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PubMed1985

[Specific modification of DNA at E. coli RNA-polymerase binding sites].

Specific modification of promoter regions of DNA has been studied. Plasmid pK56B1 DNA has been used as a model to test RNA-polymerase binding with DNA under various conditions. RNA-polymerase is shown to form specific complexes with DNA which are stable in solutions with a moderate ionic strength (0.1-0.2 M NaCl), under pH 5-8 in the presence of 0.5 M O-methylhydroxylamine of O-delta-aminooxybutylhydroxylamine. Escherichia coli JM103 cells have been transfected with DNAs treated with 0.5 M O-methylhydroxylamine at 37 degrees C, pH 5.2. The inactivation effects of the mutagen on single-stranded DNA of bacteriophage M13 m p1, double-stranded form of this bacteriophage (replicative form-RF) and on the complex of RNA-polymerase with RF DNA have been compared. The obtained data confirmed the specificity of reagent action with DNA sites binding with the enzyme. Selectivity of promoters modification has been confirmed also by the analysis of M13 m p1 DNA mutations induced in lacZ' gene by delta-aminooxybutylhydroxylamine effect on the DNA complex with DNA-polymerase.

Binding Sites↗
PubMed2006

Epigenetic tête-à-tête: the bilateral relationship between chromatin modifications and DNA methylation.

The epigenome, which comprises chromatin, associated proteins, and the pattern of covalent modification of DNA by methylation, sets up and maintains gene expression programs. It was originally believed that DNA methylation was the dominant reaction in determining the chromatin structure. However, emerging data suggest that chromatin can affect DNA methylation in both directions, triggering either de novo DNA methylation or demethylation. These events are particularly important for the understanding of cellular transformation, which requires a coordinated change in gene expression profiles. While genetic alterations can explain some of the changes, the important role of epigenetic reprogramming is becoming more and more evident. Cancer cells exhibit a paradoxical coexistence of global loss of DNA methylation with regional hypermethylation.

Acetylation↗
PubMed1984

Transformation of the Hprt gene with DNA from spermatogenic cells. Implications for the evolution of X chromosome inactivation.

DNA-mediated transformation of hypoxanthine guanine phosphoribosyl transferase (HPRT)-deficient cells was used to assess the state of the chromosome Hprt gene in spermatogenic cells. It had been shown previously that DNA from the inactive X chromosome of somatic cells functions poorly or not at all in HPRT transformation, indicating that DNA modification is involved in somatic cell X chromosome inactivation (XCI). In contrast, DNA from mature sperm does function in HPRT transformation suggesting that DNA modification may not be the basis of XCI in mature sperm. In this paper, transformation of HPRT- mouse and hamster cells has been performed to test the nature of XCI during earlier stages of spermatogenesis. DNA from these developing murine germ cells was shown to be capable of HPRT transformation, extending the observation that XCI in sperm does not appear to involve a DNA modification. We also show here that DNA from mature sperm of marsupials functions in HPRT transformation, a result consistent with a role for sperm XCI in the evolution of somatic X inactivation.

Animals↗
PubMed1988

Monitoring of interaction products of cis-diamminedichloroplatinum(II) and cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II) with DNA in cells from platinum-treated cancer patients.

The formation and stability of interaction products between the anti-cancer drug cis-diamminedichloroplatinum(II) (cis-DDP) and DNA were studied in buccal epithelial and urinary cells from ten cancer patients who received cis-DDP-based therapy. Buccal cells were collected 1 h before and 1-2 h after i.v. infusions with cis-DDP. The interaction products were visualized in an immunocytochemical peroxidase assay, using an antiserum against cis-DDP-modified calf thymus DNA. The nuclear staining density was measured by microdensitometry. Nuclear staining densities in buccal cells after infusions of greater than or equal to 20 mg/m2 cis-DDP were always higher than pretreatment values. Repeated sampling from individual patients treated for 2-5 consecutive days with daily doses of 20-70 mg/m2 cis-DDP indicated that cis-DDP-DNA binding in buccal cells increased in proportion to the cumulative total dose of cis-DDP. The variation in dose-density response between patients was 17%. Apparent adduct loss in buccal cells from four patients, as measured 8-17 days after the last infusion, amounted to 67-86%. Platinum-induced DNA modifications could also be detected in buccal cells from two cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II)-treated patients. In vitro experiments with human buccal cells and lymphocytes indicated linear relationships between DNA modification and either cis-DDP concentration or incubation time. Nuclear staining densities in pretreatment buccal cells from ten cancer patients treated in vitro with 33 microM cis-DDP for 1 h revealed that interpatient variation in in vitro DNA modification by cis-DDP was low. No quantitative correlation was found between in situ and in vitro DNA modification.

Carboplatin↗
PubMed1984

Resistance of DNA from filamentous and unicellular cyanobacteria to restriction endonuclease cleavage.

Chromosomal DNA from nine species of filamentous cyanobacteria as diverse as Nostoc, Gloeotrichia and Plectonema is suggested to be extensively modified (methylated) by its resistance to cleavage by a number of restriction endonucleases. A remarkably similar pattern of DNA modification in these species contrasts with the known heterogeneity of their type II restriction endonuclease content. In particular, Nostoc PCC 73102, which lacks detectable sequence-specific endonucleases, is shown to possess extensive DNA modification. The use of isoschizomers demonstrates the presence of a methylase in the filamentous strains analogous to the dam enzyme of Escherichia coli. As a preliminary to assessing the significance of the DNA modification, a study of susceptibility to restriction endonuclease cleavage of the genomes of five unicellular cyanobacteria revealed considerable variation between the different strains. The significance of the DNA modification patterns elucidated is discussed in terms of the restriction endonuclease content and cellular differentiation of the relevant cyanobacterial strains.

Base Sequence↗
PubMed1993

Biomarkers of aging: correlation of DNA I-compound levels with median lifespan of calorically restricted and ad libitum fed rats and mice.

I-compounds are species-, tissue-, genotype-, gender-, and diet-dependent bulky DNA modifications whose levels increase with animal age. While a few of these DNA modifications represent oxidation products, the majority of I-compounds appear to be derived from as yet unidentified endogenous DNA-reactive intermediates other than reactive oxygen species. Circadian rhythms of certain I-compounds in rodent liver imply that levels of these DNA modifications are precisely regulated. Caloric restriction (CR), the currently most effective method available to retard aging and carcinogenesis, has been previously shown to elicit significant elevations of I-compound levels in tissue DNA from Brown-Norway (BN) and F-344 rats as compared to age-matched ad libitum fed (AL) animals. The present investigation has extended this work by examining liver and kidney DNA I-compound levels in three genotypes of rats (F-344, BN, and F-344 x BN) and two genotypes of mice (C57BL/6N and B6D2F1) under identical experimental conditions in order to determine whether correlations exist between I-compound levels, measured in middle-aged animals, and median lifespan. Levels of a number of liver and kidney I-compounds were found to display genotype- and diet-dependent, statistically significant positive linear correlations with median lifespan in both species. In particular, the longer-lived hybrid F-344 x BN rats and B6D2F1 mice tended to exhibit higher I-compound levels than the parent strains. CR enhanced I-compound levels substantially in both rats and mice. Thus, I-compounds, measured at middle age, reflected the functional capability ('health') of the organism at old age, suggesting their predictive value as biomarkers of aging. The positive linear correlations between levels of certain I-compounds (designated as type I) and lifespan suggest that these modifications may be functionally important and thus not represent endogenous DNA lesions (type II), whose levels would be expected to correlate inversely with lifespan.

Aging↗
PubMed1988

Modification of DNA ends and detection of restriction enzyme recognition sequences in their ligated junctions.

A program has been developed for the modelling of modifications in DNA ends, for the construction of ligated junctions, and for the analysis in these junctions of new restriction enzyme recognition sequences. This program allows the analysis of restriction enzyme specificities in ligated junctions of cohesive or blunt DNA ends. Cohesive ends are considered in their natural configuration or after modification by possible blunt-ending procedures. The program also allows the modelling of partial filling-in for 5'-single-stranded ends. This program has proven useful for the design of sequences with new restriction sites or to predict or confirm the sequence of junctions created by the ligation of modified ends.

Base Sequence↗
PubMed1975

Host specificity of DNA in haemophilus influenzae: The physiological and genetical bases of instability of restriction and modification of DNA in strain Rd.

Further investigations of the instability of restriction and modification properties of H. influenzae Rd strain were carried out. It has been shown that the instable properties of hsd Hind1 system are maintained even after transfer of this system to another H. influenzae strain. The expression of hsd Hind1 system is very sensitive to various physiological changes which do not influence the other hsd systems present in the same Rd strain. The instability of hsd Hind1 system is postulated to be connected with some regulator gene(s).

DNA Restriction Enzymes↗
PubMed1995

Improvements in the analytical method for 8-hydroxydeoxyguanosine in nuclear DNA.

Modifications at two points in the sequence of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) analysis have contributed to a more accurate and simplified determination of 8-OH-dG in DNA. The first was an improvement in the detection limit for 8-OH-dG in high-performance liquid chromatography analysis and the second was a pronase digestion and ethanol precipitation method (pronase/ethanol method) for DNA isolation which could minimize artificial formation of 8-OH-dG. Since the changes in background current from electrochemical detection are regularly periodical, it was possible to reduce this background change by connecting a pressure damper, degassing the eluent before use and finally subtracting its theoretical function. After this background correction, the detection limit for 8-OH-dG was improved one order of magnitude, from 20 fmol (5.68 pg) to 1.76 fmol (0.5 pg). Therefore, 0.005 8-OH-dG/10(5) dG can be detected from 50 micrograms DNA. This improvement will allow the analysis of small samples, tissues from needle biopsies, < 5 ml whole blood, etc., and will contribute to the accuracy of 8-OH-dG measurements. The pronase/ethanol method resulted in lower levels of 8-OH-dG than the phenol method in analyses of both rat liver and calf thymus DNA, even after 6 h incubation at 45 degrees C. The level obtained by the pronase/ethanol method with butylated hydroxytoluene was approximately equal to or lower than the 8-OH-dG levels reported in normal rat liver. The pronase/ethanol method for DNA isolation can replace the phenol or other methods in 8-OH-dG analysis. This method also omits the use of highly toxic organic solvents.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2004

Structures of protein domains that create or recognize histone modifications.

DNA is packed together with histone proteins in cell nuclei to form a compact structure called chromatin. Chromatin represents a scaffold for many genetic events and shows varying degrees of condensation, including a relatively open form (euchromatin) and a highly condensed form (heterochromatin). Enzymes such as histone acetyltransferases (HATs) and methylases covalently label the amino-termini of histones, thereby creating a 'histone code' of modifications that is interpreted by the recruitment of other proteins through recognition domains. Ultimately, this network of interacting proteins is thought to control the degree of chromatin condensation so that DNA is available when it is required for genomic processes. Reviewed here are the structures of HAT and SET domains, which mediate the acetylation and methylation of histones, respectively, and bromodomains and chromodomains, which recognize the modified histones. How these structures have increased our understanding of DNA regulation is also discussed.

Acetylation↗
PubMed1986

Sequence-specific modification of DNA by 6-hydroxybenzo[a]pyrene.

6-Hydroxybenzo[a]pyrene cleaved phi X174 supercoiled DNA to open circular DNA in the presence of heavy metal ions. It induced an alkali-labile modification in DNA via an oxygen-radical-mediated reaction; the most frequent alkali-labile sites were on the 3' side of the pyrimidine residues of the pyrimidine cluster.

Bacteriophage phi X 174↗
PubMed1993

Modification of DNA bases in chromatin of intact target human cells by activated human polymorphonuclear leukocytes.

We investigated whether phorbol-12-acetate-13-myristate (PMA)-activated human polymorphonuclear leukocytes (PMNs) induce base modifications in target cell DNA in vivo. Human PMNs produced 9.4 +/- 0.8 (SD) nmol of H2O2/10(6) cells during 50 min of exposure to 2 micrograms/ml PMA and 13.7 +/- 2.8 nmol/10(6) cells during exposure to PMA plus 5 mM NaN3. Neither nonstimulated PMNs, nor PMA alone, nor NaN3 alone induced base modifications in chromatin-associated DNA of human Ad293 cells above control levels, when assayed by gas chromatography/mass spectrometry with selected-ion monitoring. However, a 60-min exposure to 1.7 +/- 0.4 x 10(6) PMNs/ml in the presence of 2 micrograms/ml PMA induced a 2-3-fold increase in the level of all modified bases detected by gas chromatography/mass spectrometry with selected-ion monitoring. The guanine-derived products 8-hydroxyguanine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and the adenine-derived product 4,6-diamino-5-formamidopyrimidine were induced to the highest levels among those bases detected. These data demonstrate that exposure to activated PMNs causes DNA base modifications in target cells in vivo typical of those induced by hydroxyl radical attack. The induction of potentially promutagenic modified bases may contribute to the mutagenicity of activated PMNs.

Cell Line↗
PubMed2005

Specific 3'-terminal modification of DNA with a novel nucleoside analogue that allows a covalent linkage of a nuclear localization signal and enhancement of DNA stability.

We report a straightforward method for the site-specific modification of long double-stranded DNA by using a maleimide adduct of deoxycytidine. This novel nucleoside analogue was efficiently incorporated at the 3'-termini of DNA by terminal deoxynucleotidyl transferase (TdT). Thiol-containing compounds can be covalently linked to the maleimide moieties. We added a nuclear localization signal peptide to the 3'-terminal of a 350 bp-long DNA that encoded short-hairpin RNA, and these modifications resulted in the enhancement of silencing activity by RNA interference. This enhancement is mainly attributed to increased stability of the template DNA.

Amino Acid Sequence↗
PubMed1990

Age-dependent covalent DNA alterations (I-compounds) in rat liver mitochondrial DNA.

Rat liver mitochondrial (mt) DNA was investigated for the presence of I-compounds, a recently discovered type of DNA modifications which is detected and measured via 32P-postlabeling. These DNA modifications were previously shown to accumulate in an age-dependent manner in total cellular DNA of various tissues of untreated rodents. In the present work, mt DNA of 1-, 3-, 6-, and 9-month-old female Sprague-Dawley rats was found by 32P-postlabeling also to contain I-compounds that increase with age. Most of the I-compounds were identical for mt and nuclear (nu) DNA. A cluster of 2 non-polar I-spots (termed M-compounds) was mitochondria-specific and increased about 8-fold from 1 to 9 months, attaining a RAL value of 44 X 10(-9) or 1 modification in 2.3 X 10(7) DNA nucleotides at 9 months. Quantitative differences between chromatographically identical spots were seen mainly for a low-polarity fraction of I-compounds, which exhibited 2 times higher overall levels in mt DNA versus nu DNA over the age range studied. Total I-compound levels increased during this time 6.9- and 5.1-fold in nuclei and mitochondria, respectively. The M-compound level was close to 10% of total mt DNA I-compound levels. M-compounds may conceivably be derived from potentially DNA-reactive electron carriers of the mt electron-transport chain, while I-compounds common to both mt and nu DNA presumably originate in extramitochondrial sources. The similarity of mitochondrial and nuclear I-compound profiles and amounts implies possible regulatory mechanisms in I-compound formation and repair. Mt DNA maps showed additional 32P-labeled material which may have been associated with DNA damage caused by oxygen free radicals known to be generated by the mt electron-transport chain. Age-dependent increases of mt DNA modifications are potentially related to mt mutations and may be linked to age-related degenerative changes in mitochondria.

Aging↗
PubMed2004

Histone modifications and DNA double-strand break repair.

The roles of different histone modifications have been explored extensively in a number of nuclear processes, particularly in transcriptional regulation. Only recently has the role of histone modification in signaling or facilitating DNA repair begun to be elucidated. DNA broken along both strands in the same region, a double-strand break, is damaged in the most severe way possible and can be the most difficult type of damage to repair accurately. To successfully repair the double-strand break, the cell must gain access to the damaged ends of the DNA and recruit repair factors, and in the case of homologous recombination repair, the cell must also find, colocalize, and gain access to a suitable homologous sequence. In the repair of a double-strand break, the cell must also choose between homologous and non-homologous pathways of repair. Here, we will briefly review the mechanisms of double-strand-break repair, and discuss the known roles of histone modifications in signaling and repairing double-strand breaks.

Acetylation↗
PubMed1998

Identifying 5-methylcytosine and related modifications in DNA genomes.

Intense interest in the biological roles of DNA methylation, particularly in eukaryotes, has produced at least eight different methods for identifying 5-methylcytosine and related modifications in DNA genomes. However, the utility of each method depends not only on its simplicity but on its specificity, resolution, sensitivity and potential artifacts. Since these parameters affect the interpretation of data, they should be considered in any application. Therefore, we have outlined the principles and applications of each method, quantitatively evaluated their specificity,resolution and sensitivity, identified potential artifacts and suggested solutions, and discussed a paradox in the distribution of m5C in mammalian genomes that illustrates how methodological limitations can affect interpretation of data. Hopefully, the information and analysis provided here will guide new investigators entering this exciting field.

5-Methylcytosine↗
PubMedDate not supplied

[Affinity modification of DNA polymerase I from Escherichia coli and its Klenow fragment with nucleotide imidazolides].

Affinity modification of E. coli DNA polymerase I and its Klenow fragment by imidazolides of dNMP (Im-dNMP) and dNTP was studied. DNA polymerase activity of DNA polymerase I was reduced by both Im-dNMP and Im-dNTP. However Im-dNTP does not inactivate of the Klenow fragment. The level of covalent labelling of both enzymes by radioactive Im-dNTP did not exceed 0.01 mol of reagent per mol of enzyme. But the deep inactivation of DNA polymerase I by Im-dNTP was observed. It is likely that this inactivation is due to the formation of intramolecular ether followed by phosphorylation of the carboxyl group. This assumption is strongly supported by the increase of the isoelectrical point of DNA polymerase I after its incubation with Im-dNTP in conditions of enzyme inactivation. All data permit us to suggest that the affinity modification of both enzymes by Im-dNMP and covalent labeling by Im-dNTP takes place without complementary binding of dNTP moiety with the template. However inactivation of DNA polymerase I by Im-dNTP occurs only if the dNTP-moiety is complementary to the template in the template.primer complex. It was shown that His residue was phosphorylated by Im-dNMP and Tyr or Ser residues between Met-802 and Met-848 were phosphorylated by Im-dNTP. We suppose that there are two states of DNA polymerase active site for the binding of dNTPs. One of them is independent on the template, in the other state the dNTP hydrogen bond with the template is formed.

Autoradiography↗