DNA methylation analysis: speedup of bisulfite-mediated deamination of cytosine in the genomic sequencing procedure

Understanding the biological consequences of DNA methylation is a current focus of intensive studies. A standard method for analyzing the methylation at position 5 of cytosines in genomic DNA involves chemical modification of the DNA with bisulfite, followed by PCR amplification and sequencing. Bisulfite deaminates cytosine, but it deaminates 5-methylcytosine only very slowly, thereby allowing determination of the methylated sites. The deamination is usually performed using sodium bisulfite solutions of 3–5 M concentration with an incubation period of 12–16 hr at 50 °C. We demonstrate here that this deamination can be speeded up significantly. We prepared a solution of 10 M bisulfite concentration of pH 5.4 and used it to treat DNA at temperatures up to 90 °C. In an experiment, in which denatured DNA was treated with 9 M bisulfite for 10 min at 90 °C, deamination of cytosines occurred to an extent of 99.6%, while 5-methylcytosine residues in the DNA were deaminated at less than 10%. Using a plasmid DNA fragment, we observed that the DNA can serve as a template for PCR amplification after the bisulfite treatment. This new procedure is expected to offer a significantly improved genomic sequencing method, leading to the promotion of research on understanding the biological and medical significance of DNA methylation.     

Spatial distribution of AT- and GC-rich DNA within interphase cell nuclei of Triatoma infestans Klug

Heterochromatin bodies in single- and multichromocentered interphase cell nuclei of Triatoma infestans, a vector of Chagas disease, have been suggested to contain AT-rich DNA, based on their positive response to Q-banding and Hoechst 33248 treatment. No information exists on whether GC-rich DNA is also present in these nuclei and whether it plays a role on chromatin condensation. Considering that methodologies more precise than those previously used to determine DNA base composition in situ are currently available, and that the spatial distribution of chromatin areas differing in composition in interphase cell nuclei of different species is a matter of interest, the localization of AT- and GC-rich DNA in T. infestans nuclei is revisited here. The methodologies used included DAPI/AMD and CMA(3)/Distamycin differential staining, Feulgen-DNA image analysis following Msp I and Hpa II enzymatic digestion, 5-methylcytidine immunodetection, AgNOR response, confocal microscopy, and the 5-aza-2'-deoxycytidine (5-AZA) demethylation assay. The results identified the presence of AT-rich/GC-poor DNA in chromocenters and evenly distributed AT and GC sequences in euchromatin. A GC-rich DNA zone encircling the chromocenters was also found but it could not be associated with NOR regions. To corroborate the DNA AT-richness in T. infestans nuclei, bioinformatic analyses were also performed. Methylated cytosine was evident at some points of the chromocenters' edge in single- and multichromocentered nuclei and at the euchromatin of multichromocentered nuclei and could be transiently affected by the 5-AZA treatment. The present results suggest that in the particular case of chromocenters of the hemipteran T. infestans, cytosine methylation is not a relevant factor involved in chromatin condensation.     

DNA碱基分子胞嘧啶和胸腺嘧啶的太赫兹光谱研究

利用太赫兹时域光谱技术获得了DNA碱基分子胞嘧啶和胸腺嘧啶在0.1―3.5 THz的特征吸收谱,发现胞嘧啶在2.53 THz的特征吸收细节信息。采用考虑了周期性边界条件的赝势平面波密度泛函方法对胞嘧啶分子晶体进行了结构优化和晶格动力学计算,模拟重现其太赫兹特征吸收光谱,并成功辨识了胞嘧啶在0.1―3.5 THz的所有特征吸收峰。研究结果表明,这些重要的生物分子在太赫兹频段表现出鲜明的光谱特性,胞嘧啶分子3.5 THz以下的吸收特性均来源于由分子间氢键支配的外振动模式。     

A method for evaluating correlation times for tumbling and internal motion in macromolecules using cross-relaxation rate constants from proton NMR spectra

A method of estimating the correlation times and extent of internal motion of macromolecules using ¹H NMR is proposed. The method relies on measuring the cross-relaxation rate constant between resolved, identified protons separated by a fixed distance, for example 2, 3 protons of tyrosine residues or 4, 5 protons of tryptophan residues in proteins, and the 5, 6 protons of cytosine residues in DNA. For a rigid body, the cross-relaxation rate constant yields directly an estimate of the tumbling time. Deviation of its dependence on viscosity and temperature from expectations for a rigid body allows one to estimate the degree to which internal motions contribute to the relaxation. The method is illustrated for Ribonuclease A and a 20 base pair fragment of DNA corresponding to the trp operator of Escherichia coli. The calculated correlation time of RNAse A is about 8 ns at 298 K, in good agreement with expectations from hydrodynamic measurements. Tyrosine 25 has significant internal motion, characterized by an apparent amplitude of 50–60°, a correlation time of about 5 ns, and low activation energy. The correlation time of the fragment of DNA is about 6.4 ns at 298 K, in agreement with expectations for a rigid rod. The apparent activation energy was 3.8 kcal/mol, close to the value for the dependence of the viscosity of D₂O on temperature. Further, the same result was obtained regardless of the position of the base in the sequence, indicating that bending motions are of small amplitude on the nanosecond time scale for short fragments of DNA.     

DNA strand breaks induced by concerted interaction of H radicals and low-energy electrons

We propose a mechanism of DNA single strand breaks induced by low-energy electrons. Density functional theory calculations have been performed on a neutral, hydrogenated, and/or negatively charged nucleotide of cytosine in the gas phase to identify barriers for the phosphate-sugar O–C bond cleavage. Attachment of the first excess electron induces intermolecular proton transfer to cytosine. The resulting neutral radical of hydrogenated cytosine binds another excess electron, and the excess charge is localized primarily on the C6 atom. A barrier encountered for proton transfer from the C2’ atom of the adjacent sugar unit to the C6 atom of cytosine is 3.6 and 5.0 kcal/mol, based on the MPW1K and B3LYP electronic energies corrected for zero-point vibrations, respectively. The proton transfer is followed by a barrier-free sugar-phosphate C–O bond cleavage. The proton transfer is impossible for the neutral nucleotide, as there is no local minimum for the product. In the case of anionic and hydrogenated nucleotides the same barrier determined at the B3LYP level is as large as 29.3 and 22.4 kcal/mol respectively. This illustrates that the consecutive hydrogenation and electron attachment make the nucleotide of cytosine susceptible to a strand break. The rate of the C–O bond cleavage in the anion of hydrogenated nucleotide of cytosine is estimated to be ca. 10¹⁰  s^-1. The proposed mechanism proceeds through bound anionic states, not through metastable states with finite lifetimes and discrete energy positions with respect to the neutral target. The results suggest that at least for DNA without hydration even very low-energy electrons may cleave the DNA backbone.     

紫外辐射对小牛胸腺DNA水溶液影响的拉曼光谱研究

报道了小牛胸腺DNA水溶液经9,20,40min紫外辐射的拉曼光谱图,紫外线的辐射照度为1868W·m-2。实验结果表明波长为2537nm的紫外光在起主要作用的紫外辐射对DNA的损伤是严重的,短短10min的紫外辐射就使1094cm-1这个强峰分裂成几个小峰,说明DNA的构象受到破坏,DNA的构型发生变化,部分单双键发生了断裂,出现了各种各样由于DNA键断裂产生的多核苷酸;4种碱基均受到不同程度的影响,碱基间的氢键造成断裂,其中嘧啶、嘌呤碱基受到的损伤较为严重;紫外辐射对脱氧核糖也产生了破坏。另外,该实验也表明,在水溶液中,DNA以B型结构为主,局部的A型结构仍然存在。     

CG methylation in DNA transcription

A simple model of DNA is considered in which the nucleotides cytosine (C) and guanine (G) are not assumed to be identical, and in which macroscopic thermodynamic quantities may be calculated exactly. The H bonds between the C and G nucleotides are assumed to be Morse potentials. We discuss the statistical mechanics of the DNA molecule in the configuration (5...GGG ...3; 3...CCC ...5), which may be copied by RNA polymerase into a messenger RNA (mRNA) strand (5...CCC ...3). This model suggests that replacements of C by 5-methylcytosine (5mC) may be a secondary effect in the inhibition of genetic expression, not interfering directly with the formation of an open state. An experimental test is suggested. The implications of this result are discussed for a related system, in which the enzyme methylase is known to methylate almost exclusively those Cs that are followed by Gs as a regulatory strategy employed by some eukaryotes.     

水溶液中DNA紫外辐射损伤的分子机制研究

以激光喇曼光谱分析技术,对不同紫外辐射时间条件下ＤＮＡ在水溶液中产生的损伤状况进行研究,结果表明,体外条件下单纯ＤＮＡ水溶液中由紫外辐射直接造成Ｃ→Ｔ的可能性（具体反映在Ｃ→Ｕ这一过程中）是不存在的。胞嘧啶有Ｃ４原子处氨基减少可能,但并无假设衍生形成的尿嘧啶的Ｃ４Ｏ增色表现,不支持胞嘧啶脱氨基直接转变为酮式（成为尿嘧啶）的模型。胞嘧喧Ｎ４Ｈ变形振动的减色似可能证明胞嘧啶的亚氨基化,６１６ｃｍ＾－１     

The reactivity of the 5‐formylcytosine with hydroxyl radical: A theoretical perspective

Hydroxyl radical (?OH) damages DNA/RNA by attacking pyrimidine nucleobases through the addition reaction and H‐atom abstraction. It may attack on the new cytosine derivative (5‐formylCytosine [5‐fCyt]) causing DNA oxidative damage, whereas the study of the related mechanism is still in its infancy. In the present work, 2 distinct mechanisms of ?OH‐mediated 5‐fCyt at the complete basis set methods (CBS‐QB3) and CBS‐QB3/polarized continuum model approaches have firstly been explored, the addition reaction (paths R1 ~ R3) and the abstraction reaction (paths R4 ~ R6), respectively, and it shows that the addition of ?OH to the C5═C6 double bond of 5‐fCyt is more favourable than other reactions, indicating that the ?OH addition to the C5 and C6 atoms have relatively high probability to happen. The proportion of the C5 and C6 adducts is large and may be detectable experimentally, which is in agreement with the conclusions of ?OH‐mediated cytosine reaction reported experimentally and theoretically. These hint that the new DNA base (5‐fCyt) is easily damaged when exposed the surrounding of ?OH environment. Therefore, the reducing free radical production or the addition of some antioxidants should be taken in embryonic stem cells to resistance DNA damage. Our results provide some evidences between 5‐fCyt and tumor development for the experimental scientists.     

Phase transitions for a continuous system of classical particles in a box

A continuous classical system involving an infinite number of distinguishable particles is analyzed along the same lines as its quantum analogue, considered in [1]. A commutativeC*-algebra is set up on the phase space of the system, and a representation-dependent definition of equilibrium involving the static KMS condition is given. For a special class of interactions the set of equilibrium states is realized as a convex Borel set whose extremal states are characterized by solutions to a system of integral equations. By analyzing these integral equations, we prove the absence of phase transitions for high temperature and construct a phase transition for low temperature. The construction also provides an example of a translation-invariant state whose decomposition at infinity yields states that are not translation-invariant. Thus we have an example in the classical situation of continuous symmetry breaking.This article is a part of the author's doctoral thesis, which was submitted to the mathematics department at Duke University     

Effects of cytosine methylation on DNA charge transport

The methylation of cytosine bases in DNA commonly takes place in the human genome and its abnormality can be used as a biomarker in the diagnosis of genetic diseases. In this paper we explore the effects of cytosine methylation on the conductance of DNA. Although the methyl group is a small chemical modification, and has a van der Waals radius of only 2 ?, its presence significantly changes the duplex stability, and as such may also affect the conductance properties of DNA. To determine if charge transport through the DNA stack is sensitive to this important biological modification we perform multiple conductance measurements on a methylated DNA molecule with an alternating G:C sequence and its non-methylated counterpart. From these studies we find a measurable difference in the conductance between the two types of molecules, and demonstrate that this difference is statistically significant. The conductance values of these molecules are also compared with a similar sequence that has been previously studied to help elucidate the charge transport mechanisms involved in direct DNA conductance measurements.     

On acceleration of charged particles in intensity minima of laser fields

Using Klíma-Bogoliubov-Zubarev method, the acceleration of charged particles (electrons) in moving intensity minima of interference laser fields is investigated. The electromagnetic field intensity threshold for a prospective slipping-through of the particle through the potential barrier is found both analytically and numerically. Millimetre and submillimetre electromagnetic waves should be emitted because of forced electron oscillations in potential wells.This work has been initiated by gratefully acknowledged discussions with Professor H. Hora during the author's stay at the University of N. S. W., Sydney. The author thanks this University for the kind hospitality.     

胞嘧啶-胸腺嘧啶二核苷SP光损伤反应的理论研究

在DNA光损伤产物中,有一种特殊的UV诱导生成的嘧啶二聚体(SP)．尽管胞嘧啶C和胸腺嘧啶T在分子结构上非常相似,在实验中还没有发现DNA相邻的C和T之间形成类似的SP产物．用密度泛函理论,以DNA中的片段CpT为模型分子,在B3LYP/6-311++G(d,p)//B3LYP/6-31G(d,p)计算水平上从理论的角度揭示 了阻碍CpT生成SP类似物的关键因素． 与生成类似SP产物相比较,CpT更倾向于通过光物理失活途径回到基态反应物     

DNA damage induced by laser-hematoporphyrin derivative

Irradiation caused DNA single-strand breaks in S180 cells in the presence oflaser-hematoporphyrin derivative(HPD).The number of single strand breaks was 3.69 ×10~(10)break dolton~(-1).The analysis of base composition of DNA showed that the effect of laser-HPDirradiation on guanine was the highest,being 5-12 times as high as those of the three others(adenine,cytosine and thymine).It was different from the nature of DNA damage caused by γ-ray irradiation.     

Preface

The Zimm and Bragg theory of the helix ? coil transition has been modified to explain order ? order transitions in polynucleotides, in particular B?Z? ψ(-)?coil transitions in poly(dG-dC) and poly(dG-me⁵dC). Lambda anomalies in specific heat measurements around the transition point have also been explained by a further modification of the same theory. Theoretical results are found to be in good agreement with the experimental data. The nucleation parameter is consistent with the stabilization/destabilization of the ordered states under various environmental conditions, for example, methylation of cytosine residue at the C5 position or a change in the cationic concentration of the solvent.     

Structural analysis of base mispairing in DNA containing oxidative guanine lesion

Classical molecular dynamics methods were used to analyze the importance of 8-oxoguanine (8-oxoG) pairing with other DNA bases in order to determine the impact of oxidative guanine lesions on DNA structure. Six lesioned molecules, each containing 8-oxoG mispaired with one of the four normal bases on the the opposite strand at the center of 40-mer DNA, and one non-damaged DNA molecule, were simulated for 2 nanoseconds of real time. The 8-oxoG lesioned bases were found to incorporate opposite all normal bases. There are observed conformational and energetical differences among these parings. 8-oxoG in anti-form creates firm hydrogen bonds with cytosine and this bonding has a strong attractive electrostatic interaction energy similar to that of a native base pair-guanine to cytosine. Meanwhile, it does not form a stable base pair with purine bases (adenine and guanine) nor with the pyrimidine base thymine. On the other hand, the 8-oxoG in syn-form was found to pair with adenine.      

The gravitational field of planes in general relativity

From the requirement of the plane symmetry the form of the exterior and static gravitational field of the plane shell is found. Its geometry is demonstrated by embedding the selected sections into the plane space. The exterior solution is linked up with the plane and with the couple of plane-parallel planes at rest. It is shown that the existence of strong pressures in the planes (which are necessary for the manifestation of the rest of both planes) makes it difficult to compare it directly with the corresponding classic case. If the pressure is relaxed, the planes will move towards one another by hyperbolic motion. It is shown that the hyperbolic acceleration of the plane is the same as in the classical case. The gravitational field outside the plane remains static so that no gravitational radiation arises.This work was done during the author's study-stay at the Department of Theoretical Physics, Charles University, Prague. For its realization he thanks Professor M. Brdika from Prague and Professor O. Litzman from Brno. He also wishes to thank Dr. K. Kucha from Charles University for directing the whole work. He is very obliged to him for valuable and stimulating discussions. He also thanks Dr. K. Kunc from Brno for performing the numerical computations on the SAAB computer.     

Potential Energy Surface of Cytosine and Tunneling Between Its Normal and Trans-imino Tautomer

The potential energy surface of H（13） proton in base cytosine of the DNA molecules is calculated at the Caussian 98 MP2/6-311C（d,p） level. Two potential wells are found. One corresponds to the normal cytosine, while the other corresponds to its trans-imino tautomer. The estimated tunneling probability of the H（13） proton from one well to another well shows that the life time of the proton staying in one of these wells is about 600 yrs. It is too long to let tautomers of cytosine be in thermodynamical equilibrium in a room temperature gas phase experiment.     

Potential Energy Surface of Cytosine and Tunneling Between Its Normal and Trans-imino Tautomer

The potential energy surface of H(13) proton in base cytosine of the DNA molecules is calculated at the Gaussian 98 MP2/6-311G(d,p) level. Two potential wells are found. One corresponds to the normal cytosine, while the other corresponds to its trans-imino tautomer. The estimated tunneling probability of the H(13) proton from one well to another well shows that the life time of the proton staying in one of these wells is about 600 yrs. It is too long to let tautomers of cytosine be in thermodynamical equilibrium in a room temperature gas phase experiment.     

Molecular data for a biochemical model of DNA damage: Electron impact ionization and dissociative ionization cross sections of DNA bases and sugar-phosphate backbone

As part of the database for building up a biochemical model of DNA radiation damage, electron impact ionization cross sections of sugar-phosphate backbone and DNA bases have been calculated using the improved binary-encounter dipole (iBED) model. It is found that the total ionization cross sections of - and -deoxyribose-phosphate, two conformers of the sugar-phosphate backbone, are close to each other. Furthermore, the sum of the ionization cross sections of the separate deoxyribose and phosphate fragments is in close agreement with the - and -deoxyribose-phosphate cross sections, differing by less than 10%, an indication that a building-up principle may be applicable. Of the four DNA bases, the ionization cross section of guanine is the largest, then in decreasing order, adenine, thymine, and cytosine. The order is in accordance with the known propensity of oxidation of the bases by ionizing radiation. Dissociative ionization (DI), a process that both ionizes and dissociates a molecule, is investigated for cytosine. The DI cross section for the formation of H and (cytosine-H1)⁺, with the cytosine ion losing H at the 1 position, is also reported. The threshold of this process is calculated to be 16.9 eV. Detailed analysis of ionization products such as in DI is important to trace the sequential steps in the biochemical process of DNA damage.