Nucleic Acid Residues Research Articles

Assignments and hydrogen bond sensitivities of UV resonance Raman bands of the C8‐deuterated guanine ring

AbstractIsotope‐edited Raman spectroscopy, a combination of site‐selective isotopic labeling and Raman difference spectroscopy, is a useful method for studying the structure and interaction of individual nucleic acid residues in oligonucleotides. To obtain basic data for applying isotope‐edited Raman spectroscopy to guanine residues, we studied the vibrational modes of UV resonance Raman bands of the C8‐deuterated guanine ring by examining the wavenumber shifts upon seven isotopic substitutions (2‐13C, 2‐15N, 6‐18O, 7‐15N, 8‐13C, 9‐15N and 1′‐13C). The hydrogen bond sensitivities of the Raman bands were also investigated by comparing the Raman spectra recorded in several solvents of different hydrogen bonding properties. Some of the Raman bands were found to be markers of hydrogen bonding at specific donor or acceptor sites on the guanine ring. The Raman bands, which shift on C8‐deuteration, remain in the difference spectrum between the unlabeled and C8‐deuterated guanine rings. Among them, a negative peak around 1525 cm−1 and a strong positive/negative peak pair around 1485/1465 cm−1 serve as markers of hydrogen bonding at N7 and C6O, respectively. Another weak positive/negative peak pair around 1025/1040 cm−1 is sensitive to hydrogen bonding at the proton donor sites (N1—H and N2—H2). The applicability of the hydrogen bond markers has been tested by using a 22‐mer oligonucleotide duplex containing eight guanine residues and its analog in which a single guanine residue is C8‐deuterated. The difference spectrum shows that the hydrogen bonding state of the guanine residue at the labeled position is consistent with the Watson–Crick base pair structure of DNA. Isotope‐edited Raman spectroscopy is a useful tool for studying the hydrogen bonding state of selected guanine residues in oligonucleotides. Copyright © 2002 John Wiley & Sons, Ltd.

Oligonucleotide directed misfolding of RNA inhibits Candida albicans group I intron splicing.

RNA is becoming an important therapeutic target. Many potential RNA targets require secondary or tertiary structure for function. Examples include ribosomal RNAs, RNase P RNAs, mRNAs with untranslated regions that regulate translation, and group I and group II introns. Here, a method is described to inhibit RNA function by exploiting the propensity of RNA to adopt multiple folded states that are of similar free energy. This method, called oligonucleotide directed misfolding of RNA (ODMiR), uses short oligonucleotides to stabilize inactive structures. The ODMiR method is demonstrated with the group I intron from Candida albicans, a human pathogen. The oligonucleotides, (L)(TACCTTTC) and T(L)CT(L)AC(L)GA(L)CG(L)GC(L)C, with L denoting a locked nucleic acid residue, inhibit 50% of group I intron splicing in a transcription mixture at about 150 and 30 nM oligonucleotide concentration, respectively. Both oligonucleotides induce misfolds as determined by native gel electrophoresis and diethyl pyrocarbonate modification. The ODMiR approach provides a potential therapeutic strategy applicable to RNAs with secondary or tertiary structures required for function.

The crystallographic structure of brome mosaic virus

The structure of brome mosaic virus (BMV), the type member of the bromoviridae family, has been determined from a single rhombohedral crystal by X-ray diffraction, and refined to an R value of 0.237 for data in the range 3.4–40.0 Å. The structure, which represents the native, compact form at pH 5.2 in the presence of 0.1 M Mg 2+, was solved by molecular replacement using the model of cowpea chlorotic mottle virus (CCMV), which BMV closely resembles. The BMV model contains amino acid residues 41–189 for the pentameric capsid A subunits, and residues 25–189 and 1–189 for the B and C subunits, respectively, which compose the hexameric capsomeres. In the model there are two Mg ions and one molecule of polyethylene glycol (PEG). The first 25 amino acid residues of the C subunit are modeled as polyalanine. The coat protein has the canonical “jellyroll” β-barrel topology with extended amino-terminal polypeptides as seen in other icosahedral plant viruses. Mass spectrometry shows that in native BMV virions, a significant fraction of the amino-terminal peptides are apparently cleaved. No recognizable nucleic acid residue is visible in the electron density maps except at low resolution where it appears to exhibit a layered arrangement in the virion interior. It is juxtaposed closely with the interior surface of the capsid but does not interpenetrate. The protein subunits forming hexameric capsomeres, and particularly dimers, appear to interact extensively, but the subunits otherwise contact one another sparsely about the 5-fold and quasi 3-fold axes. Thus, the virion appears to be an assembly of loosely associated hexameric capsomeres, which may be the basis for the swelling and dissociation that occurs at neutral pH and elevated salt concentration. A Mg ion is observed to lie exactly on the quasi-3-fold axis and is closely coordinated by side-chains of three quasi-symmetry-related residues glutamates 84, with possible participation of side-chains from threonines 145, and asparagines 148. A presumptive Mg 2+ is also present on the 5-fold axis where there is a concentration of negatively charged side-chains, but the precise coordination is unclear. In both cases these cations appear to be essential for maintenance of virion stability. Density that is contiguous with the viral interior is present on the 3-fold axis at the center of the hexameric capsomere, where there is a pore of about 6 Å diameter. The density cannot be attributed to cations and it was modeled as a PEG molecule.

Inhibition of HIV-1 Tat-Dependent Trans Activation by Steric Block Chimeric 2‘-O-Methyl/LNA Oligoribonucleotides

The HIV-1 trans-activation responsive element (TAR) RNA 59-residue stem-loop interacts with the HIV trans-activator protein Tat and other cellular factors to stimulate transcriptional elongation from the viral long terminal repeat (LTR). Inhibition of these interactions blocks full-length HIV transcription and hence replication. We have found that three types of 12-residue oligonucleotide analogues, namely, a 2'-O-methyl oligoribonucleotide (OMe), a chimeric oligonucleotide containing 7xOMe and 5x5-methyl C locked nucleic acid (LNA) residues, and a peptide nucleic acid (PNA), inhibit Tat-dependent in vitro transcription in HeLa cell nuclear extract equally efficiently (50% inhibition at 100-200 nM) and sequence specifically. The results are correlated with surprisingly similar binding strengths to a model 39-residue TAR under transcription conditions. A 12-mer containing 11 contiguous LNA residues was less effective in both Tat-dependent transcription inhibition and TAR 39 binding. Anti-TAR 3'-carboxyfluorescein- (FAM-) labeled OMe and OMe/LNA chimeric 12-mers were also efficient Tat-dependent in vitro transcription inhibitors as were 3'-FAM-labeled OMe oligonucleotides containing some phosphorothioate (PS) linkages. By use of a HeLa cell line containing stably integrated plasmids expressing firefly luciferase under HIV-LTR/Tat dependence as well as a Renilla luciferase constitutive control, we showed submicromolar, selective, dose-dependent, and sequence-dependent intracellular inhibition of Tat-TAR trans activation by the anti-TAR 3'-FAM 12-residue 7xOMe/5xLNA oligonucleotide when delivered by cationic lipid. No intracellular activity was observed for the corresponding anti-TAR 3'-FAM OMe 12-mer. An alternating PS-containing 3'-FAM OMe 12-mer oligonucleotide exhibited partial inhibition of trans-activation activity, but this was correlated with a similar effect on control gene expression, suggesting nonspecific inhibition.

Characterization of individual adenine residues in DNA by a combination of site-selective C8-deuteration and UV resonance Raman difference spectroscopy

Raman spectroscopy has been combined with site-selective isotopic labeling techniques to obtain structural information on a selected nucleic acid residue in oligonucleotides. In the difference spectrum between the unlabeled and site-selectively labeled oligonucleotides, the Raman signals from the residue at the labeled position show up, while the signals from the residues at unlabeled positions are canceled out. To demonstrate the utility of this new method, we have prepared a self-complementary tetradecamer DNA, d(AGTGCTCG AGC ACT) 2, containing single C8-deuterated adenine at position 9 (A9) or 12 (A12). UV (257 nm) resonance Raman difference spectra between the unlabeled and labeled oligonucleotides in solution reveal slightly different microenvironments around A9 and A12 as expected for a double-stranded helical structure of the tetradecamer DNA. In the presence of an antitumor antibiotic, actinomycin D, which intercalates into the 5′-GC-3′ sequence of DNA, the Raman signals of A9 on the 5′-side of the intercalation site become significantly weaker, indicating an increased base stacking with adjacent guanine bases. In contrast, the Raman signals of A12 on the 3′-side are not affected by the binding of actinomycin D. This observation provides the first experimental evidence that the intercalation of actinomycin D induces an asymmetric structural alteration of DNA in solution.

Crystal structure of HIV-1 reverse transcriptase in complex with a polypurine tract RNA:DNA.

We have determined the 3.0 A resolution structure of wild-type HIV-1 reverse transcriptase in complex with an RNA:DNA oligonucleotide whose sequence includes a purine-rich segment from the HIV-1 genome called the polypurine tract (PPT). The PPT is resistant to ribonuclease H (RNase H) cleavage and is used as a primer for second DNA strand synthesis. The 'RNase H primer grip', consisting of amino acids that interact with the DNA primer strand, may contribute to RNase H catalysis and cleavage specificity. Cleavage specificity is also controlled by the width of the minor groove and the trajectory of the RNA:DNA, both of which are sequence dependent. An unusual 'unzipping' of 7 bp occurs in the adenine stretch of the PPT: an unpaired base on the template strand takes the base pairing out of register and then, following two offset base pairs, an unpaired base on the primer strand re-establishes the normal register. The structural aberration extends to the RNase H active site and may play a role in the resistance of PPT to RNase H cleavage.

X-ray crystal structure of a locked nucleic acid (LNA) duplex composed of a palindromic 10-mer DNA strand containing one LNA thymine monomer

Locked nucleic acid (LNA), a recently introduced nucleic acid analogue with a bicyclic 2′-O,4′-C-methylene linked furanose sugar, exhibits enhanced affinities for DNA and RNA relative to the corresponding oligodeoxyribonucleotides and oligoribonucleotides; we report the first crystal structure of an LNA unit incorporated in an oligonucleotide duplex. The structure at 1.4 A resolution of the DNA–LNA decamer duplex with one LNA thymine monomer per strand provides a detailed view of the conformation and hydration of locked nucleic acid residues in a duplex A-form.

Characterization of a Transcription Factor Binding Site, Specifically Activating MIA Transcription in Melanoma

We have previously isolated the protein MIA, which is secreted from melanoma cells, and identified highly restricted expression patterns in melanocytic tumors. Preliminary studies of the human MIA gene provided evidence that the promoter is specifically activated in melanoma cells but is silent in nonmelanocytic cells and benign melanocytes. In this study we aimed to identify cis-regulatory promoter elements that mediate promoter activation during malignant transformation of melanocytes. We therefore subcloned 1.4 kb of the murine MIA promoter and a series of 5' deletion constructs into a luciferase reporter plasmid and identified the most active cis-regulatory element between nucleic acids -230 and -130. Cloning oligomeric fragments of this promoter region in front of a minimal TK promoter revealed a 30 bp enhancer element mediating expression of the reporter gene in melanoma cells but not in melanocytes or nonmelanocytic cells. Gel mobility shift assays and southwestern blots led to the identification of specific DNA-protein complexes in melanoma cells. Fine mutational analysis of the cis-regulatory promoter element showed that two critical nucleic acid residues are essential for both transcriptional activity and formation of the band shift complexes. By an initial small-scale affinity purification we were able to isolate a protein approximately 32 kDa in size from melanoma cells, which we refer to as MATF ("melanoma-associated transcription factor"). Our study identified for the first time MATF, a transcription factor that is upregulated or activated during malignant transformation of melanocytes.

Proton-coupled electron transfer in the oxidation of guanines by an aromatic pyrenyl radical cation in aqueous solutions

Electron transfer reactions between nucleic acid residues in DNA and strong oxidants are often the critical initial steps that initiate oxidative, irreversible DNA damage. Employing laser flash photolysis transient absorption spectroscopic techniques, we investigated the characteristics of electron transfer reactions in aqueous solutions between the 2′-deoxynucleoside 5′-monophosphates, dGMP, dAMP, dCMP and dTMP and a representative one-electron oxidant. The latter was a radical cation of a pyrene derivative with enhanced water-solubility, 7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene (BPT). The BPT radical cation BPT•+, was generated by intense nanosecond laser pulse (308 or 355 nm, 50–70 mJ pulse−1 cm−2) by a non-linear consecutive two-photon absorption process. No electron transfer reactions were observed with dAMP, dTMP and dCMP, consistent with their unfavorable redox potentials. However, BPT•+ efficiently oxidized dGMP with a rate constant kb=1.7±0.1)×109 M−1 s−1, which is smaller than the diffusion-controlled value by a factor of only ∽3. The dGMP(-H)• neutral radicals formed on time scales of a few microseconds, were identified by their characteristic transient absorption spectrum (λmax∽310 mn). The rate constant of electron transfer from dGMP to BPT•+ was smaller in D2O by a factor of ∽1.5 than in H2O. This kinetic isotope effect indicates that the electron transfer reaction from dGMP to BPT•+ is accompanied by the deprotonation of dGMP•+, and therefore appears to be a proton-coupled electron transfer reaction.

Aspects of oligonucleotide and peptide sequencing with MALDI and electrospray mass spectrometry

Biopolymer sequencing with mass spectrometry has become increasingly important and accessible with the development of matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). Here we examine the use of sequential digestion for the rapid identification of proteolytic fragments, in turn highlighting the general utility of enzymatic MALDI ladder sequencing and ESI tandem mass spectrometry. Analyses were performed on oligonucleotides ranging in size from 2 to 50 residues, on peptides ranging in size from 7 to 44 residues and on viral coat proteins. MALDI ladder sequencing using exonuclease digestion generated a uniform distribution of ions and provided complete sequence information on the oligonucleotides 2–30 nucleic acid residues long. Only partial sequence information was obtained on the longer oligonucleotides. C-terminal peptide ladder sequencing typically provided information from 4 to 7 amino acids into the peptide. Sequential digestion, or endoprotease followed by exoprotease exposure, was also successfully applied to a trypsin digest of viral proteins. Analysis of ladder sequenced peptides by LCMS generated less information than in the MALDI-MS analysis and ESI-MS2 normally provided partial sequence information on both the small oligonucleotides and peptides. In general, MALDI ladder sequencing offered information on a broader mass range of biopolymers than ESI-MS2 and was relatively straightforward to interpret, especially for oligonucleotides.

Beta2-adrenoceptor polymorphisms are in linkage disequilibrium, but are not associated with asthma in an adult population.

To determine the association between the beta2-adrenoceptor polymorphisms at amino acids 16 and 27 and markers of allergic disease and asthma per se in a random adult population, and to determine the degree of linkage disequilibrium existing between polymorphisms at amino acid positions 16, 27, 164 and nucleic acid residue 523. We measured serum IgE, skin-prick test positivity, atopy, bronchial hyperreactivity, wheeze and asthma (self-reported and doctor-diagnosed), and determined beta2-adrenoceptor genotype by allele specific oligonucleotide hybridization, in 630 adults aged between 18 and 70, selected from the electoral role in a local health authority in Nottingham. Homozygotes for the Glycine 16 polymorphism had a significantly higher incidence of atopy (chi2=6.44 (Pearson's), P=0.04). We also observed a significant association between the Glycine 16 allele and atopy (chi2=4.13 (Pearson's), P=0.04), when we assumed the Glycine 16 allele to operate in a dominant mode. No other significant associations between beta2-adrenoceptor polymorphisms and markers of allergic disease and asthma per se were observed. Marked linkage disequilibrium exists between the beta2-adrenoceptor polymorphisms at amino acid 16 and 27 (D=0.38, chi2 P<0.0001), and between the beta2-adrenoceptor polymorphisms at amino acid 27 and nucleic acid residue 523 (C-A) (D=0.36, chi2 P<0.0001). There is no consistent association between beta2-adrenoceptor polymorphisms and the risk of developing allergic disease or asthma per se in this adult sample. Marked linkage disequilibrium exists between the amino acid 16 and 27 polymorphisms, and also between the amino acid 27 polymorphism and the nucleic acid residue 523 (C-A) polymorphism. This polymorphism accounts for the Ban 1 RFLP previously described at the beta2-adrenoceptor locus on chromosome 5q 31.

Parameterization and efficient implementation of a solvent model for linear-scaling semiempirical quantum mechanical calculations of biological macromolecules

A method is developed to include solvation effects in linear-scaling semiempirical quantum calculations. Favorable scaling of computational effort for large molecules is achieved using a preconditioned conjugate gradient technique in conjunction with a linear-scaling recursive bisection method for evaluation of electrostatic interactions. The method requires approximately 30% computational overhead relative to gas-phase calculations. Effective atomic radii for biological macromolecules are derived from fitting to experimental and theoretical solvation energies for small molecules homologous to amino- and nucleic acid residues.

In vitro selection of aptamers: the dearth of pure reason

In vitro selection experiments are now routinely used to identify functional nucleic acid residues and structures, and have become a tool for studying molecular recognition, molecular biology, and molecular evolution. Technical innovations that have been made during the past year include the use of modified monomers to increase stability and photocross-linking reagents to improve affinity. These advances should dramatically increase the utility of aptamers in the future.

Molecular cloning and expression of a delta-opioid receptor from rat brain.

We have isolated and characterized a rat delta-opioid receptor. The deduced amino acid sequence (372 aa) closely resembles the murine delta-opioid receptor, DOR-1. In fact, 97% of the amino acid residues are conserved between the two species, while 93% of the nucleic acid residues are identical. A 6 kb mRNA was detected in rat cortex using rat DOR-1 as a probe. When expressed in COS cells, the clone shows high-affinity opioid binding with selectivity for delta-opioids. The rat delta-opioid receptor cDNA clone will be a useful tool for studying the function of delta-opioid receptor in rats.

Mutations in the gene encoding for the beta 2-adrenergic receptor in normal and asthmatic subjects.

It has long been hypothesized that a defective beta 2-adrenergic receptor (beta 2AR) may be a pathogenic factor in bronchial asthma. We examined the gene encoding the beta 2AR to assess the frequency of polymorphisms in 51 patients with moderate to severe asthma and 56 normal subjects. Nine different point mutations were found in both heterozygous and homozygous forms at nucleic acid residues 46, 79, 100, 252, 491, 523, 1053, 1098, and 1239. No mutations resulting in large deletions or frame shifts were detected. Of these nine polymorphisms, four were found to cause changes in the encoded amino acids at residues 16, 27, 34, and 164. The most frequent polymorphisms were arginine 16 to glycine (Arg16-->Gly) and glutamine 27 to glutamic acid (Gln27-->Glu). The other two polymorphisms, valine 34 to methionine, and threonine 164 to isoleucine, occurred in only four subjects. The incidence of beta 2AR homozygous polymorphisms was no greater in asthmatic patients as compared with controls (Arg16-->Gly: 53% versus 59%, Gln27-->Glu: 24% versus 29%, respectively; P = NS). Some subjects were found to have both of these polymorphisms simultaneously, but there was no difference in incidence between the two groups, with 23% of asthmatics and 28% of normal subjects being homozygous for both polymorphisms. The apparently normal subjects with both polymorphisms did not have subclinical hyperreactive airways disease as determined by methacholine challenge testing. In the asthma group, one mutation (Arg16-->Gly) identified a subset of patients with a distinct clinical profile.(ABSTRACT TRUNCATED AT 250 WORDS)

Degree of linear polarization method for determination of intrinsic optical anisotropy of southern bean mosaic virus

AbstractSouthern bean mosaic virus (SBMV) is a spherical plant virus. It possesses an intrinsic (structural) anisotropy due to the orientation of valence bonds of the amino acid and/or nucleic acid residues. According to the light scattering theory of Rayleigh and Gans for optically anisotropic spheres, the degree of linear polarization of the scattered light depends on the intrinsic anisotropy, the relative refractive index of the sphere to the solvent, the scattering angle, and the inclination angle of linearly polarized incident light to the scattering plane. By using the theoretical expression of the degree of linear polarization, the intrinsic anisotropy parameter of southern bean mosaic virus in the compact spherical state was calculated with the appropriate experimental values. This novel method should be useful in elucidating the internal structure of spherical viruses and other spherical complexes of macromolecules. © 1993 John Wiley &amp; Sons, Inc.

Relative specificities in binding of Watson-Crick base pairs by third strand residues in a DNA pyrimidine triplex motif.

The specificity of binding of Watson-Crick base pairs by third strand nucleic acid residues via triple helix formation was investigated in a DNA pyrimidine triplex motif by thermal melting experiments. The host duplex was of the type A10-X-A10: T10-Y-T10, and the third strand T10-Z-T10, giving rise to 16 possible triplexes with Z:XY inserts, 4 duplexes with the Watson-Crick base pairs (XY) and 12 duplexes with mismatch pairs (XZ), all of whose stabilities were compared. Two Z:XY combinations confirm the primary binding of AT and GC target pairs in homopurine.homopyrimidine sequences by T and C residues, respectively. All other Z:XY combinations in the T:AT environment result in triplex destabilization. While some related observations have been reported, the present experiments differ importantly in that they were performed in a T:AT nearest neighbor environment and at physiological ionic strength and pH, all of which were previously untested. The conclusions now drawn also differ substantially from those in previous studies. Thus, by evaluating the depression in Tm due to base triplet mismatches strictly in terms of third strand residue affinity and specificity for the target base pair, it is shown that none of the triplet combinations that destabilize qualify for inclusion in the third strand binding code for the pyrimidine triplex motif. Hence, none of the mismatch triplets afford a general way of circumventing the requirement for homopurine.homopyrimidine targets when third strands are predominated by pyrimidines, as others have suggested. At the same time, the applicability of third strand binding is emphasized by the finding that triplexes are equally or much more sensitive to base triplet mismatches than are Watson-Crick duplexes to base pair mismatches.

Nucleic Acid Content and Residue Determination in Tissues of Chicks Born From 2,4-Dichlorophenoxyaetic Butyl Ester Treated Eggs

The effects of 2,4-dichlorophenoxyacetic butyl ester (2,4-D b.e.) on protein and nucleic acid contents of different tissues of chicks born from externally treated hen's eggs were studied. Residues of 2,4-D b.e. were also determined and quantified by gas liquid chromatography (GLC). A diminished DNA content was observed in skeletal muscle and stomach, an increased one was found in cerebrum and cerebellum, whereas no variations were observed in the other analysed tissues. Protein levels were found decreased in kidney, cerebrum and heart. Besides, the GLC determinations revealed the presence of the 2,4-D b.e. in all the studied tissues finding the highest concentrations in kidney. 2,4-D did not appear to be a significant metabolite of 2,4-D b.e. in this experimental system. The dissimilar effects of 2,4-D b.e. on protein and nucleic acid contents of the studied tissues would be related to a different tissue susceptibility to the drug and/or to drug distribution.

Reaction mechanisms of trans-9,10-dihydroxy-anti-7,8-epoxy-7,8,9,10-tetrahydro-3-methylcho lan threne with DNA in aqueous solutions. Conformation of adducts.

The reaction mechanisms of trans-9,10-dihydroxy-anti-7,8-epoxy-7,8,9,10-tetrahydro-3-methylcholanth rene (anti-3-MCDE) in aqueous phosphate buffer solutions (pH 7, 24 degrees C) containing double-stranded DNA, and the structure of the covalent adduct formed, were studied by utilizing the techniques of absorption, fluorescence and linear dichroism spectroscopy. The results were compared with those obtained with the widely studied trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaPDE). The reaction mechanisms are similar in both cases since 3-MCDE also appears to form physical complexes which give rise to an acceleration of the rate of reaction of the diol-epoxide (by as much as a factor of 30 at pH 7, depending on the DNA and salt concentrations). While in the case of 3-MCDE the physical association constant is about four times lower, and the overall rate of reaction is at least five times smaller (depending on the DNA concentration) than in the case of BaPDE, the level of covalent binding is two times higher (18 +/- 2% of the 3-MCDE molecules appear as covalent adducts, the remainder forming hydrolysis products). In a novel extension of flow linear dichroism techniques, it is shown that the orientations of small aromatic moieties bound to DNA whose absorption spectra overlap with those of the nucleic acid residues below 300 nm can be studied; the dichroism at 265 nm resulting from the anthracene-like aromatic residue of 3-MCDE covalently bound to DNA suggests that its long axis tends, on average, to be tilted away from the average orientations of the planes of the nucleic acids. This conformation is similar to the one observed with adducts derived from the covalent binding of the highly tumorigenic (+) enantiomer of BaPDE, while the adducts derived from other biologically less active stereoisomers of BaPDE are characterized by conformations in which the aromatic moieties tend to be more parallel to the planes of the nucleic acid bases. Differences in the biological activities of the various polycyclic aromatic diol-epoxides are discussed in terms of these conformations and the abilities of these molecules to bind covalently to DNA.

DNA precursor pool: a significant target for N-methyl-N-nitrosourea in C3H/10T1/2 clone 8 cells.

Synchronized C3H/10T1/2 clone 8 cells were treated in vitro with a nontoxic dose of N-methyl-N-nitrosourea during their S phase. Chromatographic isolation of the deoxyribonucleotide DNA precursor pool and measurement of the precursor content per cell showed that a nucleic acid residue in the precursor pool is 190-13,000 times more susceptible to methylation than a residue in the DNA duplex, depending on the site of methylation. This conclusion comes from measurements indicating that, for example, the N-1 position of adenine in dATP is 6.3 times more methylated than the same position in the DNA, even though the adenine content of the pool is only a fraction (0.0005) of the adenine content of the DNA helix. The comparative susceptibility between pool and DNA was found to vary with the site of methylation in the order the N-1 position of adenine greater than phosphate greater than the N-3 position of adenine greater than the O6 position of guanine greater than the N-7 position of guanine. The significance of these results for chemical mutagenesis and carcinogenesis is discussed.