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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 [...]

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

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

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