inosine-triphosphate and 2--deoxyinosine-triphosphate

Topics: Deoxyribonucleotides; DNA Restriction Enzymes; DNA, Recombinant; Inosine Triphosphate; Mutagenesis; Polymerase Chain Reaction; Templates, Genetic

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.

Topics: Deoxyribonucleases, Type II Site-Specific; DNA; DNA Restriction Enzymes; DNA-Directed DNA Polymerase; Hydrogen Bonding; Inosine Triphosphate; Nucleic Acid Conformation; Structure-Activity Relationship; Substrate Specificity; Vinyl Compounds

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.

Topics: Deoxyguanine Nucleotides; DNA Footprinting; DNA Primers; DNA Probes; DNA-Binding Proteins; Inosine Triphosphate; Reverse Transcription; RNA, Ribosomal, 18S; Sequence Analysis, DNA

An alternative method to combine mutagenesis PCR with dITP and fragmentation by endonuclease V for directed evolution was developed. In comparison to the routine protocol for directed evolution, dITP was used as mutation reagent in the mutagenesis PCR. Subsequently, the incorporated dITP in the PCR products could represent as being the target of endonuclease V. Finally, the mutated dsDNA was fragmented by endonuclease V and then shuffled via assembly and reamplification as is usually done. In this study, the gene encoding kanamycin resistance has been used as reporter to verify the novel method for directed evolution. However, the mutation frequency could be easily adjusted by the amount of dITP used in the mutagenesis PCR reaction. Besides, this protocol yielded the mutation types with an obvious bias to transition substitutions as the normal error-prone PCR did. Conclusively, this novel method for directed evolution has been demonstrated to be efficient, reproducible, and easy to handle in actual practice. Using this protocol, we have successfully constructed a random mutation library for the gene encoding a serine alkaline protease.

Topics: Cloning, Molecular; Deoxyribonuclease (Pyrimidine Dimer); Directed Molecular Evolution; DNA; DNA Fragmentation; DNA Shuffling; Escherichia coli; Gene Library; Inosine Triphosphate; Kanamycin; Mutagenesis; Mutation; Polymerase Chain Reaction; Reproducibility of Results; Sequence Analysis, DNA

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,β-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.

Topics: Amino Acid Sequence; Catalytic Domain; Cations, Divalent; Crystallography, X-Ray; Deoxyuracil Nucleotides; Inosine Triphosphate; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Pyrophosphatases; Saccharomyces cerevisiae; Sequence Alignment; Substrate Specificity

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.

Topics: Acid Anhydride Hydrolases; Animals; Cell Proliferation; Cells, Cultured; Chromosomal Instability; Inosine Diphosphate; Inosine Nucleotides; Inosine Triphosphatase; Inosine Triphosphate; Mice; Mice, Knockout; Phenotype; Pyrophosphatases

In this paper, we describe the synthesis and thermal stabilities of the triplexes containing either 2'-deoxyinosine (1) or 2'-deoxyxanthosine (3) in their second strands. It was found that the triplexes with the 2'-deoxy-5-methylcytidine(dM)*1:dC and dM*1:dA base triplets are thermally stable, but those containing the dM*1:T and dM*1:dG base triplets are unstable under both neutral and slightly acidic conditions. On the other hand, it was found that the oligonucleotide containing 3 could form thermally stable triplexes with the oligonucleotides that involve four natural bases opposite the sites of 3. The rank of the thermal stabilities of the triplexes was as follows: the triplex containing the dM*3:dC base triplet > that containing the dM*3:dA base triplet > that containing the dM*3:T base triplet > that containing the dM*3:dG base triplet.

Topics: Base Composition; Deoxyribonucleosides; DNA; Hot Temperature; Inosine Triphosphate; Nucleic Acid Conformation; Nucleic Acid Denaturation; Structure-Activity Relationship

A hypothetical 21.0 kDa protein (ORF O197) from Escherichia coli K-12 was cloned, purified, and characterized. The protein sequence of ORF O197 (termed EcO197) shares a 33.5% identity with that of a novel NTPase from Methanococcus jannaschii. The EcO197 protein was purified using Ni-NTA affinity chromatography, protease digestion, and gel filtration column. It hydrolyzed nucleoside triphosphates with an O6 atom-containing purine base to nucleoside monophosphate and pyrophosphate. The EcO197 protein had a strong preference for deoxyinosine triphosphate (dITP) and xanthosine triphosphate (XTP), while it had little activity in the standard nucleoside triphosphates (dATP, dCTP, dGTP, and dTTP). These aberrant nucleotides can be produced by oxidative deamination from purine nucleotides in cells; they are potentially mutagenic. The mutation protection mechanisms are caused by the incorporation into DNA of unwelcome nucleotides that are formed spontaneously. The EcO197 protein may function to eliminate specifically damaged purine nucleotide that contains the 6-keto group. This protein appears to be the first eubacterial dITP- and XTPhydrolyzing enzyme that has been identified.

Topics: Acid Anhydride Hydrolases; Amino Acid Sequence; Base Sequence; Cloning, Molecular; DNA, Bacterial; Escherichia coli; Escherichia coli Proteins; Hydrolysis; Inosine Triphosphate; Methanococcus; Molecular Sequence Data; Nucleoside-Triphosphatase; Ribonucleotides; Sequence Homology, Amino Acid

A common problem in automated DNA sequencing when applying the Sanger chain termination method is ambiguous base calling caused by band compressions. Band compressions are caused by anomalies in the migration behavior of certain DNA fragments in the polyacrylamide gel because of intramolecular base pairing between guanine and cytosine residues. To reduce such undesired secondary structures, several modifications of the sequencing reaction parameters have been performed previously. Here, we have applied mixtures of the nucleotide analogs 7-deaza-dGTP and dITP instead of dGTP in the cycle sequencing reaction and in combination with varying buffer conditions. Band compressions were particularly well resolved, and reading length was optimal when a ratio of 7-deaza-dGTP:dITP of 4:1 was used in the in vitro DNA synthesis with AmpliTaq FS DNA polymerase. We conclude that the incorporation of both nucleotide analogs at these particular ratios leads to heterogeneous DNA chains that result in a reduction or elimination of intramolecular base pairing and thus a higher accuracy in the base assignment.

Topics: Base Pairing; Deoxyguanine Nucleotides; DNA, Single-Stranded; Electrophoresis, Polyacrylamide Gel; Inosine Triphosphate; Nucleic Acid Conformation; Sequence Analysis, DNA; Taq Polymerase; Templates, Genetic

The ability to selectively amplify RNA in the presence of genomic DNA of analogous sequence is cumbersome and requires implementation of critical controls for genes lacking introns. The convenient approaches of either designing oligonucleotide primers at the splice junction or differentiating the target sequence based on the size difference obtained by the presence of the intron are not possible. Our strategy for the selective amplification of RNA targets is based on the enzymology of a single thermostable DNA polymerase and the ability to modulate the strand separation temperature requirements for PCR amplification. Following reverse transcription of the RNA by recombinant Thermus thermophilus DNA polymerase (rTth pol), the resulting RNAxDNA hybrid is digested by the RNase H activity of rTth pol, allowing the PCR primer to hybridize and initiate second-strand cDNA synthesis. Substitution of one or more conventional nucleotides with nucleotide analogs that decrease base stacking interactions and/or hydrogen bonding (e.g. hydroxymethyldUTP or dITP) during the first- and second-strand cDNA synthesis step reduces the strand separation temperature of the resultant DNAxDNA duplex. Alteration of the thermal cycling parameters of the subsequent PCR amplification, such that the strand separation temperature is below that required for denaturation of genomic duplex DNA composed of standard nucleotides, prevents the genomic DNA from being denatured and therefore amplified.

Topics: DNA-Directed DNA Polymerase; HL-60 Cells; Humans; Inosine Triphosphate; Polymerase Chain Reaction; Recombinant Proteins; RNA, Bacterial; Thermus thermophilus

Conditional cold-sensitive mutations in Era, an essential Escherichia coli GTPase, were isolated. Localized random polymerase chain reaction (PCR) mutagenesis employing Taq and T7 DNA polymerases under error prone amplification conditions was exploited to generate mutations in the era gene. A plasmid exchange technique was used to identify conditional cold-sensitive mutations in Era that give rise to defective cell growth below 30 degrees C. Three recessive missense mutations in Era, N26S, A156D, and E200K, were isolated. All three mutations are located at residues conserved in Era homologues from Streptococcus mutans and Coxiella burnetti.

Topics: Amino Acid Sequence; Bacterial Proteins; Base Sequence; Cloning, Molecular; Cold Temperature; Coxiella burnetii; DNA-Directed DNA Polymerase; DNA, Bacterial; Escherichia coli; Escherichia coli Proteins; Genes, Recessive; GTP Phosphohydrolases; GTP-Binding Proteins; Inosine Triphosphate; Molecular Sequence Data; Mutagenesis; Plasmids; Polymerase Chain Reaction; RNA-Binding Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Species Specificity; Streptococcus mutans; Taq Polymerase

Topics: Base Composition; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; DNA; DNA Nucleotidylexotransferase; DNA-Directed DNA Polymerase; Inosine Triphosphate; Plasmids; Sequence Analysis, DNA

Topics: Deoxyguanine Nucleotides; Genes, p53; Inosine Triphosphate; Polymerase Chain Reaction; Protein Structure, Secondary; Sequence Analysis

Topics: Base Sequence; Deoxyguanine Nucleotides; DNA; DNA-Directed DNA Polymerase; Electrophoresis, Polyacrylamide Gel; Inosine Triphosphate; Molecular Sequence Data; Nucleotide Mapping