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It has been widely accepted that 5-methylcytosine is the only form of DNA methylation in mammalian genomes. Here we identify [N.sup.6]-methyladenine as another form of DNA modification in mouse embryonic stem cells. Alkbh1 encodes a demethylase for [N.sup.6]-methyIadenine. An increase of W6-methyladenine levels in Alkbh1-deficient cells leads to transcriptional silencing. W6-methyladenine deposition is inversely correlated with the evolutionary age of LINE-1 transposons; its deposition is strongly enriched at young (6 million years old) L1 elements. The deposition of [N.sup.6]-methyladenine correlates with epigenetic silencing of such LINE-1 transposons, together with their neighbouring enhancers and genes, thereby resisting the gene activation signals during embryonic stem cell differentiation. As young full-length LINE-1 transposons are strongly enriched on the X chromosome, genes located on the X chromosome are also silenced. Thus, [N.sup.6]-methyladenine developed a new role in epigenetic silencing in mammalian evolution distinct from its role in gene activation in other organisms. Our results demonstrate that [N.sup.6]- methyladenine constitutes a crucial component of the epigenetic regulation repertoire in mammalian genomes., DNA methylation is a crucial component of epigenetic regulation that controls many important aspects of mammalian biology, such as imprinting, X chromosome inactivation and tumorigenesis (1,2). The prevailing dogma states [...]

Natriuretic peptide receptors A (NPR-A) and B (NPR-B) mediate most effects of natriuretic peptides by synthesizing cGMP. ATP increases the activity of these receptors by an unknown mechanism. We recently reported that a nonhydrolyzable form of ATP, adenylyl imidodiphosphate (AMPPNP), stabilizes but is not required for the activation of NPR-A and NPR-B in membranes from highly overexpressing cells. Here, we repeated these studies on receptors expressed in endogenous settings. Kinetic analysis indicated that both AMPPNP and ATP dramatically decrease the apparent [K.sub.m] of both receptors for GTP but had little effect on the [V.sub.max]. The [EC.sub.50] for AMPPNP decreased as substrate concentration increased whereas the magnitude of the effect was greater at lower GTP concentrations. ATP increased the activity of a mutant receptor containing glutamates substituted for all known phosphorylation sites similarly to the wild-type receptor, consistent with a phosphorylation independent mechanism. Finally, the putative ATP binding sites were investigated. Mutation of the ATP modulatory domain region had no effect, but mutation of K535A dramatically diminished ANP-dependent cyclase activity in a manner that was unresponsive to ATP. Mutation of the highly conserved 630-KSS to AAA (all alanines) resulted in an expressed receptor that had no detectable guanylyl cyclase activity. We conclude that ATP is not required for the initial activation of NPRs but does increase activity over time by reducing the apparent [K.sub.m] for GTP. guanylyl cyclase; cyclic guanosine monophosphate; guanosine triphosphate; adenosine triphosphate; Michaelis-Menten constant; heart failure; hypertension; bone growth

The intrinsic properties of hypoxanthine are studied by examining the gas-phase acidity and proton affinity by using quantum mechanical calculations and gas-phase mass spectrometric methods. The acidity in gas phase is compared with that in solution and it is shown that a nonpolar environment has increased the differences in acidity among hypoxanthine, adenine and guanine.

Some adenine methyltransferases have been shown not only to protect specific DNA restriction sites from cleavage by a restriction endonuclease, but also to play a role in various bacterial processes and sometimes in bacterial virulence. This study focused on a type I restriction--modification system (designated yrml) of Y. pseudotuberculosis. This system is composed of three adjacent genes which could potentially encode an [N.sup.6]-adenine DNA methylase (YamA), an enzyme involved in site-specific recognition (YrsA) and a restriction endonuclease (YreA). Screening of 85 isolates of Y. pestis and Y. pseudotuberculosis indicated that the yrml system has been lost by Y. pestis and that yamA (but not yrsA or yreA) is present in all Y. pseudotuberculosis strains tested, suggesting that it may be important at some stages of the epidemiological cycle of this species. To further investigate the role of yamA in Y. pseudotuberculosis survival, multiplication or virulence, a [DELTA]yamA mutant of Y. pseudotuberculosis IP32953 was constructed by allelic exchange with a kanamycin cassette. The fact that [DELTA]yamA mutants were obtained indicated that this gene is not essential for Y. pseudotuberculosis viability. The IP32953[DELTA]yamA mutant strain grew as well as the wild-type in a rich medium at both 28 [degrees]C and 37 [degrees]C. It also grew normally in a chemically defined medium at 28 [degrees]C, but exhibited a growth defect at 37 [degrees]C. In contrast to the Dam adenine methyltransferase, a mutation in yamA did not impair the functions of DNA repair or resistance to detergents. However, the [DELTA]yamA mutant exhibited a virulence defect in a mouse model of intragastric infection. The in silico analysis indicated that the chromosomal region carrying the Y. pseudotuberculosis yrml locus has been replaced in Y. pestis by a horizontally acquired region which potentially encodes another methyltransferase. YamA might thus be dispensable for Y. pestis growth and virulence because this species has acquired another gene fulfilling the same functions.

Salmonella enterica serovar Typhimurium that lacks the DNA adenine methylase (Dam) ectopically expresses multiple genes that are preferentially expressed during infection, is attenuated for virulence, and confers heightened immunity in vaccinated hosts. The safety of dam mutant Salmonella vaccines was evaluated by screening within infected mice for isolates that have an increased capacity to cause disease relative to the attenuated parental strain. Since dam mutant strains are sensitive to the DNA base analog 2-aminopurine (2-AP), we screened for 2-AP-resistant (2-[AP.sup.r]) isolates in systemic tissues of mice infected with dam mutant Salmonella. Such 2-[AP.sup.r] derivatives were isolated following intraperitoneal but not oral administration and were shown to be competent for infectivity via intraperitoneal but not oral infection of naive mice. These 2-[AP.sup.r] derivatives were deficient in methyl-directed mismatch repair and were resistant to nitric oxide, yet they retained the bile-sensitive phenotype of the parental dam mutant strain. Additionally, introduction of a mutH null mutation into dam mutant cells suppressed the inherent defects in intraperitoneal infectivity and nitric oxide resistance, as well as overexpression of SpvB, an actin cytotoxin required for Salmonella systemic survival. These data suggest that restoration of intraperitoneal virulence of dam mutant strains is associated with deficiencies in methyl-directed mismatch repair that correlate with the production of systemically related virulence functions.

Homoadenine self-duplexes are formed when pyrene units are covalently bound to bases positioned at specific distances. The results of several spectroscopic experiments have suggested that the unique signal at 580 nm arises mainly from intermolecular self-duplex formation between the [A.sup.Py]-substituted oligodeoxyadenylates.

Anionic states of nucleic acid bases are involved in DNA damage by low-energy electrons and in charge transfer through DNA. Previous gas phase studies of free, unsolvated nucleic acid base parent anions probed only dipole-bound states, which are not present in condensed phase environments, but did not observe valence anionic states, which for purine bases are thought to be adiabatically unbound. Contrary to this expectation, we have demonstrated that some thus far ignored tautomers of adenine, which result from enamine-imine transformations, support valence anionic states with electron vertical detachment energies as large as 2.2 eV, and at least one of these anionic tautomers is adiabatically bound. Moreover, we predict that the new anionic tautomers should also dominate in solutions and should be characterized by larger values of electron vertical detachment energy than the canonical valence anion. All of the newfound anionic tautomers might be formed in the course of dissociative electron attachment followed by a hydrogen atom attachment to a carbon atom, and they might affect the structure and properties of DNA and RNA exposed to low-energy electrons. The new valence states observed here, unlike the dipole-bound state, could exist in condensed phases and might be relevant to radiobiological damage. The discovery of these valence anionic states of adenine was facilitated by the development of (i) an experimental method for preparing parent anions of nucleic acid bases for photoelectron experiments, and (ii) a combinatorial/quantum chemical approach for identification of the most stable tautomers of organic molecules. nucleic acid bases | photoelectron spectroscopy | tautomers | valente anion

Comparative genomic analysis has revealed limited strain diversity between Salmonella pathogenic and nonpathogenic isolates. Thus, some of the relative virulence and host-immune response disparities may be credited to differential gene regulation rather than gross differences in genomic content. Here we show that altered levels of Salmonella DNA adenine methylase (Dam) resulted in acute defects in virulence-associated gene expression, motility, flagellin synthesis, and bile resistance in the Salmonella pathogenic strain 14028 but not in avirulent laboratory strain LT2. The defects in motility exhibited by 14028 in response to altered Dam levels was not dependent on the presence of the regulatory protein, RpoS. The transitioning between flagellar types (phase variation) was also differentially regulated in 14028 versus LT2 in response to dam levels, resulting in distinct differences in flagellin expression states. These data suggest that differential gene regulation may contribute to the relative virulence disparities observed between Salmonella serovars that are closely related at the DNA level.

Escherichia coli MutY and MutS increase replication fidelity by removing adenines that were mis-incorporated opposite 7,8-dihydro-8-oxo-deoxyguanines (8-oxoG), G, or C. MutY DNA glycosylase removes adenines from these mismatches through a short-patch base excision repair pathway and thus prevents G:C-to-T:A and A:T-to-G:C mutations. MutS binds to the mismatches and initiates the long-patch mismatch repair on daughter DNA strands. We have previously reported that the human MutY homolog (hMYH) physically and functionally interacts with the human MutS homolog, hMutS[alpha] (Y. Gu et al., J. Biol. Chem. 277:11135-11142, 2002). Here, we show that a similar relationship between MutY and MutS exists in E. coli. The interaction of MutY and MutS involves the Fe-S domain of MutY and the ATPase domain of MutS. MutS, in eightfold molar excess over MutY, can enhance the binding activity of MutY with an A/8-oxoG mismatch by eightfold. The MutY expression level and activity in mutS mutant strains are sixfoid and twofold greater, respectively, than those for the wild-type cells. The frequency of A:T-to-G:C mutations is reduced by two- to threefold in a mutS mutY mutant compared to a mutS mutant. Our results suggest that MutY base excision repair and mismatch repair defend against the mutagenic effect of 8-oxoG lesions in a cooperative manner.

Translation initiation requires the precise positioning of a ribosome at the start codon. The major signals of bacterial mRNA that direct the ribosome to a translational start site are the Shine-Dalgarno (SD) sequence within the untranslated leader and the start codon. Evidence for the presence of many non-SD-led genes in prokaryotes provides a motive for studying additional interactions between ribosomes and mRNA that contribute to translation initiation. A high incidence of adenines has been reported downstream of the start codon for many Escherichia coli genes, and addition of downstream adenine-rich sequences increases expression from several genes in E. coli. Here we describe site-directed mutagenesis of the E. coli aroL, pncB, and cysJ coding sequences that was used to assess the contribution of naturally occurring adenines to in vivo expression and in vitro ribosome binding from mRNAs with different SD-containing untranslated leaders. Base substitutions that decreased the downstream adenines by one or two nucleotides decreased expression significantly from aroL-, pncB-, and cysJ-lacZ fusions; mutations that increased downstream adenines by one or two nucleotides increased expression significantly from aroL- and cysJ-lacZ fusions. Using primer extension inhibition (toe-print) and filter binding assays to measure ribosome binding, the changes in in vivo expression correlated closely with changes in in vitro ribosome binding strength. Our data are consistent with a model in which downstream adenines influence expression through their effects on the mRNA-ribosome association rate and the amount of ternary complex formed. This work provides evidence that adenine-rich sequence motifs might serve as a general enhancer of E. coli translation.

Distinct photophysical behavior of nucleobase adenine and its constitutional isomer, 2-aminopurine, has been studied by using quantum chemical methods, in particular an accurate ab initio multiconfigurational second-order perturbation theory. After light irradiation, the efficient, ultrafast energy dissipation observed for nonfluorescent 9H-adenine is explained here by the nonradiative internal conversion process taking place along a barrierless reaction path from the initially populated [sup.1]([pi][pi]*[L.sub.a]) excited state toward a low-lying conical intersection (CI) connected with the ground state. In contrast, the strong fluorescence recorded for 2-aminopurine at 4.0 eV with large decay lifetime is interpreted by the presence of a minimum in the [sup.1]([pi][pi]*[L.sub.a]) hypersurface lying below the lowest CI and the subsequent potential energy barrier required to reach the funnel to the ground state. Secondary deactivation channels were found in the two systems related to additional CIs involving the [sup.1]([pi][pi]*[L.sub.b]) and [sup.1](n[pi]*) states. Although in 9H-adenine a population switch between both states is proposed, in 7H-adenine this may be perturbed by a relatively larger barrier to access the [sup.1](n[pi]*) state, and, therefore, the [sup.1]([pi][pi]*[L.sub.b]) state becomes responsible for the weak fluorescence measured in aqueous adenine at [approximately equal to] 4.5 eV. In contrast to previous models that explained fluorescence quenching in adenine, unlike in 2-aminopurine, on the basis of the vibronic coupling of the nearby [sup.1]([pi][pi*]) and [sup.1](n[pi*]) states, the present results indicate that the [sup.1](n[pi]*) state does not contribute to the leading photophysical event and establish the prevalence of a model based on the CI concept in modern photochemistry. conical intersections | DNA photophysics | fluorescence quenching | quantum chemistry | ultrafast decay

The 'affinity maturation' of the humoral immune response is driven by antigen-activated somatic hypermutation (SHM) of the genes that encode antibody variable regions and the subsequent antigenic selection of mutant clones. The molecular mechanism of SHM is yet to be completely elucidated. SHM affects cytosine-guanine (C/G) and adenine--thymine (A/T) pairs with approximately equal frequency in vivo. The proposition that error-prone DNA-dependent DNA synthesis explains A/T-targeted hypermutagenesis seems to have mainstream support within the hypermutation research community at present. A major feature of SHM in vivo is that C/G hypermutation is strand unbiased, whereas A/T hypermutation is strand biased. We show that the 'DNA-based polymerase error' model of A/T-targeted hypermutagenesis does not explain this important aspect of SHM.

Resonance Raman and UV-vis spectroscopy is used to investigate the interactions between the active site in unmanipulated photolyase and UV-damaged DNA. The substrate binding of unmanipulated Escherichia coli photolyase induces changes in both the resonance Raman and the absorption spectrum of flavin adenine dinucleotide (FADH.).

The conformational molecular dynamics of the flavin adenine dinucleotide (FAD) cofactor in aqueous solution are studied. The study shows that FAD displays a highly heterogeneous intensity decay, resulting in lifetime spectra with two major components.

Byline: E. G. Malygin (1), L. G. Ovechkina (1), A. A. Evdokimov (1), V. V. Zinoviev (1) Keywords: DNA-methyltransferase; oligonucleotides; pre-steady-state kinetics; protein subunit crosslinking; fluorescence; recognition specificity Abstract: Interaction of T4 DNA-(N6-adenine)-methyltransferase was studied with a variety of synthetic oligonucleotide substrates containing the native recognition site GATC or its modified variants. The data obtained in the decisecond and second intervals of the reaction course allowed for the first time the substrate methylation rates to be compared with the parameters of the steady-state reaction. It was established that the substrate reaction proceeds in two stages. Because it is shown that in steady-state conditions T4 MTase forms a dimeric structure, the following sequence of events is assumed. Upon collision of a T4 MTase monomer with an oligonucleotide duplex, an asymmetrical complex forms in which the enzyme randomly oriented relative to one of the strands of the specific recognition site catalyzes a fast transfer of the methyl group from S-adenosylmethionine to the adenosine residue (k .sub.1 = 0.21 s.sup.--1). Simultaneously, a second T4 MTase subunit is added to the complex, providing for the continuation of the reaction. In the course of a second stage, which is by an order of magnitude slower (k .sub.2 = 0.023 s.sup.--1 for duplex with the native site), the dimeric T4 MTase switches over to the second strand and the methylation of the second residue, target. The rate of the methyl group transfer from donor, S-adenosylmethionine, to DNA is much higher than the overall rate of the T4 MTase-catalyzed steady-state reaction, although this difference is considerably less than that shown for EcoRI MTase. Base substitutions and deletions in the recognition site affect the substrate parameters in different fashions. When the GAT sequence is disrupted, the proportion of the initial productive enzyme--substrate complexes is usually sharply reduced. The flipping of the adenosine residue to be modified in the recognition site upon interaction with the enzyme, revealed by fluorescence titration, supports the existing notions about the involvement of such a DNA deformation in reactions catalyzed by various DNA-MTases. Author Affiliation: (1) Institute of Molecular Biology, State Research Center of Virology and Biotechnology VECTOR, Kol'tsovo, Novosibirsk Region, 630559, Russia Article History: Registration Date: 08/10/2004

A density functional theory (DFT) study are performed in order to explore the influences of oxygen at the C8 position on the intramolecular proton transfer between the 6-oxo form and 6-hydro form of C8 -oxidative adenine. The 8-oxo form with the oxygen at the C8 position is found to be energetically favored by the purines.

Addition of phosphoryl chloride/formamide solution to 6-(glycosylamino)-5-nitrosopyrimidines resulted in the formation of 8-amino-9-glycosylpurines in favorable amounts. The simple one-step method made it possible to tranform seven 6-(glycosylamino)-5-nitrosopyrimidines to their appropriate glycosylpurines at room temperature. Formamide served as the supplier of the carbon(8)-amino group of the purine and as the reducing agent of the nitroso group of the pyrimidine. The role of the formamide thereby involved simultaneous oxidation-reduction in the process.

The Caulobacter crescentus DNA adenine methyltransferase CcrM and its homologs in the [alpha]-Proteobacteria are essential for viability. CcrM is 34% identical to the yhdJ gene products of Escherichia coli and Salmonella enterica. This study provides evidence that the E. coli yhdJ gene encodes a DNA adenine methyltransferase. In contrast to an earlier report, however, we show that yhdJ is not an essential gene in either E. coli or S. enterica.

The quantum chemical study results from key tertiary interaction patterns in RNA reveal A-minor interactions to be formed by adenine interacting through its sugar edge with a WC base pair from a canonical double helix while the P-interaction was found to bring together a G/U wobble base pair and a GC WC base pair.

Twelve tautomers of the DNA base adenine are investigated at the BP86/TZ2P and BP86/QZ4P levels of density functional theory and the vertical and adiabatic ionization energies of all tautomers are determined as the difference in energy between the radical cation and the corresponding neutral system. The implications of the existence of the tautomeric forms of adenine for the DNA replication are discussed.

A novel approach utilizing ATP-agarose affinity chromatography is employed to demonstrate that the intracellular domain of guanylyl cyclase C (GC-C) and the kinase homology domain (KHD) bind ATP. Results shows direct ATP binding to the pseudokinase domain of receptor guanylyl cyclase C, as well as to identify dramatic conformational changes that occur in this domain ATP binding, akin to those in catalytically active protein kinases.

DNA alkylation is examined by pyrrole (Py)-imidazole (Im) hairpin polyamides that possess 1,2,9,9a-tetrahydrocyclopropal[1,2-c]benz[1,2-e]indol-4-one (CBI) or cyclopropapyrroloindole (CPI) as DNA alkylating moieties. The high selectivity of the CBI conjugate gives additional sequence specificity relative to CPI conjugates that would be useful for the biological applications.

Experiments on hole transport in DNA are interpreted as showing that a hole introduced onto a guanine (G) followed by a series of adenines (As) in a DNA duplex travels. Estimation of energy fluctuations in the environment and conformational changes of the DNA find them to be large enough so that GGG and even G can still act as hole traps but trapping on the former is still very much more likely because of its lower energy.

The excited-state dynamics of adenine and thymine dimers and the adenine-thymine base pair were investigated by femtosecond pump-probe ionization spectroscopy with excitation wavelengths of 250-272 nm. It was found that the excited state dynamics of the adenine (A)-thymine (T) base pair were similar to those of the homodimers and indicated a conserved relaxation mechanism in the two chromophores.

Combined experimental and computational study of adenine ion dissociations to shed some light on the formation, structures, and stabilities of distonic and ylid ions derived from adenine. The deprotonation of the amino group in the ionized adenine moiety in DNA is expected to produce a highly reactive radical that is able to abstract a hydrogen atom from thymine and amino acid residues exothermically.

Adenine and its derivatives form a large family of biological compounds that offer a variety of hydrogen-bonding functional groups. Watson-Crick interaction, observed in DNA and Hoogsteen bonding, dominating in the complexes of adenine with Rebek imides are the two most often occurring types of hydrogen bonding involving adenine.

The residues that are located at positions corresponding to Leu159 and Ala131 in adenine phosporibosyltransferase (hAPRT) are responsible for the base specificities of type 1 PRTases are proposed. The comparative analysis shown provides structural information for the mechanism by which mutations in the human APRT lead to DHA-urolithiasis.

The 298 K gas-phase acidities of all possible deprotonation sites of the most important tautomers of adenine and guanine were calculated by using direct and isodesmic reaction-based methods. The gas-phased acidities (GPAs) predicted by both methods are similar at the B3LYP/ aug--cc--pVTZ//B3LYP/6-31+G(d) level of theory.

The reactions of [PtCl(en)(ACRAMTU-S)](NO3)2, the prototype of a new class of cytotoxic DNA-targeted agents, with 2'-deoxyguanosine (dGuo) and random-sequence native DNA by in-line liquid chromatography/mass spectrometry (LC/MS) and NMR spectroscopy are investigated to identify the covalent adducts form by platinum. Findings indicate that it is possible to direct platinum away from its natural target by disfavoring cross-link formation and tethering of the metal to an adenine-affinic, minor groove-directed intercalator.

The energetics of the dehalogenation of adenine-halouracil base pairs, upon attachment of low-energy electrons, is investigated by use of density functional theory. The findings indicate that the radiosensitization properties of halouracils should be less effective in double-stranded DNA than in single-stranded DNA.

Two different processes for the hole transfer across the identical (A)(sub n)-bridge in DNA are demonstrated. It is shown that the mechanism of hole transfer in DNA strongly depends on the redox nature of the oxidant, whether it produces only G(super .+) or both A(super .+) and G(super .+).

A computational investment is conducted on the conformational surfaces of three nucleoside analogues, where an adenine is attached to a diol of tetrahydrofuran, a diol of cyclopentane, and a diol of cyclopentine. In each system, the lowest-energy conformer displayed a conformational lock into the south position by an internal hydrogen bond between O2'H of the five-membered ring and the N3 nitrogen of adenine.

2-Aminopurine is a highly fluorescent analogue of adenine that can be incorporated synthetically into DNA with little perturbation of the native double-helical structure. The sensitive dependence of the quantum yield of 2-Aminopurine on nuclear acid conformation has made it an invaluable probe for DNA structure, dynamics and interactions.