Differential methylation of mitochondrial and nuclear DNA in cultured mouse, hamster and virus-transformed hamster cells. In vivo and in vitro methylation

Information has been lacking as to whether mitochondrial DNA of animal cells is methylated. The methylation patterns of mitochondrial and nuclear DNAs of several mammalian cell lines have therefore been compared by four methods: (1) in vivo transfer of the methyl group from [methyl-³H]methionine; (2) in vivo incorporation of [³²P]orthophosphate and a combination of (1) and (2); (3) in vivo incorporation of [³H]deoxycytidine; (4) in vitro methylation of DNAs with ³H-labeled S-adenosylmethionine as methyl donor and DNA methylase preparations from L cell nuclei. The cell lines were mouse L cells, $\text{BHK21}\text{C13}$, $\text{C13}\text{B4}$ (baby hamster kidney cells transformed by the Bryan strain of Rouse sarcoma virus), and PyY (BHK cells transformed by polyoma virus). DNA bases were separated chromatographically, using 5-methylcytosine, 6-methylaminopurine and, in some cases, 7-methylguanine as markers.Mitochondrial DNA was found to be significantly less methylated than nuclear DNA with respect to 5-methylcytosine in all cell types studied and by all methods used. The relative advantages and disadvantages of each method have been discussed. The level of 5-methylcytosine in mitochondrial DNA as compared with that in nuclear DNA was estimated as one-fourth to one-fourteenth in various cell lines. The estimated 5-methylcytosine content per circular mitochondrial DNA molecule (mol. wt 10 × 10⁶) was about 12 methylcytosine residues for L cells and 24, 30 and 36 methylcytosine residues for BHK, B4 and PyY cells, respectively. Relative to cytosine residues, the estimate was one 5-methylcytosine per 500 cytosine residues of mitochondrial DNA and one 5-methylcytosine per 36 cytosine residues of nuclear DNA from L-cells. The values for methylcytosine of mitochondrial DNA are presumed to be maximal. PyY cells as compared with other cells had the highest methylcytosine content of both mitochondrial and nuclear DNA as estimated by method (3). No methylation of nuclear DNA was observed in confluent L cells.Evidence for the presence of DNA methylase activity associated with mitochondrial fractions was obtained. This activity could be distinguished from other cellular DNA methylase activity by differential response to mercaptoethanol. Radioactivity from ³H-labeled S-adenosylmethionine was found only in 5-methyl-cytosine of DNA.     

Direct detection of methylation in genomic DNA

The identification of methylated sites on bacterial genomic DNA would be a useful tool to study the major roles of DNA methylation in prokaryotes: distinction of self and nonself DNA, direction of post-replicative mismatch repair, control of DNA replication and cell cycle, and regulation of gene expression. Three types of methylated nucleobases are known: N⁶-methyladenine, 5-methylcytosine and N⁴-methylcytosine. The aim of this study was to develop a method to detect all three types of DNA methylation in complete genomic DNA. It was previously shown that N⁶-methyladenine and 5-methylcytosine in plasmid and viral DNA can be detected by intersequence trace comparison of methylated and unmethylated DNA. We extended this method to include N⁴-methylcytosine detection in both in vitro and in vivo methylated DNA. Furthermore, application of intersequence trace comparison was extended to bacterial genomic DNA. Finally, we present evidence that intrasequence comparison suffices to detect methylated sites in genomic DNA. In conclusion, we present a method to detect all three natural types of DNA methylation in bacterial genomic DNA. This provides the possibility to define the complete methylome of any prokaryote.     

Bioinformatics and functional analysis define four distinct groups of AlkB DNA-dioxygenases in bacteria

The iron(II)- and 2-oxoglutarate (2OG)-dependent dioxygenase AlkB from Escherichia coli (EcAlkB) repairs alkylation damage in DNA by direct reversal. EcAlkB substrates include methylated bases, such as 1-methyladenine (m¹A) and 3-methylcytosine (m³C), as well as certain bulkier lesions, for example the exocyclic adduct 1,N⁶-ethenoadenine (εA). EcAlkB is the only bacterial AlkB protein characterized to date, and we here present an extensive bioinformatics and functional analysis of bacterial AlkB proteins. Based on sequence phylogeny, we show that these proteins can be subdivided into four groups: denoted 1A, 1B, 2A and 2B; each characterized by the presence of specific conserved amino acid residues in the putative nucleotide-recognizing domain. A scattered distribution of AlkB proteins from the four different groups across the bacterial kingdom indicates a substantial degree of horizontal transfer of AlkB genes. DNA repair activity was associated with all tested recombinant AlkB proteins. Notably, both a group 2B protein from Xanthomonas campestris and a group 2A protein from Rhizobium etli repaired etheno adducts, but had negligible activity on methylated bases. Our data indicate that the majority, if not all, of the bacterial AlkB proteins are DNA repair enzymes, and that some of these proteins do not primarily target methylated bases.     

Phosphorus and osmium as elemental tags for the determination of global DNA methylation—A novel application of high performance liquid chromatography inductively coupled plasma mass spectrometry in epigenetic studies

The hyphenation of high performance liquid chromatography with inductively coupled plasma mass spectrometry (HPLC–ICP-MS) is proposed in this work as a novel approach for the evaluation of DNA methylation, defined as the ratio between methylated cytosine and total cytosine bases in DNA. In the first part, reversed phase separation of 5-methyl-2′-deoxycytidine monophosphate (5mdCMP) and four deoxynucleotides with specific ICP-MS detection on ³¹P had been explored. In further development, selective labeling of 5-methylcytosine in ssDNA was carried out using potassium osmate (K₂OsO₄) in the presence of strong oxidant (K₃Fe(CN)₆) and N,N,N′,N′-tetramethylethylenediamine (TEMED). The sample was then cleaned up and introduced to size exclusion chromatography–ICP-MS for specific detection at ³¹P and ¹⁸⁹Os and for evaluation of the molar ratio between Os and P eluted in DNA molecular mass fraction. The quantification of the two elemental tags was achieved by external calibration with phosphoric acid and Os(VI)-TEMED, respectively. The amount of Os in DNA fraction corresponded to methylated cytosines, while P signal was directly proportional to the total amount of DNA and could be recalculated to the amount of cytosine bases. The two procedures were tested by analyzing salmon testes DNA and a commercial oligonucleotide of known composition. For comparative purposes, these same samples were digested to deoxynucleosides and analyzed by reversed phase HPLC with spectrophotometric detection (DAD) at 280 nm. The results obtained using two procedures based on ICP-MS detection were in good agreement and also in agreement with the results obtained by HPLC–DAD procedure. In conclusion, ICP-MS specific detection at internal or external element tags seems to be an interesting alternative for the evaluation of global DNA in epigenetic studies.     

Methylation of parental and progeny DNA strands in Physarum polycephalum

Although 5-methylcytosine comprises 4 to 8% of the cytosine residues in the major nuclear DNA of Physarum polycephalum (Evans &; Evans, 1970), only 1 % of the cytosine residues of progeny DNA become methylated during replication. Further methylation occurs during the same and subsequent mitotic cycles, so that 6 to 7 cycles after its synthesis, 5-methylcytosine comprises 5 to 7% of the DNA-cytosine residues of a single generation of DNA. The extent of methylation occurring during the S period has been measured by the determination of the specific activity of the precursor (S-adenosylmethionine) and the product (DNA-5-methylcytosine) and by comparison of the radioactivity in DNA-cytosine and DNA-5-methylcytosine after incorporation of [¹⁴C]deoxycytidine. Continuing methylation of parental DNA has been shown, by density shift experiments and by the conversion of prelabeled DNA-cytosine to DNA-5-methylcytosine. The DNA-5-methylcytosine once formed was found to be stable.     

Determination of valproic acid and its metabolites using gas chromatography with mass-selective detection: application to serum and urine samples from sheep

An improved method for the quantitative determination of valproic acid (VPA) and sixteen of its metabolites has been developed using gas chromatography-mass spectrometry with selected-ion monitoring. The method is applicable to serum or urine and all metabolites are measured in a single chromatographic run of 29.5 min. Ions selected for quantitative purposes were the characteristic [M-57]⁺ ions of the tert.-butyldimethylsilyl (tBDMS) derivatives. The method utilizes heptadeuterated VPA as well as six heptadeuterated metabolites as internal standards [i.e. 2-[²H₇]propyl-2-pentenoic acid (2-ene[²H₇]VPA), 2-[²H₇]propyl-4-pentenoic acid (4-ene[²H₇]VPA), 2-[²H₇]propyl-3-oxopentanoic acid (3-keto[²H₇]VPA), 2-[²H₇]propyl-4-oxopentanoic acid (4-keto[²H₇]VPA), 2-[²H₇]propyl-3-hydroxypentanoic acid (3-OH[²H₇]VPA), 2-[²H₇]propyl-5-hydroxypentanoic acid (5-OH[²H₇]VPA)]. The method demonstrates very good accuracy and precision over a large range of concentrations for VPA and all metabolites measured in both human and sheep biological fluids. The assay was applied to the analysis of VPA and metabolites in serum and urine samples collected from three non-pregnant ewes following intravenous bolus administration of a mixture of VPA and [¹³C₄]VPA. Sheep were observed to produce measurable quantities of the majority of metabolites found in humans, with the notable exception of the di-unsaturated compounds (i.e. 2,3′-diene VPA and 2,4-diene VPA). The pharmacokinetics and metabolism of VPA and [¹³C₄]VPA appear to be equivalent in the sheep model. The elimination half-life of VPA and [¹³C₄] VPA in the ewe were estimated to be approximately 3.5 ± 0.4 and 3.2 ± 0.4 h, respectively.     

Biochemical effects of binding [(H2O)(NH3)5RuII]2+ to DNA and oxidation to[(NH3)5RuIII]n—DNA.

The reaction of [(H₂O)(NH₃)₅Ru^II]²⁺ with calf thymus and salmon sperm DNA has been studied over a wide fange of transition metal ion concentrations. Kinetic studies revealcd a biphasic reaction with an initial fairly rapid coordination of the metal ion being followed by slower reactions. Binding studies were done under pseudo-equilibrium conditions following completion of the initial rapid reaction. Spectra and HPLC of acid-hydrolyzed samples of [(NH3)₅Ru^II]n-DNA prepared by incubation of [(H₂O)(NH₃)₅Ru^II]²⁺ with DNA (where [P_DNA] = 1.5 mM and reactant [Ru^II]/[P_DNA] ratios were in the fange 0.1 to 0.3) followed by air oxidation showed the predominant binding site on helical DNA to be in the major groove at the N-7 of guanine. The equilibrium constant for [(H₂O)(NH₃)₅Ru^II]²⁺ binding to the G⁷ site in helical CT DNA is 5.1 x 103. Differential pulse voltammetry exhibited a single peak at 48 mV, which is attributed to the reduction of Rum on the G⁷ sites.At [Run]/[P_DNA] <0.5, Tm values for the DNA decreased linearly with increasing ruthenium concentration and an increase in the intensity of the 565 nm dG→ Ru(III) charge transfer band was noted upon melting. The UV and CD spectra of these samples indicated no extensive destacking or alteration in geometry (B family) compared to unsubstituted DNA. At [Run]/[P_DNA]〉 0.5 or when single-stranded DNA was used, increased absorbance at 530 nm and 480 nm suggested additional binding to the exocyclic amine sites of adenine and cytosine residues. HPLC and individual spectrophotometric identification of the products derived from hydrolysis of these spec~es yielded both [(Gua)(NH₃)₅Ru^III] and [(Ade)(NH₃)₅Ru^III]. Earlier studies have established the cytidine and adenosine binding sites of [(NH₃)₅Ru^III] to be at their exocyclic amines (C4 and A6). Coordination to these positions indicates disruption of the double helix since these amines are involved in hydrogen bonding on the interior of B-DNA.Agrose gel electrophoresis of superhelical pBR322 plasmid DNA after exposure to various complexes of [(nh₃)₅Ruⁱⁱⁱ] in the presence of a reductant and air generally revealed moderately efficient cleavage of the DNA, presumably due to the generation of hydroxyl radical via Fenton's chemistry. However, similar studies involving [(NH₃)₅Ru^III] directly coordinated to the DNA showed no strand cutting above background. Polyacrylamide gel electrophoresis of a 381 bp, 3′-³²P-labeled fragment of pBR322 plasmid DNA containing low levels of bound [(NH₃)₅ Ru^III] further indicated negligible DNA cutting by the coordinated metal ion.     

Intragenome distribution of 5-methylcytosine in DNA of healthy and wilt-infected cotton plants (Gossypium hirsutum L.)

Fractionation of DNA of healthy and wilt-infected cotton plants has been carried out according to the reassociation kinetics and the content of GC and 5-methylcytosine in the resulting fractions has been studied. The genome of cotton plant was found to be methylated quite unevenly. The GC rich (GC=64.7 mole%) fraction of highly reiterated sequences (C ₀ t=0–3.7×10^-2) has a high content of 5-methylcytosine (5.8 mole%), whereas the methylation degree of the fraction of unique sequences (C ₀ t487) is very low (the 5-methylcytosine content is about 0.5 mole%). In plants being infected with wilt, the 5-methylcytosine content in DNA of cotton leaves decreases two-fold; no other changes in the structure and molecular population of DNA has been found. The sharp change in the 5-methylcytosine content in DNA of infected plants takes place at the expense of the decrease in the 5-methylcytosine content in fractions of highly reiterated sequences. The methylation degree of unique sequences (structural genes) remains unchanged.     

The kinetics of DNA methylation in cultures of a mouse adrenal cell line

Direct measurements of the methylation of newly-synthesized DNA were made in cultures of a clonal mouse adrenal cortex cell line, Y129OS3, by (1) following the incorporation of radioactivity from methionine-(methyl)-C¹⁴ into a segment of DNA which had been density-labeled with bromouracil and (2) labeling DNA cytosine with C¹⁴-deoxycytidine and then following the appearance of radioactivity in DNA 5-methylcytosine. The results establish that during exponential growth the DNA of this cell line is methylated entirely within a few minutes of its synthesis. Using the second technique described above accurate, sensitive measurements of DNA methylation levels can be made by comparing radioactivity in 5-methylcytosine to radioactivity in cytosine plus 5-methylcytosine. In this cell line 5-methylcytosine accounts for 4.3 ± 0.2% of the DNA cytosine. Some apparent contradictions between these results and those of other workers are discussed.     

Structural dynamics of double-stranded DNA with epigenome modification

Modification of cytosine plays an important role in epigenetic regulation of gene expression and genome stability. Cytosine is converted to 5-methylcytosine (5mC) by DNA methyltransferase; in turn, 5mC may be oxidized to 5-hydroxymethylcytosine (5hmC) by ten-eleven translocation enzyme. The structural flexibility of DNA is known to affect the binding of proteins to methylated DNA. Here, we have carried out a semi-quantitative analysis of the dynamics of double-stranded DNA (dsDNA) containing various epigenetic modifications by combining data from imino ¹H exchange and imino ¹H R_1ρ relaxation dispersion NMR experiments in a complementary way. Using this approach, we characterized the base-opening (k_open) and base-closing (k_close) rates, facilitating a comparison of the base-opening and -closing process of dsDNA containing cytosine in different states of epigenetic modification. A particularly striking result is the increase in the k_open rate of hemi-methylated dsDNA 5mC/C relative to unmodified or fully methylated dsDNA, indicating that the Watson–Crick base pairs undergo selective destabilization in 5mC/C. Collectively, our findings imply that the epigenetic modulation of cytosine dynamics in dsDNA mediates destabilization of the GC Watson–Crick base pair to allow base-flipping in living cells.     

A unique family of Mrr-like modification-dependent restriction endonucleases

Mrr superfamily of homologous genes in microbial genomes restricts modified DNA in vivo. However, their biochemical properties in vitro have remained obscure. Here, we report the experimental characterization of MspJI, a remote homolog of Escherichia coli’s Mrr and show it is a DNA modification-dependent restriction endonuclease. Our results suggest MspJI recognizes ^mCNNR (R = G/A) sites and cleaves DNA at fixed distances (N₁₂/N₁₆) away from the modified cytosine at the 3′ side (or N₉/N₁₃ from R). Besides 5-methylcytosine, MspJI also recognizes 5-hydroxymethylcytosine but is blocked by 5-glucosylhydroxymethylcytosine. Several other close homologs of MspJI show similar modification-dependent endonuclease activity and display substrate preferences different from MspJI. A unique feature of these modification-dependent enzymes is that they are able to extract small DNA fragments containing modified sites on genomic DNA, for example ∼32 bp around symmetrically methylated CG sites and ∼31 bp around methylated CNG sites. The digested fragments can be directly selected for high-throughput sequencing to map the location of the modification on the genomic DNA. The MspJI enzyme family, with their different recognition specificities and cleavage properties, provides a basis on which many future methods can build to decode the epigenomes of different organisms.     

Kinetics of deamination and Cu(II)/H2O2/Ascorbate-induced formation of 5-methylcytosine glycol at CpG sites in duplex DNA

Mutation in p53 tumor suppressor gene is a hallmark of human cancers. Six major mutational hotspots in p53 contain methylated CpG (mCpG) sites, and C →T transition is the most common mutation at these sites. It was hypothesized that the formation of 5-methylcytosine glycol induced by reactive oxygen species, its spontaneous deamination to thymine glycol and the miscoding property of the latter may account, in part, for the ubiquitous C →T mutation at CpG site. Here, we assessed the kinetics of deamination for two diastereomers of 5-methylcytosine glycol in duplex DNA. Our results revealed that the half-lives for the deamination of the (5S,6S) and (5R,6R) diastereomers of 5-methylcytosine glycol in duplex DNA at 37°C were 37.4 ± 1.6 and 27.4 ± 1.0 h, respectively. The deamination rates were only slightly lower than those for the two diastereomers in mononucleosides. Next, we assessed the formation of 5-methyl-2′-deoxycytidine glycol in the form of its deaminated product, namely, thymidine glycol (Tg), in methyl-CpG-bearing duplex DNA treated with Cu(II)/H₂O₂/ascorbate. LC-MS/MS quantification results showed that the yield of Tg is similar as that of 5-(hydroxymethyl)-2′-deoxycytidine. Together, our data support that the formation and deamination of 5-methylcytosine glycol may contribute significantly to the C →T transition mutation at mCpG dinucleotide site.     

Improved automated solid-phase microsequencing of peptides using DABITC

The methylated purines O⁶-methyl- and 7-methylguanine were isolated from mouse liver DNA hydrolysates by means of a column cleanup employing a Sep Pak C-18 reverse-phase cartridge. The purine bases were eluted from the cartridge with methanol, evaporated to dryness, and then dissolved in mobile phase for liquid chromatographic analysis by normalphase chromatography. The system consisted of a LiChrosorb Si 60 column with a watersaturated mobile phase of 20% methanol in chloroform containing 0.001% H₃PO₄. The two methylated bases eluted before adenine or guanine. For extremely low-level (<300 pmol) quantitation, the peaks corresponding to O⁶-methyl- and 7-methylguanine were collected and then analyzed by reverse-phase chromotography with a LiChrosorb RP-18 column and a mobile phase of 5% methanol in pH 7 phosphate buffer (for 7-methylguanine) or 9.5% methanol/buffer (for O⁶-methylguanine). Comparisons were made with fluorescence detection and with scintillation counting (in animal studies where [¹⁴C]dimethylnitrosamine was used). Minimum detectable levels at 254 nm were about 3 ng (3:1 signal to noise ratio) for each of the title compounds. As low as 10 pmol/mg of each could be detected in DNA hydrolysates. Recoveries of O⁶-methyl- and 7-methylguanine from DNA spiked at 750 pmol/mg were greater than 80%.     

Recognition and cleavage of 5-methylcytosine DNA by bacterial SRA-HNH proteins

SET and RING-finger-associated (SRA) domain is involved in establishment and maintenance of DNA methylation in eukaryotes. Proteins containing SRA domains exist in mammals, plants, even microorganisms. It has been established that mammalian SRA domain recognizes 5-methylcytosine (5mC) through a base-flipping mechanism. Here, we identified and characterized two SRA domain-containing proteins with the common domain architecture of N-terminal SRA domain and C-terminal HNH nuclease domain, Sco5333 from Streptomyces coelicolor and Tbis1 from Thermobispora bispora. Both sco5333 and tbis1 cannot establish in methylated Escherichia coli hosts (dcm⁺), and this in vivo toxicity requires both SRA and HNH domain. Purified Sco5333 and Tbis1 displayed weak DNA cleavage activity in the presence of Mg²⁺, Mn²⁺ and Co²⁺ and the cleavage activity was suppressed by Zn²⁺. Both Sco5333 and Tbis1 bind to 5mC-containing DNA in all sequence contexts and have at least a preference of 100 folds in binding affinity for methylated DNA over non-methylated one. We suggest that linkage of methyl-specific SRA domain and weakly active HNH domain may represent a universal mechanism in competing alien methylated DNA but to maximum extent minimizing damage to its own chromosome.     

Copper(I)-organophosphine complexes of bis(3,5-dimethylpyrazol-1-yl)dithioacetate ligand

Copper(I) complexes have been synthesized from the reaction of CuCl, monodentate tertiary phosphines PR₃ (PR₃ = P(C₆H₅)₃; P(C₆H₅)₂(4-C₆H₄COOH); P(C₆H₅)₂(2-C₆H₄COOH); PTA, 1,3,5-triaza-7-phosphaadamantane; P(CH₂OH)₃, tris(hydroxymethyl)phosphine) and lithium bis(3,5-dimethylpyrazolyl)dithioacetate, Li[LCS₂]. Mono-nuclear complexes of the type [LCS₂]Cu[PR₃] have been obtained and characterized by elemental analyses, FT-IR, ESI-MS and multinuclear (¹H, ¹³C and ³¹P) NMR spectral data; in these complexes the ligand behaves as a κ³-N,N,S scorpionate system. One exception to this stoichiometry was observed in the complex [LCS₂]Cu[P(CH₂OH)₃]₂, where two phosphine co-ligands are coordinated to the copper(I) centre. The solid-state X-ray crystal structure of [LCS₂]Cu[P(C₆H₅)₃] has been determined. The [LCS₂]Cu[P(C₆H₅)₃] complex has a pseudo tetrahedral copper site where the bis(3,5-dimethylpyrazolyl)dithioacetate ligand acts as a κ³-N,N,S donor.     

Study of the cytotoxicity and particle action in human cancer cells of titanocene-functionalized materials with potential application against tumors

Titanocene dichloride [Ti(η⁵-C₅H₅)₂Cl₂] (1), has been grafted onto dehydrated hydroxyapatite (HAP), Al₂O₃ and two mesoporous silicas MSU-2 (Michigan State University Silica type 2) and HMS (Hexagonal Mesoporous Silica), to give the novel materials HAP/[Ti(η⁵-C₅H₅)₂Cl₂] (S1) (1.01 wt.% Ti), Al₂O₃/[Ti(η⁵-C₅H₅)₂Cl₂] (S2) (2.36 wt.% Ti), HMS/[Ti(η⁵-C₅H₅)₂Cl₂] (S3) (0.75 wt.% Ti) and MSU-2/[Ti(η⁵-C₅H₅)₂Cl₂] (S4) (0.74 wt.% Ti), which have been characterized by powder X-ray diffraction, X-ray fluorescence, nitrogen gas sorption, multinuclear magic angle spinning NMR spectroscopy, IR spectroscopy, thermogravimetry analysis, UV spectroscopy, scanning electronic microscopy and transmission electronic microscopy. The cytotoxicity of the titanocene-functionalized materials toward human cancer cell lines from five different histogenic origins: 8505 C (anaplastic thyroid cancer), A253 (head and neck cancer), A549 (lung carcinoma), A2780 (ovarian cancer) and DLD-1 (colon cancer) has been determined. M₅₀ values (quantity of material needed to inhibit normal cell growth by 50%) and Ti-M₅₀ values (quantity of anchored titanium needed to inhibit normal cell growth by 50%) indicate that the activity of S1-S4 against studied human cancer cells depended on the surface type as well as on the cell line. In addition, studies on the titanocene release and the interaction of the materials S1-S4 with DNA show that the cytotoxic activity may be due to particle action, because no release of titanium complexes has been observed in physiological conditions, while electrostatic interactions of titanocene-functionalized particles with DNA have been observed.     

Differential binding of Escherichia coli McrA protein to DNA sequences that contain the dinucleotide m5CpG

The Escherichia coli McrA protein, a putative C⁵-methylcytosine/C⁵-hydroxyl methylcytosine-specific nuclease, binds DNA with symmetrically methylated HpaII sequences (Cm5CGG), but its precise recognition sequence remains undefined. To determine McrA’s binding specificity, we cloned and expressed recombinant McrA with a C-terminal StrepII tag (rMcrA-S) to facilitate protein purification and affinity capture of human DNA fragments with m5C residues. Sequence analysis of a subset of these fragments and electrophoretic mobility shift assays with model methylated and unmethylated oligonucleotides suggest that N(Y > R) m5CGR is the canonical binding site for rMcrA-S. In addition to binding HpaII-methylated double-stranded DNA, rMcrA-S binds DNA containing a single, hemimethylated HpaII site; however, it does not bind if A, C, T or U is placed across from the m5C residue, but does if I is opposite the m5C. These results provide the first systematic analysis of McrA’s in vitro binding specificity.     

Quantitative high-pressure liquid chromatographic analysis of methylated purines in DNA of rats treated with chemical carcinogens

A rapid, sensitive method for the quantitative measurement of certain major and modified purines in DNA of carcinogen-treated animals is presented. DNA hydrolysates are analyzed by high-pressure liquid chromatography combined with fluorescence detection and electronic integration of peaks. Limits of detection are approximately 7 ng for 7-methylguanine and 150 pg for O⁶-methylguanine. Between 100 and 250 μg target organ DNA from animals treated with several carcinogens was shown to contain readily detectable amounts of these methylated bases. The method provides results comparable to those obtained with conventional methods using radioactively labeled carcinogens.     

One-dimensional separation of nine DNA bases on strong cation-exchange thin-layers.

In contrast to the large number of base modifications in RNA, only a limited number of modified bases have been identified in DNA. 5-Methyl-cytosine and N⁶-methyl-adenine have been found to be minor components. On the other hand, in some bacteriophages complete replacement of one of the pyrimidines occurs: 5-methylcytosine or 5-hydroxymethyl-cytosine replaces cytosine and uracil and 5-hydroxymethyl-uracil substitutes for thymine (1). ¹N-thyminylputrescine (2) and dihydroxypentyluracil (3) have been isolated from bacteriophages ΦW-14 and SP-15. The present paper does not deal with these compounds.