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At least 1,513 records · Page 84Linked to original sources
PubMed2006

Histone post-translational modifications and the response to DNA double-strand breaks.

The packaging of DNA into chromatin creates a number of significant barriers to the detection of DNA lesions and their timely and accurate repair. Eukaryotic cells have evolved a number of enzymes that modulate chromatin structure and facilitate DNA repair. Recent research illustrates how nucleosome remodelling enzymes cooperate with both DNA-damage-inducible and constitutive histone modifications to promote many facets of the cellular response to DNA damage.

Acetylation↗
PubMed2004

Detection of endonuclease III- and 8-oxoguanine glycosylase-sensitive base modifications in gamma-irradiated DNA and cells by the aldehyde reactive probe (ARP) assay.

Ionizing radiation generates diverse DNA lesions that differentially induce cell death and mutations. In the present study, calf thymus DNA (400 microg/ml) and HeLa cells were irradiated by (60)Co gamma-rays, and abasic (AP) sites and endonuclease (Endo)III- and 8-oxoguanine glycosylase (hOGG1)-sensitive base modifications in DNA were quantitated by the aldehyde reactive probe (ARP) assay. The irradiation of calf thymus DNA in phosphate buffer generated 91 Endo III- and 100 hOGG1-sensitive base modifications and 110 AP sites per 10(6) base pairs (bp) per Gy. The yield of the lesions in Tris buffer was 41- to 91-fold lower than that in phosphate, demonstrating a radioprotective effect of Tris. The HeLa cell chromosomal DNA contained 12 Endo III- and 3.8 hOGG1-sensitive base modifications and less than 1 AP sites per 10(6) bp as endogenous damage, and their level was increased by irradiation. The yields of the damage at 1 Gy (roughly equivalent to the lethal dose of HeLa cells [1.6-1.8 Gy]) were 0.13 Endo III, 0.091 hOGG1, and 0.065 AP sites per 10(6) bp, showing that irradiation with a lethal dose brought about only a marginal increase in base damage relative to an endogenous one. A comparison of the present data with those reported for DNA strand breaks supports the primary importance of double-strand breaks and clustered lesions as lethal damages formed by ionizing radiation.

Base Pair Mismatch↗
PubMed1986

Specific BPDE I modification of replicating and parental DNA from early S phase human foreskin fibroblasts.

Replicating DNA was modified by BPDE I to a greater extent than parental DNA when human fibroblast cells were treated with the carcinogen for 30 min in early S phase. Synchronized cells were exposed to 5-bromodeoxyuridine and treated with non-radioactive BPDE I and [methyl-3H]thymidine in early S phase. The density- and tritium-labeled, replicated DNA was separated from parental DNA in a CsCl gradient. The individual carcinogen-DNA adduct levels in both samples were quantitated by using the 32P-postlabeling method. The total modification of replicated DNA was 1.4-2.4 times greater than parental DNA. This difference was mainly reflected by differences in the main adducts, identified as the 3', [5'-32P]bisphosphates of 7R and 7S-BPDE I-dG. Confirmation of the identity of these two specific carcinogen-DNA adducts was accomplished by co-chromatography on t.l.c. with 3H-labeled 3',5'-bisphosphate adducts. The two 3H- and 32P-labeled adducts were isolated and dephosphorylated. The resultant 3H-labeled deoxyribonucleoside adducts were analyzed on h.p.l.c. and identified by co-chromatography with authentic standards. These results suggest that preferential modification of replicating DNA occurs when human cells are treated with BPDE I in early S phase. The ultimate result of this specific modification is the expression of a transformed phenotype.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1996

Pyridoxal 5'-phosphate inhibition of adenovirus DNA polymerase.

Pyridoxal phosphate modification of adenovirus DNA polymerase results in loss of DNA polymerase activity, whereas the 3' --> 5' exonuclease activity is unaffected. Inhibition by pyridoxal phosphate is time-dependent, displays saturation kinetics, and is reversible in the presence of excess primary amine unless the pyridoxal phosphate-enzyme adduct is first reduced with NaBH4. Thus, inhibition is the consequence of Schiff base formation between the aldehyde moiety of pyridoxal phosphate and primary amino groups on the enzyme. In addition to inhibiting DNA polymerase activity, pyridoxal phosphate also inhibited the ability of the enzyme to initiate viral DNA replication, by transfer of dCMP onto the preterminal protein. Neither template-primer nor dNTP protect against pyridoxal phosphate inhibition, but the combination of template-primer and complementary substrate dNTP protected both initiation and DNA polymerase activities. Thus, it is likely that both the dCMP transfer activity required for initiation and DNA polymerase activity are carried out at the same site of the enzyme.

Adenoviridae↗
PubMedDate not supplied

Avoidance of DNA methylation. A virus-encoded methylase inhibitor and evidence for counterselection of methylase recognition sites in viral genomes.

The ocr+ gene of bacterial virus T7 codes for the first protein recognized to inhibit a specific group of DNA methylases. The recognition sequences of several other DNA methylases, not susceptible to Ocr inhibition, are significantly suppressed in the virus genome. The bacterial virus T3 encodes an Ado-Met hydrolase, destroying the methyl donor and causing T3 DNA to be totally unmethylated. These observations could stimulate analogous investigations into the regulation of DNA methylation patterns of eukaryotic viruses and cells. For instance, an underrepresentation of methylation sites (5'-CG) is also true for animal DNA viruses. Moreover, we were able to disclose some novel properties of DNA restriction-modification enzymes concerning the protection of DNA recognition sequences in which only one strand can be methylated (e.g., type III enzyme EcoP15) and the primary resistance of (unmethylated) DNA recognition sites towards type II restriction endonuclease EcoRII.

Base Sequence↗
PubMed1992

EcoRI DNA methyltransferase-DNA interactions.

We present a novel strategy with synthetic hemimethylated DNA substrates containing uracil for thymine and inosine for guanosine replacements and EcoRI DNA methyltransferase to characterize the importance of major groove hydrophobic groups to the sequence-specific modification of DNA. The bacterial Mtase uses S-adenosyl-L-methionine to methylate the double-stranded DNA site 5'GAATTC3' at the N6 position of the central adenosine of each strand. Uracil substitution in either strand at the outer thymine (5'GAATUC3') causes 2.2- and 1.7-fold improvements in specificity (kcat/KmDNA). The fact that the specificity constant for the substrate containing uracil in both strands is identical to the value expected for noninteracting substitutions suggests that no significant methyltransferase-DNA interactions are altered beyond the site of either substitution. Similar analysis of the internal thymine (5'GAAUTC3') also shows these methyl groups to make a negative contribution to specificity, although the observed nonadditivity with the doubly modified substrate clearly shows methyltransferase-DNA interactions beyond the site of substitution to be affected in this case. To further probe the effect of analogue incorporation on methyltransferase-DNA interactions beyond the site of substitution, the relatively "silent" and additive uracil changes (5'GAATUC3') were combined with inosine for guanosine substitutions (e.g., 5'IAATTC3') known to have significant negative effects on specificity. In contrast to the additivity observed with the outer thymines, these studies show significant changes in methyltransferase-DNA interactions caused by the removal of the thymine methyls. Our results implicate a complex and flexible methyltransferase-DNA interface in which subtle structural changes in the substrate are transmitted over the entire canonical site.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1980

Use of an indicator sequence of human DNA to study DNA damage by methylbis(2-chloroethyl)amine.

A highly reiterated sequence of human DNA was used to determine the sites of modification of DNA caused by the anti-tumor drug methylbis(2-chloroethyl)amine (HN2, mechlorethamine, nitrogen mustard) upon treatment of cells in culture and of purified DNA. The lengths of the breakage products of the DNA treated with HN2 were compared to the lengths of DNA scission products produced by chemical reactions used for DNA sequence determination. HN2 was found to create alkali-labile lesions at positions of guanine. The distribution of the guanine modifications was the same for DNA extracted from cells treated with HN2 and for purified DNA treated with HN2. However, the extent of damage was at least 2-fold greater when purified DNA was used as the substrate. Several nitrogen mustard analogues also produced alkali-labile lesions at positions of guanine.

Alkylating Agents↗
PubMed1999

Overexpression of Ogg1 in mammalian cells: effects on induced and spontaneous oxidative DNA damage and mutagenesis.

Chinese hamster ovary cell lines (AA8 and AS52) were stably transfected to overexpress hOgg1 protein, the human DNA repair glycosylase for 7,8-dihydro-8-oxoguanine (8-oxoG). In the transfectants, the repair rate of 8-oxoG residues induced by either potassium bromate or the photosensitizer [R]-1-[(10-chloro-4-oxo-3-phenyl-4H-benzo[a]quinolizin-1-yl)-carbo nyl ]-2-pyrrolidinemethanolplus light was up to 3-fold more rapid than in the parental cells. However, the improved repair had little effect on the mutagenicity of potassium bromate in the guanine phosphoribosyl transferase (gpt) locus of the OGG1-transfected AS52 cells. The steady-state (background) levels of DNA base modifications sensitive to Fpg protein, which include 8-oxoG, in cells not exposed to a damaging agent were not reduced by the overexpression of Ogg1 protein. Moreover, the spontaneous mutation rates in the gpt locus were similar in OGG1-transformed and vector-only-transformed cells. The results demonstrate the potential of Ogg1 protein to remove its substrate modifications from most of the chromosomal DNA. They indicate, on the other hand, that the Ogg1 protein alone may not be rate limiting for the repair of the residual substrate modifications observed in cells under normal growth conditions.

Animals↗
PubMed1997

Reactive oxygen species-induced DNA damage and its modification: a chemical investigation.

The main purpose of this study was to determine whether well-known reactive oxygen species (ROS)-generating agents can induce DNA damage in a simple chemical system with or without Fenton reaction components (iron and reducing agents), and to explore whether antioxidants which normally exist in the cellular environment can modify such damage, i.e. to determine chemical reactions of relevance to biological systems. A neutral electrophoresis technique was used to investigate DNA double stranded breaks (DSBs) caused by chemical treatments of lambda-DNA in eppendorf tubes by various ROS-generating compounds and the degree of DNA damage was categorised by analysis of enhanced digital images. Double strand breaks were induced by hydroquinone (HQ), benzoquinone (BQ), benzenetriol (BT), hydrogen peroxide (H2O2), bleomycin (BLM) and sodium ascorbate (Vit C). DNA damage was modulated by various agents including catalase (CAT), superoxide dismutase (SOD), desferoxamine mesylate (DFO), ferrous chloride (FeCl2), reduced glutathione (GSH), trolox, silymarin and myricetin. Individual chemicals (except BLM) at the concentration of 1 mM did not induce large numbers of DSBs without iron [Fe(II) or Fe(III) at 25 microM]. GSH enhanced the damaging effect of HQ, BT and Vit C, did not alter the non-damaging effect of H2O2, but had a small protective effect on BLM. When compared with the non-enzyme protein, bovine serum albumin (BSA), SOD had a protective effect against BT, H2O2 and BLM; in the presence of GSH, SOD diminished the effect of HQ, BQ and Vit C but enhanced the effect of BT, H2O2 and BLM. With both GSH and Fe and compared with BSA, SOD enhanced the effect of HQ, BQ and BLM, ameliorated the effect of H2O2, and did not affect the others. CAT showed a protective effect for almost all examined compounds, but had little effect on BLM. With GSH alone, DFO enhanced the effect of HQ, BQ, H2O2 and ameliorated the effect of BT, BLM and Vit C and trolox was largely protective. With GSH and Fe, DFO was protective for all compounds except doxorubicin (Dox), trolox was protective for all compounds except Dox and BLM, silymarin was protective except that it had little effect on BLM and Dox, but myricetin did not show any protective effect. In conclusion, the results from the present study have further highlighted the adverse potential of reducing agents and redox cycling agents, and also the need for a cautious view of antioxidants.

Antioxidants↗
PubMed2005

Modulating gene expression in stem cells without recombinant DNA and permanent genetic modification.

Future therapeutic applications of stem cells in regenerative medicine require efficient techniques for modulating gene expression. Conventionally, this is achieved through the use of recombinant DNA, which invariably leads to permanent genetic alteration to the cell. Overwhelming safety and ethical concerns are likely to preclude the application of genetically modified stem cells in human clinical therapy for the foreseeable near future. An alternative may be to adopt a "milieu-based" approach to influence gene expression, by exposing stem cells to a cocktail of exogenous cytokines, growth factors, and extracellular matrix. Nevertheless, the non-specific pleiotropic effects exerted by various cytokines, growth factors, and extracellular matrix would make this a relatively inefficient approach. Moreover, a "milieu-based" approach is likely to require extended durations of in vitro culture, which might delay autologous transplantation of adult stem cells to the patient and might alter their immunogenicity through prolonged exposure to xenogenic proteins within the culture milieu. The obvious solution would be to deliver proteins, RNA, or their synthetic analogs, such as peptide nucleic acid, directly into the cell to modulate gene expression. Currently, two promising delivery platforms are available: (1) protein transduction domains, and (2) immunoliposomes. Because such molecules have a limited active half-life in the cytosol and are obviously not incorporated into the genetic code of the cell, these would only exert a transient modulatory effect on gene expression. Nevertheless, a transient effect may be preferable for clinical therapy, since this would ultimately avoid permanent genetic alteration to the cell.

Animals↗
PubMed1997

Radiation-induced double-strand modification in calf thymus DNA in the presence of 1,2-dihydroxy-9,10-anthraquinone and its Cu(II) complex.

When cells or DNA is exposed to ionizing radiation, the radicals produced in the irradiated sample will modify the base-pair region of the double strands. Effects of 1,2-dihydroxy-9,10-anthraquinone (DHA) and its Cu(II) complex on the radiation-induced modification of double-strands in calf thymus DNA were studied using ethidium bromide as a fluorescent probe. Our results show that the Cu(II)-DHA complex is more efficient in modifying the base-pair region in double-stranded DNA compared to free DHA.

Animals↗
PubMed1984

Immunological methods for detection of carcinogen-DNA adducts.

Considerable advances have been made during recent years, with regard to the detection and quantification of carcinogen- or mutagen-induced, structural modifications in the DNA of mammalian cells, by the introduction of immunoanalytical methods, in particular in conjunction with monoclonal antibodies (Mab). Antibodies are characterized by an outstanding capacity for the specific recognition of subtle alterations of molecular structure. They can, therefore, be used as sensitive detection probes in assays for DNA modifications caused by low levels of DNA-reactive (e.g., environmental) agents. Depending on the purpose of analysis, various types of immunoassays can be performed. The competitive radioimmunoassay (RIA) represents a routinely applicable, reproducible and sensitive assay for the detection of defined carcinogen-DNA adducts in hydrolysates of cellular DNA, in body fluids or in urine. Depending on their particular design, enzyme immunoassay (EIA) may have exceptionally low detection limits, due to the enzymatic amplification of the measured radioactivity or colour intensity. Similarly, recently established immuno-slot-blot (ISB) techniques are also characterized by very high sensitivity. Immunocytological assays (ICA) use Mab in conjunction with electronically intensified immunofluorescence for detection of modified DNA components in the nuclei of individual cells. Finally, single modified deoxynucleosides can be detected and localized in individual DNA molecules by immuno-electron microscopy (IEM).

Antibodies↗
PubMed1997

Age-related negative associations between parameters of cytogenetic damage and ex vivo (+/-)-anti-benzo(a)pyrene diolepoxide-induced unscheduled DNA synthesis in smoking humans.

Chemical or physical modification of DNA may cause an increase in genomic mutations or other genetic alterations, which may ultimately result in the onset of cancer. To avoid these deleterious effects of DNA damage, humans possess DNA repair mechanisms. Decreased DNA repair, induced ex vivo by UV light or ionizing radiation in human peripheral blood lymphocytes (PBLs), has been associated with aging. The aim of this study was to investigate whether repair of DNA damage, after ex vivo exposure of PBLs obtained from smokers (n = 20) to (+/-)-anti-benzo(a)pyrene diolepoxide [(+/-)-anti-BPDE], which is a mixture of reactive metabolites from the environmental carcinogen benzo(a)pyrene, is also associated with age. Furthermore, age-related associations between ex vivo (+/-)-anti-BPDE-induced DNA repair and the frequency of endogenous cytogenetic damage (sister chromatid exchange frequencies and micronuclei frequencies) in PBLs were evaluated. A statistically significant negative association was observed between ex vivo (+/-)-anti-BPDE-induced unscheduled DNA synthesis and age of the donors. Also, parameters of endogenous lymphocytic cytogenetic damage were negatively associated with ex vivo (+/-)-anti-BPDE-induced unscheduled DNA synthesis and positively associated with age in this population. It is concluded that, with increasing age, a decrease in lymphocytic excision repair capacity may be responsible for increased lymphocytic DNA damage in smokers.

Adult↗
PubMed1994

Modification of plasmid and bacteriophage DNA by aromatic amines: effects on survival, template activity, and mutagenicity.

The carcinogenic and mutagenic effects of the aromatic amines are believed to depend on their covalent modification of DNA, primarily through the formation of adducts at C8 of guanine. The actual biologic and biochemical responses to these adducts can be envisioned as the consequence of the abilities of the cell to repair the lesions, with or without fidelity, and the introduction of errors through bypass of the adducts by polymerases. A key question is whether changes in DNA sequence arise through the participation of common repair processes that cause mutations independent of adduct structure. Alternatively, do mutations arise through miscoding during polymerase bypass at the site of the adducts and are, therefore, more likely to produce sequence changes that are more characteristic of adduct structure? This question has been approached using single, site-specific, or randomly introduced aromatic amine DNA adducts in bacterial cells, and in vitro studies with DNA polymerases that employ site-specifically modified templates. The results of both approaches demonstrate that these adducts are distinguished readily by virtue of their structures, thus supporting the conclusion that mutagenic effects of the aromatic amines arise from their structures rather than from their triggering a common inaccurate repair response.

Amines↗
PubMed2006

Epigenetics and airways disease.

Epigenetics is the term used to describe heritable changes in gene expression that are not coded in the DNA sequence itself but by post-translational modifications in DNA and histone proteins. These modifications include histone acetylation, methylation, ubiquitination, sumoylation and phosphorylation. Epigenetic regulation is not only critical for generating diversity of cell types during mammalian development, but it is also important for maintaining the stability and integrity of the expression profiles of different cell types. Until recently, the study of human disease has focused on genetic mechanisms rather than on non-coding events. However, it is becoming increasingly clear that disruption of epigenetic processes can lead to several major pathologies, including cancer, syndromes involving chromosomal instabilities, and mental retardation. Furthermore, the expression and activity of enzymes that regulate these epigenetic modifications have been reported to be abnormal in the airways of patients with respiratory disease. The development of new diagnostic tools might reveal other diseases that are caused by epigenetic alterations. These changes, despite being heritable and stably maintained, are also potentially reversible and there is scope for the development of 'epigenetic therapies' for disease.

Arginine↗
PubMed1985

Convenient modification of the method for oligonucleotide-directed in vitro mutagenesis of cloned DNA.

A new modification of the oligonucleotide-mediated mutagenesis technique has been developed. The proposed methodology has been used to produce specific base changes in the double-stranded plasmid DNA. For this purpose, special cloning vectors have been constructed using the synthetic oligodeoxyribonucleotides. The developed method allows the production of mutant DNA from those of the wild-type with a yield of 10-20%.

Base Sequence↗
PubMed2005

Dynamic DNA methylation and histone modifications contribute to lentiviral transgene silencing in murine embryonic carcinoma cells.

Embryonic stem cells are subjected to a dynamic genome regulation during development. Here we report that the ectopic lentiviral transgenes are quickly silenced in murine embryonic carcinoma P19 cells. The silencing was correlated with CpG hypermethylation in the transgene promoter. Using high-resolution sodium bisulfite genome sequencing, we detected distinct DNA methylation kinetics in different proviral regions. DNase I sensitivity and chromatin immunoprecipitation assays revealed condensed chromatin structure and histone code switch during silencing. Longitudinal analysis of nonsilenced and silenced identical single-cell clones revealed that the silencing was coupled with CpG methylation in the promoter, as well as a global histone H3 deacetylation. Interestingly, the primer binding site and the packaging signal region appeared to serve as a DNA methylation initiation center which was rapidly hypermethylated regardless of transgene silencing and chromatin modifications. Analysis of cellular genes 45 to 50 kbp upstream and downstream of the integration site indicated that transcriptional activities of the flanking host genes were not affected. Genetic modifications of stem cells have great therapeutic potentials and our results picture a dynamic embryonic genome response to ectopic transgene integration that may have important implications in the future safety and efficacy modifications of stem cells.

Animals↗