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PubMed2003

Engineering of restriction endonucleases: using methylation activity of the bifunctional endonuclease Eco57I to select the mutant with a novel sequence specificity.

Type II restriction endonucleases (REs) are widely used tools in molecular biology, biotechnology and diagnostics. Efforts to generate new specificities by structure-guided design and random mutagenesis have been unsuccessful so far. We have developed a new procedure called the methylation activity-based selection (MABS) for generating REs with a new specificity. MABS uses a unique property of bifunctional type II REs to methylate DNA targets they recognize. The procedure includes three steps: (1) conversion of a bifunctional RE into a monofunctional DNA-modifying enzyme by cleavage center disruption; (2) mutagenesis and selection of mutants with altered DNA modification specificity based on their ability to protect predetermined DNA targets; (3) reconstitution of the cleavage center's wild-type structure. The efficiency of the MABS technique was demonstrated by altering the sequence specificity of the bifunctional RE Eco57I from 5'-CTGAAG to 5'-CTGRAG, and thus generating the mutant restriction endonuclease (and DNA methyltransferase) of a specificity not known before. This study provides evidence that MABS is a promising technique for generation of REs with new specificities.

Amino Acid Motifs↗

PubMed2003

Illegitimate DNA integration in mammalian cells.

Foreign DNA integration is one of the most widely exploited cellular processes in molecular biology. Its technical use permits us to alter a cellular genome by incorporating a fragment of foreign DNA into the chromosomal DNA. This process employs the cell's own endogenous DNA modification and repair machinery. Two main classes of integration mechanisms exist: those that draw on sequence similarity between the foreign and genomic sequences to carry out homology-directed modifications, and the nonhomologous or 'illegitimate' insertion of foreign DNA into the genome. Gene therapy procedures can result in illegitimate integration of introduced sequences and thus pose a risk of unforeseeable genomic alterations. The choice of insertion site, the degree to which the foreign DNA and endogenous locus are modified before or during integration, and the resulting impact on structure, expression, and stability of the genome are all factors of illegitimate DNA integration that must be considered, in particular when designing genetic therapies.

Animals↗

PubMed1998

Molecular analysis of new subtypes of ELE/RET rearrangements, their reciprocal transcripts and breakpoints in papillary thyroid carcinomas of children after Chernobyl.

A high prevalence of RET rearrangements is found in papillary thyroid carcinomas (PTC) of children from Belarus after the Chernobyl reactor accident. The ELE/RET rearrangement (PTC3) is prevailing. Aberrant types of ELE/RET rearrangement have been found with a truncated ELE1 gene: As compared with the common form (PTC3r1) one aberrant type is shorter by one 144 bp exon (PTC3r2) (three cases); in the second atypic form (PTC3r3) the ELE1 part is 18 bp shorter than in PTC3r1. In agreement with the observation that the oncogenic RET is generated by a paracentric inversion at chromosome 10, we found not only ELE/RET, but also RET/ELE transcripts in these tumors. Sequencing of the breakpoint regions at the genomic DNA level revealed DNA modifications that might be relevant for illegitimate recombination after DNA doublestrand breaks. The high prevalence of ELE/RET rearrangements and various subtypes appears to be typical for radiation-induced thyroid carcinomas of children after the Chernobyl reactor accident.

Artificial Gene Fusion↗

PubMed1989

Reduced accumulation of I-compounds in liver DNA of rats fed a choline-devoid diet.

Groups of rats were fed for 1, 4 or 7 months choline-devoid or choline-supplemented diets, that provided approximately 50% of the methionine, and 0.15% or 150% of the choline requirements of young, growing rats. Liver DNA was isolated and analyzed by the nuclease P1-enhanced version of the 32P-postlabeling assay, which detects aromatic/hydrophobic DNA adducts and I-compounds (adduct-like DNA modifications shown to accumulate in tissue of aging rats). DNA adducts and qualitative differences in the patterns of I-compounds were not observed in rats fed the two diets. However, in rats fed the choline-devoid diet there was a drastic reduction in the accumulation of I-compounds, compared with that in rats fed the control diet. These results extend previous evidence of a lack of relevant DNA adducts in the liver of rats fed the choline-devoid diet, and suggest the possibility of a role of I-compounds in the carcinogenicity of this diet.

Animals↗

PubMed1991

DNA adduct formation in liver and kidney of male Syrian hamsters treated with estrogen and/or alpha-naphthoflavone.

Chronic administration of estrogens to male Syrian hamsters induces kidney tumors. Co-administration of estrogen plus alpha-naphthoflavone (ANF) suppresses this kidney carcinogenesis but induces liver tumors instead. In an attempt to elucidate the mechanism of the switch from estrogen-induced kidney to liver carcinogenesis in response to ANF treatment, patterns of kidney and liver DNA adducts were investigated by 32P-postlabeling analysis and compared to controls. Chronic treatment of hamsters with ANF alone or in combination with estradiol resulted in a flavone-specific DNA adduct pattern in livers of these animals. These spots, adducts 1 and 2, on 32P-postlabeling maps were taken as evidence of covalent ANF-DNA modifications. The kidney-specific estrogen-induced indirect DNA adducts, observed previously in hamsters treated chronically with estrogen, occurred in renal but not hepatic DNA of animals treated with estradiol alone or in combination with ANF. Pretreatment of hamsters with ANF for 3 days decreased by 75-80% the hepatic and renal diethylstilbestrol (DES)-DNA adducts, which are formed after injection of a single large dose of DES. It is concluded from these changes in DNA adduct patterns and levels that estrogen quinone-DNA adduction may play an etiological role in estrogen-induced kidney cancer. The prevention of estrogen-induced kidney tumors by ANF co-treatment may be a consequence of the decrease in renal concentrations of these adducts in response to ANF. Hepatic concentrations of estrogen quinone-DNA adducts also decrease, but ANF-DNA adducts, observed only in liver, may assume an etiological role in the induction of hepatomas.

Animals↗

PubMed1992

Peroxidative activation of o-phenylhydroquinone leads to the formation of DNA adducts in HL-60 cells.

Using 32P-postlabeling we studied DNA adduct formation in HL-60 cells treated with the o-phenylphenol metabolites o-phenylhydroquinone (o-PHQ) and o-phenylbenzoquinone (o-PBQ). Treatment with 25-500 microM o-PHQ for 8 h produced one principal and three minor adducts with a relative distribution of 80, 10, 6 and 4%. The relative adduct levels from these treatments were 0.26-2.31 adducts/10(7) nucleotides. Treatment with 25-250 microM o-PBQ for 2 h resulted in a similar level of DNA modification and adduct distribution. Reaction of purified calf thymus DNA with o-PBQ produced one DNA adduct, which did not correspond to the major adduct produced in HL-60 cells. These results show that o-PHQ and o-PBQ can form DNA adducts. Peroxidase activation of o-phenylphenol may therefore play a role in the carcinogenic effect of this compound.

Benzoquinones↗

PubMed1990

Omeprazole and the gastric mucosa.

The potential for either omeprazole or its sulphenamide to interact with DNA was investigated by incubating tritiated omeprazole at either neutral or acidic pH with either purified prokaryotic (Escherichia coli) or purified eukaryotic (salmon sperm) DNA. Each incubation was carried out for 30 min. The DNA was separated on agarose gels, stained with ethidium bromide, and the radioactivity in the DNA determined. No radioactivity was associated with the DNA on the gel with either prokaryotic or eukaryotic DNA or with either protocol. No interaction with the DNA was found, thus excluding the possibilities of base modification, DNA strand breakage or intercalation. It was concluded that neither omeprazole nor its gastric metabolites are genotoxic.

Animals↗

PubMed2004

Sodium nitroprusside and peroxynitrite effect on hepatic DNases: an in vitro and in vivo study.

BACKGROUND: It has been documented that nitric oxide (NO) donor sodium nitroprusside (SNP) and authentic peroxynitrite are capable of promoting apoptosis in a number of different cell types. Various endonucleases have been proposed as candidates responsible for the internucleosomal cleavage of the genomic DNA observed during apoptosis, but the main effect is attributed to the alkaline-DNases (Mg2+- and caspase-dependent) and acid-DNase. The aim of this study was to examine an in vivo and in vitro possibility for alkaline- and acid-DNases to be activated by SNP and peroxynitrite. RESULTS: The effect on liver tissue alkaline and acid DNase activity together with the markers of tissue and plasma oxidative and nitrosative stress (lipid peroxidation, SH group content, carbonyl groups and nitrotyrosine formation) was investigated in plasma and liver tissue. The activity of liver alkaline DNase increased and that of acid DNase decreased after in vivo treatment with either SNP or peroxynitrite. A difference observed between the in vivo and in vitro effect of oxide donor (i.e., SNP) or peroxynitrite upon alkaline DNase activity existed, and it may be due to the existence of the "inducible" endonuclease. After a spectrophotometric scan analysis of purified DNA, it was documented that both SNP and peroxynitrite induce various DNA modifications (nitroguanine formation being the most important one) whereas DNA fragmentation was not significantly increased. CONCLUSION: Alkaline DNase activation seems to be associated with the programmed destruction of the genome, leading to the fragmentation of damaged DNA sites. Thus, the elimination of damaged cells appears to be a likely factor in prevention against mutation and carcinogenesis.

Journal Article↗

PubMed1990

Footprinting studies of sequence recognition by mithramycin.

The binding of mithramycin to DNA has been investigated using a variety of chemical and enzymic footprinting probes. Mithramycin failed to affect DNA modification by several chemical agents which react in the DNA major groove, suggesting that the drug binds via the minor groove. The pattern of reaction with diethylpyrocarbonate was modified by the antibiotic at the binding site and in surrounding regions, consistent with drug-induced structural changes. Hydroxyl radical and DNase I footprinting confirms that the drug binds best to GpG, especially when this is located within a GC-rich environment. Cleavage by DNase II and micrococcal nuclease is inhibited around drug binding sites and suggests a local stabilization of the DNA helix.

Base Composition↗

PubMed1979

[Use of liposomes for incorporation of alkylating derivatives of mono- and oligonucleotides into mammalian cells].

It was shown that within the liposomes mono- and oligonucleotides and their alkylating derivatives penetrate the cells of Ehrlich ascite carcinoma and peritoneal exudate of the mice. Inside the cells the alkylating reagents are mainly utilized for modification of proteins (42--76%), RNA (5--16%) and DNA (3--9%). Presumably DNA modification is largely dependent on the penetration of the reagents into the nuclei. No significant differences in alkylation of the cell components by oligoadenylate derivatives, capable of complementary interactions with nucleic acids and mononucleotide derivatives, incapable of such interactions, were observed.

Alkylating Agents↗

PubMed1993

Ormaplatin sensitivity/resistance in human ovarian cancer cells made resistant to cisplatin.

The human ovarian cancer cell lines A2780 and A2780/CP70 were studied to investigate the cellular basis for their relative sensitivities to tetrachloro(DL-trans)-1,2-diamminecyclohexaneplatinum(IV) (ormaplatin). Cells were exposed to ormaplatin for 1 h in all experiments. As assessed by colony formation assays, the A2780/CP70 cell line [50% inhibitory dose (IC50) = 3.6 microM] was 9.5-fold more resistant to ormaplatin than the A2780 cell line [IC50 = 0.38 microM]. For cisplatin, the IC50 doses were 40 and 3 microM, respectively. Both cell lines were treated with ormaplatin at doses ranging from 0.10 to 40 microM, for the purpose of studying drug accumulation and efflux, and DNA adduct formation and repair. When these cell lines were treated at their respective IC50 doses, drug accumulation was greater in the resistant cells. When treated at equal microM doses, the sensitive cells formed 8-fold more DNA adduct than the resistant cells. When cells were treated with ormaplatin so as to achieve equivalent levels of platinum-DNA modification, sensitive cells removed 53% of the platinum-DNA damage in the first 6 h after drug exposure, compared to 68% in the resistant cells. We conclude that in human ovarian cancer cells made resistant to cisplatin, there is moderate cross-resistance to ormaplatin. This cross-resistance is not explained by differences in drug accumulation but is associated with reduced platinum-DNA adduct formation, which may be attributable in part to cytosolic inactivation of drug.

Cisplatin↗

PubMed1989

Singlet oxygen as an ultimately reactive species in Salmonella typhimurium DNA damage induced by methylene blue/visible light.

The specific recognition of various DNA modifications by repair enzymes is exploited for the analysis of DNA damage induced by visible light in the presence of methylene blue in Salmonella typhimurium. The relative frequencies of various endonuclease-sensitive sites and strand breaks are determined in the plasmid pAQ1 of the treated bacteria and are compared with those observed after exposure of isolated DNA to various conditions. This comparison of damage profiles indicates that the cellular DNA damage by illumination in the presence of methylene blue is caused predominantly by the direct action of singlet oxygen. Indirect mechanisms, e.g. involving a generation of superoxide and hydroxyl radicals or the activation of cellular nucleases, do not contribute very much. The damage is dominated by base modifications. These are subject to an efficient repair that is not mediated by uvrABC proteins and therefore most probably involves recognition by specific glycosylases. Revertant frequencies observed under these conditions in the strains TA1535, TA100, TA2638 and TA104 indicate a pronounced mutagenicity of the lesions induced. On the other hand, the DNA damage does not contribute significantly to the cytotoxicity caused by the treatment as an excision repair deficiency (uvrB) has no influence on cell killing.

DNA Damage↗

PubMed1988

Age dependency of base modification in rabbit liver DNA.

Age-related modifications of DNA bases have been observed in the liver of the New Zealand white (NZW) rabbit (Oryctolagus cuniculus), a lagomorph with a median life span in captivity of 5-7 yr. The ages of the animals studied ranged from 6 wk to 9 yr. After the DNA had been extracted from the liver cell nuclei and hydrolyzed with acid, the bases were analyzed by column chromatography with Cellulofine gels (GC-15-m). Two peaks in the chromatogram, which eluted before the four DNA bases, contained modified bases. Those materials, which were obtained in relatively large amounts from old animals, were highly fluorescent, and were shown to be crosslinked base products by mass spectrometry. The yield of crosslinked products versus rabbit age (greater than 0.5 yr) can be fitted by an exponential function (correlation coefficient: 0.76 +/- 0.09).

Aging↗

PubMed1993

beta-D-glucosyl-hydroxymethyluracil: a novel modified base present in the DNA of the parasitic protozoan T. brucei.

We have previously shown that the DNA of the unicellular eukaryote T. brucei contains about 0.1% of a novel modified base, called J. The presence of J correlates with a DNA modification associated with the silencing of telomeric expression sites for the variant surface antigens of trypanosomes. Here we show that J is 5-((beta-D-glucopyranosyloxy)-methyl)-uracil (shortened to beta-D-glucosyl-hydroxymethyluracil), a base not previously found in DNA. We discuss putative pathways for the introduction of this base modification at specific positions in the DNA and the possible contribution of this modification to repression of surface antigen gene expression.

Animals↗

PubMed2004

Plant chromatin -- epigenetics linked to ATP-dependent remodeling and architectural proteins.

Studies in organisms belonging to different eukaryotic kingdoms have revealed that the structural state of chromatin is controlled by interactions of DNA, small RNAs and specific proteins, linked to a self-reinforcing complex network of biochemical activities involving histone and DNA modifications and ATP-dependent nucleosome remodeling. However, these findings must now be reinterpreted in light of the recent discovery of the highly dynamic character of interphase chromosomes exemplified by the constant flux of enzymatic and structural proteins through both eu- and heterochromatin and by short- and long-range chromosome movements in the nucleus. The available data on chromosome organization in Arabidopsis thaliana and links between proteins influencing chromatin structure and DNA and histone modifications documented in this model plant provide strong supportive evidence for the dynamic nature of chromosomes.

Adenosine Triphosphate↗

PubMed1995

Lipid hydroperoxide-induced endogenous DNA adducts in hamsters: possible mechanism of lipid hydroperoxide-mediated carcinogenesis.

DNA of livers or kidneys of rodents contain endogenously modified nucleotides of as yet unknown structure or function. As part of our examination of the origin of endogenous DNA modifications, we examined polar adducts of hamster liver and kidney DNA by 32P-postlabeling analysis (central and upper cuts) and compared them to adducts generated by incubation of DNA with [9Z,11E,(13S)]-13-hydroperoxyoctadecadienoic acid or with malondialdehyde. The central and upper cuts of DNA adduct maps of intact female retired breeder hamsters contained one and three adducts called C1, U1, U2, and U3, respectively, which cochromatographed with similar adducts in DNA incubated with the hydroperoxide or with malondialdehyde. Concentrations of adducts C1, U1, and U2 in organs of 1-month-old hamsters were lower than those in the 5-month-old female retired breeder hamsters. Adduct concentrations increased in tandem with lipid peroxide concentrations in livers and kidneys of hamsters treated with carbon tetrachloride compared to controls. A linear dependence of the concentrations of adducts on those of lipid peroxides was demonstrated independent of the treatment conditions or the organ examined. The polar endogenous adducts U1, U2, and U3 were identified by cochromatography in five different chromatographic systems with adducts induced by linoleic hydroperoxide or malondialdehyde. Adducts U1, U2, or U3 were also formed by incubating malondialdehyde with dAMP or with dGMP, respectively. The dependence of adduct concentrations in vivo on lipid peroxide levels are taken as evidence that decomposition products of lipid peroxides such as malondialdehyde induced endogenous DNA adducts in intact animals. Moreover, these adducts may mediate carcinogenic processes induced by chemicals which raise lipid peroxide levels.

Age Factors↗

PubMed1990

MamI, a novel class-II restriction endonuclease from Microbacterium ammoniaphilum recognizing 5'-GATNN decreases NNATC-3'.

A new site-specific class-II restriction endonuclease, MamI, has been discovered in the nonsporulating Gram+ Microbacterium ammoniaphilum. MamI recognition sequence and cleavage positions were deduced using experimental and computer-assisted mapping and sequencing approaches. MamI cleavage specificity corresponds to: [formula: see text] The novel 43-kD enzyme recognizes a palindromic hexanucleotide interrupted by four ambiguous nucleotides. MamI cleavage positions are located in the center of the recognition sequence resulting in blunt-ended fragments after cleavage in the presence of Mg2+ ions. MamI is inhibited by N6-methyladenine residues. In case of overlapping sequences of MamI and Escherichia coli-coded DNA modification methyltransferase M.EcodamI (5'-[formula: see text]-3'), cleavage of DNA isolated from E. coli wild-type cells will be inhibited. By applying incubation conditions forcing star activity, relaxing of MamI sequence specificity is observed (MamI*).

Base Sequence↗

PubMed1990

Sulfite enhancement of diolepoxide mutagenicity: the role of altered glutathione metabolism.

Sulfur dioxide is a cocarcinogen for benzo[a]pyrene in the respiratory tract of rats and hamsters. Sulfur dioxide exists under physiological conditions as the sulfite ion. Sulfite enhances the mutagenic potency of (+-)-7r,8t-dihydroxy-9t,-10t- epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) and 7r,8t-dihydroxy-9c10c-epoxy-7,8,9,10-tetrahydrobenzo[a]py ren e (syn-BPDE) in Salmonella typhimurium strains TA98 and TA100. This enhancement of diolepoxide mutagenicity is observed with sulfite concentrations between 1 and 20 mM, and the concentration dependence is identical for the two diolepoxides. Half-maximal enhancement of mutagenicity occurs at approximately 5 mM sulfite. Sulfite is neither toxic nor mutagenic to the bacteria under these conditions. The enhancement of diolepoxide mutagenicity requires that the bacteria be exposed to sulfite prior to the addition of the diolepoxide. Simultaneous addition of sulfite and diolepoxide significantly decreases the enhancing effect, and addition 15 min after the diolepoxide virtually abolishes the effect. This is consistent with sulfite serving to increase the efficiency of processes leading to DNA modification by the diolepoxides, rather than some effect subsequent to DNA adduct formation. Direct evidence for this hypothesis was provided by determining the effect of sulfite on mutagenicity and DNA binding in TA98 using [3H]anti-BPDE. Exposure of the bacteria to 10 mM sulfite for 5 min prior to the addition of the labeled mutagen led to as much as 170% increase in DNA binding levels relative to parallel incubations without sulfite. Corresponding increases in mutagenicity were seen as well. As sulfite can affect the glutathione/glutathione-S-transferase systems, the primary cellular defense against BPDE, the effect of sulfite on these pathways in Salmonella was determined. When strain TA98 was treated with N-acetoxy-2-acetamidofluorene, a direct-acting mutagen not scavenged by glutathione, prior addition of 10 mM sulfite to the bacteria had no effect on resultant viability or mutagenicity. Assessment of the bacterial glutathione levels revealed that 10 mM sulfite treatment results in an 82% decrease in the concentration of the cosubstrate. We were, however, unable to detect diolepoxide-glutathione conjugates in any of our incubations. Moreover, the presence of sulfite leads to significant trapping of the diolepoxide in the form of sulfonate derivatives. Based on these data, we conclude that the depletion of glutathione does indeed play a role in the enhancement of diolepoxide mutagenicity in S. typhimurium.(ABSTRACT TRUNCATED AT 400 WORDS)

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