Formation of cytosine glycol and 5,6-dihydroxycytosine in deoxyribonucleic acid on treatment with osmium tetroxide.

OsO4 selectively forms thymine glycol lesions in DNA. In the past, OsO4-treated DNA has been used as a substrate in studies of DNA repair utilizing base-excision repair enzymes such as DNA glycosylases. There is, however, no information available on the chemical identity of other OsO4-induced base lesions in DNA. A complete knowledge of such DNA lesions may be of importance for repair studies. Using a methodology developed recently for characterization of oxidative base damage in DNA, we provide evidence for the formation of cytosine glycol and 5,6-dihydroxycytosine moieties, in addition to thymine glycol, in DNA on treatment with OsO4. For this purpose, samples of OsO4-treated DNA were hydrolysed with formic acid, then trimethylsilylated and analysed by capillary gas chromatography-mass spectrometry. In addition to thymine glycol, 5-hydroxyuracil (isobarbituric acid), 5-hydroxycytosine and 5,6-dihydroxyuracil (isodialuric acid or dialuric acid) were identified in OsO4-treated DNA. It is suggested that 5-hydroxyuracil was formed by formic acid-induced deamination and dehydration of cytosine glycol, which was the actual oxidation product of the cytosine moiety in DNA. 5-Hydroxycytosine obviously resulted from dehydration of cytosine glycol, and 5,6-dihydroxyuracil from deamination of 5,6-dihydroxycytosine. This scheme was supported by the presence of 5-hydroxyuracil, uracil glycol and 5,6-dihydroxyuracil in OsO4-treated cytosine. Treatment of OsO4-treated cytosine with formic acid caused the complete conversion of uracil glycol into 5-hydroxyuracil. The implications of these findings relative to studies of DNA repair are discussed.     

Photochemical demonstration of stacked C.C+ base pairs in a novel DNA secondary structure

The secondary structure of the alternating polydeoxynucleotide sequence poly[d(C-T)] was studied as a function of pH by ultraviolet absorbance and circular dichroism spectroscopy and by the analysis of UV-induced photoproducts. As the pH was lowered, poly[d(C-T)] underwent a conformational transition that was characterized by changes in the long-wavelength region (280-320 nm) of the CD spectrum. These changes have previously been interpreted as evidence for the formation of a core of stacked, protonated C X C+ base pairs in a double-helical complex of poly[d(C-T)], with the thymidyl residues being looped out into the solvent [Gray, D. M., Vaughan, M., Ratliff, R. L., & Hayes, F. N. (1980) Nucleic Acids Res. 8, 3695-3707]. In the present work, poly[d(C-T)] was labeled with [U-14C]cytosine and [methyl-3H]thymine and irradiated at pH values both above and below the conformational transition point (monitored by CD spectroscopy). The distribution of radioactivity in uracil means value of uracil dimers, uracil means value of thymine dimers (the deamination products of cytosine means value of cytosine and cytosine means value of thymine dimers, respectively), and thymine-means value of thymine dimers was then determined. As the pH was decreased, we found an increase in the yield of uracil means value of uracil dimers and a decrease in the yield of uracil means value of thymine dimers, which occurred concomitantly with the change in the CD spectrum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Translocation of a discrete piece of deoxyribonucleic acid carrying an amp gene between replicons in Eschericha coli.

Uptake and intracellular transformation of pyrimidines supplying cells of the yeast Rhodotorula glutinis with nitrogen have been studied. The amine nitrogen of cytosine was found to be the easiest to utilize. The presence in the medium of inorganic ammonia along with cytosine had a slight effect on cytosine deaminase (EC 3.5.4.1) activity. The uracil produced entered into the nutrient medium with no fission break of the pyridmidine ring. In the absence of any other source of nitrogen, the cells of the yeast R. glutinis utilized nitrogen of the pyrimidine ring of oxypyrimidines. Catabolism of uracil followed the reductive pattern, with release of carbon dioxide; this was accompanied by synthesis of the key enzyme of pyrimidine catabolism, dihydrouracil dehydrogenase (EC 1.3.1.1), whose activity rose 10-fold. With thymidne as the sole source of nitrogen, the lag-phase growth of the yeast cells was maximum. Catabolism of the pyrimidine ring of thymine was possibly preceded by its transformation into uracil. With no source of nitrogen easily utilized, the uridine 5'-monophosphate content in the generally acid-soluble pool rose. Our discussion of the regulation of catabolism of exogenous pyrimidine bases by the yeast R. glutinis takes into account the fact that transformations of pyrimidine bases are determined by how easily the cells can use a particular base as a source of nitrogen.     

Nitrous acid damage to duplex deoxyribonucleic acid: distinction between deamination of cytosine residues and a novel mutational lesion.

The rate of nitrous acid deamination of labeled cytosine residues in native Escherichia coli deoxyribonucleic acid was monitored in vitro by release of acid-soluble counts after treatment with uracil deoxyribonucleic acid glycosylase. The reaction exhibited a lag and was not stimulate by several agents previously shown to enhance base substitution mutagenesis during nitrous acid treatment of duplex deoxyribonucleic acid. We conclude that a significant proportion of nitrous acid induced mutagenic lesions are novel lesions and not cytosine deaminations.     

Alanine-scanning mutagenesis reveals a cytosine deaminase mutant with altered substrate preference

Suicide gene therapy of cancer is a method whereby cancerous tumors can be selectively eradicated while sparing damage to normal tissue. This is accomplished by delivering a gene, encoding an enzyme capable of specifically converting a nontoxic prodrug into a cytotoxin, to cancer cells followed by prodrug administration. The Escherichia coli gene, codA, encodes cytosine deaminase and is introduced into cancer cells followed by administration of the prodrug 5-fluorocytosine (5-FC). Cytosine deaminase converts 5-FC into cytotoxic 5-fluorouracil, which leads to tumor-cell eradication. One limitation of this enzyme/prodrug combination is that 5-FC is a poor substrate for bacterial cytosine deaminase. The crystal structure of bacterial cytosine deaminase (bCD) reveals that a loop structure in the active site pocket of wild-type bCD comprising residues 310-320 undergoes a conformational change upon cytosine binding, making several contacts to the pyrimidine ring. Alanine-scanning mutagenesis was used to investigate the structure-function relationship of amino acid residues within this region, especially with regard to substrate specificity. Using an E. coli genetic complementation system, seven active mutants were identified (F310A, G311A, H312A, D314A, V315A, F316A, and P318A). Further characterization of these mutants reveals that mutant F316A is 14-fold more efficient than the wild-type at deaminating cytosine to uracil. The mutant D314A enzyme demonstrates a dramatic decrease in cytosine activity (17-fold) as well as a slight increase in activity toward 5-FC (2-fold), indicating that mutant D314A prefers the prodrug over cytosine by almost 20-fold, suggesting that it may be a superior suicide gene.     

Metabolism of pyrimidine bases and nucleosides by Pseudomonas fluorescens biotype F

Pyrimidine metabolism in Pseudomonas fluorescens biotype F, and its ability to grow in liquid culture on pyrimidines and related compounds was investigated. It was found that uracil, uridine, cytosine, cytidine, deoxycytidine, dihydrouracil, dihydrothymine, beta-alanine or beta-aminoisobutyric acid could be utilized by this pseudomonad as a sole nitrogen source. Only uridine, cytidine, beta-alanine, beta-aminoisobutyric acid or ribose were capable of supporting its growth as a sole source of carbon. In solid medium, the pyrimidine analogue 5-fluorouracil or 5-fluorouridine could prevent P. fluorescens biotype F growth at a low concentration while a 20-fold higher concentration of 5-fluorocytosine, 5-fluorodeoxyuridine or 6-azauracil was necessary to block its growth. The pyrimidine salvage enzymes cytosine deaminase, nucleoside hydrolase, uridine phosphorylase, thymidine phosphorylase and cytidine deaminase were assayed. Only cytosine deaminase and nucleoside hydrolase activities could be detected under the assay conditions used. The effect of growth conditions on cytosine deaminase and nucleoside hydrolase levels in the micro-organism was explored. Cytosine deaminase activity was shown to increase if glycerol was substituted for glucose as the sole carbon source or if asparagine replaced (NH4)2SO4 as the sole nitrogen source in each respective medium. In contrast, nucleoside hydrolase activity remained virtually unchanged whether the carbon source in the medium was glucose or glycerol. A decrease in nucleoside hydrolase activity was witnessed when asparagine was present in the medium instead of (NH4)2SO4 as the sole source of nitrogen.     

A possible prebiotic synthesis of thymine: uracil-formaldehyde-formic acid reaction.

When uracil is reacted with formaldehyde and formic acid in dilute aqueous solutions at 100-140 degrees C, 5-hydroxymethyluracil (5-HMU), methylenebiuracil (MBU) and thymine are formed. It has been shown that 5-HMU is an intermediate in the formation of MBU and thymine. In the presence of formic acid, 5-HMU gives MBU, thymine and in some cases uracil. The formation of thymine is generally favoured under acidic conditions, although small amounts of this base could also be obtained when the reactions were carried out under mildly basic conditions. A hydride ion transfer mechanism is suggested for some of these reactions. These results have relevance to the formation of thymine under prebiotic conditions.     

The metabolism of pyrimidines by proteolytic clostridia.

Uracil was used by growing cultures of Clostridium sporogenes, and by proteolytic strains of C. botulinum types A and B. Uracil was not used by C. bifermentans; C. botulinum, type B (non-proteolytic); C. botulinum, type F (non-proteolytic); C. botulinum, type E; C. butyricum; C. cochlearium; C. difficile; C. histolyticum; C. oedematiens, type A; C. paraputrificum; C. scatologenes; C. specticum; C. sordellii; C. sticklandii; C. tertium; C. tetani; C. tetanomorphum; C. welchii, types A, B, C, E and 4 untyped strains. The growth of C. sporogenes was not increased by uracil; it was reduced to dihydrouracil. Experiments with washed cells of C. sporogenes showed that the uracil-reducing system was inducible. Washed cell suspensions incubated under hydrogen with uracil, thymine, iso-barbituric acid, 5-amino uracil and cytosine consumed 1 mole H2/mole pyrimidine. The reduction product of cytosine was dihydrouracil indicating that it was deaminated before reduction. The reduction products of the remaining pyrimidines were the corresponding dihydro derivatives. Extracts of C. sporogenes reduced uracil in the presence of NADPH2 but not NADH2.     

Understanding the thermodynamic stability of an RNA hairpin and its mutant.

The thermodynamic stability of RNA hairpin loops has been a subject of considerable interest in the recent past (Wimberly et al., 1991). There have been experimental reports indicating that the hairpins with a C(UUCG)G loop sequence are thermodynamically very stable (Wimberly et al., 1991). We used the solution structure of GGAC(UUCG)GUCC (Cheong et al., 1990; Varani et al., 1991) as the starting conformation in our attempt to understand its thermodynamic stability. We carried out molecular dynamics/free energy simulations to understand the basis for the destabilization of the C(UUCG)G loop by mutating cytosine (C7)-->uracil. Because of the limited length of simulation and the presence of kinetic barriers (solvent intervention) to the uracil-->cytosine mutation, all of our computed free energy differences are based on multiple forward simulations. Based on these calculations we find that the cytosine-->uracil mutation in the loop destabilizes it by approximately 1.5kcal/mol relative to that of the reference state, an A-form RNA but with cytosine (C7) looped out. This is the same sign and magnitude as that observed in the thermodynamic studies carried out by Varani et al.(1991). We have carried out free energy component analysis to understand the effect of mutating the cytosine residue to uracil on the thermodynamic stability of the C(UUCG)G hairpin loops. Our calculations show that the most significant contribution to the stability is from the phosphate group linking U5 and U6, which favors the cytosine residue over uracil by about 6.0 kcal/mol. The residues U5, U6, and G8 in the loop region also contribute significantly to the stability. The contributions from the salt and solvent compensate each other, indicating the dynamic nature of interactions of the environment with the nucleic acid system and the coupling between these two components.     

Repair of nitrous acid damage to DNA in Escherichia coli.

A number of mutant strains of Escherichia coli have been examined for their sensitivity to nitrous acid and in some instances to methylmethanesulfonate. All ung- mutants tested are abnormally sensitive to nitrous acid. Since the ung mutation is phenotypically expressed as a defect in uracil DNA glycosidase, this observation supports the contention that treatment of cells with nitrous acid causes deamination of cytosine to uracil. In addition the observed sentitivity indicates that the ung gene is involved in the repair of uracil in DNA. Studies with other mutants suggest that both exonuclease III and DNA polymerase I of E. coli are involved in the repair of nitrous acid damage in vivo.     

Application of a universal solvation model to nucleic acid bases: comparison of semiempirical molecular orbital theory, ab initio Hartree-Fock theory, and density functional theory

The free energies of solvation of six nucleic acid bases (adenine, cytosine, hypoxanthine, guanine, thymine, and uracil) in water and chloroform are calculated using CM2 class IV charges and SM5.42R atomic surface tensions. Using any of three approximations to the electronic wave function (AM1, Hartree-Fock, or DFT), we obtain good agreement with experiment for five cases where the experimental results are known for the partition coefficients between the two solvents. Decomposition of the solvation effects into bulk electrostatic contributions and first-solvation-shell effects shows that the partitioning is dominated by the former, and this illustrates the importance of using accurate partial atomic charges for modeling these molecules in aqueous solution.     

癌症新型的诊疗靶点－APOBEC

APOBEC（“载脂蛋白质B mRNA编辑催化多肽”）是一类进化保守的胞苷脱氨酶家族。在人体内,已知含有保守的DNA胞嘧啶脱氨酶结构域的基因共有11种,包括AID、APOBEC1、APOBEC2、APOBEC3基因家族APOBEC3A、APOBEC3B、APOBEC3C、APOBEC3DE、APOBEC3F、APOBEC3G、APOBEC3H（分别称为A3A、A3B、A3C、A3D、A3F、A3G和A3H）和APOBEC4。APOBEC利用其脱氨酶活性通过与RNA和/或DNA结合,催化mRNA或使DNA中的胞嘧啶核苷酸转变为尿嘧啶,或者胞嘧啶核苷酸转变为胸腺嘧啶核苷酸,进而完成各自不同的功能。目前研究发现,AID及APOBEC3（A3s）的7种脱氨酶在人类的天然免疫和适应性免疫防御过程中发挥重要的作用,且在口腔癌,肺癌（腺癌和鳞状细胞癌）,结直肠癌和乳腺癌等的诊疗过程中具有重要的潜在应用价值。AID可以通过将胞嘧啶脱氨基成尿嘧啶,来启动SHM (体细胞超突变)和CSR (类别转换重组),进而在抗体多样性方面发挥作用。它的异常表达能够使B细胞淋巴瘤等恶性肿瘤的发病频率显著增加。而A3A、A3B通过胞嘧啶到尿嘧啶转换,以及自身表达量上调而在乳腺癌和肺癌诊疗中起作用。A3G通过APOBEC3G/miR 29/MMP2为了解结直肠癌肝转移和开发治疗晚期结肠癌的有效疗法开辟了新的途径。综上所述,本文将以AID,A3A,A3B,A3G为例子,对APOBEC在癌症诊断和治疗方面的应用进行综述,以期为进一步药物研究和临床应用等提供参考。     

癌症新型的诊疗靶点－APOBEC

APOBEC（“载脂蛋白质B mRNA编辑催化多肽”）是一类进化保守的胞苷脱氨酶家族。在人体内,已知含有保守的DNA胞嘧啶脱氨酶结构域的基因共有11种,包括AID、APOBEC1、APOBEC2、APOBEC3基因家族APOBEC3A、APOBEC3B、APOBEC3C、APOBEC3DE、APOBEC3F、APOBEC3G、APOBEC3H（分别称为A3A、A3B、A3C、A3D、A3F、A3G和A3H）和APOBEC4。APOBEC利用其脱氨酶活性通过与RNA和/或DNA结合,催化mRNA或使DNA中的胞嘧啶核苷酸转变为尿嘧啶,或者胞嘧啶核苷酸转变为胸腺嘧啶核苷酸,进而完成各自不同的功能。目前研究发现,AID及APOBEC3（A3s）的7种脱氨酶在人类的天然免疫和适应性免疫防御过程中发挥重要的作用,且在口腔癌,肺癌（腺癌和鳞状细胞癌）,结直肠癌和乳腺癌等的诊疗过程中具有重要的潜在应用价值。AID可以通过将胞嘧啶脱氨基成尿嘧啶,来启动SHM (体细胞超突变)和CSR (类别转换重组),进而在抗体多样性方面发挥作用。它的异常表达能够使B细胞淋巴瘤等恶性肿瘤的发病频率显著增加。而A3A、A3B通过胞嘧啶到尿嘧啶转换,以及自身表达量上调而在乳腺癌和肺癌诊疗中起作用。A3G通过APOBEC3G/miR 29/MMP2为了解结直肠癌肝转移和开发治疗晚期结肠癌的有效疗法开辟了新的途径。综上所述,本文将以AID,A3A,A3B,A3G为例子,对APOBEC在癌症诊断和治疗方面的应用进行综述,以期为进一步药物研究和临床应用等提供参考。     

In vivo reshaping the catalytic site of nucleoside 2'-deoxyribosyltransferase for dideoxy- and didehydronucleosides via a single amino acid substitution

Nucleoside 2'-deoxyribosyltransferases catalyze the transfer of 2-deoxyribose between bases and have been widely used as biocatalysts to synthesize a variety of nucleoside analogs. The genes encoding nucleoside 2'-deoxyribosyltransferase (ndt) from Lactobacillus leichmannii and Lactobacillus fermentum underwent random mutagenesis to select variants specialized for the synthesis of 2',3'-dideoxynucleosides. An Escherichia coli strain, auxotrophic for uracil and unable to use 2',3'-dideoxyuridine, cytosine, and 2',3'-dideoxycytidine as a source of uracil was constructed. Randomly mutated lactobacilli ndt libraries from two species, L. leichmannii and L. fermentum, were screened for the production of uracil with 2',3'-dideoxyuridine as a source of uracil. Several mutants suitable for the synthesis of 2',3'-dideoxynucleosides were isolated. The nucleotide sequence of the corresponding genes revealed a single mutation (G --> A transition) leading to the substitution of a small aliphatic amino acid by a nucleophilic one, A15T (L. fermentum) or G9S (L. leichmannii), respectively. We concluded that the "adaptation" of the nucleoside 2'-deoxyribosyltransferase activity to 2,3-dideoxyribosyl transfer requires an additional hydroxyl group on a key amino acid side chain of the protein to overcome the absence of such a group in the corresponding substrate. The evolved proteins also display significantly improved nucleoside 2',3'-didehydro-2',3'-dideoxyribosyltransferase activity.     

Synthesis of prebiotic molecules — Role of some carbonyl compounds in prebiotic chemistry

Methyleneaminoacetonitrile(MAAN) resulting from the interaction of formaldehyde, ammonia and hydrogen cyanide on hydrolysis under mildly alkaline conditions gives a number of amino acids and peptides. Various aldehydes react with glycine to give corresponding hydroxyalkyl amino acids, which on reduction with formic acid are converted to reduced amino acids. Formaldehyde reacts with uracil to give 5-hydroxymethyl uracil which on reduction with formic acid yields thymine. Pyrrole formed by heating serine reacts with aldehydes to form porphyrins. Clays do not seem to influence most of these reactions, except the uracil-formaldehyde — formic acid reaction which results in enhanced yield of thymine.     

Isolation of certain oligonucleotides obtained by degradation of deoxyribonucleic acid

1. Pyrimidine oligodeoxynucleotides containing one thymine nucleotide residue and up to five cytosine nucleotide residues were isolated by chromatography on DEAE-Sephadex after degrading calf-thymus DNA with diphenylamine in aqueous formic acid.     

Cytosine-to-Uracil Deamination by SssI DNA Methyltransferase

The prokaryotic DNA(cytosine-5)methyltransferase M.SssI shares the specificity of eukaryotic DNA methyltransferases (CG) and is an important model and experimental tool in the study of eukaryotic DNA methylation. Previously, M.SssI was shown to be able to catalyze deamination of the target cytosine to uracil if the methyl donor S-adenosyl-methionine (SAM) was missing from the reaction. To test whether this side-activity of the enzyme can be used to distinguish between unmethylated and C5-methylated cytosines in CG dinucleotides, we re-investigated, using a sensitive genetic reversion assay, the cytosine deaminase activity of M.SssI. Confirming previous results we showed that M.SssI can deaminate cytosine to uracil in a slow reaction in the absence of SAM and that the rate of this reaction can be increased by the SAM analogue 5’-amino-5’-deoxyadenosine. We could not detect M.SssI-catalyzed deamination of C5-methylcytosine (^m5C). We found conditions where the rate of M.SssI mediated C-to-U deamination was at least 100-fold higher than the rate of ^m5C-to-T conversion. Although this difference in reactivities suggests that the enzyme could be used to identify C5-methylated cytosines in the epigenetically important CG dinucleotides, the rate of M.SssI mediated cytosine deamination is too low to become an enzymatic alternative to the bisulfite reaction. Amino acid replacements in the presumed SAM binding pocket of M.SssI (F17S and G19D) resulted in greatly reduced methyltransferase activity. The G19D variant showed cytosine deaminase activity in E. coli, at physiological SAM concentrations. Interestingly, the C-to-U deaminase activity was also detectable in an E. coli ung ⁺ host proficient in uracil excision repair.     

Proteomic examination of Ralstonia eutropha in cellular responses to formic acid

In this study, Ralstonia eutropha was used to elucidate protein changes in response to formic acid. Sixty-three differentially expressed proteins in relation to formic acid in R. eutropha were found with 1-D PAGE and nano-LC-MS/MS. Among the proteins with decreased expression, four were involved in the shikimate pathway and three proteins in the pyrimidine biosynthesis pathway. With the increased expression of proteins, a dramatic change occurred in the induction of ion transporters in relation to maintenance of the acid-base balance. A detoxification process of formic acid in the bacteria might be related to a membrane enzyme, formate hydrogenylase. Three proteins in polyhydroxyalkanoate synthesis were enhanced and five proteins in glutathione biosynthesis increased in response to formic acid. Three enzymes in mevalonate biosynthesis and heat shock proteins were also elevated in the cells. Therefore, formic acid might have an inhibitory effect on aromatic amino acid production and pyrimidine biosynthesis in R. eutropha. R. eutropha cells seemed to attempt to overcome the effects of formic acid by increasing ion transporters and proteins that metabolized formic acid.     

Amino acid sequence determination of the ADP,ATP carrier from beef heart mitochondria. The sequence of the C-terminal acidolytic fragment

The primary structure of the C-terminal region (94 residues) of the ADP,ATP carrier of beef heart mitochondria is described. CNBr cleavage results in a large peptide (CB1) with Mr 22 000 and several small peptides (CB2 to CB8). Peptide separation was achieved by gel chromatography with 80% formic acid or with an ethanol/formic acid mixture. The amino acid sequence of the small CNBr peptides was determined by solid-phase techniques. Hydrolysis in formic acid cleaves the carrier protein into an Mr 23 000 fragment (A1) with the blocked N-terminus and an Mr 10 000 fragment (A2) starting with proline. The alignment of two CNBr fragments was possible by degradation of A2 by solid-phase methods for 34 steps. The remaining CNBr fragments were arranged by sequencing the tryptic peptides of citraconylated A2.     

Acetic anhydride requirement in the colorimetric determination of tryptophan

Nucleic acid research frequently necessitates the analytical resolution of nucleic acid derivatives. Thin-layer chromatography (tlc), for its simplicity, short development time, and superior resolving power, is often preferable to other methods (1–3). Although the literature contains a large number of methods that have been devised for the separation of purine and pyrimidine derivatives (4,5) no tlc technique has hitherto been described for the concomitant separation of bases, nucleosides, nucleoside 5′-monophosphates, nucleoside 3′-monophosphates, nucleoside diphosphates, and nucleoside triphosphates.The present communication deals with methods devised for the simultaneous separation of the above-mentioned pyrimidine derivatives. They enable the resolution of either the six uracil derivatives or the six cytosine derivatives, on commercial cellulose tlc sheets. Alternatively, the six pyrimidine derivatives can be separated on cellulose layers 0.75 mm thick. Since formic acid extracts of bacterial cells do not interfere with the separation, these methods can be used for the direct estimation of extracts of biological materials.