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Chemico-biological Interactions 1985Rat liver DNA was treated in vitro with benzo[a]pyrene-diolepoxide (BPDE), the ultimate carcinogenic metabolite derived from the polycyclic hydrocarbon benzo[a]pyrene....
Rat liver DNA was treated in vitro with benzo[a]pyrene-diolepoxide (BPDE), the ultimate carcinogenic metabolite derived from the polycyclic hydrocarbon benzo[a]pyrene. On incubation of the reacted DNA, apurinic sites developed which gave rise to strand breakage in alkaline solution. The reduction in molecular weight produced by these breaks was measured by analytical ultracentrifugation. In the case of anti-BPDE this depurination was shown to occur in two stages. The first was mainly due to attack at the 7-position of guanine, to yield an adduct which was lost from the DNA within a few hours. The second stage was due to much slower loss of the major N2-guanine adduct. The separated enantiomers, (+)- and (-)-anti-BPDE, and syn-BPDE all caused depurination to various extents. It is argued that although these processes are important in a study of the action of BPDE on DNA in vitro, their contribution to the biological activity of BPDE is probably negligible.
Topics: 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; Animals; Benzopyrenes; Cell Transformation, Neoplastic; Chemical Phenomena; Chemistry; DNA; DNA, Single-Stranded; Hydrogen-Ion Concentration; Liver; Purines; Rats
PubMed: 3922635
DOI: 10.1016/s0009-2797(85)80095-8 -
Nucleosides, Nucleotides & Nucleic Acids Feb 2003The removal of 4,4'-dimethoxytrityl (DMTr) groups from oligonucleotides at low pH and the acid lability of the glycosidic linkage of purine nucleotides constitute an...
The removal of 4,4'-dimethoxytrityl (DMTr) groups from oligonucleotides at low pH and the acid lability of the glycosidic linkage of purine nucleotides constitute an inherent conflict in preparative oligonucleotide chemistry. The use of a mildly acidic NaOAc buffer (10 mM, pH 3.0-3.2) allows adjustment of the pH in a range where the progress of the DMTr removal reaction can be monitored conveniently by HPLC and the optimum reaction time can be calculated. As a result, oligonucleotides with minimum depurination are obtained.
Topics: Adenine; Chromatography, High Pressure Liquid; Guanine; Hydrogen-Ion Concentration; Models, Chemical; Oligodeoxyribonucleotides; Oligonucleotides, Antisense; Purines; Trityl Compounds
PubMed: 12744600
DOI: 10.1081/NCN-120019499 -
Chemical Communications (Cambridge,... Jan 2012Development of a simple label-free fluorescence hybridization assay to monitor the depurination activity of toxic ribosome inactivating proteins by using a fluorescent...
Development of a simple label-free fluorescence hybridization assay to monitor the depurination activity of toxic ribosome inactivating proteins by using a fluorescent ligand that specifically pseudo base pairs with a cytosine residue opposite an abasic site is described. This method could be potentially implemented in screening platforms for the discovery of small molecules that inhibit the activity of these toxins.
Topics: Animals; Fluorescent Dyes; In Situ Hybridization, Fluorescence; Naphthyridines; Nucleic Acid Hybridization; Plant Proteins; Plants; Purines; RNA, Ribosomal; Rats; Ribosome Inactivating Proteins, Type 1; Saporins
PubMed: 22105782
DOI: 10.1039/c1cc16667k -
Plant Molecular Biology Dec 1992Mirabilis antiviral protein (MAP) is a single-chain ribosome-inactivating protein (RIP) isolated from Mirabilis jalapa L. It depurinates the 28S-like rRNAs of...
Mirabilis antiviral protein (MAP) is a single-chain ribosome-inactivating protein (RIP) isolated from Mirabilis jalapa L. It depurinates the 28S-like rRNAs of prokaryotes and eukaryotes. A specific modification in the 25S rRNA of M. jalapa was found to occur during isolation of ribosomes by polyacrylamide/agarose composite gel electrophoresis. Primer extension analysis revealed the modification site to be at the adenine residue corresponding to A4324 in rat 28S rRNA. The amount of endogenous MAP seemed to be sufficient to inactivate most of the homologous ribosomes. The adenine of wheat ribosomes was also found to be removed to some extent by an endogenous RIP (tritin). However, the amount of endogenous tritin seemed to be insufficient for quantitative depurination of the homologous ribosomes. Endogenous MAP could shut down the protein synthesis of its own cells when it spreads into the cytoplasm through breaks of the cells. Therefore, we speculate that MAP is a defensive agent to induce viral resistance through the suicide of its own cells.
Topics: Adenine; Antiviral Agents; Base Sequence; Consensus Sequence; DNA; Molecular Sequence Data; N-Glycosyl Hydrolases; Plant Proteins; Plants, Toxic; Polymerase Chain Reaction; RNA, Ribosomal; Ribosome Inactivating Proteins; Ribosome Inactivating Proteins, Type 1; Ribosomes; Nicotiana
PubMed: 1463845
DOI: 10.1007/BF00028897 -
Nature May 1981
Topics: Apurinic Acid; Bacteriophage phi X 174; Base Sequence; DNA Polymerase I; DNA Replication; DNA, Viral; Escherichia coli; Mutation; Virus Replication
PubMed: 6453298
DOI: 10.1038/291349a0 -
Basic Life Sciences 1982The possible consequences of depurination for both spontaneous and induced mutagenesis were investigated using in vitro and in vivo assays. Depurination of synthetic...
The possible consequences of depurination for both spontaneous and induced mutagenesis were investigated using in vitro and in vivo assays. Depurination of synthetic polynucleotide templates such as poly [d(A-T)] or poly [d(G-C)] leads to increased misincorporation of noncomplementary nucleotides when these templates are copied by prokaryotic and eukaryotic DNA polymerases. The ability of Escherichia coli DNA polymerase I to copy over apurinic sites was demonstrated using single-stranded circular DNA of bacteriophage 0X174 as a template and starting DNA synthesis at a fixed point. Analysis of the newly synthesized 0X174 restriction fragments on neutral and alkaline sucrose gradients shows that synthesis proceeded past apurinic sites. When using depurinated 0X174 DNA containing the am3 amber mutation as a template for copying by E. coli DNA polymerase I, an increased reversion to wild type is observed after transfection into E. coli spheroplasts. The enhancement in reversion frequency is proportional to the extent of depurination, suggesting that depurination is also mutagenic during copying natural DNA in vitro. When noncopied depurinated 0X174 am3 DNA is transfected in E. coli spheroplasts, no increase in reversion frequency is observed above background level. However, when the spheroplasts are derived from bacteria in which the SOS response had been induced by UV irradiation, a substantial increase is observed for depurinated molecules, whereas no increase is observed for nondepurinated templates, suggesting in vivo mutagenesis at depurinated sites. In each of the different assay systems investigated, the increase in misincorporation or reversion frequency is a linear function of the number of sites and is abolished by treatment of the depurinated templates with alkali, which rapidly induces strand breakage at apurinic sites.
Topics: Apurinic Acid; Carcinogens; DNA Repair; DNA Replication; DNA, Single-Stranded; DNA-Directed DNA Polymerase; Escherichia coli; Mutation; Polynucleotides; Templates, Genetic
PubMed: 7052053
DOI: 10.1007/978-1-4613-3476-7_12 -
Nucleic Acids Research Nov 1983Sterically hindered N6-dialkylformamidine protected deoxyadenosine is more stable to acidic depurination than N6-benzoyldeoxyadenosine and is potentially a valuable...
Sterically hindered N6-dialkylformamidine protected deoxyadenosine is more stable to acidic depurination than N6-benzoyldeoxyadenosine and is potentially a valuable protecting group in the synthesis of deoxyoligonucleotides.
Topics: Adenosine; Amidines; Deoxyadenosines; Formamides; Indicators and Reagents; Kinetics; Oligodeoxyribonucleotides; Oligonucleotides; Structure-Activity Relationship
PubMed: 6606157
DOI: 10.1093/nar/11.22.8031 -
Biological Chemistry Aug 2000Among five ribosome-inactivating proteins tested only saporin-S6 could efficiently release the adenine from adenosine 20 of the synthetic oligoribonucleotide (SRD RNA)... (Comparative Study)
Comparative Study
Among five ribosome-inactivating proteins tested only saporin-S6 could efficiently release the adenine from adenosine 20 of the synthetic oligoribonucleotide (SRD RNA) mimic of the sarcin/ricin domain of rat 28S rRNA with a Km of 9 microM and a kcat of approximately 0.4 min(-1) at pH 7.6. The optimal pH for the depurination activity of saporin-S6 is 5.0. However, saporin-S6 lost its site-specificity of depurination on SRD RNA around the optimal pH. The non-specific depurination activity of saporin-S6 was dependent on the enzyme concentration and pH conditions. These results are valuable to understand the diversity and the depurination mechanism of ribosome-inactivating proteins.
Topics: Adenine; Animals; Binding Sites; Deoxyribonucleases; Dose-Response Relationship, Drug; Electrophoresis, Agar Gel; Hydrogen-Ion Concentration; Immunotoxins; Kinetics; Molecular Mimicry; N-Glycosyl Hydrolases; Oligoribonucleotides; Plant Proteins; RNA, Ribosomal, 28S; Rats; Ribosome Inactivating Proteins, Type 1; Saporins; Substrate Specificity
PubMed: 11030435
DOI: 10.1515/BC.2000.098 -
Toxins Jun 2018Ribosome-inactivating proteins (RIPs) are potent toxins that inactivate ribosomes by catalytically removing a specific adenine from the α-sarcin/ricin loop (SRL)... (Review)
Review
Ribosome-inactivating proteins (RIPs) are potent toxins that inactivate ribosomes by catalytically removing a specific adenine from the α-sarcin/ricin loop (SRL) of the large rRNA. Direct assays for measuring depurination activity and indirect assays for measuring the resulting translation inhibition have been employed to determine the enzyme activity of RIPs. Rapid and sensitive methods to measure the depurination activity of RIPs are critical for assessing their reaction mechanism, enzymatic properties, interaction with ribosomal proteins, ribotoxic stress signaling, in the search for inhibitors and in the detection and diagnosis of enteric infections. Here, we review the major assays developed for measuring the catalytic activity of RIPs, discuss their advantages and disadvantages and explain how they are used in understanding the catalytic mechanism, ribosome specificity, and dynamic enzymatic features of RIPs.
Topics: Animals; Biological Assay; Catalysis; Ribosome Inactivating Proteins
PubMed: 29899209
DOI: 10.3390/toxins10060240 -
IUBMB Life Feb 2012Depurinating DNA adducts formed by aromatic hydrocarbons and catechol estrogen quinones play a major role in cancer initiation. Most of these adducts depurinate...
Depurinating DNA adducts formed by aromatic hydrocarbons and catechol estrogen quinones play a major role in cancer initiation. Most of these adducts depurinate instantaneously, but some guanine adducts depurinate from DNA with half-lives of hours. We report here, that after 10 h at 37 °C, reaction of estradiol-3,4-quinone (E(2)-3,4-Q) with ds-DNA to yield N7Gua and N3Ade adducts was complete and more efficient than with ss-DNA. When E(2)-3,4-Q reacted with t-RNA, no adducts were detected after 10 h, and the level of N3Ade and N7Gua adducts after 10 days was less than half that with ss-DNA after 10 h. Reaction of E(2)-3,4-Q and dG yielded 4-OHE(2)-1-N7dG, which spontaneously depurinated to yield 4-OHE(2)-1-N7Gua. To investigate the mechanism of depurination, E(2)-3,4-Q was reacted with carbocyclicdeoxyguanosine, in which the ring oxygen of the deoxyribose moiety is substituted with CH(2) , and depurination was observed. The results from this experiment demonstrate that the oxocarbenium ion mechanism plays the major role in depurination and provides the first experimental evidence for this mechanism. A newly discovered β-elimination mechanism also plays a minor role in depurination. Understanding why the depurinating estrogen-DNA adducts come from DNA, and not from RNA, underscores the critical role that these adducts play in initiating cancer.
Topics: Carcinogens; Cell Transformation, Neoplastic; Cyclopentanes; DNA Adducts; DNA Damage; DNA, Single-Stranded; Deoxyguanosine; Estradiol; Humans; Hydrogen-Ion Concentration; Kinetics; Purines; RNA, Transfer
PubMed: 22162200
DOI: 10.1002/iub.586