The present studies were undertaken to determine the mechanism by which attachment of the carcinogen N-2-acetylaminofluorene to guanosine residues in nucleic acids distors their structure and function. Oligonucleotides were modified with N-acetoxy-2-acetylaminofluorene, repurified, and their base compositions analyzed. Evidence is presented that acetylaminofluorene residues bound to guanosines in GpUpU, ApApG, or poly (U,G) inactivates their function in codon recognition. Circular dichroism spectra suggest that this is caused by gross conformational changes in these compounds involving both a rotation about the glycosidic bond of guanosine residues bearing N-2-acetylaminofluorene, as well as stacking interactions between the drug and bases adjacent to the substituted guanosine.

Acetates↗

PubMed1969

Codon recognition by enzymatically mischarged valine transfer ribonucleic acid.

Direct evidence for the adaptor hypothesis has been obtained by examining the codon recognition of a purified Escherichia coli valine transfer ribonucleic acid which was enzymatically mischarged with phenylalanine labeled with carbn-14 by reaction with purified phenylalanyl-transfer ribonucleic acid synthetase from Neurospora crassa. The mischarged transfer ribonucleic acid recognized the valine codons but failed to recognize the phenylalanine codon when tested in trinucleotide-directed ribosomal binding assay.

Bacterial Proteins↗

PubMedDate not supplied

Mutation and repair induced by the carcinogen 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) in the dihydrofolate reductase gene of Chinese hamster ovary cells and conformational modeling of the dG-C8-PhIP adduct in DNA.

Three experiments using 20 microM 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) were performed to induce mutations in the dihydrofolate reductase (DHFR) gene of a hemizygous Chinese hamster ovary (CHO) cell line (UA21). Metabolized forms of this chemical primarily bind at the C-8 position of guanine in DNA. In total, 21 independent induced mutants were isolated and 20 were characterized. DNA sequencing showed that the preferred mutation type found in 75% of the induced DHFR- clones was G.C-->T.A single and tandem double transversions. In addition to base substitutions, one mutant carried a-1 frameshift and another one had lost the entire locus by deletion. The induced changes affected purine targets on the nontranscribed strand of the gene in nearly all of the mutants sequenced (18/19). At the time that the first two experiments were performed, the initial adduct levels were quantitated in treated cells at the mutagenic dose by 32P-postlabeling. While the induced frequency of mutation was relatively low (approximately 5 x 10(-6), the adduct levels after a 1-h exposure of UA21 cells to 20 microM N-OH-PhIP were relatively high (13 adducts x 10(-6) nucleotides). This latter method was then employed to learn if the induced mutation frequency correlated with rapid overall genome repair of PhIP-DNA adducts. Total adduct levels, determined using DNA samples from treated cells collected after intervals of time, were reduced by about 50% after 6 h, and about 70% after 24 h. Since overall genome repair in CHO cells is relatively slow compared with preferential gene repair, the removal of dG-C8-PhIP adducts was apparently efficient. In order to better understand the mutational and repair results, we performed computational modeling to determine the lowest energy structure for the major dG-C8-PhIP adduct in a repetitively mutated duplex sequence opposite dA. Results of this analysis indicate that the PhIP-modified base resembles previous structural determinations of (deoxyguanosin-8-yl)-aminofluorene; the carcinogen is in the B-DNA minor groove and its adopts a syn conformation mispaired with an anti A. The implications of this conformational distortion in DNA structure for damage recognition by cellular repair enzymes are discussed.

Animals↗

PubMedDate not supplied

Solution conformation of the N-(deoxyguanosin-8-yl)aminofluorene adduct opposite deoxyinosine and deoxyguanosine in DNA by NMR and computational characterization.

Two-dimensional proton NMR and energy minimization computations have been employed to characterize the conformations of the N-(deoxyguanosin-8-yl)aminofluorene adduct [(AF)G] positioned opposite deoxyguanosine in one, and opposite deoxyinosine in another DNA undecamer duplex in aqueous solution. The two oligomer duplexes used in this study are d[C1-C2-A3-T4-C5-(AF)G6-C7-T8-A9-C10-C11].[G12-G13-T14 -A15-G16-X17-G18- A19-T20-G21-G22], where X17 was deoxyinosine in one duplex and deoxyguanosine in another. The exchangeable and nonexchangeable protons of the DNA are well resolved and narrow in the NMR spectra of the duplexes, and the base and sugar nucleic acid protons were assigned by NOESY and COSY data sets. All nine of the nonexchangeable aminofluorene ring protons were also assigned for the duplex that has deoxyinosine across from the modification site, and the (AF)G-I structure was employed to model the (AF)G-G one. The NOE distance restraints establish that the glycosidic torsion angle at (AF)G6 is syn. All other glycosidic torsion angles are anti, Watson-Crick type A.T and G-C base pairing is intact throughout the duplex except at the site of modification, and the helix maintains an overall B-DNA conformation. The syn orientation at the (AF)G6 places the aminofluorene ring in the B-DNA minor groove in a conformation similar to that found previously when the (AF)G was positioned opposite deoxyadenosine [Norman et al. (1989) Biochemistry 28, 7462-7476].

2-Acetylaminofluorene↗