DNA甲基化-非甲基化碱基间堆积作用的理论研究

运用二级Mфller-Plesset(MP2)理论方法和cc-pVDZ基组优化了6-甲基鸟嘌呤(O6-MethylG),4-甲基胸腺嘧啶(O4-MethylT)以及5-甲基胞嘧啶(C5-MethylC)与DNA碱基鸟嘌呤(G),腺嘌呤(A),胞嘧啶(C),胸腺嘧啶(T)之间的堆积构型.在MP2/aug-cc-pVXZ//MP2/cc-pVDZ(X=D,T)水平上,采用完全基组外推方法校正了堆积碱基对间的相互作用能,并用完全均衡校正法(CP)校正了基组重叠误差(BSSE).MP2计算结果表明,DNA碱基甲基化使得嘧啶-嘧啶、嘧啶-嘌呤堆积碱基间的平行旋转角发生明显改变,并使堆积碱基间的相互作用能增大.在MP2/cc-pVDZ计算级别上得到了各堆积碱基对的全电子波函数,并用分子中的原子理论(AIM)分析了堆积碱基对间的弱相互作用.AIM分析结果显示,甲基化增强了堆积碱基间的π-π作用,且甲基氢与相邻碱基间形成H2CH…X(X=O,N,CH3,NH2)等类型的氢键.甲基化损伤使碱基间重叠程度增大、π-π作用增强以及堆积碱基间形成多个氢键,是堆积作用能增加的主要原因.     

Detection of 5-methylcytosine and 5-hydroxymethylcytosine in DNA via host–guest interactions inside α-hemolysin nanopores

Cytosine methylation and hydroxymethylation are both important epigenetic modifications of DNA in mammalian cells. Therefore, profiling DNA (hydroxy)methylation across the genome is vital for understanding their roles in gene regulation. Here, we report a nanopore-based approach for quick and reliable detection of 5-methylcytosine and 5-hydroxymethylcytosine in DNA at the single-molecule level. The single-stranded DNA containing 5-methylcytosine or 5-hydroxymethylcytosine was first selectively modified on the epigenetic base to attach a host–guest complex. Threading of the modified DNA molecules through α-hemolysin nanopores causes unbinding of the host–guest complex and generates highly characteristic current signatures. Statistical analysis of the signature events affords quantitative information about 5-methylcytosine and 5-hydroxymethylcytosine in DNA. Our results suggest that other DNA modifications could also be detected with the developed method. Furthermore, we anticipate our nanopore sensing strategy to be generally useful in biochemical analysis and to find applications in the early diagnosis of diseases.     

Electrochemical detection of DNA methylation using a glassy carbon electrode modified with a composite made from carbon nanotubes and β-cyclodextrin

We describe a method for detecting DNA methylation. It is based on direct oxidation of DNA bases at a glassy carbon electrode (GCE) modified with film of a multiwalled carbon nanotube-β-cyclodextrin composite. This nano-structured film causes a strong enhancement on the oxidation current of DNA bases due to its large effective surface area and extraordinary electronic properties. Well-defined peaks were obtained as a result of electro-oxidation of guanine (at 0.67 V), adenine (at 0.92 V), thymine (at 1.11 V), cytosine (at 1.26 V), and 5-methylcytosine (at 1.13 V; all data vs. saturated calomel electrode (SCE)). The potential difference between 5-methylcytosine and cytosine (130 mV) is large enough to enable reliable simultaneous determination and analysis. The interference by thymine can be eliminated by following the principle of complementary pairing between purine and pyrimidine bases in DNA. The modified electrode was successfully applied to the evaluation of 5-methylcytosine in a fish sperm DNA, the methylation level of cytosine was found to be 7.47 %, and the analysis process took less than 1 h.     

Activation barriers for DNA alkylation by carcinogenic methane diazonium ions

Methylation reactions of the DNA bases with the methane diazonium ion, which is the reactive intermediate formed from several carcinogenic methylating agents, were examined. The SN2 transition states of the methylation reactions at N7, N3, and O6 of guanine; N7, N3, and N1 of adenine; N3 and O2 of cytosine; and O2 and O4 of thymine were calculated using the B3LYP density functional method. Solvation effects were examined using the conductor-like polarizable continuum method and the combined discrete/SCRF method. The transition states for reactions at guanine N3, adenine N7, and adenine N1 are influenced by steric interactions between the methane diazonium ion and exocyclic amino groups. Both in the gas phase and in aqueous solution, the methylation reactions at N atoms have transition states that are looser, and generally occur earlier along the reaction pathways than reactions at O atoms. The forming bonds in the transition states in water are 0.03 to 0.13 A shorter than those observed in the gas phase, and the activation energies are 13 to 35 kcal/mol higher. The combined discrete/SCRF solvation energy calculations using base-water complexes with three water molecules yield base solvation energies that are larger than those obtained from the CPCM continuum method, especially for cytosine. Reactivities calculated using barriers obtained with the discrete/SCRF method are consistent with the experimentally observed high reactivity at N7 of guanine.     

Methylation reaction for four DNA base molecules by methanediazonium ions

The methylation reactions at ten nucleophilic sites in four DNA base molecules with methanediazonium ions (CH₃N₂ ⁺) have been theoretically investigated including solvent effects at the B3LYP/6-31G^** and MP2/6-31G^** levels. The results show that all the methylation reactions have relatively small activation energy (<33.5 kJ/mol), and the methylation process is exothermic reaction and easy to occur. This study shows that the ultimate carcinogen CH₃N₂ ⁺ by NDMA can easily methylate DNA base molecules and form carcinogenic products. Supported by the Shanghai Municipal Education Commission (Grant No. YYY-07015) and Shanghai Institute of Technology (Grant No. YJ2007-36)     

Theoretical studies on the inactivation mechanism of γ-aminobutyric acid aminotransferase

The inactivation mechanism of γ-aminobutyric acid aminotransferase (GABA-AT) in the presence of γ-vinyl-aminobutyric acid, an anti-epilepsy drug, has been studied by means of theoretical calculations. Density functional theory methods have been applied to compare the three experimentally proposed inactivation mechanisms (Silverman et al., J. Biol. Chem., 2004, 279, 363). All the calculations were performed at the B3LYP/6-31+G(d,p) level of theory. Single point solvent calculations were carried out in water, by means of an integral equation formalism-polarizable continuum model (IEFPCM) at the B3LYP/6-31+G(d,p) level of theory. The present calculations provide an insight into the mechanistic preferences of the inactivation reaction of GABA-AT. The results also allow us to elucidate the key factors behind the mechanistic preferences. The computations also confirm the importance of explicit water molecules around the reacting center in the proton transfer steps.     

Redefinition of rubisco carboxylase reaction reveals origin of water for hydration and new roles for active-site residues

Crystallographic, mutagenesis, kinetic, and computational studies on Rubisco over three decades have revealed much about its catalytic mechanism and the role played by several active-site residues. However, key questions remain unanswered. Specific details of the carboxylase and oxygenase mechanisms, required to underpin the rational re-engineering of Rubisco, are still speculative. Here we address critical gaps in knowledge with a definitive comprehensive computational investigation of the mechanism of carboxylase activity at the Rubisco active site. Density functional theory calculations (B3LYP/6-31G(d,p)) were performed on active-site fragment models of a size up to 77 atoms, not previously possible computationally. All amino acid residues suspected to play roles in the acid-base chemistry in the multistep reaction, and interacting directly with the central Mg (2+) atom and the reactive moiety of substrate and intermediates, were included. The results provide a firm basis for us to propose a novel mechanism for the entire sequence of reactions in the carboxylase catalysis and to define precise roles for the active-site residues, singly and in concert. In this mechanism, the carbamylated LYS201 plays a more limited role than previously proposed but is crucial for initiating the reaction by acting as a base in the enolization. We suggest a wider role for HIS294, with involvement in the carboxylation, hydration, and C2-C3 bond-scission steps, consistent with the suggestion of Harpel et al. (1998) but contrary to the consensus view of Cleland et al. (1998). In contrast to the common assumption that the water molecule for the hydration step comes from within the active site, we propose that the Mg-coordinated water is not dissociated at the start of the gas-addition reaction but rather remains coordinated and is used for the hydration of the C3 carbon atom. New roles are also proposed for LYS175, GLU204, and HIS294. The mechanism suggests roles in the gas-addition step for residues in three spatially distinct regions of the active site, HIS294 and LYS334 in the C-terminal domain of the large subunit (LSU), but also hitherto unsuspected roles for a cluster of three residues (ASN123, GLU60, and TYR20) in the N-terminal domain of the partner LSU of the dimer containing the active site. Our new mechanism is supported by existing experimental data, provides new convincing interpretations of previously puzzling data, and allows new insights into mutational strategies for improving Rubisco activity.     

Modeling zinc in biomolecules with the self consistent charge-density functional tight binding (SCC-DFTB) method: applications to structural and energetic analysis

Parameters for the zinc ion have been developed in the self-consistent charge density functional tight-binding (SCC-DFTB) framework. The approach was tested against B3LYP calculations for a range of systems, including small molecules that contain the typical coordination environment of zinc in biological systems (cysteine, histidine, glutamic/aspartic acids, and water) and active site models for a number of enzymes such as alcohol dehydrogenase, carbonic anhydrase, and aminopeptidase. The SCC-DFTB approach reproduces structural and energetic properties rather reliably (e.g., total and relative ligand binding energies and deprotonation energies of ligands and barriers for zinc-assisted proton transfers), as compared with B3LYP/6-311+G** or MP2/6-311+G** calculations.     

Quantum mechanics study and Monte Carlo simulation on the hydrolytic deamination of 5-methylcytosine glycol

The efficient formation of 5-methylcytosine glycol (mCg) and its facile deamination to thymine glycol (Tg) may account for the prevalent C → T transition mutation found at methylated CpG site (mCpG) in human p53 gene, a hallmark for many types of human tumors. In this work, the hydrolytic deamination of mCg was investigated at the MP2 and B3LYP levels of theory using the 6-311G(d,p) basis set. In the gas phase, three pathways were explored, paths A-C, and it indicates that the direct deamination of mCg with H(2)O by either pathway is unlikely because of the high activation free energies involved in the rate-determining steps, the formation of the tetrahedral intermediate for paths A and B as well as the formation of the Tg tautomer for path C. In aqueous solution, the role of the water molecules in the deamination of mCg with H(2)O was analyzed in two separate parts: the direct participation of one water molecule in the reaction pathway, called the water-assisted mechanism; and the complementary participation of the aqueous solvation. The water-assisted mechanism was carried out for mCg and the cluster of two water molecules by quantum mechanical calculations in the gas phase. This indicates that the presence of the auxiliary water molecule significantly contributes to decreasing all the activation free energies. The bulk solution effect on the water-assisted mechanism was included by free energy perturbation implemented on Monte Carlo simulations, which is found to be substantial and decisive in the deamination mechanism of mCg. In this case, the water-assisted path A is the most plausible mechanism reported for the deamination of mCg, where the calculated activation free energy (22.6 kcal mol(-1) at B3LYP level of theory) agrees well with the experimentally determined activation free energy (24.8 kcal mol(-1)). The main striking results of the present DFT computational study which is in agreement with previous experimental data is the higher rate of deamination displayed by mCg residues with respect to 5-methylcytosine (mC) bases, which supports that the deamination of mCg contributes significantly to the C → T transition mutation at mCpG dinucleotide site.     

NIR-SERS studies of DNA and DNA bases attached on polyvinyl alcohol (PVA) protected silver grass-like nanostructures

In this work, polyvinyl alcohol (PVA) protected silver grass-like nanostructure (PVA–Ag–GNS) with near infrared surface-enhanced Raman scattering (NIR-SERS) activity was prepared and employed to detect DNA and DNA bases. The PVA–Ag–GNS demonstrated high NIR-SERS activity and good optical reproducibility in the detection of adsorbates such as the case of crystal violet, DNA and DNA bases. By using of the tested molecule of thymine, the PVA–Ag–GNS shows a high enhancement factor (EF) of ∼10⁸. For NIR-SERS detection of DNA molecules, Raman signals from the DNA bases of guanine (630 cm⁻¹) and adenine (720 cm⁻¹) are greatly enhanced. For DNA molecules NIR-SERS detection, Raman signals from the DNA bases of guanine (630 cm⁻¹), adenine (720 cm⁻¹) and cytosine (1010 cm⁻¹) are greatly enhanced. The experimental results show that the NIR-SERS spectrum of DNA is dominated by guanine mode, which is followed by adenine and cytosine modes, respectively. Meanwhile, the NIR-SERS signal intensities of the DNA bases increase in the order of thymine (T) < cytosine (C) < adenine (A) < guanine (G). One can conclude that the adsorption strength of the DNA bases in DNA molecule with the silver surface is in the order T < C < A < G, which is different from that of the four DNA bases in individual molecule adsorbed on silver surface (T < A < G < C). On the other hand, the geometry optimization and calculated wavenumber of the complexes of adenine–Ag, guanine–Ag, cytosine–Ag and thymine–Ag for the ground states are performed with DFT, B3LYP functional and the LanL2DZ basis set. The calculated wavenumbers match well with the experimental results. According to our experiment and calculations, DNA base molecules adsorbed on silver surface via the intra-annular nitrogen atom which is adsorbed on the silver nanoparticle and formed metal–molecule complexes by the available lone pair.     

DNA Methylation: Site-Specific Methylations Cause Gene Inactivation

In the molecular biology of eukaryotic organisms, the elucidation of mechanisms involved in the regulation of gene expression has assumed an important role. All cells of an organism carry the same genes, but differ in the patterns of genes they express. There is an increasing amount of evidence that cancer cells exhibit a pattern of gene expression which can be very different from that of normal cells. One of the molecular signals that has been recognized in the regulation of gene expression in eukaryotes is the modified nucleotide 5-methylcytosine (5-mC). Through experiments in well-characterized eukaryotic systems, evidence has been adduced that the introduction of 5-mC into highly specific sequences, particularly into the 5′ and promoter regions of a gene, can cause gene inactivation. Viral and other eukaryotic systems have helped in the recognition of this cause-and-effect relationship. Inactive genes are frequently hypermethylated in the promoter region; active genes are hypomethylated. However, these correlations are not always as simple and straightforward. The biochemical mechanisms by which site-specific DNA methylations cause gene inactivation have not yet been determined. It is plausible to postulate that promoter methylations could somehow affect the binding of cellular enzymes involved in recognizing the promoter of a gene. Structural alterations of DNA promoter sequences arising from DNA methylations could also be important. DNA methylation is likely to represent a long-term inactivation signal, since it is presently thought that patterns of DNA methylation can be changed only by DNA replication and specific inhibition of post-replicative maintenance methylation.     

A quantum mechanical investigation of possible mechanisms for the nucleotidyl transfer reaction catalyzed by DNA polymerase beta

Several quantum mechanical (QM) and hybrid quantum/molecular mechanical (QM/MM) studies have been employed recently to analyze the nucleotidyl transfer reaction in DNA polymerase beta (pol beta). Our examination reveals strong dependence of the reported mechanism on the initial molecular model. Thus, we explore here several model systems by QM methods to investigate pol beta's possible pathway variations. Although our most favorable pathway involves a direct proton transfer from O3'(primer) to O2alpha(Palpha), we also discuss other initial proton-transfer steps--to an adjacent water, to triphosphate, or to aspartic units--and the stabilizing effect of crystallographic water molecules in the active site. Our favored reaction route has an energetically undemanding initial step of less than 1.0 kcal/mol (at the B3LYP/6-31G(d,p) level), and involves a slight rearrangement in the geometry of the active site. This is followed by two major steps: (1) direct proton transfer from O3'(primer) to O2alpha(Palpha) leading to the formation of a pentavalent, trigonal bipyramidal Palpha center, via an associative mechanism, at a cost of about 28 kcal/mol, and (2) breakage of the triphosphate unit (exothermic process, approximately 22 kcal/mol) that results in the full transfer of the nucleotide to the DNA and the formation of pyrophosphate. These energy values are expected to be lower in the physical system when full protein effects are incorporated. We also discuss variations from this dominant pathway, and their impact on the overall repair process. Our calculated barrier for the chemical reaction clearly indicates that chemistry is rate-limiting overall for correct nucleotide insertion in pol beta, in accord with other studies. Protonation studies on relevant intermediates suggest that, although protonation at a single aspartic residue may occur, the addition of a second proton to the system significantly disturbs the active site. We conclude that the active site rearrangement step necessary to attain a reaction-competent geometry is essential and closely related to the "pre-chemistry" avenue described recently as a key step in the overall kinetic cycle of DNA polymerases. Thus, our work emphasizes the many possible ways for DNA polymerase beta's chemical reaction to occur, determined by the active site environment and initial models.     

Computational study on 1,1,3,3-tetramethylguanidine-catalyzed cyanosilylation mechanism of hypnone

The reaction mechanism of cyanosilylation of hypnone catalyzed by 1,1,3,3-tetramethylguanidine (TMG) was investigated using the density functional theory at the Becke three-parameter hybrid functional combined with Lee–Yang–Parr correlation functional (B3LYP)/6-31G(d), B3LYP/6-31G(d, p) and B3LYP/6-311++G(d, p) levels. The results show that the title reaction occurs through two processes, the formation of the intermediate five through the interaction of TMG with trimethylsilyl cyanide (TMSCN) and the reaction between the intermediate five and hypnone. The formation of intermediate five controls the whole reaction with a Gibbs free energy barrier of 31.84 kcal/mol. In addition, the results indicate that the catalyst TMG significantly promotes the title reaction and changes the mechanism. The results are in reasonable agreement with the experimental observations. Our results reveal that the overall reaction is stepwise and exoergonic in solvent-free conditions at room temperature.     

A DFT study of reactions of methyldiazonium ion with DNA/RNA nucleosides: Investigating effect of sugar moiety on methylation pattern of bases

Methyldiazonium ion ( ) is an ultimate carcinogen that can methylate multiple sites in DNA/RNA. In present contribution, density functional theory calculations using the B3LYP and M06‐2X functionals and the 6‐31G(d,p) and aug‐cc‐pVDZ basis sets are carried out to study methylation reactions of at the different nucleophilic sites of DNA/RNA bases and their nucleosides. Total 12 nucleophilic sites, that is, the N2, N3, N7, and O6 sites of guanine; the N1, N3, N6, and N7 sites of adenine; O2 and N3 sites of cytosine and the O2 and O4 sites of thymine and uracil have been considered for study. Thus, a total of 30 reactions have been studied here. The polarizable continuum model is used for solvation calculations. The N7 site of guanine, N7(G), is found to be most reactive in all the reactions studied here, which is in agreement with experiment. However, the calculated reactivity of toward the N7(G) site in aqueous media follows the order: guanine > deoxyguanosine > guanosine. The reactivities of many other sites including the O6(G), O2(C), and N3(A) sites are also modified in going from DNA/RNA bases to their nucleosides and from DNA to RNA nucleosides. Thus, we note that the presence of sugar moiety significantly modifies the methylation pattern of bases caused by . © 2014 Wiley Periodicals, Inc.     

A new photoproduct of 5-methylcytosine and adenine: a theoretical study

Both the cycloaddition mechanism of 5-methylcytosine with adenine and the deamination mechanism of the cycloaddition product have been studied using density functional theory method. The results suggest that the cycloaddition reaction could occur more easily through photochemical reaction pathway than through thermal reaction pathway. The obtained four-member ring structure could be easily transformed to an eight-member ring structure through bond cleavage of C₅–C₆ (the energy barrier is <2 kcal/mol). Then hydrolytic deamination reaction takes place with water assistance. The hydroxyl group of one water molecule attacks the C₄^′ atom and the hydrogen atom of another water molecule attacks N₃^′ atom to form a tetrahedral intermediate. Subsequently, the hydrogen atom of hydroxyl group transfers to N₈^′ to produce ammonia, and the amino group of the former 5-methylcytosine changes to carboxyl oxygen. Our calculations explain the phenomena that 5-methylcytosine and adenine could obtain the same photoproduct as thymine and adenine from theoretical aspects.     

Improved synthesis and mutagenicity of oligonucleotides containing 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine

5-Formylcytosine (fC or (5-CHO)dC) and 5-carboxylcytosine (caC or (5-COOH)dC) have recently been identified as constituents of mammalian DNA. The nucleosides are formed from 5-methylcytosine (mC or (5-Me)dC) via 5-hydroxymethylcytosine (hmC or (5-HOMe)dC) and are possible intermediates of an active DNA demethylation process. Here we show efficient syntheses of phosphoramidites which enable the synthesis of DNA strands containing these cytosine modifications based on Pd(0)-catalyzed functionalization of 5-iododeoxycytidine. The first crystal structure of fC reveals the existence of an intramolecular H-bond between the exocyclic amine and the formyl group, which controls the conformation of the formyl substituent. Using a newly designed in vitro mutagenicity assay we show that fC and caC are only marginally mutagenic, which is a prerequisite for the bases to function as epigenetic control units.     

An Expedient Method for Regioselective Methylation of Catechol Coumarins

An expedient and efficient method for selective methylation of catechol coumarins by working with different alkalis under appropriate reaction conditions was reported. Esculetins were selectively methylated at position 6 and position 7 in good yields using CH₃I in the presence of NaH and Na₂CO₃, respectively. However, daphnetins showed less regioselectivity under the same reaction conditions. Furthermore, the site preference for the methylation reaction was interpreted by the density functional theory at B3LYP/6-31+G(d) level.     

细胞内第二信使3',5'-环核苷酸酯质子迁移机理的理论研究

应用密度泛函理论B3LYP/6-31G**计算方法对气相中细胞内第二信使3¢,5¢-环核苷酸酯(cAMPm)质子迁移机理进行了理论研究,此外,在相同水平上模拟了水分子作催化剂的反应机理。计算结果表明cAMPm两种构象Bm 和Dm之间的转化经过一个环状过渡态,其中,两分子水参与的H 迁移反应的势能面最低,反应更容易进行。我们的计算结果为研究相关的磷酸二酯的H质子迁移反应提供了理论依据。     

Solvent structure and hammerhead ribozyme catalysis

Although the hammerhead ribozyme is regarded as a prototype for understanding RNA catalysis, the mechanistic roles of associated metal ions and water molecules in the cleavage reaction remain controversial. We have investigated the catalytic potential of observed divalent metal ions and water molecules bound to a 2 A structure of the full-length hammerhead ribozyme by using X-ray crystallography in combination with molecular dynamics simulations. A single Mn(2+) is observed to bind directly to the A9 phosphate in the active site, accompanying a hydrogen-bond network involving a well-ordered water molecule spanning N1 of G12 (the general base) and 2'-O of G8 (previously implicated in general acid catalysis) that we propose, based on molecular dynamics calculations, facilitates proton transfer in the cleavage reaction. Phosphate-bridging metal interactions and other mechanistic hypotheses are also tested with this approach.     

A Molecular Tool Targeting the Base-Flipping Activity of Human UHRF1

During DNA replication, ubiquitin-like, containing PHD and RING fingers domains 1 (UHRF1) plays key roles in the inheritance of methylation patterns to daughter strands by recognizing through its SET and RING-associated domain (SRA) the methylated CpGs and recruiting DNA methyltransferase 1 (DNMT1). Herein, our goal is to identify UHRF1 inhibitors targeting the 5′-methylcytosine (5mC) binding pocket of the SRA domain to prevent the recognition and flipping of 5mC and determine the molecular and cellular consequences of this inhibition. For this, we used a multidisciplinary strategy combining virtual screening and molecular modeling with biophysical assays in solution and cells. We identified an anthraquinone compound able to bind to the 5mC binding pocket and inhibit the base-flipping process in the low micromolar range. We also showed in cells that this hit impaired the UHRF1/DNMT1 interaction and decreased the overall methylation of DNA, highlighting the critical role of base flipping for DNMT1 recruitment and providing the first proof of concept of the druggability of the 5mC binding pocket. The selected anthraquinone appears thus as a key tool to investigate the role of UHRF1 in the inheritance of methylation patterns, as well as a starting point for hit-to-lead optimizations.