Enzymatic synthesis of hypermodified DNA polymers for sequence-specific display of four different hydrophobic groups.

A set of modified 2'-deoxyribonucleoside triphosphates (dNTPs) bearing a linear or branched alkane, indole or phenyl group linked through ethynyl or alkyl spacer were synthesized and used as substrates for polymerase synthesis of hypermodified DNA by primer extension (PEX). Using the alkyl-linked dNTPs, the polymerase synthesized up to 22-mer fully modified oligonucleotide (ON), whereas using the ethynyl-linked dNTPs, the enzyme was able to synthesize even long sequences of >100 modified nucleotides in a row. In PCR, the combinations of all four modified dNTPs showed only linear amplification. Asymmetric PCR or PEX with separation or digestion of the template strand can be used for synthesis of hypermodified single-stranded ONs, which are monodispersed polymers displaying four different substituents on DNA backbone in sequence-specific manner. The fully modified ONs hybridized with complementary strands and modified DNA duplexes were found to exist in B-type conformation (B- or C-DNA) according to CD spectral analysis. The modified DNA can be replicated with high fidelity to natural DNA through PCR and sequenced. Therefore, this approach has a promising potential in generation and selection of hypermodified aptamers and other functional polymers.

Cytotoxic and Mutagenic Properties of C3'-Epimeric Lesions of 2'-Deoxyribonucleosides in Escherichia coli Cells.

Reactive oxygen species (ROS), resulting from endogenous metabolism and/or environmental exposure, can induce damage to the 2-deoxyribose moiety in DNA. Specifically, a hydrogen atom from each of the five carbon atoms in 2-deoxyribose can be abstracted by hydroxyl radical, and improper chemical repair of the ensuing radicals formed at the C1', C3', and C4' positions can lead to the stereochemical inversion at these sites to yield epimeric 2-deoxyribose lesions. Although ROS-induced single-nucleobase lesions have been well studied, the biological consequences of the C3'-epimeric lesions of 2'-deoxynucleosides, i.e., 2'-deoxyxylonucleosides (dxN), have not been comprehensively investigated. Herein, we assessed the impact of dxN lesions on the efficiency and fidelity of DNA replication in Escherichia coli cells by conducting a competitive replication and adduct bypass assay with single-stranded M13 phage containing a site-specifically incorporated dxN. Our results revealed that, of the four dxN lesions, only dxG constituted a strong impediment to DNA replication, and intriguingly, dxT and dxC conferred replication bypass efficiencies higher than those of the unmodified counterparts. In addition, the three SOS-induced DNA polymerases (Pol II, Pol IV, and Pol V) did not play any appreciable role in bypassing these lesions. Among the four dxNs, only dxA directed a moderate frequency of dCMP misincorporation. These results provided important insights into the impact of the C3'-epimeric lesions on DNA replication in E. coli cells.

Synthesis and nonenzymatic template-directed polymerization of 2'-amino-2'-deoxythreose nucleotides.

Threose nucleic acid (TNA) is a potential alternative genetic material that may have played a role in the early evolution of life. We have developed a novel synthesis of 2'-amino modified TNA nucleosides (2'-NH2-TNA) based on a cycloaddition reaction between a glycal and an azodicarboxylate, followed by direct nucleosidation of the cycloadduct. Using this route, we synthesized the thymine and guanine 2'-NH2-TNA nucleosides in seven steps with 24% and 12% overall yield, respectively. We then phosphorylated the guanine nucleoside on the 3'-hydroxyl, activated the phosphate as the 2-methylimidazolide, and tested the ability of the activated nucleotide to copy C4 RNA, DNA, and TNA templates by nonenzymatic primer extension. We measured pseudo-first-order rate constants for the first nucleotide addition step of 1.5, 0.97, and 0.57 h(-1) on RNA, DNA, and TNA templates, respectively, at pH 7.5 and 4 °C with 150 mM NaCl, 100 mM N-(hydroxylethyl)imidazole catalyst, and 5 mM activated nucleotide. The activated nucleotide hydrolyzed with a rate constant of 0.39 h(-1), causing the polymerization reaction to stall before complete template copying could be achieved. These extension rates are more than 1 order of magnitude slower than those for amino-sugar ribonucleotides under the same conditions, and copying of the TNA template, which best represented a true self-copying reaction, was the slowest of all. The poor kinetics of 2'-NH2-TNA template copying could give insight into why TNA was ultimately not used as a genetic material by biological systems.

Feeding the deoxyribonucleoside salvage pathway to rescue mitochondrial DNA.

Mutations in an increasing number of nuclear genes involved in deoxyribonucleotide homeostasis cause disorders associated with somatic mitochondrial DNA (mtDNA) abnormalities. Dysfunction of the products of these genes leads to limited availability of substrates for mtDNA replication and results in mtDNA depletion, multiple deletions or point mutations; mtDNA depletion is the molecular feature linked to greatest clinical severity. In this review, we discuss recent results demonstrating that enhancement of the salvage pathways by increasing the availability of deoxyribonucleosides needed for each specific genetic defect prevents mtDNA depletion. Hence, we propose administration of selected deoxyribonucleosides and/or inhibitors of their catabolism as a pharmacological strategy to treat these diseases.

Survival of DNA damage in yeast directly depends on increased dNTP levels allowed by relaxed feedback inhibition of ribonucleotide reductase.

In eukaryotes, DNA damage elicits a multifaceted response that includes cell cycle arrest, transcriptional activation of DNA repair genes, and, in multicellular organisms, apoptosis. We demonstrate that in Saccharomyces cerevisiae, DNA damage leads to a 6- to 8-fold increase in dNTP levels. This increase is conferred by an unusual, relaxed dATP feedback inhibition of ribonucleotide reductase (RNR). Complete elimination of dATP feedback inhibition by mutation of the allosteric activity site in RNR results in 1.6-2 times higher dNTP pools under normal growth conditions, and the pools increase an additional 11- to 17-fold during DNA damage. The increase in dNTP pools dramatically improves survival following DNA damage, but at the same time leads to higher mutation rates. We propose that increased survival and mutation rates result from more efficient translesion DNA synthesis at elevated dNTP concentrations.

Interaction of DNA containing Fapy.dA or its C-nucleoside analogues with base excision repair enzymes. Implications for mutagenesis and enzyme inhibition.

Fapy.dA is produced in DNA as a result of oxidative stress. Recently, this lesion and its C-nucleoside analogues were incorporated in chemically synthesized oligonucleotides at defined sites. The interaction of DNA containing Fapy.dA or nonhydrolyzable analogues with Fpg and MutY is described. Fpg efficiently excises Fapy.dA (K(m) = 1.2 nM, k(cat) = 0.12 min(-1)) opposite T. The lesion is removed as efficiently from duplexes containing Fapy.dA:dA or Fapy.dA:dG base pairs. Multiple turnovers are observed for the repair of Fapy.dA mispairs in a short period of time, indicating that the enzyme does not remain bound to the product duplex. MutY does not incise dA from a duplex containing this nucleotide opposite Fapy.dA, nor does it exhibit an increased level of binding compared to DNA composed solely of native base pairs. MutY also does not incise Fapy.dA when the lesion is opposite dG. These data suggest that Fapy.dA could be deleterious to the genome. Fpg strongly binds duplexes containing the beta-C-nucleoside analogue of Fapy.dA (beta-C-Fapy.dA) opposite all native nucleotides (K(D) < 27 nM), as well as the alpha-C-nucleoside (alpha-C-Fapy.dA) opposite dC (K(D) = 7.1 +/- 1.5 nM). A duplex containing a beta-C-Fapy.dA:T base pair is an effective inhibitor (K(I) = 3.5 +/- 0.3 nM) of repair of Fapy.dA by Fpg, suggesting the C-nucleoside may have useful therapeutic properties.

Depletion of the other genome-mitochondrial DNA depletion syndromes in humans.

We present the current knowledge on the genetic and phenotypic aspects of mitochondrial DNA depletion syndromes. The human mitochondrial DNA encodes 13 of the 82 structural proteins of the mitochondrial electron transport chain. The replication and maintenance of the mtDNA require a large number of nuclear encoded enzymes and balanced nucleotide pools. Mitochondrial nucleotide synthesis is of major importance because of the constant need for nucleotides for mtDNA maintenance even in quiescent cells. As de novo enzymes are not present in the mitochondria, synthesis is accomplished via the salvage pathway. Defective mtDNA synthesis and maintenance manifest by multiple deletions or by depletion of the mitochondrial genome. Patients with multiple deletions typically present with progressive external ophthalmoplegia, ptosis and, exercise intolerance after the first decade of life. mtDNA depletion is usually an infantile disease characterized by severe muscle weakness, hepatic failure, or renal tubulopathy with fatal outcome. Linkage analysis in families with multiple mtDNA deletions reveal mutations in proteins that participate in mtDNA replication, the mitochondrial DNA polymerase gene, and the Twinkle gene, a putative mitochondrial helicase and in factors which play a role in mitochondrial nucleotide metabolism, the adenine nucleotide translocator, and the thymidine phosphorylase gene. We have recently identified mutations in an additional two essential proteins in the nucleotide salvage pathway, the mitochondrial deoxyribonucleoside kinases. The phenotype was distinctive for each gene, with hepatic failure and encephalopathy associated with mutations in the deoxyguanosine kinase gene and isolated devastating myopathy as the sole manifestation of thymidine kinase 2 deficiency. The tissue selectivity of these disorders and especially the exclusive muscle involvement in thymidine kinase 2 mutations is puzzling. The normal sequence of the remaining mtDNA copies in spite of a serious mitochondrial nucleotide imbalance is also unexpected. We propose several tissue-specific protective mechanisms and a time window, likely encompassing fetal life and even early infancy, during which nuclear nucleotide synthesis provides mitochondrial needs in all organs. We also speculate on future genes to be discovered in other phenotypes of mtDNA depletion.

Minimising the secondary structure of DNA targets by incorporation of a modified deoxynucleoside: implications for nucleic acid analysis by hybridisation.

Some regions of nucleic acid targets are not accessible to heteroduplex formation with complementary oligonucleotide probes because they are involved in secondary structure through intramolecular Watson-Crick pairing. The secondary conformation of the target may be destabilised to assist its interaction with oligonucleotide probes. To achieve this, we modified a DNA target, which has self-complementary sequence able to form a hairpin loop, by replacing dC with N:4-ethyldeoxycytidine (d(4Et)C), which hybridises specifically with natural dG to give a G:(4Et)C base pair with reduced stability compared to the natural G:C base pair. Substitution by d(4Et)C greatly reduced formation of the target secondary structure. The lower level of secondary structure allowed hybridisation with complementary probes made with natural bases. We confirmed that hybridisation could be further enhanced by modifying the probes with intercalating groups which stabilise the duplex.

In vitro evolution of the hammerhead ribozyme to a purine-specific ribozyme using mutagenic PCR with two nucleotide analogues.

The conventional hammerhead ribozyme cleaves RNA 3' to nucleotide triplets with the general formula NUH, where N is any nucleotide, U is uridine and H is any nucleotide except guanosine. In order to isolate hammerhead ribozyme sequences capable of cleaving 3' to the GUG triplet, we performed a mutagenic selection protocol starting with the conventional sequence of an NUH-cleaving ribozyme. The 22 nucleotides in the core and the stem-loop II region were subjected to mutagenic PCR using the two nucleotide analogues 6-(2-deoxy-beta-d-ribofuranosyl)-3,4-dihydro-8H-pyrimido-[4,5-C)][1, 2] oxazin-7-one and of 8-oxo-2'-deoxyguanosine. After five repetitions of the selection cycle, several clones showed cleavage activity. One sequence, having one deletion, showed at least a 90 times higher in trans cleavage rate than the starting ribozyme. It cleaved 3' to GUG and GUA. The sequence of this ribozyme is essentially identical with that obtained previously by selection for AUG cleavage starting with a randomised core and stem-loop II region. This identical result of two independent selection procedures supports the notion that sequences for NUR cleavage, where R is a purine nucleotide, are not compatible with the classical hammerhead structure, and that the sequence space for this cleavage specificity is very limited. The cleavage of NUR triplets is not restricted to the sequence of the substrate that was used for selection but is sequence-independent for in trans cleavage, although the sequence context influences the value for the cleavage rate somewhat. Analysis of cleavage activities indicates the importance of A at position L2.5 in loop II.

Nucleoside analogue substitutions in the trinucleotide DNA template recognition sequence 3'-(CTG)-5' and their effects on the activity of bacteriophage T7 primase.

Bacteriophage T7 primase catalyzes the synthesis of the oligoribonucleotides pppACC(C/A) and pppACAC from the single-stranded DNA template sites 3'-d[CTGG(G/T)]-5' and 3'-(CTGTG)-5', respectively. The 3'-terminal deoxycytidine residue is conserved but noncoding. A series of nucleoside analogues have been prepared and incorporated into the conserved 3'-d(CTG)-5' site, and the effects of these analogue templates on T7 primase activity have been examined. The nucleosides employed include a novel pyrimidine derivative, 2-amino-5-(beta-2-deoxy-D-erythro-pentofuranosyl)pyridine (d2APy), whose synthesis is described. Template sites containing d2APy in place of the cryptic dC support oligoribonucleotide synthesis whereas those containing 3-deaza-2'-deoxycytidine (dc(3)C) and 5-methyl-6-oxo-2'-deoxycytidine (dm(5ox)C) substitutions do not, suggesting that the N3 nitrogen of cytidine is used for a critical interaction by the enzyme. Recognition sites containing 4-amino-1-(beta-2-deoxy-D-erythro-pentofuranosyl)-5-methyl-2,6[1H, 3H]-pyrimidione (dm(3)2P) or 2'-deoxyuridine (dU) substitutions for dT support oligoribonucleotide synthesis whereas those containing 5-methyl-4-pyrimidinone 2'-deoxyriboside (d(2H)T) substitutions do not, suggesting the importance of Watson-Crick interactions at this template residue. Template sites containing 7-deaza-2'-deoxyguanosine (dc(7)G) or 2'-deoxyinosine (dI) in place of dG support oligoribonucleotide synthesis. The reduced extent to which dc(7)G is successful within the template suggests a primase-DNA interaction. Inhibition studies suggest that the primase enzyme binds "null" substrates but cannot initiate RNA synthesis.

Misincorporation of 2'-deoxyoxanosine into DNA: a molecular basis for NO-induced mutagenesis derived from theoretical calculations.

A wide range of theoretical methods, including high level ab initio, density functional, self-consistent reaction field, molecular dynamics and thermodynamic integration calculations, have been used to analyze the mutagenic properties of oxanosine. The major tautomeric forms in the gas phase and aqueous solution have been determined. The ability of oxanosine to recognize thymine and cytosine in the gas phase and in the DNA environment has been compared with that of guanine. A physicochemical explanation for the mutagenic properties of oxanosine is suggested.

Tyrosine 222, a member of the YXDD motif of MuLV RT, is catalytically essential and is a major component of the fidelity center.

Tyrosine 222 of MuLV RT is an invariant residue of the highly conserved YXDD motif in the reverse transcriptase class of enzymes. The residue X is Met 184 in HIV-1 RT and Val 223 in MuLV RT. This residue has been implicated in the fidelity of DNA synthesis, whereas the role of the preceding tyrosine in this aspect, as well as in the catalytic mechanism of MuLV RT, remains to be elucidated. We have substituted Tyr 222 with Phe, Ser, and Ala by site-directed mutagenesis and have characterized the properties of the individual mutant enzymes. The results show that Tyr-->Phe substitution did not affect the polymerase activity of the enzyme, while Tyr-->Ser and Tyr-->Ala substitutions significantly reduced the polymerase activity. The pyrophosphorolysis activities of these mutants showed the same trend as the polymerase activities, suggesting an essential role for Y222 in the catalytic mechanism of MuLV RT. One of the most interesting observations of Y-->F substitution was the significantly increased fidelity of DNA synthesis on RNA templates. In addition, a limited extent of ribonucleotide incorporation on RNA template that was consistently noted with the wild-type enzyme was reduced with the Y222F mutant. The resistance to all four ddNTPs, however, persisted in the wild type and Y222 mutants on the RNA template. A ternary complex model of MuLV RT shows that (a) the aromatic ring of Tyr/Phe is positioned between the terminal and penultimate primer bases and (b) the phenolic OH group is seen within hydrogen bonding distance with the base moieties of two template and penultimate primer nucleotides. We propose that the base stacking interaction of Tyr 222 stabilizes the primer terminus position which is essential for the catalytic reaction. However, the weaker stacking interaction of Y compared to F, due to polarization of the pi-charge toward the phenoxyl-OH as well as the resonating character of its H-bond center, may provide slight flexibility to the position of the template base which may be responsible for the error-proneness of MuLV RT.

Misincorporation of 2'-deoxyoxanosine 5'-triphosphate by DNA polymerases and its implication for mutagenesis.

2'-Deoxyoxanosine (dOxo) is a novel DNA lesion produced by the reaction of 2'-deoxyguanosine (dGuo) with nitrous acid and nitric oxide [Suzuki, T., Yamaoka, R., Nishi, M., Ide, H., and Makino, K. (1996) J. Am. Chem. Soc. 118, 2515-2516]. In this work, 2'-deoxyoxanosine 5'-triphosphate (dOTP) was prepared by nitrous acid treatment of 2'-deoxyguanosine 5'-triphosphate (dGTP), and its incorporation into DNA by DNA polymerases was investigated to elucidate the substrate and mutagenic properties of dOTP. Primed M13mp18 DNA was replicated by Escherichia coli DNA polymerase I Klenow fragment (Pol I Kf) in the presence of three normal dNTPs and dOTP or 2'-deoxyxanthosine 5'-triphosphate (dXTP), another major product of reaction of dGTP with nitrous acid and nitric oxide. dOTP substituted for dGTP and to a lesser extent for dATP, while dXTP substituted slightly for dGTP but not for dATP. Neither dOTP nor dXTP substituted for dCTP and dTTP. The similar results were obtained for the incorporation by T7 DNA polymerase deficient in 3'-5' exonuclease [T7(exo-)]. To quantify the substitution efficiency, kinetic parameters for incorporation of dOTP and dXTP opposite template C or T by Pol I Kf (exo-) were determined and compared with those for dGTP using oligodeoxynucleotide templates. Incorporation efficiencies (f = Vmax/Km) of dOTP (f = 0.28% min-1 microM-1) and dXTP (f = 0.10% min-1 microM-1) opposite template C were much lower than that of dGTP (f = 1506% min-1 microM-1). Frequencies of mutagenic incorporation of dOTP opposite template T were dependent on the nearest neighbor base pairs, and 1.6-3.9-fold higher than those for dGTP with the nearest neighbors containing G.C pairs. dXTP was not incorporated opposite template T with all four nearest neighbors. These data suggest that formation of dOTP, but not dXTP, from dGTP with nitrous acid or nitric oxide in the intracellular nucleotide pool would result in the elevation of the mutation frequency.

Determinants of ribose specificity in RNA polymerization: effects of Mn2+ and deoxynucleoside monophosphate incorporation into transcripts.

The catalytic specificity of T7 RNA polymerase (RNAP) for ribonucleoside triphosphates vs deoxynucleoside triphosphates {(kcat/Km)rNTP/(kcat/Km)dNTP} during transcript elongation is approximately 80. Mutation of tyrosine 639 to phenylalanine reduces specificity by a factor of approximately 20 and largely eliminates the Km difference between rNTPs and dNTPs. The remaining specificity factor of approximately 4 is kcat-mediated and is nearly eliminated if Mn2+ is substituted for Mg2+ in the reaction. Mn2+ substitution does not significantly affect the Km difference between rNTPs and dNTPs. Mn2+ substitution also enhances the activity of poorly active mutant enzymes carrying nonconservative substitutions in the active site, and its effects are generally consistent with the Mn2+-catalyzed reaction being less restrictive in its requirements for alignment of the reactive groups. In addition to discrimination occurring at the level of nucleoside monophosphate (NMP) incorporation, it is also found that transcripts containing deoxynucleoside monophosphates (dNMPs) are more poorly extended than transcripts of canonical structure, though a severe barrier to transcript extension is seen only when the 3' region of the transcript is heavily substituted with dNMPs. The barrier to extension of transcripts heavily substituted with dNMPs is reduced for sequences known to be amenable to forming A-like helices and is larger for sequences that resist transformation from B-form DNA.DNA structures. The barrier to extension of dNMP-substituted transcripts is also reduced by solution conditions known to destabilize B-form DNA and to stabilize A-form structures. These observations imply a requirement for a non-B-form, possibly A-like, conformation in the transcript.template hybrid that is disrupted when the transcript is of predominantly deoxyribose structure.

International Commission for Protection Against Environmental Mutagens and Carcinogens. Deoxyribonucleoside triphosphate levels: a critical factor in the maintenance of genetic stability.

DNA precursor pool imbalances can elicit a variety of genetic effects and modulate the genotoxicity of certain DNA-damaging agents. These and other observations indicate that the control of DNA precursor concentrations is essential for the maintenance of genetic stability, and suggest that factors which offset this control may contribute to environmental mutagenesis and carcinogenesis. In this article, we review the biochemical and genetic mechanisms responsible for regulating the production and relative amounts of intracellular DNA precursors, describe the many outcomes of perturbations in DNA precursor levels, and discuss implications of such imbalances for sensitivity to DNA-damaging agents, population monitoring, and human diseases.

[Transposition of the deo operon structural genes in Escherichia coli K-12 to plasmid RP4 using bacteriophage mu]. / Transpozitsiia strukturnykh genov deo-operona Escherichia coli K-12 s pomoshch'iu bakteriofaga Mu na plazmidu RP4.

Transposition of the structural genes of the deo operon of Escherichia coli K-12 into plasmid RP4 by means of temperate bacteriophage Mu was carried out. Some variants of composite RP4-deo-Mu plasmids were obtained and the expression of the deo genes integrated into the RP4 plasmid genome was studied. It was shown that the expression of these genes remains under the control of the chromosomal regulatory genes (deoR and cytR); although the activity of thymidine phosphorilase in the strain E. coli which contains hybrid plasmid is 4-6 fold greater than that in strains of E. coli with chromosomal localization of the deo operon.