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At least 487 records · Page 27Linked to original sources
PubMed1984

Perturbations of enzymic uracil excision due to guanine modifications in DNA.

Phage PBS2 DNA, which contains uracil in place of thymine, was used as substrate for purified Bacillus subtilis uracil:DNA glycosylase. Incubation of this DNA with the ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene resulted in the production of N-(deoxyguanosin-8-yl)acetylaminofluorene. A decreased Vmax resulted from the reaction of the glycosylase with this arylamidated substrate. Addition of a 2-fold excess of control PBS2 DNA following initiation of the reaction with the modified substrate showed delayed dissociation of the enzyme from the arylamidated DNA. This shows that the presence of a carcinogen-modified DNA base can reduce the capacity for uracil excision. Therefore, interference with enzymic release of uracil from DNA may be an indirect mechanism of mutagenesis by carcinogen:DNA adducts.

2-Acetylaminofluorene↗
PubMed1982

Preparation of psoralen-cross-linked R-loops and generation of large deletions by their repair in vivo.

A technique for site-selective introduction of psoralens into DNA has been developed. Irradiation of Escherichia coli 16 S rRNA with aminomethyltrimethyl psoralen (AMT) at 390 nm leads to the incorporation of AMT monoadducts in double-stranded regions of the RNA, but no cross-links. The AMT-containing RNA hybridizes efficiently to a supercoiled plasmid, pCS3, a derivative of pBR313 containing an insert of part of the E. coli rrnC cistron including the 3' 60% of the 16 S rDNA. When the resulting hybrids are re-irradiated at 360 nm, psoralen cross-links form between the rRNA and the rDNA, resulting in covalent R-loops. These were partially purified and used to transform E. coli after various nuclease treatments, employing an ampicillin selection to make cell survival dependent on successful repair of the psoralen-containing region. S1 nuclease-treated AMT-containing covalent R-looped plasmids show efficient transformation, and 9 mutants with large deletions were isolated by random screening of 100 colonies. These fell into 3 specific classes, each of which appears to start some place within the AMT-cross-linked rDNA. The structure of one mutant has been analyzed by DNA sequencing which shows a deletion which extends approximately 6800 nucleotides from nucleotide 868 of 16 S rDNA to nucleotide 315 on the tetracycline gene. Some hints of symmetry exist around the point of the deletion, but the pattern is not a striking one. This technique should be useful in making covalent D-loops as well as R-loops. It offers a number of potential applications for site-specific DNA modification and studies of DNA repair.

DNA↗
PubMed1981

Photoreaction of thymidine with primary amines. Application to specific modification of DNA.

Irradiation of calf thymus DNA with 254-nm light in the presence of n-butylamine resulted in the formation of l-n-butylthymine. The progress of the reaction can be easily monitored by the appearance of a new absorption band at 300 nm by UV spectroscopy. Model experiments on TpdA indicated that strand scission may occur preferentially at thymines during the photoreaction between DNA and n-butylamine. Because of its high selectively to Thyd, this type of photoreaction with n-butylamine may be used as a new method for selective release of thymine moieties from DNA.

Amines↗
PubMed1996

"Small is beautiful": major modifications in DNA structure or dynamics by small substituents or ligands.

This short review assembles the contributions of the author's laboratory to the structural aspects of DNA. DNA was modified by small ligands and/or substituents. There are three aspects to this work: a) Protonation of guanosine and DNA and the formation of triple- and quadruple-strands of guanosine, its nucleotides, their polymers and DNA. b) Substitution of the 2'-position of deoxyribose by the most polar atom, fluorine: studies on 2'-deoxy-2'-fluro-nucleosides, -nucleotides and their polymers, studied both by structural and biological methods. c) The effect of introducing the methyl group in the large groove of DNA: NMR studies of oligonucleotides containing N6-methylated adenine residues, and enzymatic and molecular biology work on Dam methylase are reported.

Adenine↗
PubMed1995

Structural and function modification of DNA by mitomycin C. Mechanism of the DNA sequence specificity of mitomycins.

Mitomycin C (MC) is a clinically used antitumor agent, which upon reductive activation activates and cross-links DNA. The covalent products of alkylation and cross-linking by the unnatural synthetic enantiomer of MC (ent-MC) were isolated as drug-deoxyguanosine monoadducts and drug-deoxyguanosine bisadducts and were fully characterized structurally. Specificity of alkylation and cross-linking of guanines in the CpG.CpG sequence was observed by ent-MC, similarly to that observed previously by MC. These findings define the mechanism of recognition of the CpG.CpG sequence of DNA by the mitomycins in the minor groove. In contrast, the natural MC metabolite, 2,7-diaminomitosene (2,7-DAM) which lacks the aziridine alkylator function is shown to recognize and alkylate guanines only the GpG.CpC sequence in the major groove, by a different mechanism. Thoughts on the molecular evolution of the basic mitomycin structure as a very efficient lethal DNA cross-linker are discussed.

Antibiotics, Antineoplastic↗
PubMed1996

Mutagenic DNA base modifications are correlated with lesions in nonneoplastic hepatic tissue of the English sole carcinogenesis model.

Hydroxyl radical-induced mutagenic base modifications have been linked to neoplasia in a number of biological systems, including English sole from chemically contaminated urban environments. However, virtually no information exists on the relationship between the mutagenic base modifications and preneoplastic and other lesions found in tumor-free tissues prone to cancer. We studied six hepatic lesions in immature, neoplasm-free English sole exposed to an urban and reference environment and established correlations between the lesion incidence and concentrations of the mutagenic base modifications 8-hydroxyguanine and 8-hydroxyadenine. The lesions were putatively preneoplastic basophilic foci, hepatocellular karyomegaly, megalocytic hepatosis, hepatocellular vacuolar change, hyalin droplet formation, and apoptosis. With the exception of hepatocellular vacuolar change, significant positive correlations were found between the lesions and the mutagenic base modifications. The hydroxyl radical may be a common etiological factor in the formation of the base modifications and hepatic lesions.

Adenine↗
PubMedDate not supplied

[Free radical mechanisms of radiation modification of DNA sugar fragment].

Analysis of the DNA and modeling its certain fragments substances radiolysis data allows to attribute the cardinal role to the primary 2-deoxyribosyl radicals in the processes of the breaks (C'3, C'5) and alkali-labile sites (C'1, C'2, C'4) in DNA strands, of the free bases appearance (C'3, C'5). The higher yield of bases destruction in DNA is explained by the transferring of a damage (unpaired electron) from 2-deoxyribosyl to a base within one nucleotide.

DNA↗
PubMed1993

Damage to DNA in cervical epithelium related to smoking tobacco.

OBJECTIVE: To determine whether tobacco smoking causes increased DNA modification (adducts) in human cervical epithelium. DESIGN: Comparison of DNA adducts measured by the technique of postlabelling with phosphorus-32 in normal ectocervical epithelium of smokers and non-smokers. A questionnaire on smoking habit and a urinary cotinine assay were used to identify smokers and non-smokers. SETTING: Cytology unit in large teaching hospital. SUBJECTS: 39 women (11 current smokers, seven former smokers, and 21 who had never smoked) undergoing gynaecological treatment (colposcopy or hysterectomy). Nineteen members of staff who did not smoke as controls. INTERVENTIONS: Biopsy of normal ectocervical epithelium. Urine sample. MAIN OUTCOME MEASURES: Measurement of DNA adducts in cervical epithelial tissue of smokers and non-smokers. Smoking habit derived from results of questionnaire and urinary cotinine:creatinine ratio. Proportion of adducts in women with abnormal and normal results of cervical smear test. RESULTS: DNA samples from smokers (identified from questionnaire) had significantly higher median proportions of DNA adducts that non-smokers (4.62 (95% confidence interval 4.04 to 7.74) v 3.47 (2.84 to 4.78) adducts/10(8) nucleotides; p = 0.048). Exclusion of women whose urinary cotinine:creatinine ratio did not confirm their self reported smoking habit (smoker or non-smoker) increased this difference (4.7 (3.85 to 8.08) v 3.52 (2.32 to 4.95) adducts/10(8) nucleotides; p = 0.03). Women who had abnormal results of cervical smear tests had significantly higher proportions of adducts than those with normal results (4.7 (3.90 to 8.13) v 3.47 (3.06 to 5.36) adducts/10(8) nucleotides; p = 0.03). CONCLUSIONS: Tobacco smoking by women leads to increased modification of DNA in cervical epithelium, suggesting biochemical evidence consistent with smoking as a cause of cervical cancer.

Adult↗
PubMed1977

Secondary structural modifications as a consequence of in vitro acetylation and phenanthrylation of DNA by the ultimate carcinogen N-acetoxy-N-2-acetylaminophenanthrene.

The acetic acid ester of the proximate carcinogen N-hydroxy-N-2-acetylaminophenanthrene was reacted in vitro with native and heat-denatured calf thymus DNA under various conditions. We showed that besides the phenanthrylation of the DNA bases there is an acetylation reaction of the DNA during its reaction with the ultimate carcinogen. Heat-denatured DNA is 5 to 10 times more acetylated than native DNA. This result suggests that most of the acetylation sites are nonreactive in the double-helical structure of DNA. On the other hand, the phenanthrylation of the bases is shown not to depend on the DNA secondary structure, suggesting that the phenanthrylation sites of the bases are accessible in the grooves of the DNA double helix. The influence of the DNA dynamic structure on the reactions of acetylation and phenanthrylation has been investigated by increasing the ionic strength of the incubation buffer. The melting temperature of different DNA samples, which have been reacted with different concentrations of N-acetoxy-N-2-acetylaminophenanthrene, decreases as the extent of the DNA modifications increases. This thermal destabilization of the double helix is tentatively attributed to the phenanthrylation rather than to the acetylation reaction.

Acetylation↗
PubMed1993

Comparison of sequence preference of tomaymycin- and anthramycin-DNA bonding by exonuclease III and lambda exonuclease digestion and UvrABC nuclease incision analysis.

The DNA bonding sites of two pyrrolo[1,4]benzodiazepine derivatives--tomaymycin (Tma) and anthramycin (Atm)--were identified by exonuclease III (exo III) digestion, lambda exonuclease (lambda exo) digestion, and UvrABC nuclease incision analysis. exo III digestion stalls 4-5 bases 3' to a drug-DNA adduct. While this method can recognize most of the Atm-and Tma-DNA modification sites, it is complicated in that exo III digestion is also stalled by certain unmodified sequences and by drug bound to the opposite strand. lambda exo digestion stalls 1-2 bases 5' to a drug-DNA adduct. The lambda exo method also recognizes most of the drug-DNA bonding sites and renders a cleaner background; however, it is also affected by opposite-strand drug bonding. Due to their intrinsic digestion polarities, these two exonucleases tend to be stalled by the drug-DNA adduct at one end of the DNA molecule. Purified UvrA, UvrB, and UvrC proteins acting together make dual incisions 6-8 bases 5' and 4 bases 3' to a Atm- or Tma-DNA adduct. This nuclease complex recognizes all the Tma- and Atm-DNA bonding sites identified by exonuclease digestion methods, and all the UvrABC incisions can be attributed to drug modifications in the incised DNA strand. The degree of UvrABC nuclease incision increases with increasing drug concentrations for DNA modification. Using the UvrABC incision method, we have identified the sequence preference of Tma- and Atm-DNA adduct formation in three DNA fragments, and we have found that these two drugs have different preferred sites for adduction. Both Tma- and Atm-DNA bonding is strongly influenced by the 5' and 3' neighboring bases; the orders of preferred 5' and 3' bases for Tma are A > G, T > C, and A, C > G, T, and for Atm the orders are A > G > T > C and A > G > T, C. The preferred triplets for Tma bonding are -AGA- > -GGC-, -TGC-, and AGC- and for Atm are -AGA-, -AGG- > -GGA-, -GGG-.

Anthramycin↗
PubMed1976

The effect of nitroimidazole and nitroxyl radiosensitizers on the post-irradiation synthesis of DNA.

The modification of DNA damage by three radiosensitizing drugs, present during gamma-irradiation of hypoxic Chinese hamster cells, was investigated. Both 2-methyl-5-nitroimidazole-1-ethanol (metronidazole) and 1-(2-nitro-1-imidazole)-3-methoxy-2-propranol (Ro-07-0582) were found to cause large increases in the yield of DNA single-strand breaks (SSB); triacetoneamine-N-oxyl (TAN) was found to have only a small effect on SSB production. The three drugs tested did not inhibit the rejoining of SSB. A pulse label and chase procedure was used to examine post-irradiation DNA synthesis. TAN present during irradiation under hypoxia was found to cause interruptions in subsequent DNA synthesis. Metronidazole and Ro-07-0582 had no effect on post-irradiation DNA synthesis. In addition, the effects of pre- and post-irradiation exposure to TAN were investigated, since these treatments have shown increased cell-killing in survival studies. TAN pre- and post-treatments were found to have no significant effect on subsequent DNA synthesis.

Cells, Cultured↗
PubMed1979

Standardization of the alkaline elution procedure using X-ray-damaged nuclear DNA.

Structural modification of DNA were induced by X-irradiation of crude hepatic nuclei at various dose ranges to standardize DNA damage evaluated by the alkaline elution technique. This quantitative assay can be used as reference for DNA damage induced by the in vivo administration of mutagens and/or carcinogens involved in the environment.

Alkalies↗
PubMed2004

Prereplicative purine methylation and postreplicative demethylation in each DNA duplication of the Escherichia coli replication cycle.

Escherichia coli plasmid DNA activated for initiation of duplication is in a stable low linking number supercoiled conformation. Low linking number DNA is methylated at the internal purines of a frequent 5'-Pyr-Pyr-Pur-Pur tetramer with a 5'-Pyr-Pur-3' axis of symmetry and is cut at the axis of symmetry by pneumococcal restriction enzyme DpnI when methylated in both strands. Purine methylation is of adenine in one strand and guanine in the other. Methylation of one of the two purines is removed during the cell cycle, presumably before the reverse shift to the B-supercoiled conformation. The topological transition was reconstituted in vitro only with DNA unmethylated at purines. Methylation-restriction analyses coupled with the chemical properties of low-linking number DNA and B-DNA respectively, suggest that removal of guanine methylation is essential for the low-linking number to B-DNA transition and hence for the deactivation of replication. Demethylation of methylguanine could explain the presence in E. coli of the two-member inducible operon known as ada. Characteristics of ada suggest a cascade of chemical DNA modifications that reverse prereplicative guanine methylation. Guanine demethylation could provide a model for the pivotal role played by de novo methylation in replication and for the essential role of "repair" enzyme ExoIII in demethylation leading to the reversal of replicative DNA activation and other processes that affect DNA function.

Animals↗
PubMedDate not supplied

[Preparation of photoreactive oligonucleotide duplexes and their application for photoaffinity modification of DNA-binding proteins].

To introduce photoreactive dNTP residues to the 3'-end of a mononucleotide gap, base-substituted photoreactive deoxynucleoside triphosphate derivatives, (5-[N-(2,3,5,6-tetrafluoro-4-azidobenzoyl)-trans-3-aminopropenyl-1]- and 5-(N-[N-(4-azido-2,5-difluoro-3-chloropyridine-6-yl)-3-aminopropionyl]- trans-3-aminopropenyl-1)-2'-deoxyuridine 5'-triphosphates, were used as substrates in the DNA polymerase beta-catalyzed reaction. The resulting nick, containing a modified base at the 3'-end, was sealed by T4 phage DNA ligase. This approach enables the preparation of DNA duplexes bearing photoreactive groups at predetermined position(s) of the nucleotide chain. Using the generated photoreactive DNA duplexes, the photoaffinity modifications of DNA polymerase beta and human replicative protein A (hRPA) were carried out. It was shown that DNA polymerase beta and hRPA subunits were modified with the photoreactive double-stranded DNA considerably less effectively than by the nicked DNA. In the case of double-stranded DNA, the hRPA p70 subunit was preferentially labeled, implying a crucial role of this subunit in the protein-DNA interaction.

DNA Polymerase beta↗