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PubMed1989

Base and conformational specificity of an amine modification of DNA.

We have investigated the site and conformational preference of the reaction of a formaldehyde/amine reagent with DNA. Previous investigations of this laboratory have established that this reagent will react with native DNA, placing a positively charged amine moiety on the duplex that will survive exhaustive dialysis. The resulting adduct is duplex and base stacked in character, possessing B backbone geometry with a higher average winding angle and exhibiting remarkable stability with respect to the A-form, Z-form, or the single-strand denaturated species. In this current investigation, we have found that the stability of the adduct is dramatically reduced if the DNA is converted to mononucleotides, thus obviating the usual approach of nuclease digestion and chromatography for the identification of the modified nucleotides. Using indirect approaches, we have established that the reactive site that survives removal of the equilibrium concentrations of CH2O and amine is the exocyclic amino group of the guanine bases. This conclusion is based on (1) the positive correlation between GC content and the extent of adduct formation under standard reaction conditions (27 degrees C, 0.63M CH2O, 0.007M n-butylamine, pH 7); (2) decreases in the level of substitution of amine in DNA, which has this site blocked by trinitrobenzene modification; and (3) failure of poly(dI-dC) to retain amine upon dialysis. Raman spectra of the derivatized poly(dG-dC) show enhanced 2'-endo B character, with no marked shifts in the position of any of the lines, indicating the absence of any ring structures involving the N7 and the 06 of G. In standard reaction mixtures, other sites may react but this phenomenon appears to be minimal under conditions that do not favor fluctuational opening of base pairs. In the latter case, excess loading of amine on high GC content polymers produces a CD spectrum that is similar to one produced by poly(dA-dT) in the "X"-form [M. Vorlickova, E. Minyat, and J. Kypr (1984) Biopolymers 23, 1-4]. This conformation is lost, however, upon removal of excess reagents by dialysis and cannot be reestablished, in the absence of unbound amine and formaldehyde. The reaction is specific for the B-form of polynucleotides as demonstrated by the failure of poly(dG-m5dC) in the stable Z-form to exhibit substantial reaction. The B-form of this polymer will react readily with the retention of 0.23 moles amine/mole nucleotide under our standard reaction conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Amines↗
PubMed1991

Cloning the BstVI restriction-modification system in Escherichia coli.

A standard DNA modification methyltransferase (MTase) selection protocol was followed to clone the BstVI restriction and modification system from Bacillus stearothermophilus in Escherichia coli. Both genes were contained in a 4.4-kb EcoRI fragment from B. stearothermophilus V chromosomal DNA. The heterologous expression of these genes did not depend on their orientation in the vector, suggesting that the genes are expressed in E. coli under the control of promoters located on the cloned fragment. Subcloning experiments demonstrated that the bstVIR gene was expressed in the absence of its cognate MTase.

Cloning, Molecular↗
PubMed1991

Molecular dosimetry of sister chromatid exchange induction in 9L cells treated with 6-thioguanine.

The induction of sister chromatid exchanges (SCE) in 9L cells treated with 6-thioguanine (6-TG) has been investigated. A 24 h treatment with 0.2 microM 6-TG induced approximately 28 SCE/metaphase. The dose-response curve was linear at doses below 0.2 microM and had a slope of 139 SCE/metaphase/microM 6-TG. At concentrations of 0.023 to 1 microM, incorporation of 6-TG in DNA was linear with dose. The slope of the dose-response curve was 4135 mumol 6-TG/mol DNA/microM 6-TG. Comparison of these results with those obtained in our previous studies of the monofunctional alkylating agent ethylnitrosourea and the bifunctional alkylating agent 3-(4-amino-2-methyl-5-pyrimidinyl)methyl-1-(2-chloroethyl)-1-nitrosourea suggest that to accurately estimate the effectiveness of particular DNA modifications at inducing SCE, the extent of formation of these DNA modifications must be known.

Alkylating Agents↗
PubMed1984

Use of short DNA oligonucleotides for determination of DNA sequence modifications induced by benzo[a]pyrene diol epoxide.

Various organic agents that alkylate DNA are known to induce mutations in bacterial and animal cells. The precise nature and location of modified DNA sequences in such mutants are often difficult to ascertain. In this report, a 10-base-pair oligomer (BamHI linker) is treated with (+/-)-trans-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide and inserted into replicative form DNA of phage M13 by ligation at a specific restriction site. Escherichia coli are transfected with the recombinant DNA containing the alkylated target, progeny viral plaques are selected, and their DNAs are subjected to DNA sequence analysis at the region of oligomer insertion. For the alkylated inserts used in this study, the DNA sequence analysis of progeny viral DNA showed that nucleotide deletions were present in every clone examined. These deletions occurred primarily, but not exclusively, at G-C cluster regions, varied from 1 to 24 base pairs in length, and included both target and nontarget nucleotides. A second type of repair, which restores most of the original nucleotide bases in the alkylated insert, is also implied by the DNA sequence data obtained.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1998

High levels of endogenous DNA adducts (I-compounds) in pig liver. Modulation by high cholesterol/high fat diet.

I (indigenous)-compounds are bulky endogenous DNA adducts which are detected by 32P-postlabeling in unexposed animals. I-compound levels in rodents depend on age, species, strain, gender, tissue, diet, and chemical exposure. There are two classes of I-compounds, type I and type II. While many type I I-compounds may not reflect DNA damage, type II I-compounds have been identified as oxidative DNA lesions some of which can be produced in vitro under Fenton reaction conditions. In rats, caloric restriction (CR) increases the levels of many type I I-compounds compared with ad libitum fed animals, while high fat diet has the opposite effect. Here, we have tested whether hepatic DNA of a non-rodent mammal, the pig, contains I-compounds and whether feeding a high cholesterol/high fat (HC/HF) diet modulates their levels, assuming this would affect the formation of lipid-related precursors and cause oxidative stress. Male Yorkshire pigs aged 2 months old, were fed either control or HC/HF diet (control diet supplemented with 2% cholesterol and 19% lard) for 2 months. Pig liver DNA contained at least 19 type I and five type II I-compounds. Among the former, only five matched corresponding spots in rat liver DNA, while all the latter DNA lesions were detected in both species. The levels of both types of DNA modifications were six to eight-fold higher in pig DNA. HC/HF diet reduced levels of many type I I-compounds up to several fold but had little effect on the oxidative lesions. Several type I I-compounds showed negative linear correlations with serum cholesterol levels, while this association was positive for total type II I-compounds. The substantially elevated steady-state levels of bulky endogenous DNA adducts in the species with the longer life expectancy were surprising. Thus, for the first time, an intimate link between nutritional status and endogenous DNA modifications has been established in a non-rodent system. We propose that in order to explain our observations, differences in diet composition, antioxidant defenses, and DNA repair, as well as cytochrome P450 modulation of precursor levels and hormonal effects need to be considered.

Animals↗
PubMed1998

Duck hepatitis B virus inactivation and 8-methoxypsoralen photoadduct formation in human platelet concentrates.

Photochemical inactivation (PCI) of virus and bacteria in platelet concentrates (PC) has been demonstrated using 8-methoxypsoralen (8-MOP) and long-wavelength UV light (UVA). To study inactivation of blood-borne virus, we have employed duck hepatitis B virus (DHBV), a model for human hepatitis B virus. A specific hepatocyte culture infectivity assay, with PCR detection, could measure 5-6 log10 virus kill. The DHBV inactivation in PC was dependent on UVA dose, was enhanced when plasma was reduced from 100% to 20% and was limited by 8-MOP solubility in the reduced-plasma medium. Optimum conditions for PCI were 100 micrograms/mL 8-MOP in 20% plasma and 80% synthetic platelet storage medium. A radiolabeling assay for 8-MOP photoadducts in hepatocytes seeded into PC confirmed that DHBV inactivation reflected DNA modification and indicated that adduct formation was insensitive to minor variations in conditions. Kinetic modeling indicated that optimum adduct formation was a compromise between 8-MOP dark binding and optical transmittance and that plasma proteins competed for 8-MOP binding. The PCI results in various media correlated with corresponding DNA modification densities and were compared to statistical models incorporating DHBV characteristics and predictions of 8-MOP crosslink formation between DNA strands. Behavior was consistent with one or a small number of lethal modifications per DNA strand, including monoadducts, but probably not crosslinks alone. A minor subpopulation of DHBV was found to be somewhat more difficult to inactivate, consistent with three-fold lower modification, due possibly to single-stranded DNA character or host repair of photoadducts.

Blood Platelets↗
PubMed1985

Metabolism and specific benzopyrene metabolite modification of DNA in early S by human lung epithelial and fibroblast cells leading to the expression of an abnormal phenotype.

Examination of the high pressure liquid chromatographic profiles of ethyl acetate extractable benzo [a] pyrene (B(a)P)-metabolites from human lung fibroblast and type II epithelial cells after S phase entry indicated that B(a)P-7,8-diol and 9,10-diol species were produced following the oxygenation of B(a)P. These metabolites were detected intracellularly and in the extracellular growth medium. Both cell types appeared to release extracellularly, elevated amounts of the B(a)P-7,8-diol species. It was interesting to note of the 4 pmol of oxygenated metabolites localized intracellularly, in the fibroblast, that we identified two major metabolites, B(a)P-9,10 and -7,8-diol species. Lung epithelial cells metabolize intracellular B(a)P extensively, greater than or equal to 93% of the parent B(a)P. No tetrols were detected intracellularly or extracellularly in the treated fibroblast cells. The treated epithelial cells produced both tetrols and sulfate conjugates. The extent of observed modification of early S phase nuclear DNA of lung epithelial cells was 7.5 +/- 4.9 adducts per 10(6) bases and 4.2 +/- 2.7 adducts per 10(6) bases in lung fibroblasts. The major adduct formed in both cell types was 7 beta-BPDE-I-dG. Under conditions for transformation, both the B(a)P treated lung epithelial cells and lung fibroblasts treated in early S with either B(a)P or BPDE-I yielded populations that exhibited properties of anchorage independent growth and cellular invasiveness. Metabolism and the presence internally of metabolites did not correlate with the extent of modification of DNA in early S.

Benzo(a)pyrene↗
PubMed2004

Molecular diagnostic applications of DNA methylation technology.

Cancer arises due to the accumulation of DNA modifications that give cells a selective growth advantage. One common DNA modification is promoter hypermethylation associated with loss of expression of a tumor suppressor gene. The methylation status of a specific sequence or the pattern of methylation across the genome can be readily measured, and these sequences and analytical methods are being rapidly developed for molecular diagnostic applications. Detection of certain methylation events can be used for early detection of tumors, and analysis of patterns of methylation across the genome might provide information on disease subtype, aggressiveness, and treatment response. DNA methylation-based molecular diagnostic assays are particularly attractive because of the stability of the target analyte (DNA) and the potential sensitivity of the assays. As the field matures, methylation-based assays will make a major contribution to the field of molecular diagnostics, providing tools to fill unmet needs in current diagnostic and treatment plans for many types of cancer.

Biomarkers, Tumor↗
PubMed1976

Salmonella typhimurium SA host specificity system is based on deoxyribonucleic acid-adenine methylation.

We have determined the nature of the deoxyribonucleic acid (DNA) modification governed by the SA host specificity system of Salmonella typhimurium. Two lines of evidence indicate that SA modification is based on methylation of DNA-adenine residues. (i) The SA+ locus of Salmonella was transferred into Escherichia coli B, a strain that does not contain 5-methylcytosine in its DNA; although the hybrid strain was able to confer SA modification, its DNA still did not contain 5-methylcytosine. (ii) the N6-methyladenine content of phage L DNA was measured after growth in various host strains; phage lacking SA modification contained fewer N6-methyladenine residues per DNA. We also investigated the possibility, suggested by others (32), that SA modification protects phage DNA against restriction by the RII host specificity system. Phages lambda, P3, and L were grown in various SA+ and SA- hosts and tested for their relative plating ability on strains containing or lacking RII restriction; the presence or absence of SA modification had no effect on RII restriation. In vitro studies revealed, however, that Salmonella DNA is protected against cleavage by purified RII restriction endonuclease (R-EcoRII). This protection is not dependent on SA modification; rather, it appears to be due to methylation by a DNA-cytosine methylase which has overlapping specificity with the RII modification enzyme, but which is not involved in any other known host specificity system.

Adenine↗
PubMed1995

DNA base modifications and antioxidant enzyme activities in human benign prostatic hyperplasia.

The authors have studied DNA base damage and activities of antioxidant enzymes in human benign prostatic hyperplasia (BPH) tissues and surrounding disease-free tissues removed from prostate glands of 15 patients. In these tissues, endogenous levels of various typical hydroxyl radical-induced products of DNA bases and activities of catalase and superoxide dismutase were measured. The majority of patients had higher levels of DNA base lesions and lower activities of enzymes in BPH tissues than in normal prostate tissues. When activities of both enzymes were lower in BPH tissues than in normal tissues, the increases in the amounts of DNA base lesions over control levels were most prominent. In the case of similar enzyme activities in both BPH and normal tissues, no changes in levels of DNA base lesions were observed. These results suggest a possible association between antioxidant enzyme activities and levels of DNA base lesions in BPH tissues. Some of the identified DNA lesions are known to be premutagenic and may play a role in carcinogenesis. Although a possible link between BPH and prostate cancer is controversial, BPH patients with both decreased antioxidant enzyme activities and increased levels of DNA lesions may be at risk of developing prostate cancer.

Antioxidants↗
PubMed1995

DNA base modifications and membrane damage in cultured mammalian cells treated with iron ions.

We investigated DNA base damage in mammalian cells exposed to exogenous iron ions in culture. Murine hybridoma cells were treated with Fe(II) ions at concentrations of 10 microM, 100 microM, and 1 mM. Chromatin was isolated from treated and control cells and analyzed by gas chromatography/mass spectrometry for DNA base damage. Ten modified DNA bases were identified in both Fe(II)-treated and control cells. The quantification of modified bases was achieved by isotope-dilution mass spectrometry. In Fe(II)-treated cells, the amounts of modified bases were increased significantly above the background levels found in control cells. Dimethyl sulfoxide at concentrations up to 1 M in the culture medium did not significantly inhibit the formation of modified DNA bases. A mathematical simulation used to evaluate the plausibility of DNA damage upon Fe(II) treatment predicted a dose-dependent response, which agreed with the experimental results. In addition, Fe(II) treatment of cells increased the cell membrane permeability and caused production of lipid peroxides. The nature of DNA base lesions suggests the involvement of the hydroxyl radical in their formation. The failure of dimethyl sulfoxide to inhibit their formation indicates a site-specific mechanism for DNA damage with involvement of DNA-bound metal ions. Fe(II) treatment of cells may increase the intracellular iron ion concentration and/or cause oxidative stress releasing metal ions from their storage sites with subsequent binding to DNA. Identified DNA base lesions may be promutagenic and play a role in pathologic processes associated with iron ions.

Animals↗
PubMed1984

Modification of DNA by reducing sugars: a possible mechanism for nucleic acid aging and age-related dysfunction in gene expression.

Reducing sugars react nonenzymatically with protein amino groups to initiate a process called nonenzymatic browning. Long-lived proteins, such as collagen and the lens crystallins, accumulate sufficient modification in vivo that they acquire many of the chemical properties characteristic of aged proteins. We have obtained evidence that nucleic acids also can undergo nonenzymatic modification by sugars. Incubation of DNA or nucleotides with glucose 6-phosphate (Glc-6-P) produces spectral changes similar to those described for nonenzymatic browning proteins. The occurrence of chemical modification was verified by measuring the transfection efficiency of viral DNA after incubation with glucose and Glc-6-P. A loss of transfection potential occurred that was first order with respect to time and sugar concentration. The rate of inactivation by Glc-6-P was 25 times that of glucose; 8 days of incubation with 150 mM Glc-6-P decreased transfection by 4 orders of magnitude. Glc-6-P also produced strand scission in a time- and concentration-dependent manner. We conclude that glucose, Glc-6-P, and possibly other sugars can react with DNA to produce significant structural and biological alterations. Since nucleic acids are long-lived molecules in the resting cell, the accumulation of these addition products might be a mechanism for the decreased genetic viability characteristic of the aged organism.

Aging↗
PubMed1999

Combined prenatal toxicity of 6-mercaptopurine riboside and hydroxyurea in mice.

Hydroxyurea (HU) and 6-mercaptopurine riboside (6-MPr) are used as cytostatic chemotherapeutics. Their teratogenic potential in experimental animals has been well known for several decades. Generally, it is assumed that the toxicity of both agents is due to an interference with enzymes of DNA synthesis. In the case of 6-MPr, it was speculated that the teratogenicity in rodents might be paralleled by or even correlated to an incorporation of 6-thioguanine into the DNA of the embryos. In this study, the interaction between these two compounds with regard to teratogenicity in NMRI mice was investigated. Dose-response data of 6-MPr (s.c.-treatment) were published earlier. First, a dose-response study with HU alone (i.p.-treatment) was performed. From these data, the doses for the combination study were derived: HU 250 mg/kg (NOAEL dose) and 6-MPr 16 mg/kg (strongly teratogenic dose). In all experimental groups the substances were administered to the dams once on day 11 of gestation. Combination effects were investigated applying various dosing regimens. In group I treatment was simultaneous, in group II HU was administered 2 h before 6-MPr, in group III 2 h after 6-MPr. The differences in the overall frequency of gross structural abnormalities were moderate. However, when analysing the effects in more detail (single abnormalities), group III exhibited great differences: 1) 6-MPr co-treatment increased the frequency of HU effects (skull defects) and 2) HU co-treatment decreased the frequency of 6-MPr effects (limb defects) when compared to the findings of the dose-response studies. In addition, the influence of HU on the 6-MPr-induced DNA modification was determined by measuring the incorporation of 6-thioguanine into the DNA of day-11 embryos. As expected, the HU co-treatment corresponding to the group III dosing regimen of the teratogenicity experiment decreased the incorporation rate by ca. 40%. This was in parallel to the decrease in the frequency of 6-MPr effects in the teratogenicity III group. This finding may be considered as a further indication that in the case of 6-MPr, DNA modification is accompanying teratogenicity.

Abnormalities, Drug-Induced↗
PubMed1987

Developmental expression and modification of genes.

Differentiating tissue is characterized by a specific repertoire of proteins out of which some are developmentally controlled. This review describes modifications in the structure of genes which encode developmentally regulated proteins. Evidence is provided for changes in chromatin conformation and DNA methylation of specific genes-either change can be observed in various stages of some vertebrates. The involvement of hormones in regulating DNA modifications is suggested, and interrelationships between DNA modifications and gene expression are discussed.

5-Methylcytosine↗
PubMedDate not supplied

Synthesis and characterization of a new 5-thiol-protected deoxyuridine phosphoramidite for site-specific modification of DNA.

A new nucleotide analogue was developed for site-specific incorporation of a reactive thiol group into DNA. This creates a unique site for the post-synthetic modification of that nucleotide with a variety of molecular tags, such as photo-cross-linkers and fluorescent or spin-label moieties. 5'-O-(4,4'-Dimethoxytrityl)-5-[S-(2,4-dinitrophenyl)thio]-2'-deoxyuridin e 3'-O-(2-cyanoethyl N,N'-diisopropylphosphoramidite) was synthesized and incorporated at internal positions in several oligonucleotides using automated DNA synthesis and standard phosphoramidite chemistry. The coupling yield of the analogue was comparable to the coupling yield for a standard phosphoramidite, and no significant differences were observed in the overall yields of the dinitrophenyl-labeled oligonucleotides compared to the corresponding unmodified oligonucleotides. Characterization of the dinitrophenyl-modified oligonucleotides included enzymatic degradation, HPLC chromatography, and gel electrophoresis. Deprotection of the mercaptan group with beta-mercaptoethanol yielded an oligonucleotide containing 5-mercaptodeoxyuridine which was then selectively modified, without purification, by reaction with 5-(iodoacetamido)fluorescein. Incorporation of the dinitrophenyl-modified oligonucleotide into double-stranded DNA was achieved using the polymerase chain reaction. CHaracterization of the dinitrophenyl-labeled product by immunodetection with anti-dinitrophenyl antibodies confirmed the stability of the protecting group to the thermocycling and thus established the use of this thiol-protected mercaptodeoxyuridine phosphoramidite for preparation of site-specifically modified DNA.

Alkylation↗
PubMed1978

Modification of DNA by the benzo[a]pyrene metabolite diol-epoxide r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene.

The structural modification of double-stranded circular DNA of simian virus 40 and plasmid ColE1 by in vitro binding of r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene was studied. Stepwise hydrolysis with endonuclease S1 and DNase followed by DNA base analysis by thin-layer chromatography provided evidence that binding to adenine caused the local denaturation of DNA, whereas the more than 10-fold greater binding to guanine did not create such local denaturation. Of the two synthetic double-stranded polymers, poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC), bound to the diol-epoxide, only the former showed a marked hydrolysis after endonuclease S1 treatment, whereas binding occurred 24-fold more on the latter.

Benzopyrenes↗
PubMed1984

High-affinity monoclonal antibodies for the specific recognition and quantification of deoxynucleosides structurally modified by N-nitroso compounds.

The applicability of conventional radiochromatographic procedures to the detection and quantification of specific, carcinogen-induced structural modifications in the DNA of mammalian cells is limited by the necessity of using radioactively labelled agents and by the relatively large amounts of DNA required for analysis of low levels of DNA modification. Recently developed immunoanalytical methods have improved this situation considerably. High-affinity monoclonal antibodies (MAB), in combination with radio- and enzyme-immunoassays, now permit the sensitive detection of alkyldeoxynucleosides in small samples of hydrolysed DNA from tissues and cultured cells exposed previously to non-radioactive (e.g., environmental) alkylating N-nitroso carcinogens. Furthermore, MAB can be used to quantify by direct immunofluorescence (and with the aid of computer-based image analysis of electronically intensified fluorescence signals) specific alkylation products in the DNA of individual cells. With this method, the present detection limit for, e.g. O6-ethyl-2'-deoxyguanosine (O6-EtdGuo) is of the order of 7 X 10(2) O6-EtdGuo molecules per diploid genome. Therefore, cells (e.g. from biopsy material) can now be monitored directly for the presence of specific carcinogen-DNA adducts, or with respect to their capacity to remove enzymatically such modified structures from DNA. In combination with transmission electron microscopy, MAB also permit the direct visualization of specific carcinogen-modified sites in DNA. Thus, O6-EtdGuo can be localized in double-stranded DNA molecules by the binding of a MAB specifically directed against this ethylation product.

Antibodies, Monoclonal↗