Transcription of the two unlinked structural genes URA1 and URA3 of Saccharomyces cerevisiae is positively regulated by the gene product PPR1. We have used S1 digestion and primer extension mapping to investigate the RNAs produced in different genetic backgrounds: wild-type, ppr1 deletion mutants, constitutively induced and non-inducible ppr1 mutants. Results show that each structural gene specifies multiple messenger RNA classes with different 5'-terminal sequences. The basal level of these transcripts does not require a functional PPR1 gene. Induction of URA1 results from an even increase of the level of synthesis of all the transcripts in contrast to that of URA3 which is effected by selectively increasing the levels of synthesis of one subset of transcripts. The PPR1-mediated control was also studied in the foreign genetic background of Schizosaccharomyces pombe using autonomously replicating hybrid plasmids carrying the gene URA1 or URA3 along with the regulatory gene PPR1, either in a constitutive or non-inducible allelic form. The 5' ends of the transcripts URA1 and URA3 made in S. pombe map upstream from the initiation sites used in S. cerevisiae. In contrast to S. cerevisiae, in S. pombe the URA3 but not URA1 transcripts respond to the PPR1-induction. We have identified a minimal control region for the PPR1-specific induction of URA1, that includes sequences located between the T-A-T-A box and the translation start codon. This region contains sequence features in common with URA3. There is an extensive alternating Pu:Py region including the T-A-T-A box of both promoters and an eight base-pair exact homology; further downstream, there is another 11 base-pair highly conserved sequence which either overlaps or lies in close proximity to the unregulated start sites of URA1 in S. pombe and of URA3 in S. cerevisiae. A positive regulatory model taking into accounts all these observations is presented.

Base Sequence↗

PubMed1985

DNA binding and mutation spectra of the carcinogen N-2-aminofluorene in Escherichia coli. A correlation between the conformation of the premutagenic lesion and the mutation specificity.

When the chemical carcinogen N-2-acetylaminofluorene binds to DNA in vivo, two major adducts are formed, both at position C-8 of the guanine residue. One of these (the acetylaminofluorene adduct) retains the acetyl group, while the other (the aminofluorene adduct) is the corresponding deacetylated form. Unlike -AAF adducts, which trigger important structural changes of the DNA secondary structure (either the insertion-denaturation model or the induction of a Z-DNA structure, depending upon the local nucleotide sequence), -AF adducts bind to the C-8 of guanine residues without causing any major conformational change of the B-DNA structure. Well-defined adducts (either -AF or -AAF) can be formed in vitro by reacting DNA with either N-hydroxy-N-2-aminofluorene or N-acetoxy-N-2-acetylaminofluorene. Specific cleavage of the phosphodiester backbone at -AF adducts can be achieved by treating -AF-modified DNA in 1 M-piperidine at 90 degrees C. This observation led us to construct the spectrum for -AF binding to a defined DNA restriction fragment. It is found that only guanine residues react to form alkali-labile lesions and that the reactivity among the different guanines is similar. In a forward mutation assay, namely the inactivation of the tetracycline resistance gene, we found previously that more than 90% of mutations induced by -AAF adducts are frameshift mutations. Using the same assay, we show here that -AF adducts induce primarily base substitution mutations (85%), mainly of the G to T transversion type. There is therefore a strong correlation between the nature of the carcinogen-induced conformational change of the DNA structure and the corresponding mutation specificity. The -AF-induced base substitution mutations depend upon the umuC gene function(s). The data obtained in our forward mutation assay are compared to the data previously obtained in the histidine reversion assay (Ames test).

2-Acetylaminofluorene↗

PubMed1985

Covalent binding of a carcinogen as a probe for the dynamics of deoxyribonucleic acid.

In order to determine the kinetic parameters of the binding to DNA of two closely related ultimate carcinogens, 2-(N-acetoxy-N-acetylamino)fluorene (N-Aco-AAF) and 2-(N-hydroxyamino)fluorene (N-OH-AF), three kinds of experiments were performed: measurement of the final amount of adduct (N-Aco-AAF and N-OH-AF), determination of the initial rate, and study of the reaction with deoxyguanosine (N-Aco-AAF only) at temperatures between 4 and 50 degrees C. The kinetic treatment of the chemical equations relies on two main assumptions: (i) binding of carcinogen to the C8 of guanine (G) could occur either with the classical B conformation or with a transient conformational state of the sugar--phosphate chain at the level of the guanine and denoted by G*; (ii) the equilibrium between G and G* is fast as compared to the chemical rate of carcinogen binding. These two assumptions have been verified by comparing experimental and calculated values of some of the data. From experimental data it is possible then to determine the characteristic independent parameters of the reaction: the constant K of the G in equilibrium G* and the enthalpy change delta H of the process, the rate constant k3 of the binding to the C8 of G, and the rate constant k1 of hydrolysis of the carcinogen with their corresponding activation enthalpies E3 and E1. Some essential results obtained are as follows: (a) The amount of G* that represents about 10% of the G at room temperature increases with temperature and is higher in denatured than in native DNA. (b) The values of delta H (approximately 9 kcal mol-1) and delta S (approximately 27 cal K-1 mol-1) of the G in equilibrium G* equilibrium are close to those associated with single base pair opening [Wartell, R.M., & Benight, A.S. (1982) Biopolymers 21, 2069]. (c) N-Aco-AAF reacts only with the G* conformation while N-OH-AF binds preferentially to the "classical" G (B conformation). Therefore, the electrophilic carcinogens behave as probes of the dynamic state of the DNA, but the rate of the G in equilibrium G* exchange is fast as compared to the binding rate of the carcinogen.(ABSTRACT TRUNCATED AT 400 WORDS)

2-Acetylaminofluorene↗

PubMed1984

In vitro enzymatic methylation of DNA substituted by N-2-aminofluorene.

Both the initial velocity and the overall methylation of DNA substituted by aminofluorene, by a rat liver DNA(cytosine-5-)-methyltransferase, are increased as compared to native DNA. The Km and Vmax of the modified DNA for the enzyme increase as a function of the extent of modification. The carcinogen may induce a secondary structure favouring the 'walking' of the enzyme along the DNA. The hypermethylation caused by this carcinogen could have a significance in gene activity, cellular differentiation and cancer induction.

Animals↗

PubMed1984

Carcinogen-induced mutation spectrum in wild-type, uvrA and umuC strains of Escherichia coli. Strain specificity and mutation-prone sequences.

Forward mutations induced by the ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF) in the tetracycline resistance gene carried on plasmid pBR322 are shown to be dependent upon the induction of the host SOS functions in wild-type and umuC Escherichia coli cells. The mutation frequency in the umuC strain is equal to about 40% of the mutation frequency observed in the umu+ background. In the excision-repair-deficient uvrA mutant strain the mutagenic response is the same as in SOS-induced wild-type cells whether or not the uvrA bacteria are SOS-induced. Equal mutation frequencies are obtained in both the wild-type and the uvrA strains for equal modification levels although the survival of AAF-modified plasmid DNA is greatly reduced in the uvrA strain as compared to the wild-type strain. Sequence analysis of the mutations reveals that more than 90% of the N-Aco-AAF-induced mutations are frameshift mutations. Two types of mutational hotspots are observed occurring either at repetitive sequences or at non-repetitive sequences. Both types of mutants appear at similar locations and frequencies in both the wild-type and the uvrA strains. On the other hand, only the non-repetitive sequence mutants are obtained in the umuC background. These non-repetitive sequence mutants preferentially occur within the sequence 5' G-G-C-G-C-C 3' (the NarI restriction enzyme recognition sequence). The analysis of the -AAF binding spectrum to the same DNA fragment shows that there is no direct correlation between the modification spectrum and the mutation spectrum. We suggest that certain sequences are "mutation-prone" in the sense that only these sequences can be efficiently mutated as the result of an active processing mediated by specific proteins. When a sequence is said to be mutation-prone it probably corresponds to a particular structure that is induced within this sequence as a result of the binding to the DNA of the mutagen. This sequence-specific conformational change is the substrate for the protein(s) that fixes the mutation. The mutagenic processing pathway(s) is part of the cellular response to DNA-damaging agents (the so-called SOS response). Two pathways for frameshift mutagenesis are suggested by the data: an umuC-dependent pathway, which is involved in the mutagenic processing of lesions within repetitive sequences; an umuC-independent pathway responsible for the fixation of mutations within specific non-repetitive sequences.

2-Acetylaminofluorene↗

PubMed1984

DNA binding spectrum of the carcinogen N-acetoxy-N-2-acetylaminofluorene significantly differs from the mutation spectrum.

The 3'----5' exonuclease activity of bacteriophage T4 DNA polymerase is found to be blocked in the vicinity of the N-2-acetylaminofluorene (-AAF) adducts to DNA. This observation allowed us to determine the binding spectrum of the -AAF adducts along a given DNA sequence. The mutation spectrum in a forward mutation assay within this same sequence has been established. Comparison between the -AAF binding spectrum and the mutation spectrum shows that there is no direct correlation.

2-Acetylaminofluorene↗

PubMed1984

pBR322 plasmid DNA modified with 2-acetylaminofluorene derivatives: transforming activity and in vitro strand cleavage by the Escherichia coli uvrABC endonuclease.

Covalently closed circular plasmid DNA was treated with three reactive derivatives of 2-acetylaminofluorene: N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF), its 7-iodo derivative (N-Aco- AAIF ) and N-hydroxy-N-2-aminofluorene (N-OH-AF), and tested as substrates for the Escherichia coli uvrABC endonuclease and for transformation frequencies on wild-type, uvrA, recA, uvrArecA and polA mutant strains. The uvrABC endonuclease reacted with all three substrates with high efficiency, implicating this enzyme in the repair of DNA containing all three types of adducts. However, only AAF- and AAIF -DNA showed greatly reduced survival on uvrA mutants (five adducts/lethal hit) relative to wild-type (20 adducts/lethal hit). AF-DNA survived equally well on uvrA mutant and wild-type cells, and at a much higher level of modification (60 adducts/lethal hit). A mutation in recA had only a minor effect on the survival of either DNA. The polA mutation reduced the survival of the AAF-treated DNA to the same extent as the uvrA mutation (five adducts/lethal hit). Also AF-DNA showed reduced survival on polA mutant cells versus wild-type. However, many more adducts (20/lethal hit) were tolerated than for AAF-DNA, indicating that AF lesions in the template do not efficiently block replication of DNA.

2-Acetylaminofluorene↗

PubMed1984

Chemically modified nucleic acids as immunodetectable probes in hybridization experiments.

Guanine residues in nucleic acids can be modified by treatment with N-acetoxy-N-2-acetylaminofluorene and its 7-iodo derivative in an in vitro nonenzymatic reaction. The modified nucleic acids (ribo or deoxyribo, single or double stranded) are recognized by specific antibodies. They can be immunoprecipitated or used as probes in hybridization experiments and detected by immunochemical techniques.

Acetoxyacetylaminofluorene↗

PubMed1983

Different levels of induction of RecA protein in E. coli (PQ 10) after treatment with two related carcinogens.

By means of an immunoradiometric assay the induction of protein RecA in E. coli PQ 10 was measured after treatment by two related carcinogens. On an adduct basis N-Acetoxy-N-2-acetylaminofluorene was shown to induce the protein RecA at a similar level as U.V. On the other hand, N-hydroxy-N-2-aminofluorene shows only a poor induction capacity of the RecA protein. The difference in the SOS inducing potential of the aminofluorene and acetylaminofluorene adducts is discussed in relation to the major difference in the local conformational change the two adducts induce in DNA.

2-Acetylaminofluorene↗

PubMed1983

In vivo transcription of a eukaryotic regulatory gene.

The PPR1 gene encodes the positive regulator of the URA1 and URA3 genes in yeast. Its transcription product is a 2.9-kb polyadenylated RNA with an extremely short half-life of 1 min. The induced or non-induced cell contains approximately 0.1 molecules of PPR1 RNA, a constitutive level which is not altered by changing the number of structural genes to be regulated. The DNA sequence of a 399-bp AccI-Bg/II fragment including 180 nucleotides of the 5'-flanking region of the gene PPR1 has been determined. By S1 mapping we present evidence that the 5' non-coding region of the PPR1 mRNA is heterogeneous in length. The 5' termini of the different RNAs map 20, 36, 45 and 50 nucleotides upstream from the translation start codon. The sequence indicates that the only open translation phase begins with an AUG codon that is preceded by two out-of-frame AUG triplets. This particular structure of the 5'-terminal sequence of the transcripts of PPR1 is discussed in relation to both their stability and translational efficiency.

Base Composition↗

PubMed1983

Enzymatic methylation of chicken erythrocyte DNA modified by two carcinogens, 2-(N-acetoxyacetylamino) fluorene and methylnitrosourea.

Both DNA-AAF and MNU-alkylated DNA are methylated less than nonmodified DNA by rat brain nuclei cytosine 5-methyltransferase purified either by chromatography on DEAE cellulose or by Dyematrex. The inhibition of methylation is proportional to the modification of the DNA, and DNA having a given percentage of bases modified with MNU is less methylated than DNA modified to the same extent with AAF. Moreover, DNA-AAF irreversibly inhibits the methylation of native DNA, whereas MNU-alkylated DNA does not inhibit the methylation of native DNA. The AAF-substituted DNA has a higher affinity for the enzyme than native DNA. However, this is probably not due to the AAF-induced local destabilization of the DNA helix, since heat-denatured DNA shows a lower affinity for the enzyme than double-stranded DNA. Addition of DNA-AAF to the enzyme preincubated with native DNA inhibits methylation, but only after a lag period. This agrees with the model in which the methylase walks along the strand to methylate cytosine residues before being detached from the DNA. AAF bound to guanine residues may block the movement of the enzyme along the helix. The in vitro hypomethylation of DNA, caused by carcinogens, could explain the in vivo observations made by several authors and could have significance in gene activity, cellular differentiation, and oncogenesis.

2-Acetylaminofluorene↗

PubMed1983

Analysis at the sequence level of mutations induced by the ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene.

The covalent binding of an ultimate carcinogen to the DNA bases or phosphate groups creates a premutational lesion that in vivo is processed by the repair, replication and recombination enzymes, and eventually may be converted into a mutation. Being interested in the way that an initial premutational event is converted into a stable heritable mutation, we have sequenced stable mutations in a gene that has formed covalent adducts in vitro with N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF, a model for the ultimate metabolite of the rat liver carcinogen 2-acetylaminofluorene, AAF). In vivo studies have shown the mutagenicity of AAF and its derivatives in both bacterial and eukaryotic systems. N-AcO-AAF reacts in vitro with DNA leading mainly to the formation of a guanine adduct, N-2-(deoxyguanosin-8-yl)-acetylaminofluorene (80%) and to at least three minor adducts. Studies by our group showed that binding of N-AcO-AAF to DNA resulted in a local distortion of the DNA helix around the C-8 adduct (the insertion-denaturation model). We describe here the analysis of forward mutations induced in the tetracycline-resistance gene of pBR322 by directing the chemical reaction of the carcinogen to a small restriction fragment (BamHI-SalI) inside the antibiotic-resistance gene. Mutants are selected for ampicillin (Ap) resistance and tetracycline (Tc) sensitivity. The plasmid DNA of such mutants was analyzed for sequence changes in the fragment where the AAF binding had been directed. We show here that the mutations are mainly frameshifts involving GC base pairs and that certain base pairs (hotspots) are affected at high frequencies.

2-Acetylaminofluorene↗

PubMed1982

Non-random binding of N-acetoxy-N-2-acetylamino-fluorene to chromatin subunits as visualized by immunoelectron microscopy.

The influence of chromatin structure on the accessibility of DNA to the model ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF) was investigated by means of an immunoelectron microscopic technique developed recently. An homogeneous population of core particles or trinucleosomes from chicken erythrocytes, was submitted to electrophilic attack by N-Aco-AAF. After DNA isolation, N-2-acetylaminofluorene (AAF) binding sites were mapped upon the DNA fragments using specific antibodies as a probe. Our results indicate a non-random binding of AAF along the DNA. Our data support the results of previous studies showing a preferential binding on the linker region.

2-Acetylaminofluorene↗

PubMed1982

Repair and mutagenesis of plasmid DNA modified by ultraviolet irradiation or N-acetoxy-N-2-acetylaminofluorene.

Plasmid DNA was modified in vitro to various extents with N-acetoxy-N-2-acetylaminofluorene or UV irradiation. The modified plasmid DNAs were then used to transform Escherichia coli strains having different repair capabilities. Both survival and mutagenesis frequencies of the plasmid were measured as a function of the number of lesions per plasmid molecule. The majority of N-2-acetylaminofluorene (AAF) adducts, like thymine dimers, were repaired by the excision (uvrA+-dependent) pathway. In rec+ strains, dose-dependent mutagenesis occurred in either AAF- or UV-modified plasmid DNA. This is in contrast with results obtained in recA- strains, in which only AAF adducts gave rise to a lower, but dose-dependent, mutagenesis frequency. In these recA- strains there was no UV mutagenesis. Unlike what is observed with phages, induction of the "SOS" functions by UV irradiation of the bacteria prior to transformation did not increase the survival or the mutagenesis of the plasmid.

2-Acetylaminofluorene↗