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At least 217 records · Page 12Linked to original sources

PubMed2006

Is the repair of oxidative DNA base modifications inducible by a preceding DNA damage induction?

In mammalian cells, 7,8-dihydro-8-oxoguanine (8-oxoG) and some other oxidative guanine modifications are removed from the DNA by base excision repair, which is initiated by OGG1 protein. We have tested whether this repair is inducible in mouse embryonic fibroblasts (MEFs), MCF-7 breast cancer cells and primary human fibroblasts by a pretreatment with the photosensitizer Ro19-8022 plus light, which generates predominantly 8-oxoG, or with methyl methanesulfonate (MMS), which generates alkylated bases and abasic sites (AP sites). The results indicate that the repair rate of the oxidative guanine modifications induced by the photosensitizer was not increased if a priming dose of the oxidative or alkylating agent was applied 6 or 18h prior to a challenging dose, although pretreatments with both agents resulted in two-fold elevated glutathione levels as an indication for an adaptive response. Similarly, the activity of total protein extracts of the cells to incise at a single 8-oxoG residue in an oligonucleotide was unchanged. It has to be concluded that the repair of 8-oxoG is not inducible by oxidative or alkylation damage.

Animals↗

PubMed1999

Hypochlorous acid-induced DNA base modification: potentiation by nitrite: biomarkers of DNA damage by reactive oxygen species.

Chronic inflammation results in increased nitric oxide formation and nitrite (NO-2) accumulation. Activated phagocytes release myeloperoxidase generating the cytotoxic agent hypochlorous acid (HOCl). Reaction of HOCl with NO-2 results in the formation of nitryl chloride (NO2Cl), a potent oxidising, nitrating and chlorinating species. Exposure of DNA to NO-2 alone (up to 250 microM) at pH 7.4 did not induce oxidative DNA base damage. However, incubation of DNA with NO-2 in the presence of HOCl led to increases in thymine glycol, 5-hydroxyhydantoin, 8-hydroxyadenine and 5-chlorouracil to levels higher than those achieved by HOCl alone. No significant increases in 8-hydroxyguanine, xanthine, hypoxanthine, 2-hydroxyadenine, FAPy guanine, FAPy adenine and 8-chloroadenine were observed. HOCl-induced depletion of FAPy guanine and 8-hydroxyguanine was reduced in the presence of NO-2. Modification of DNA by HOCl/NO-2 (presumably generating NO2Cl) produces a pattern of DNA base damage products in isolated DNA that is similar to the pattern produced by HOCl but not other reactive species.

Animals↗

PubMed1996

Tumor progression to the metastatic state involves structural modifications in DNA markedly different from those associated with primary tumor formation.

Wavenumber-absorbance relationships of infrared spectra of DNA analyzed by principal components analysis may be expressed as points in space. Each point represents a highly discriminating measure of DNA structure. Structural modifications of DNA, such as those induced by free radicals, alter vibrational and rotational motion and consequently change the spatial location of the points. Using this technology to analyze breast tumor DNA, we revealed a 94 degrees difference in direction between the progression of normal DNA-->primary tumor DNA and the progression of primary tumor DNA-->metastatic tumor DNA (P < 0.001). This sharp directional change was accompanied by a substantial increase in the structural diversity of the metastatic tumor DNA (P = 0.003), which, on the basis of the volume of the core cluster of points, could comprise as many as 11 x 10(9) different phenotypes. This suggests that the heterogeneity and varied physiological properties known to characterize malignant tumor cell populations may at least partially arise from these diverse phenotypes. The evidence suggests that the progression to the metastatic state involves structural modifications in DNA that are markedly different from the modifications associated with the formation of the primary tumor. Overall, the findings of this and earlier studies imply that the observed DNA alter-ations are a pivotal factor in the etiology of breast cancer and a formidable barrier to overcome in intervention to control the disease. In terms of cancer etiology and prediction, the technology described has potentially wide application to studies in which the structural status of DNA is an important consideration.

Breast↗

PubMed1992

Surface lysine and tyrosine residues are required for interaction of the major herpes simplex virus type 1 DNA-binding protein with single-stranded DNA.

Modification of the herpes simplex virus type 1 major DNA-binding protein (ICP8) with reagents and conditions specific for arginine, lysine, and tyrosine residues indicates that surface lysine and tyrosine residues are required for the interaction of this protein with single-stranded DNA. Modification of either of these two amino acids resulted in a loss and/or modification of binding activity as judged by nitrocellulose filter assays and gel shift. Modification specific for arginine residues did not affect binding within the limits of the assays used. Finally, quenching of the intrinsic tryptophan fluorescence of ICP8 in the presence of single-stranded DNA either suggests involvement of this amino acid in the binding reaction or reflects a conformational change in the protein upon binding.

Acetic Anhydrides↗

PubMed2003

Oxidative and alkylating damage in DNA.

Modification of cellular DNA upon exposure to reactive oxygen and nitrogen species is the likely initial event involved in the induction of the mutagenic and lethal effects of various oxidative stress agents. Evidence has been accumulated for the significant implication of singlet oxygen (1O(2)), generated as the result of UVA activation of endogenous photosensitizers as porphyrins and flavins. 7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxodGuo) has been shown to be the exclusive product of the reaction of 1O(2) with the guanine moiety of cellular DNA, in contrast to the hydroxyl radical, which reacts almost indifferently with all the nucleobases and the sugar moiety of DNA. Furthermore 8-oxodGuo is also produced by other oxidants and can be used as an ubiquitous biomarker of DNA oxidation but can not be a specific marker of any particular species. The role of DNA etheno adducts in mutagenic and carcinogenic processes triggered by known occupational and environmental carcinogens has also been studied. Much interest in etheno adducts resulted from the detection of increased levels of 1,N(6)-etheno-2'-deoxyadenosine and 3,N(4)-etheno-2'-deoxycytidine in DNA from human, rat and mouse tissues under pathophysiological conditions associated with oxidative stress. A method involving on-line HPLC with electrospray tandem mass spectrometry detection has been developed for the analysis of 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-epsilondGuo) in DNA. This methodology permits direct quantification of 20 fmol (7.4 adducts/10(8) dGuo) of the etheno adduct from approximately 350 microg of crude DNA hydrolysates. This method provides the first evidence of the occurrence of 1,N(2)-epsilondGuo as a basal endogenous lesion and may be utilized to better assess the biological consequences of etheno DNA damage under normal and pathological conditions. This work addresses the importance of isotope labeling associated with mass spectrometry technique for biomolecule damage studies.

Alkylating Agents↗

PubMed1997

Comparative 32P-postlabeling analysis of exogenous and endogenous DNA adducts in mouse skin exposed to a wood-preserving waste extract, a complex mixture of polycyclic and polychlorinated chemicals.

Wood preserving waste (WPW) sites contain numerous toxic compounds, including phenols, polycyclic aromatic hydrocarbons (PAHs), polychlorinated dibenzodioxins, and dibenzofurans. Previous in vitro and in vivo 32P-postlabeling studies showed the induction of multiple carcinogen-DNA adducts by WPW extracts. We now have tested the hypothesis in a mouse skin bioassay that a WPW extract not only causes the formation of exogenous, xenobiotic-derived DNA adducts, but also alters the levels of endogenous DNA modifications. Skin DNA of female ICR mice treated topically with an organic WPW extract was found by 32P-postlabeling to contain significantly increased levels of bulky oxidative DNA lesions (type II I-compounds), in addition to exogenous PAH-derived adducts. The mechanism of this increase is postulated to proceed through electrophilic quinoid compounds, which presumably were formed from phenols by chemical reactions of waste material or biologically by oxidative metabolism. On the other hand, the levels of another class of endogenous DNA adducts (type I I-compounds) were reduced significantly in exposed skin DNA. This effect was explained by the presence of cytochrome P450 inducers in the extract. All three types of DNA alterations observed may play a significant role in carcinogenesis. Our results imply that in addition to exogenous carcinogen-DNA adducts, alterations of endogenous DNA modifications may need to be considered in evaluating carcinogenic risk from toxic chemical wastes and the effects of remediation measures.

Acetone↗

PubMed1991

Chemical modification of DNA: quantitation of O6-methyldeoxyguanosine by tandem mass spectrometry.

Methodology is described which allows quantitation of O6-methyldeoxyguanosine generated as a product of in vitro methylation of calf thymus DNA by methyl methanesulfonate (MeMS). Quantitative precision of 10% is achieved on samples of 10(-11)-10(-12) mol generated in 0.02% yield (expressed as O6-methyldeoxyguanosine versus deoxyguanosine) when DNA is treated with the weak carcinogen MeMS. These results show the potential application of this method to the analysis of DNA chemical modifications at the low levels that are relevant to the induction of biological effects of many alkylating agents. The methodology utilizes enzymatic degradation, reverse-phase chromatography and finally analysis by tandem mass spectrometry using desorption chemical ionization. Multiple reaction monitoring was used to increase sensitivity and the CD3-labeled nucleoside was used as an internal standard for quantification.

Chromatography, High Pressure Liquid↗

PubMed1990

Mechanism of formation and 32P-postlabeling of DNA adducts derived from peroxidative activation of carcinogenic non-aminoazo dye 1-phenylazo-2-hydroxynaphthalene (Sudan I).

Horseradish peroxidase in the presence of hydrogen peroxide mediates the activation of carcinogenic 1-phenylazo-2-hydroxynaphthalene (Sudan I) to DNA-bound products in vitro. The peroxidase activating system is greater than 10 times more effective with respect to DNA modification by Sudan I than the microsomal enzymes containing cytochrome P450. The DNA-binding reaction of the Sudan I metabolite(s) formed by the peroxidase system is dependent on Sudan I and H2O2 concentration and pH. Reactive intermediate(s) or product(s) of the Sudan I oxidation by peroxidase with a short half-life are responsible for the DNA modification. DNA modified by peroxidase-activated Sudan I becomes colored and has an absorption maximum at approximately 480 nm. The modification of DNA by Sudan I metabolites(s) formed by the peroxidase system is inhibited by some compounds of physiological importance (ascorbate, glutathione, Mg2+ ions) and by radical trapping agents (nitrosobenzene, methyl viologen). 32P-Postlabeling assay of the DNA modified by Sudan I activated by the peroxidase system indicates that the covalent DNA adduct formation is the principal type of the DNA modification. Four major and several minor adducts of deoxyribonucleotide 3',5'-bisphosphate from DNA with Sudan I metabolite(s) were detected by the classical Randerath 32P-postlabelling assay as well as by the nuclease P1 version of the same method.

Animals↗

PubMed1998

Oxidative DNA base modifications as factors in carcinogenesis.

Reactive oxygen species can cause extensive DNA modifications including modified bases. Some of the DNA base damage has been found to possess premutagenic properties. Therefore, if not repaired, it can contribute to carcinogenesis. We have found elevated amounts of modified bases in cancerous and precancerous tissues as compared with normal tissues. Most of the agents used in anticancer therapy are paradoxically responsible for induction of secondary malignancies and some of them may generate free radicals. The results of our experiments provide evidence that exposure of cancer patients to therapeutic doses of ionizing radiation and anticancer drugs causes base modifications in genomic DNA of lymphocytes. Some of these base damages could lead to mutagenesis in critical genes and ultimately to secondary cancers such as leukemias. This may point to an important role of oxidative base damage in cancer initiation. Alternatively, the increased level of the modified base products may contribute to genetic instability and metastatic potential of tumor cells.

Animals↗

PubMed1994

Visible light generates oxidative DNA base modifications in high excess of strand breaks in mammalian cells.

The DNA damage induced by visible light in L1210 mouse leukaemia cells was analysed by an alkaline elution assay with specific repair endonucleases. DNA single-strand breaks and DNA modifications sensitive to FPG protein (formamidopyrimidine-DNA glycosylase), endonuclease III and exonuclease III were quantified in parallel. The light-induced cellular DNA damage was found to consist of many base modifications sensitive to FPG protein, which most probably are predominantly 7,8-dihydro-8-oxoguanine (8-hydroxyguanine) residues. Base modifications sensitive to endonuclease III are virtually absent. The yield of the FPG-sensitive base modifications is 10-fold higher than that of single-strand breaks plus AP sites (sites of base loss). The described ratios of the various modifications indicate that the damage most probably results from a reaction of DNA with singlet oxygen (type II reaction) or directly with an excited endogenous photosensitizer (type I reaction) and is not mediated by hydroxyl radicals. Experiments with cut-off filters indicate that wavelengths between 400 and 500 nm are responsible for most of the modifications. The FPG-sensitive base modifications are repaired efficiently (t1/2 approximately 1 h at 37 degrees C). This is perhaps why the light-induced DNA damage is apparently associated with only low mutagenicity.

Animals↗

PubMed1978

Effect of chemical modification of supercoiled simian virus 40 DNA on the rate of in vitro transcription.

Superhelical simian virus 40 FI DNA could be modified with the single-strand-specific reagent N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethylcarbodiimide (CMC). A limited reaction, of less than 2% of the base pairs, resulted in almost total inhibition of in vitro transcription by DNA-dependent RNA polymerase from Escherichia coli. This effect was shown to be due to DNA modification and not to inhibition of polymerase activity by the reagent. Inhibition of enzyme activity occurred if the contaminating reagent was not absorbed with another protein before polymerase addition. No inhibition was observed when DNA and polymerase were incubated together to allow the formation of pre-initiation complexes before CMC was added. Studies of template saturation with polymerase showed that the inhibition of transcription by DNA modification was due to a loss of binding ability of the enzyme to the reacted, supercoiled DNA when reaction times of less than 2 h were used.

CME-Carbodiimide↗

PubMed2026

DREAMS illuminates spatial DNA and RNA modification landscapes.

DNA and RNA modifications regulate gene expression and RNA processing, but their spatial organization in complex tissues remains elusive. Here we developed DNA RNA Elements Areal Mass Spectrometry (DREAMS), a mass spectrometry imaging platform that spatially maps diverse nucleic acid modifications simultaneously. Applying DREAMS to TET-deficient mouse brains (Tet1Δ/Δ and triple Tet1/2/3Δ/Δ), we uncover TET1's unexpected role in modulating N1-methyladenosine (m1A), a pivotal RNA modification. While DREAMS reveals broad modification landscapes altered across TET knockouts, we identify TET1-mediated changes in m1A that correlate with transcriptome alterations. Our work establishes DREAMS as a transformative tool for spatial epigenomics/epitranscriptomics and suggests that TET enzymes could influence multiple DNA and RNA modifications with potential gatekeeping roles in nucleic acid regulation.

Animals↗

PubMed1993

Macroevolution by transposition: drastic modification of DNA recognition by a type I restriction enzyme following Tn5 transposition.

We have characterized a novel mutant of EcoDXXI, a type IC DNA restriction and modification (R-M) system, in which the specificity has been altered due to a Tn5 insertion into the middle of hsdS, the gene which encodes the polypeptide that confers DNA sequence specificity to both the restriction and the modification reactions. Like other type I enzymes, the wild type EcoDXXI recognizes a sequence composed of two asymmetrical half sites separated by a spacer region: TCA(N7)RTTC. Purification of the EcoDXXI mutant methylase and subsequent in vitro DNA methylation assays identified the mutant recognition sequence as an interrupted palindrome, TCA(N8)TGA, in which the 5' half site of the wild type site is repeated in inverse orientation. The additional base pair in the non-specific spacer of the mutant recognition sequence maintains the proper spacing between the two methylatable adenine groups. Sequencing of both the wild type and mutant EcoDXXI hsdS genes showed that the Tn5 insertion occurred at nucleotide 673 of the 1221 bp gene. This effectively deletes the entire carboxyl-terminal DNA binding domain which recognizes the 3' half of the EcoDXXI binding site. The truncated hsdS gene still encodes both the amino-terminal DNA binding domain and the conserved repeated sequence that defines the length of the recognition site spacer region. We propose that the EcoDXXI mutant methylase utilizes two truncated hsdS subunits to recognize its binding site. The implications of this finding in terms of subunit interactions and the malleability of the type I R-M systems will be discussed.

Base Sequence↗

PubMed2002

Chromatin remodeling, histone modifications, and DNA methylation-how does it all fit together?

DNA methylation is important in the control of gene transcription and chromatin structure. The complexities of this process are just beginning to be elucidated in relationship to other epigenetic mechanisms. Exciting new research in the areas of histone methylation and chromatin remodeling make it clear just how important the connections between these various mechanisms and DNA methylation are for the control of chromosome structure and gene expression. Emerging evidence suggests that chromatin remodeling enzymes and histone methylation are essential for proper DNA methylation patterns. Other histone modifications, such as acetylation and phosphorylation, in turn, affect histone methylation and histone methylation also appears to be highly reliant on chromatin remodeling enzymes. This review will summarize what is likely only the beginning of a flood of new information that will ultimately link all epigenetic modifications of the mammalian genome. A model will also be put forth to account for how chromatin modifications lead to genomic DNA methylation patterns.

Acetylation↗

PubMed2003

Substrate-mediated DNA delivery: role of the cationic polymer structure and extent of modification.

DNA complex immobilization to substrates that support cell adhesion can enhance gene transfer by maintaining DNA within the cellular environment while limiting complex aggregation. This report examines the tether design (e.g., extent of functionalization) and cationic polymer structure for their effect on complex binding to the substrate and cellular transfection. DNA is complexed with cationic polymers (polylysine, PL; polyethylenimine, PEI), which are functionalized with biotin for binding to a neutravidin (NA) substrate. Surfaces densities ranging from 0.4 to 2.6 microg DNA/cm(2) were obtained for PL, and from 0.7 to 1.0 microg DNA/cm(2) for PEI. The distribution of biotin groups for PL/DNA complexes had a dual effect on cellular transfection. Increasing the fraction of PL with biotin residues decreased luciferase activity; however, increasing the number of biotin residues per PL increased luciferase activity. For PEI, the number of biotin groups present on the complex did not affect transgene expression. Release studies demonstrated that 20-30% of the immobilized DNA was released over 8 days, with 8-20% released during the first 24 h. Enzymatic degradation of cationic polymers is not necessary for transfection. Additionally, the duration of transgene expression was extended for surface-mediated delivery relative to bolus delivery.

Cations↗

PubMed1994

The interaction of intercalators and groove-binding agents with DNA triple-helical structures: the influence of ligand structure, DNA backbone modifications and sequence.

The effects of ligand structure and properties, DNA backbone modifications and DNA sequence on the interaction of a variety of well-known groove-binding agents and intercalators with DNA duplexes and triplexes have been evaluated by thermal melting experiments and molecular modeling. Both methylphosphonate and phosphorothioate substitutions generally destabilize DNA duplexes and triplexes. Modified duplexes can be strongly stabilized by both groove-binding agents and intercalators whereas triplexes are primarily stabilized by intercalators. Of the compounds tested, the intercalators coralyne and quinacrine provide the largest stabilization of the triplex dT19.dA19.dT19. Molecular modeling studies suggest that the large intercalating ring system of coralyne stacks well with the triplex bases whereas the alkylamino side chain of quinacrine fits snugly into the remaining space of the major groove of dT19.dA19.dT19 triplex and forms extensive van der Waals contacts with the thymine methyl groups that line the groove. Converting some of the T.A.T base triples to C+.G.C (e.g. dT19.dA19.dT19 to d(T4C+)3T4.d(A4G)3A4.(T4C)3T4) causes very significant decreases in observed Tm increases for compounds such as quinacrine and coralyne. Although removal of thymine methyl groups and addition of positive charge on substitution of C+.G.C for T.A.T should reduce binding of cationic intercalators, the large difference observed between the pure AT and the mixed sequence triplexes suggest that they may also have differences in structure and properties.

Base Sequence↗

PubMed1986

Modification of DNA and metabolism of ethyl carbamate in vivo: formation of 7-(2-oxoethyl)guanine and its sensitive determination by reductive tritiation using 3H-sodium borohydride.

The modification of liver DNA of mice and rats by ethyl carbamate and its putative proximate metabolite, vinyl carbamate, has been investigated. Following treatment with [ethyl-1-14C]-ethyl carbamate, the main radioactive DNA adduct was identified as 7-(2-oxoethyl)guanine by cochromatography with the authentic marker in several separation systems. After reduction by sodium borohydride (NaBH4) to 7-(2-hydroxyethyl)guanine, the radioactive material again cochromatographed with the respective marker. Reduction of modified liver DNA by 3H-NaBH4, following administration of unlabelled ethyl carbamate or vinyl carbamate, allowed the quantitation of 7-(2-oxoethyl)guanine (as 7-(2-hydroxy-2-[3H]-ethyl)guanine). Vinyl carbamate led to about 100 times as much 7-(2-oxoethyl)guanine (on a molar basis) as did ethyl carbamate. Both the formation of 7-(2-oxoethyl)guanine by ethyl carbamate and vinyl carbamate, and the much higher activity of the latter compound, strongly support the existence of the metabolic activation pathway, ethyl carbamate----vinyl carbamate----epoxyethyl carbamate, as proposed by Dahl et al. (1978, 1980). The possible role of 7-(2-oxoethyl)guanine in the initiation of the carcinogenic process is discussed in view of the structural equilibrium with its hemiacetal conformation, O6,7-(1'-hydroxyethano)guanine. In the latter conformation, it is assumed to represent a promutagenic lesion. In addition, intrastrand cross-links between modified guanine and adjacent cytosine or adenine seem possible and may have promutagenic consequences. Replication of DNA containing such lesions may lead to the induction of mutations. This may be a critical event in the initiation, and eventually progression, of the carcinogenic process as determined by ethyl carbamate and other carcinogens, such as vinyl chloride, which lead to the same DNA modification.

Animals↗

PubMed1986

Modification of calf thymus DNA by methyl methanesulfonate. Quantitative determination of 7-methyldeoxyguanosine by mass spectrometry.

Quantitation of 7-methyldeoxyguanosine (m7dG) produced in the in vitro methyl methanesulfonate (MeMS) action on calf-thymus DNA is achieved by enzymatic degradation, liquid chromatographic separation and chemical ionization mass spectrometry. The total degree of methylation, measured by uptake of [14C]MeMS was 0.35%. Mass spectral analysis shows that m7dG constitutes 84% of the total methylated product. It is also shown that tandem mass spectrometry allows detection of m7dG, as the protonated base, down to 1 pmol level, suggesting that MS/MS analysis can be the method of choice in quantitation of the adducts of in vivo DNA modifications.

Animals↗