mechanisms of substrate selectivity and catalysis of dUTPases.

NUDT16 and ITPA play a dual protective role in maintaining chromosome stability and cell growth by eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals.

Mammalian inosine triphosphatase encoded by ITPA gene hydrolyzes ITP and dITP to monophosphates, avoiding their deleterious effects. Itpa(-) mice exhibited perinatal lethality, and significantly higher levels of inosine in cellular RNA and deoxyinosine in nuclear DNA were detected in Itpa(-) embryos than in wild-type embryos. Therefore, we examined the effects of ITPA deficiency on mouse embryonic fibroblasts (MEFs). Itpa(-) primary MEFs lacking ITP-hydrolyzing activity exhibited a prolonged doubling time, increased chromosome abnormalities and accumulation of single-strand breaks in nuclear DNA, compared with primary MEFs prepared from wild-type embryos. However, immortalized Itpa(-) MEFs had neither of these phenotypes and had a significantly higher ITP/IDP-hydrolyzing activity than Itpa(-) embryos or primary MEFs. Mammalian NUDT16 proteins exhibit strong dIDP/IDP-hydrolyzing activity and similarly low levels of Nudt16 mRNA and protein were detected in primary MEFs derived from both wild-type and Itpa(-) embryos. However, immortalized Itpa(-) MEFs expressed significantly higher levels of Nudt16 than the wild type. Moreover, introduction of silencing RNAs against Nudt16 into immortalized Itpa(-) MEFs reproduced ITPA-deficient phenotypes. We thus conclude that NUDT16 and ITPA play a dual protective role for eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals.

NUDT16 is a (deoxy)inosine diphosphatase, and its deficiency induces accumulation of single-strand breaks in nuclear DNA and growth arrest.

Nucleotides function in a variety of biological reactions; however, they can undergo various chemical modifications. Such modified nucleotides may be toxic to cells if not eliminated from the nucleotide pools. We performed a screen for modified-nucleotide binding proteins and identified human nucleoside diphosphate linked moiety X-type motif 16 (NUDT16) protein as an inosine triphosphate (ITP)/xanthosine triphosphate (XTP)/GTP-binding protein. Recombinant NUDT16 hydrolyzes purine nucleoside diphosphates to the corresponding nucleoside monophosphates. Among 29 nucleotides examined, the highest k(cat)/K(m) values were for inosine diphosphate (IDP) and deoxyinosine diphosphate (dIDP). Moreover, NUDT16 moderately hydrolyzes (deoxy)inosine triphosphate ([d]ITP). NUDT16 is mostly localized in the nucleus, and especially in the nucleolus. Knockdown of NUDT16 in HeLa MR cells caused cell cycle arrest in S-phase, reduced cell proliferation, increased accumulation of single-strand breaks in nuclear DNA as well as increased levels of inosine in RNA. We thus concluded that NUDT16 is a (deoxy)inosine diphosphatase that may function mainly in the nucleus to protect cells from deleterious effects of (d)ITP.

The GTP responsiveness of PI5P4Kß evolved from a compromised trade-off between activity and specificity.

Unlike most kinases, phosphatidylinositol 5-phosphate 4-kinase ß (PI5P4Kß) utilizes GTP as a physiological phosphate donor and regulates cell growth under stress (i.e., GTP-dependent stress resilience). However, the genesis and evolution of its GTP responsiveness remain unknown. Here, we reveal that PI5P4Kß has acquired GTP preference by generating a short dual-nucleotide-recognizing motif called the guanine efficient association (GEA) motif. Comparison of nucleobase recognition with 660 kinases and 128 G proteins has uncovered that most kinases and PI5P4Kß use their main-chain atoms for adenine recognition, while the side-chain atoms are required for guanine recognition. Mutational analysis of the GEA motif revealed that the acquisition of GTP reactivity is accompanied by an extended activity toward inosine triphosphate (ITP) and xanthosine triphosphate (XTP). Along with the evolutionary analysis data that point to strong negative selection of the GEA motif, these results suggest that the GTP responsiveness of PI5P4Kß has evolved from a compromised trade-off between activity and specificity, underpinning the development of the GTP-dependent stress resilience.

Inosine Triphosphate Pyrophosphatase (ITPase): Functions, Mutations, Polymorphisms and Its Impact on Cancer Therapies.

Inosine triphosphate pyrophosphatase (ITPase) is an enzyme encoded by the ITPA gene and functions to prevent the incorporation of noncanonical purine nucleotides into DNA and RNA. Specifically, the ITPase catalyzed the hydrolysis of (deoxy) nucleoside triphosphates ((d) NTPs) into the corresponding nucleoside monophosphate with the concomitant release of pyrophosphate. Recently, thiopurine drug metabolites such as azathioprine have been included in the lists of ITPase substrates. Interestingly, inosine or xanthosine triphosphate (ITP/XTP) and their deoxy analogs, deoxy inosine or xanthosine triphosphate (dITP/dXTP), are products of important biological reactions such as deamination that take place within the cellular compartments. However, the incorporation of ITP/XTP, dITP/dXTP, or the genetic deficiency or polymorphism of the ITPA gene have been implicated in many human diseases, including infantile epileptic encephalopathy, early onset of tuberculosis, and the responsiveness of patients to cancer therapy. This review provides an up-to-date report on the ITPase enzyme, including information regarding its discovery, analysis, and cellular localization, its implication in human diseases including cancer, and its therapeutic potential, amongst others.

Effect of prenatal hypobaric hypoxia on glutamatergic signal transduction in rat brain.

Ca(2+)-mediated signal transduction of group I metabotropic glutamate receptors (ImGluR) was studied in the brain of young (15 days) and old rats (90 days) exposed to severe hypobaric hypoxia on gestation days 14-16. Changes in the concentration of bound intracellular Ca(2+) (Ca(2+) response) were evaluated after repeated application of a selective ImGluR agonist 3,5-dihydroxyphenylglycine (DHPG) to cultured brain slices. Primary application of DHPG for 2 min induced a negative Ca(2+) response in slices from 15-day-old intact animals, while repeated application caused a positive response. In slices from 90-day-old control animals, both responses were negative. In slices from rats of both age groups subjected to severe prenatal hypobaric hypoxia, both responses were mainly positive, but short-term negative components were present in adult animals. Our results suggest that severe hypobaric hypoxia changes the balance between the two constitutive signal pathways triggered by ImGluR (inosine triphosphate and diacylglycerol pathways). This procedure is followed by the increased influx of extracellular Ca(2+) (as compared to Ca(2+) release from the intracellular stores). This imbalance is particularly pronounced at the early stage of ontogeny.

Effects of amitriptyline, a tricyclic antidepressant, on smooth muscle reactivity in isolated rat trachea.

PURPOSE: This study was designed to investigate the action of amitriptyline, a tricyclic antidepressant, on airway smooth muscle reactivity and its underlying mechanisms. METHODS: In isolated rat trachea, isometric force was recorded to examine the effects of amitriptyline on the contractile response to acetylcholine (ACh), electrical field stimulation (EFS), calyculin A (a myosin light chain phosphatase inhibitor), and sphingosylphosphorylcholine (SPC; a Rhokinase activator). In addition, inositol monophosphate (IP1) accumulation was measured to examine its effects on inositol 1, 4, 5-trisphosphate (IP(3)) production during stimulation with ACh. RESULTS: Amitriptyline inhibited the contractile responses to ACh, EFS, calyculin A, and SPC, with the concentrations of amitriptyline (mean +/- SD) required to exert 50% inhibition (IC(50)) being 4.3 +/- 1.3 microM, 3.2 +/- 1.6 microM, 256.4 +/- 106.4 microM, and 98.2 +/- 21.8 microM, respectively. In addition, amitriptyline (10 microM) eliminated the ACh (10 microM)-induced IP(1) accumulation. CONCLUSION: The results suggest that amitriptyline does not influence tracheal smooth muscle reactivity at clinical concentrations (<1 microM), but attenuates the reactivity at supraclinical concentrations (> or =1 microM). The attenuated response to ACh brought about by amitriptyline is presumably due, at least in part, to the inhibition of phosphatidylinositol (PI) metabolism. The ability of amitriptyline to inhibit the calyculin Ainduced contraction suggests that amitriptyline also inhibits the Ca(2+)-calmodulin-myosin light chain pathway independently of the inhibition of PI metabolism. Finally, the difference between the IC(50) values for SPC-induced contraction and those for calyculin A-induced contraction suggests that amitriptyline may also inhibit the Rho-kinase pathway.

Metabolism and biochemical properties of nicotinamide adenine dinucleotide (NAD) analogs, nicotinamide guanine dinucleotide (NGD) and nicotinamide hypoxanthine dinucleotide (NHD).

Nicotinamide adenine dinucleotide (NAD) is an important coenzyme that regulates various metabolic pathways, including glycolysis, ß-oxidation, and oxidative phosphorylation. Additionally, NAD serves as a substrate for poly(ADP-ribose) polymerase (PARP), sirtuin, and NAD glycohydrolase, and it regulates DNA repair, gene expression, energy metabolism, and stress responses. Many studies have demonstrated that NAD metabolism is deeply involved in aging and aging-related diseases. Previously, we demonstrated that nicotinamide guanine dinucleotide (NGD) and nicotinamide hypoxanthine dinucleotide (NHD), which are analogs of NAD, are significantly increased in Nmnat3-overexpressing mice. However, there is insufficient knowledge about NGD and NHD in vivo. In the present study, we aimed to investigate the metabolism and biochemical properties of these NAD analogs. We demonstrated that endogenous NGD and NHD were found in various murine tissues, and their synthesis and degradation partially rely on Nmnat3 and CD38. We have also shown that NGD and NHD serve as coenzymes for alcohol dehydrogenase (ADH) in vitro, although their affinity is much lower than that of NAD. On the other hand, NGD and NHD cannot be used as substrates for SIRT1, SIRT3, and PARP1. These results reveal the basic metabolism of NGD and NHD and also highlight their biological function as coenzymes.

Purification and characteristics of functional properties of soluble nucleoside triphosphatase (apyrase) from bovine brain.

Soluble NTPase, differing in its properties from known proteins exhibiting NTPase activity, was purified from bovine brain to homogeneity. The enzyme has pH optimum at 7.5 and shows absolute dependence on bivalent cations and broad substrate specificity towards nucleoside-5 -tri- and -diphosphates, characteristics of apyrases. The NTPase follows Michaelis-Menten kinetics in the range of investigated substrate concentrations, the apparent K(m) values for UTP, ITP, GTP, CTP, CDP, and ATP being 86, 25, 41, 150, 500, and 260 microM, respectively. According to gel-filtration and SDS-PAGE data, the molecular mass of the enzyme is 60 kD. The NTPase is localized in the cytosol fraction and expressed in different bovine organs and tissues. Total NTPase activity of extracts of bovine organs and tissues decreases in the following order: liver > heart > skeletal muscle > lung > brain > spleen > kidney ~ small intestine. The enzyme activity can be regulated by acetyl-CoA, alpha-ketoglutarate, and fructose-1,6-diphosphate acting as activators in physiological concentrations, whereas propionate exhibits an inhibitory effect.

Endothelin receptor subtype B mediates synthesis of nitric oxide by cultured bovine endothelial cells.

Endothelins (ET) produce endothelium-dependent vasodilation through nitric oxide (NO) synthesis. The present study was designed to elucidate the cellular mechanism by which ET induces synthesis and release of endothelium-derived NO by cultured bovine endothelial cells (EC). Binding studies revealed that bovine EC membrane had the binding sites of a novel agonist (BQ3020) for non-isopeptide-selective receptor subtype (ETB). Affinity labeling studies showed a major labeled band with the apparent molecular mass of 50 kD. Northern blot analysis demonstrated the expression of mRNA for ETB receptor. BQ3020 rapidly and dose dependently induced formation of inositol-1,4,5-triphosphate and increased intracellular Ca2+ concentrations in fura-2-loaded cells. Concomitantly, BQ3020 dose dependently stimulated production of both nitrate/nitrite (NOx) and cyclic GMP; a highly significant correlation existed between NOx and cGMP production. The stimulatory effect on NOx and cGMP production by ETB agonist was inhibited by NO synthase inhibitor monomethyl-L-arginine; this effect was reversed by coaddition of L-arginine, but not D-arginine. NOx and cGMP production stimulated by BQ3020 was inhibited by pretreatment with pertussis toxin. ETB agonist-induced NOx production was blocked by a calmodulin inhibitor and an intracellular Ca2+ chelator, but not by an extracellular Ca2+ chelator or a Ca2+ channel blocker. These data suggest that endothelins stimulate ETB receptor-mediated phosphoinositide breakdown via pertussis toxin-sensitive G-protein(s), which triggers release of intracellular Ca2+, thereby activating Ca2+/calmodulin-dependent NO synthase in EC.

Enhanced ryanodine-mediated calcium release in mutant PS1-expressing Alzheimer's mouse models.

Intracellular Ca(2+) signaling involves Ca(2+) liberation through both inositol triphosphate and ryanodine receptors (IP(3)R and RyR). However, little is known of the functional interactions between these Ca(2+) sources in either neuronal physiology, or during Ca(2+) disruptions associated with Alzheimer's disease (AD). By the use of whole-cell recordings and 2-photon Ca(2+) imaging in cortical slices we distinguished between IP(3)R- and RyR-mediated Ca(2+) components in nontransgenic (non-Tg) and AD mouse models and demonstrate powerful signaling interactions between these channels. Ca(2+)-induced Ca(2+) release (CICR) through RyR contributed modestly to Ca(2+) signals evoked by photoreleased IP(3) in cortical neurons from non-Tg mice. In contrast, the exaggerated signals in 3xTg-AD and PS1(KI) mice resulted primarily from enhanced CICR through RyR, rather than through IP(3)R, and were associated with increased RyR expression levels. Moreover, membrane hyperpolarizations evoked by IP(3) in neurons from AD mouse models were even greater than expected simply from the exaggerated Ca(2+) signals, pointing to an increased coupling efficiency between cytosolic [Ca(2+)] and K(+) channel regulation. Our results highlight the critical roles of RyR-mediated Ca(2+) signaling in both neuronal physiology and pathophysiology, and point to presenilin-linked disruptions in RyR signaling as an important genetic factor in AD.

[Letter to the Editor] Incorrect assignment of affected nucleotides in footprinting/probing experiments.

Address correspondence to Sergey Belikov or Lars Wieslander, Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, SE-106 91 Stockholm, Sweden. E-mail: sergey.belikov@su.se or lars.wieslander@su.se.

Biochemical characterization of a novel hypoxanthine/xanthine dNTP pyrophosphatase from Methanococcus jannaschii.

A novel dNTP pyrophosphatase, Mj0226 from Methanococcus jannaschii, which catalyzes the hydrolysis of nucleoside triphosphates to the monophosphate and PPi, has been characterized. Mj0226 protein catalyzes hydrolysis of two major substrates, dITP and XTP, suggesting that the 6-keto group of hypoxanthine and xanthine is critical for interaction with the protein. Under optimal reaction conditions the k(ca)(t) /K(m) value for these substrates was approximately 10 000 times that with dATP. Neither endonuclease nor 3'-exonuclease activities were detected in this protein. Interestingly, dITP was efficiently inserted opposite a dC residue in a DNA template and four dNTPs were also incorporated opposite a hypoxanthine residue in template DNA by DNA polymerase I. Two protein homologs of Mj0226 from Escherichia coli and Archaeoglobus fulgidus were also cloned and purified. These have catalytic activities similar to Mj0226 protein under optimal conditions. The implications of these results have significance in understanding how homologous proteins, including Mj0226, act biologically in many organisms. It seems likely that Mj0226 and its homologs have a major role in preventing mutations caused by incorporation of dITP and XTP formed spontaneously in the nucleotide pool into DNA. This report is the first identification and functional characterization of an enzyme hydrolyzing non-canonical nucleotides, dITP and XTP.

Transfer of second messengers through gap junction connexin 43 channels reconstituted in liposomes.

Gap junction channels reconstituted in liposomes provide a pathway for the transfer of second messengers. Gap junction channels were formed in the artificial unilamellar liposomes using immunoaffinity-purified connexin 43 gap junction protein from rat brain. Sucrose-permeable and -impermeable liposomes were separated on the basis of sucrose permeability in the iso-osmolar sucrose density gradient. The liposomes permeable to sucrose were also permeable to a communicating dye molecule, Lucifer yellow. In the present study, we examined the transfer of second messengers through the connexin 43 channels reconstituted in liposomes and first report the direct evidence that the gap junction channels are permeable to second messengers including adenosine 3',5'-cyclic phosphate and inositol 1,4,5-trisphosphate.