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PubMed2004

A molecular beacon assay for measuring base excision repair activities.

The base excision repair (BER) pathway plays a key role in protecting the genome from endogenous DNA damage. Current methods to measure BER activities are indirect and cumbersome. Here, we introduce a direct method to assay DNA excision repair that is suitable for automation and industrial use, based on the fluorescence quenching mechanism of molecular beacons. We designed a single-stranded DNA oligonucleotide labelled with a 5'-fluorescein (F) and a 3'-Dabcyl (D) in which the fluorophore, F, is held in close proximity to the quencher, D, by the stem-loop structure design of the oligonucleotide. Following removal of the modified base or incision of the oligonucleotide, the fluorophore is separated from the quencher and fluorescence can be detected as a function of time. Several modified beacons have been used to validate the assay on both cell-free extracts and purified proteins. We have further developed the method to analyze BER in cultured cells. As described, the molecular beacon-based assay can be applied to all DNA modifications processed by DNA excision/incision repair pathways. Possible applications of the assay are discussed, including high-throughput real-time DNA repair measurements both in vitro and in living cells.

Animals↗
PubMed2004

The structure of the TrmE GTP-binding protein and its implications for tRNA modification.

TrmE is a 50 kDa guanine nucleotide-binding protein conserved between bacteria and man. It is involved in the modification of uridine bases (U34) at the first anticodon (wobble) position of tRNAs decoding two-family box triplets. The precise role of TrmE in the modification reaction is hitherto unknown. Here, we report the X-ray structure of TrmE from Thermotoga maritima. The structure reveals a three-domain protein comprising the N-terminal alpha/beta domain, the central helical domain and the G domain, responsible for GTP binding and hydrolysis. The N-terminal domain induces dimerization and is homologous to the tetrahydrofolate-binding domain of N,N-dimethylglycine oxidase. Biochemical and structural studies show that TrmE indeed binds formyl-tetrahydrofolate. A cysteine residue, necessary for modification of U34, is located close to the C1-group donor 5-formyl-tetrahydrofolate, suggesting a direct role of TrmE in the modification analogous to DNA modification enzymes. We propose a reaction mechanism whereby TrmE actively participates in the formylation reaction of uridine and regulates the ensuing hydrogenation reaction of a Schiff's base intermediate.

Amino Acid Sequence↗
PubMed1983

Salmonella typhimurium LT2 strains which are r- m+ for all three chromosomally located systems of DNA restriction and modification.

We describe the derivation of two strains of Salmonella typhimurium LT2 which are r- m+ for all three of the known chromosomal genes for the restriction and modification of DNA, hsdLT, hsdSA, and hsdSB; the strains were designated LB5000 and LB5010. LB5000 is a smooth derivative sensitive to phage P22; LB5010 is a galE strain sensitive to phage P1.

DNA, Bacterial↗
PubMed1988

Complementation and hybridization evidence for additional families of type I DNA restriction and modification genes in Salmonella serotypes.

Of eight Salmonella, serB-linked hsd genes for the restriction and modification of DNA transferred to Escherichia coli/Salmonella hybrids, only two--those with SM and ST (S. muenchen and S. thompson, respectively) specificities--may have weakly complemented rSB- and none complemented rK-. An A-specific DNA probe failed to hybridize to HindIII-restricted fragments of each of the hybrids, but an SB (S. typhimurium)-specific probe hybridized to DNA from the hybrid with ST specificity. These results indicate that additional families of the type I hsd genes may exist.

DNA, Bacterial↗
PubMed1997

Nuclear transfer and reprogramming.

Nuclear transfer techniques for mammalian embryos have been developed in the last decade. Embryonic nuclei from advanced stages of preimplantation development can be fully reprogrammed and the totipotency is restored when nuclei are transferred into ooplasts. Transfer of nuclei after gene expression from the embryonic genome has started does not appear to restrict the reprogramming of these nuclei. The principles of nuclear transfer are outlined with respect to nuclear remodelling, nucleocytoplasmic interactions and effects of the cell cycle. However, the molecular mechanisms involved in reprogramming donor nuclei remain unknown. It is proposed that epigenetic DNA modification, such as DNA methylation that regulates gene expression, is related to the reprogramming of transplanted nuclei.

Animals↗
PubMed2004

Triplex-forming oligonucleotides - sequence-specific DNA ligands as tools for gene inhibition and for modulation of DNA-associated functions.

The down regulation of gene expression is a promising strategy for molecular medicine and experimental biology. Molecules that bind to the DNA double helix may interfere with gene expression and, in addition to potential therapeutic applications, can be helpful for the investigation of DNA processing, chromatin package, or associated biological processes. Triplex-forming oligonucleotides (TFOs) bind to specific sequences in the DNA double helix via hydrogen bonding interactions. TFOs have been shown to down-regulate gene expression, to induce targeted genomic DNA modifications, to stimulate DNA recombination, and to modulate chromatin organization. Additionally, they may be used as carriers to position DNA-modifying agents to selected sequences. TFO-mediated effects have been mostly described in cell culture, but one study reported TFO activity in a mouse model. Critical issues regarding TFO-based technologies are the development of new oligonucleotide analogues with improved binding affinity, better target selectivity, and sufficient stability in the intracellular environment. A prerequisite for the development of such DNA-binding molecules is the availability of appropriate methods to assess their binding properties quantitatively at the desired target sequence in the genome. This review focuses on recent results regarding gene-inhibitory effects of TFOs in cell culture and methods to evaluate TFO-binding to the desired target sequence in the context of the human genome.

Animals↗
PubMed2000

Free DNA induces modification on the protein synthesis profile of human peripheral blood mononuclear cells of healthy donors.

Understanding how free DNA might act as a signal between cells is important for knowing how DNA orchestrates immune responses and for optimizing the therapeutic of cancer, infection and immunologic diseases. This communication demonstrates that DNAs from different origins (bacteria, T. cruzi, HeLa cells) and synthetic oligonucleotide containing an unmethylated CpG motif are capable of inducing alterations in the protein profile of normal human leukocytes. As far as we know there have been no similar studies regarding the comparative effects of different free DNAs on early protein synthesis of human peripheral blood mononuclear cells.

CpG Islands↗
PubMed1990

Monitoring carcinogen actions on DNA by 32P-postlabeling.

Among several recently developed analytical methods, 32P-postlabeling analysis is a highly sensitive method for the detection and measurement of covalent carcinogen-DNA adducts and other DNA modifications. Since the method does not require radioactive carcinogens, it is suitable for DNA of humans exposed to environmental or occupational genotoxicants. The basic procedure entails the enzymatic incorporation of 32P-label into monomeric or dimeric hydrolysis products of DNA, followed by chromatographic mapping and autoradiography of the 32P-labeled digestion products and quantitation by scintillation spectrometry. Microgram amounts of DNA are analyzed; thus the assay is well suited for limited amounts of cells or tissue. Various versions of the assay afford different sensitivities of adduct detection. Under optimal conditions, one aromatic or bulky/hydrophobic adduct in 10(8)-10(10) nucleotides can be detected and measured (corresponding to 0.3-30 amol adduct/microgram DNA or 0.1-10 nmol adduct/mol DNA-P). The assay has been successfully applied to a variety of mutagenic (genotoxic) as well as non-mutagenic carcinogens. In humans, the 32P-postlabeling assay has been applied to DNA specimens from cigarette smokers, iron foundry workers, and coke oven workers. Estimation of total aromatic adduct levels in exposed individuals gave values of 1 adduct in 10(6)-10(8) DNA nucleotides. These values are similar to the total levels of persistent adducts in tissues of animals after exposure to initiating or carcinogenic doses of authentic aromatic genotoxicants. Among the non-mutagenic carcinogens investigated are estrogens, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), choline-devoid diet, carbon tetrachloride, and peroxisome proliferators. In addition, age-dependent DNA modifications (I-compounds) are being detected by 32P-postlabeling in animals that have not been knowingly exposed to mutagens/carcinogens. I-compound profiles and levels are dependent on species, tissue, sex, and diet. Reduced levels of I-compounds have been consistently noted in the target organ of carcinogen-exposed animals and in resulting neoplasms, suggesting that I-compound loss may play a role in carcinogenesis.

Animals↗
PubMed1983

Differences in the DNA of the inactive X chromosomes of fetal and extraembryonic tissues of mice.

We have examined the role of DNA modification in X chromosome inactivation of fetal tissues of the mouse using DNA-mediated gene transfer for the gene hypoxanthine phosphoribosyltransferase (HPRT). Two types of tissues have been examined with respect to randomness of inactivation in 14-day mouse conceptuses: 1) fetal tissue, which undergoes random inactivation of either the maternal or paternal X; and 2) yolk sac endoderm tissue, an extraembryonic membrane, which normally undergoes nonrandom inactivation of the paternal X. Exploiting an electrophoretic variant of HPRT as a means to mark the active and inactive HPRT alleles we provide evidence that: 1) inactive X DNA of the fetus at 14 days behaves like that of both adult tissue and cell lines in that the inactive X DNA is not efficient in gene transfer; and 2) in contrast, inactive X DNA from yolk sac endoderm is functional in gene transfer. Thus, despite the similarity in single active X chromosome expression in yolk sac endoderm and somatic tissues, there appears to be a difference at the level of DNA modification between these two tissues.

Animals↗
PubMed2000

Endogenous oxidative DNA base modifications analysed with repair enzymes and GC/MS technique.

GC/MS technique was used to identify endogenous levels of oxidatively modified DNA bases. To avoid possible artefact formation we used Fpg and Endo III endonucleases instead of acid hydrolysis to liberate the base products from unmodified DNA samples. Several different DNA preparations were used: (i) commercial calf thymus DNA, (ii) DNA isolated from rat liver, (iii) DNA isolated from human lymphocytes and (iv) nuclei isolated from rat liver. In all DNA samples used in our assays the most efficiently removed bases by Fpg protein are FapyG and FapyA although 8-oxoG was also detected in all preparations. The amount of 8-oxoG in human lymphocytes and in rat liver DNA was 3 and 2 per 10(7)bases, respectively. It is reasonable to postulate that the presented method is one of the techniques which should be used to reveal the enigma of endogenous, oxidative DNA damage.

Animals↗
PubMed1997

Determination of steady-state levels of oxidative DNA base modifications in mammalian cells by means of repair endonucleases.

The alkaline elution technique in combination with various repair endonucleases (Fpg protein, endonuclease III, exonuclease III, T4 endonuclease V) was used to quantify steady-state (background) levels of oxidative base modifications in various types of mammalian cells. In human lymphocytes the number of base modifications sensitive to Fpg protein, which include 8-hydroxyguanine, was 0.25 +/- 0.05 per 10(6) base pairs. Even lower levels (0.07 +/- 0.02 per 10(6) bp) were observed in HeLa cells. The numbers of sites sensitive to the other repair endonucleases were below the detection limit (0.05 per 10(6) bp). In a direct comparison, the background level of Fpg-sensitive modifications determined by alkaline elution was much lower than the background level of 8-hydroxydesoxyguanosine (8-oxodG) determined after enzymatic DNA hydrolysis by HPLC and electrochemical detection. However, the number of additional Fpg-sensitive modifications induced by a photosensitizer plus light was similar to the additional number of 8-oxodG residues determined by HPLC with electrochemical detection. This indicates that the enzyme assay does not systematically underestimate the number of lesions and points to an artefactual generation of 8-oxodG during DNA isolation and hydrolysis.

Chromatography, High Pressure Liquid↗
PubMed1988

Release of 7-alkylguanines from haloethylnitrosourea-treated DNA by E. coli 3-methyladenine-DNA glycosylase II.

Previous studies have related DNA modification by the haloethylnitrosoureas to their antitumor activity. Repair of this damage, particularly by O6-alkylguanine-DNA alkyltransferase, has been linked to tumor resistance by several previous investigations. We report here that E. coli 3-methyladenine-DNA glycosylase II can also remove several of the DNA modifications caused by the haloethylnitrosoureas. 7-Chloroethylguanine, 7-hydroxyethylguanine, and diguan-7-ylethane are all released into the supernatant from DNA modified by N-[2-chloroethyl-1,2-14C]-N'-cyclohexyl-N-nitrosourea. Release of diguan-7-ylethane is of particular interest since this entity evidently represents a DNA intrastrand cross-link. If a similar activity is present in mammalian cells, it might be an important source of resistance to the therapeutic action of the haloethylnitrosoureas.

Chromatography, High Pressure Liquid↗
PubMed1998

Oxidative DNA damage in vivo: relationship to age, plasma antioxidants, drug metabolism, glutathione-S-transferase activity and urinary creatinine excretion.

Oxidative DNA modification has been implicated in development of certain cancers and 8-oxodG, the most abundant and mutagenic DNA modification, has for some time been considered a biomarker of this activity. Urinary excretion of 8-oxodG over 24h has been used to estimate the rate of damage to DNA, and animal studies have supported this rationale. Reported determinants include tobacco smoking, heavy exercise, environmental pollution and individual oxygen consumption. Samples from three published studies were used to determine the association of urinary 8-oxodG excretion with age, plasma antioxidants, the glutathione-S-transferase phenotype and the activity of the xenobiotic metabolising enzyme CYP1A2. In the age range 35-65 years, age was not related to urinary 8-oxodG excretion, and there were no relations to either the glutathione-S-transferase phenotype or to the plasma antioxidants: vitamin C, alpha-tocopherol, beta-carotene, lycopene or coenzyme Q10. The activity of CYP1A2 showed a significant correlation in two of the three studies, as well as a significant correlation of 0.26 (p < 0.05) in the pooled data set. Regression analysis of CYP1A2 activity on 8-oxodG indicated that 33% increase in CYP1A2 activity would correspond to a doubling of 8-oxodG excretion. This finding needs to be confirmed in independent experiments. Spot morning urine samples can under certain circumstances be used to estimate 8-oxodG excretion rate provided that creatinine excretion is unchanged (in paired experiments) or comparable (in un-paired experiments), as evaluated from the correlation between 8-oxodG excretion in 24 h urine samples and in morning spot urine samples corrected for creatinine excretion (r = 0.50, p < 0.05). We conclude that 8-oxodG excretion is determined by factors like oxygen consumption and CYP1A2 activity rather than by factors like plasma antioxidant concentrations.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1999

Monoclonal antibody against DNA adducts with osmium structural probes.

Osmium tetroxide complexes with nitrogen ligands (Os,L) have been widely used as probes of the DNA structure. A monoclonal antibody OsBP7H8 against DNA adducts with Os,L was produced in mice. OsBP7H8 does not bind to proteins or total yeast RNA modified with Os,2,2'-bipyridine (bipy) nor to the unmodified nucleic acids and proteins. The antibody recognizes DNA modified with Os,bipy (DNA-Os,bipy) or with OsO4,1,10-phenanthroline (DNA-Os,phen) but it does not cross-react with oxidized DNA and with DNA adducts of osmium tetroxide complexes with other ligands (such as pyridine, TEMED and bathophenanthroline disulfonic acid). The affinity of OsBP7H8 to DNA-Os,phen is about five-fold higher as compared to DNA-Os,bipy. The antibody can be thus applied either for recognition of single-stranded and distorted regions in DNA (after DNA modification with Os,bipy) or for detection of both single-stranded and double-stranded DNAs (after DNA modification with Os,phen). A new simplified procedure for the dot-blot analysis is proposed, not requiring the purification of DNA-osmium adduct prior to its application to the membrane.

Animals↗
PubMed1988

Inhibition of estrogen-induced renal carcinogenesis in male Syrian hamsters by tamoxifen without decrease in DNA adduct levels.

Estrogens have previously been shown to induce covalent DNA modifications specifically in the hamster kidney, the target organ of estrogen-inducible and -dependent renal carcinoma. The DNA adducts, formed by yet unknown mechanisms, have been postulated to mediate hormonal carcinogenesis in this animal model. In an attempt to study a possible involvement of estrogen receptor mechanisms in the formation of DNA adducts, 17 beta-estradiol and the antihormone tamoxifen were concomitantly administered as s.c. implants to male Syrian hamsters. 17 beta-Estradiol-treated and tamoxifen-treated animals served as positive and negative controls, respectively. The tumor incidence decreased from 100% in 17 beta-estradiol-treated controls to 25% in the group receiving tamoxifen in addition to hormone. Tamoxifen-treated animals did not develop kidney tumors and did not show any detectable DNA damage. DNA adduct levels were comparable in hamsters treated with 17 beta-estradiol and 17 beta-estradiol plus tamoxifen for 5 or 7 months. In hamsters inoculated with H-301 cells, which are derived from the estrogen-induced hamster renal carcinoma and are estrogen dependent for growth, tamoxifen decreased estrogen-dependent H-301 tumor growth. However, in cell culture, neither 17 beta-estradiol nor tamoxifen influenced H-301 cell division. It was concluded that tamoxifen inhibited the growth of estrogen-induced renal carcinoma but did not interfere with tumor initiation since it did not inhibit the formation of DNA adducts. Moreover, receptor mechanisms were most probably not involved in the induction of DNA modifications by estrogens.

Animals↗
PubMed2001

Age-related and tissue-specific accumulation of oxidative DNA base damage in 7,8-dihydro-8-oxoguanine-DNA glycosylase (Ogg1) deficient mice.

Mutations that influence the repair of oxidative DNA modifications are expected to increase the steady-state (background) levels of these modifications and thus create a mutator phenotype that predisposes to malignant transformation. We have analysed the steady-state levels and repair kinetics of oxidative DNA modifications in cells of homozygous ogg1(-/-) null mice, which are deficient in Ogg1 protein, a DNA repair glycosylase that removes the miscoding base 8-hydroxyguanine (8-oxoG) from the genome. Oxidative purine modifications including 8-oxoG were quantified by means of an alkaline elution assay in combination with Fpg protein, the bacterial functional analogue of Ogg1 protein. In primary hepatocytes of adult ogg1(-/-) mice aged 9-12 months, the steady-state level of the lesions was 2.8-fold higher than in wild-type control mice. In contrast, no difference between ogg1(-/-) and wild-type mice was observed in splenocytes, spermatocytes and kidney cells. In hepatocytes of ogg1(-/-) mice, but not in wild-type controls, the steady-state levels increased continuously over the whole lifespan. No significant accumulation of the oxidative base modifications was observed in ogg1(-/-) fibroblasts in culture when they were kept confluent for 8 days. Both in confluent and proliferating ogg1(-/-) fibroblasts, the global repair of additional oxidative base modifications induced by photosensitization was 4-fold slower than in wild-type cells. The results suggest that the consequences of an Ogg1 defect are restricted to slowly proliferating tissues with high oxygen metabolism such as liver, because of a back-up mechanism for the repair of 8-oxoG residues that is independent of transcription and replication.

Age Factors↗
PubMed1987

Guanine modification during chemical DNA synthesis.

Base modification during solid-phase phosphoramidite synthesis of oligodeoxynucleotides has been investigated. We have discovered chemical modification that converts dG and dG-containing oligomers to a fluorescent form. This modification has been linked to N,N-dimethylaminopyridine (DMAP), an acylation catalyst, which can displace phosphate triester adducts at the 6-position of guanine. Further, we have found that this fluorescent intermediate can be converted in ammonium hydroxide solution to 2,6 diaminopurine deoxyribonucleoside (2,6 DAP), a potentially mutagenic nucleoside analog. We have shown that N-methylimidazole (NMI) in place of DMAP eliminates the fluorescent species and reduces 2,6 DAP contamination.

Chromatography, High Pressure Liquid↗